Abstract

Despite extensive efforts to identify a magic bullet to prevent the occurrence or mitigate the progression of Alzheimer’s disease (AD), no effective single “pill-for-an-ill” strategy has been found, which is likely why on April 28, 2010, an NIH consensus panel on AD prevention pronounced, “There is no good evidence that Alzheimer’s disease or the other forms of dementia affecting millions of Americans are preventable…there are no modifiable issues or variables that are going to prevent Alzheimer’s or cognitive decline, and people should know that.” 

We beg to differ. A clear association has been well documented in the peer-reviewed medical literature between lifestyle habits—diet, intake of specific micronutrients, and exercise--and the occurrence of AD. This review summarizes key, clinically relevant, findings.

Statins—Not the Answer to Alzheimer’s Disease

As noted in Part I of this review, as our understanding of the etiology of AD has deepened, the evidence has become incontrovertible that no single molecular target or pharmaceutical silver bullet will prevent or cure AD. Nevertheless, it has been suggested that, via their cholesterol-lowering effects, statins might lessen risk or ameliorate progression of AD. The latest research, however, indicates that statins are not the solution for AD.

Cochrane reviews, considered the gold standard analysis of the best available information about healthcare interventions, focus primarily on randomized controlled trials (RCTs). In regards to the use of statins for AD prevention, reviewers evaluated the two large RCTs whose results have been published; neither showed any reduction in AD occurrence in patients treated with statins compared to those given placebo. Regarding treatment of AD, results from the large RCTs that have assessed this outcome have not been published, but initial analysis available from these studies indicates statins provide no benefit on the Alzheimer's Disease Assessment Scale Cognitive Subscale.²

Not only do statins provide no benefit in preventing or treating AD, it appears they may do harm. A recently published analysis of results of a University of California-San Diego survey of 171 patients (age range 34-86 years) reporting statin-associated adverse cognitive effects,  determined that cognitive ADRs were probably or definitely related to statin therapy in 128 patients (75%). Of 143 patients (84%) who reported stopping statin therapy, 128 (90%) reported improvement in cognitive problems. Median time to first-noted recovery was 2.5 weeks after statin discontinuation. Notably, in some patients, a diagnosis of dementia or Alzheimer's disease was reversed. Nineteen patients whose symptoms improved or resolved after they discontinued statin therapy, and who then underwent rechallenge with a statin, exhibited cognitive problems again (multiple rechallenges with the same outcome in some).³

Given that, as discussed in Part I of this review, disordered cerebral cholesterol metabolism contributes to AD, why aren’t statins, HMG Co-A reductase inhibitors whose efficacy in aggressively lowering cholesterol is well documented, helpful in combating AD? A number of reasons are laid out in the research:

Cholesterol production typically decreases in AD brains. This is not a good thing! Cholesterol depletion has catastrophic consequences in neuronal culture preparations: it results in loss of vesicles and exocytotic activity, inhibits neurite growth and impairs neuronal survival. Evidence indicates that loss of cholesterol negatively and gravely impacts plasma membrane stability. In addition, the transduction of extracellular signals, essential for neuronal growth and survival, breaks down when levels of cholesterol become insufficient.⁴

When cholesterol is low, Abeta preferentially localizes at the membrane surface and assumes an aggregation-prone structure, but when the membrane contains ~33% cholesterol, the Abeta peptide inserts into the membrane with an altered structure that hinders peptide aggregation.⁵

Cholesterol is an essential component of the plasma membrane of all cells, including neurons, in which it increases plasma membrane rigidity and is found concentrated in lipid rafts together with sphingolipids and gangliosides (two classes of lipids that play important roles in cell signal transduction). In the adult, cholesterol plays key roles in the maintenance of neuronal plasticity and function. In lipid rafts, cholesterol acts as a spacer between sphingolipids and as a dynamic glue that holds the rafts together. Cholesterol removal results in raft disassembly with dissociation, deregulation and/or inactivation of most raft proteins.⁵

One of the proteins affected is plasmin, a raft-resident enzyme generated from an inactive precursor called plasminogen, whose activation occurs within lipid rafts. Plasmin cleaves Abeta peptides at multiple sites, thus preventing their aggregation, and also enhances APP cleavage by alpha-secretase. The lipid raft disorganization resulting from lowering neuronal membrane cholesterol prevents plasmin formation and activity, thus increasing production of Abeta peptides and oligomers.⁶

In the hippocampus of both humans and transgenic mice, only a very small amount of APP and beta-secretase (the enzyme that cleaves APP to produce Abeta) is found in the same membrane environment; with a moderate reduction of brain cholesterol, these co-localized APP and beta-secretase fractions increase markedly in rodents and in AD patients.⁷

Statins lower cholesterol by short-circuiting one of the earliest steps in cholesterol formation, the activity of HMGCoA reductase, thus statins’ action also blocks the synthesis of downstream intermediates with important physiological functions, including production of farnesylpyrophosphate, a precursor of molecules involved in cell signaling and inflammation, and ubiquinone (CoQ10), an antioxidant molecule that also plays a key role as an electron carrier in the mitochondrial electron transport chain. (In addition, via their inhibition of cholesterol, statins also short circuit synthesis of the entire class of steroid hormones, including DHEA and testosterone. A drop in either hormone may also contribute to loss of cognitive function.) Despite statins’ claim to anti-inflammatory effects in the vasculature, these actions promote oxidative stress and inflammation in the brain.

Another neuroprotective outcome of cholesterol synthesis in the brain occurs at the final step of the pathway in which desmosterol is reduced to cholesterol, a reaction catalyzed by the product of the Dhcr24 gene, also known as Seladin-1 (SELective Alzheimer’s Disease INdicator-1). In addition to participating in cholesterol synthesis, Seladin-1 has anti-apoptotic and neuroprotective effects against oxidative and oncogenic stimuli, and is directly involved in estrogen-mediated neuroprotection. Inhibition of cholesterol synthesis further down-regulates expression of Seladin-1, whose levels are already down-regulated in the AD brain.⁵⁸

Finally, the cholesterol metabolites 24SOH-Chol and 27OH-Chol appear to regulate the activity of alpha-secretase and beta-secretase. 24SOH-Chol, the main cholesterol metabolite in the brain, reduces beta-secretase activity while increasing alpha-secretase activity.⁹

For all the above reasons, further lowering cholesterol and inhibiting its metabolism in the brain will promote rather than lessen Alzheimer’s disease, except possibly in one group of individuals—those who are carriers of the ApoE4 allele.

Statins Promote Insulin Resistance in ApoE2 and E3 Carriers, But May Benefit Those with ApoE4

It has also recently been revealed that statin users who are not carriers of the ApoE4 allele have greater insulin resistance (as indexed by greater HOMA-IR values) than ApoE4 carriers taking statins, or ApoE2 or ApoE3 carriers who are not statin users.¹⁰ These results suggest that statins could contribute to insulin resistance (and therefore risk of AD) in ApoE2 and ApoE3 carriers, but might provide protection against AD in ApoE4 carriers.

This relationship among statins, insulin resistance, and APOE genotype is likely related to the effects of each on lipid metabolism:

As noted in Part I of this review and immediately above, normal cerebral homeostasis is disrupted in AD.  ApoE is a key cholesterol transport molecule in the periphery and the primary cholesterol carrier in the brain. Lipid efflux from neurons is significantly impacted by ApoE isoform. The ability of the ApoE2 and ApoE3 isoforms to induce lipid efflux, and therefore support normal cholesterol metabolism in the brain, is 2.5- to 3.9-fold greater than that of ApoE4. One of the key ways in which ApoE4 increases risk of AD is by reducing the rate at which cholesterol is recycled in the brain.¹¹

In both the brain and the periphery, insulin is a primary regulator of lipid metabolism, stimulating lipogenesis and reducing lipolysis. In adipocytes, insulin resistance results in increased lipolysis. Thus insulin resistance produces increased levels of free fatty acids whose influx into the liver then inhibits insulin’s suppression of hepatic very low-density lipoprotein (VLDL) secretion, which normally quickly curtails post-prandial hyperlipidemia.

Insulin’s acute inhibitory effects on VLDL production allow the liver to rapidly restore levels of postprandial plasma lipids to an optimal physiologic range. In insulin-resistant individuals, however, insulin’s check on VLDL production is removed, so they experience higher and longer post-prandial increases of VLDL and other lipids with artery-damaging potential.

Epidemiological studies show increased peripheral lipid levels in mid-life can increase risk of AD  2- to 3-fold,  which partly explains why ApoE4 carriers, who, even though they are less likely to be insulin resistant (as discussed in Part I of this review ¹²), have higher total and LDL cholesterol levels than non-ApoE4 carriers, and are at increased risk for AD.

In sum, statins may increase AD risk in non-ApoE4 carriers by reducing brain cholesterol levels as well as increasing insulin resistance in these individuals. Yet, in carriers of the ApoE4 allele, statins may reduce risk of AD by lowering peripheral total and LDL cholesterol in this genotype.

Don’t Be SAD, Tour the Mediterranean

In the last few years, research into the diet and nutrition relationship to disease has focused on dietary patterns instead of single nutrients for two key reasons: important interactions occur among components of a diet, and, more importantly, people do not eat isolated nutrients. Thus, researchers have developed dietary scores to estimate adherence to particular dietary patterns and their relationships to disease. One dietary pattern has consistently been associated with significantly reduced incidence of chronic degenerative diseases, including AD: the Mediterranean diet.¹³

Defining the Mediterranean Diet

First of all, “diet” in its typical connotation is a misnomer when applied to the Mediterranean diet, which is not a specific food regimen, but a collection of eating habits traditionally followed by people in the different countries bordering the Mediterranean Sea. Characterized by high consumption of vegetables, fruit, legumes, and complex carbohydrates (whole grains); moderate consumption of fish; the use of olive oil as the main source of fat; and low-to-moderate consumption of alcohol in the form of a little red wine with meals, this eating pattern has been reported to provide an optimal intake of antioxidant vitamins, polyunsaturated fats, and other beneficial nutrients for the prevention of chronic degenerative diseases, including AD.¹⁴ 

From the numerous recent studies investigating the association between nutrition and AD, the unsurprising discovery is that the Standard American Diet (SAD) promotes AD while a Mediterranean-style diet delivers neuroprotection against all forms of cognitive decline, including AD. The latest research confirming the Mediterranean diet’s cognitive benefits follows earlier studies indicating this dietary pattern is optimal for the prevention of key risk factors for AD, including cardio- and cerebro-vascular diseases (atherosclerosis, hypertension, stroke); disorders involving insulin resistance (e.g., MetS and type 2 diabetes); and obesity.

A recent meta-analysis involving 12 studies including a total of 1,574,299 subjects, followed for time periods ranging from 3 to 18 years, investigated the association between adherence to a Mediterranean diet and health status. The finding: greater adherence to a Mediterranean diet is associated with substantial improvement in health status, as indicated by significant reductions in overall mortality (9%), mortality from cardiovascular diseases (9%), incidence of or mortality from cancer (6%), and incidence of Parkinson's disease and Alzheimer's disease (13%).¹⁴

The Mediterranean Diet’s Anti-inflammatory / Anti-Alzheimer’s Effects

One reason for these healthy statistics, also discussed in the above meta-analysis, is that greater adherence to the Mediterranean diet is associated with beneficial effects on numerous inflammatory and coagulation markers (including significantly lower levels of homocysteine, C-reactive protein, interleukin-6, white blood cell count, and fibrinogen), lipids, and blood pressure – all important risk factors for cardiovascular diseases and for AD.

The Mediterranean diet’s anti-inflammatory effects have recently been further confirmed in a multicenter longitudinal study involving 1,003 myocardial infarction survivors from several European regions. In this study, each unit of increasing adherence to the “Mediterranean diet score” [further discussed below] correlated with a reduction of 3.1% in mean levels of C-reactive protein and of 1.9% in mean levels of interleukin-6.¹⁵

Homocysteine

Homocysteine serves as a good example of the ways in which the Mediterranean diet lessens AD risk. The presence of high levels of this intermediate compound of the methylation cycle is a common finding in patients affected by cognitive impairment and AD.¹⁶¹⁷ 

High homocysteine levels are associated with increased risk of cardiovascular and cerebrovascular ischemic diseases, which are well recognized risk factors for AD.¹⁸   Insufficiencies of B6, B12 or folic acid, all of which are necessary cofactors for homocysteine metabolism, have also been correlated with increased risk of cognitive decline and AD.^18192021

In vitro studies have reported that homocysteine and deficiencies of folic acid impair DNA repair in hippocampal neurons, increasing susceptibility to toxicity from beta-amyloid protein.²²  Homocysteine also potentiates the accumulation of soluble beta-amyloid in the brain, thus contributing to AD both indirectly via vascular damage and directly as a neurotoxin.  Homocysteine is corrosive, degrading the main structural components of the artery: collagen and elastin.²⁴²⁵  In both the vasculature and brain, homocysteine and the product of its spontaneous oxidation, homocysteic acid, activate NMDA receptors, increasing intracellular levels of ionized calcium and ROS.²⁶  

 The Mediterranean diet has been shown to significantly lower levels of homocysteine—an unsurprising outcome since this dietary pattern provides not only a variety of foods rich in B vitamins, but polyphenolic antioxidants, including resveratrol (in red wine and grape skins) and hydroxytyrosol (one of the main phenols in extra virgin olive oil), which have demonstrated protective effects against homocysteine-induced vascular damage.²⁷²⁸

Neuroprotective Fats

The brain is highly susceptible to oxidative damage because of its high metabolic rate and its abundance of oxidizable material, e.g., the polyunsaturated fatty acids that form the plasma membranes of neural cells.  AD is characterized by a chronic inflammatory process around amyloid plaques, and increased levels of free radicals and pro-inflammatory cytokines are present in the brain of patients affected by AD. 

Thus the SAD, with its typical pro-inflammatory omega-6:omega-3 ratio of ≥20:1, promotes AD, while the Mediterranean diet, which produces an anti-inflammatory omega-6:omega-3 ratio of 2:1 and has been shown to substantially lower the plasma ratio of omega-6 to omega-3 fatty acids within as little as 4 weeks, reduces oxidative stress and inflammation, lessening risk of AD.²⁹³⁰³¹

Olive oil, the hallmark source of fat in the Mediterranean diet -- albeit a monounsaturated fat (MUFA), whose main component (55-83%) is the omega-9 fatty acid, oleic acid, rather than a polyunsaturated fat (PUFA) -- has also recently been associated with lowered risk of cognitive decline in a French three-city study involving 6,947 subjects followed for 4 years.  Mechanistic explanations, in addition to the anti-inflammatory effects of olive oil phenols noted above, are provided by two recent papers: an article reporting that a virgin olive oil based breakfast repressed in vivo expression of several pro-inflammatory genes in mononuclear cells from patients with MetS , and the summary of the II international conference on olive oil and health. This consensus report references studies indicating that MUFA-rich membranes, although less fluid than those formed with PUFA,  are also less vulnerable to oxidative damage, and suggests this translates to olive oil’s beneficial effects in maintaining the structural integrity of mitochondria and neuronal membranes, thereby promoting healthy neuronal transmission. ³⁶

A Glass of Red Wine

Another hallmark of the Mediterranean diet is a small amount of alcohol in that daily glass of red wine enjoyed with meals. In addition to the resveratrol provided by red wine (which improves blood flow to the brain,³⁷ and delivers protection against AD both via its direct antioxidant effects and via activation of sirtuin 1³⁸), elderly individuals who are not carriers of the ApoE4 allele also benefit from the light-to-moderate intake of alcohol, which is associated with a 45% reduction in AD risk. 

Several papers have now reported likely molecular mechanisms for these benefits: in vitro, moderate ethanol preconditioning of hippocampal slices interferes with various glial-mediated neurotoxic responses to Abeta, and also causes an almost 3-fold increase in brain levels of heat shock protein 70, a protective molecular chaperone that inhibits induction of superoxide radical.⁴⁰⁴¹⁴²

ApoE4 carriers can still benefit from phenolic-rich grape juice, which another recent paper indicates has comparable beneficial postprandial effects on endothelial function (specifically, flow mediated dilation) to red wine.⁴³

Where the Tire Meets the Road—Estimating Patient Adherence

The adherence score seen in the research (originally proposed by Trichopoulou et al.⁴⁴) was developed to enable researchers to estimate subjects’ global dietary pattern in relation to the typical characteristics of the Mediterranean diet. A value of 0 or 1 is assigned to each dietary component, whether protective/Mediterranean or potentially harmful/SAD. For each of the beneficial food groups (vegetables, fruits, legumes, whole grains, fish, moderate intake of red wine during meals, and the ratio of daily consumption of monounsaturated fats [from olive oil, nuts and seeds] to saturated fats), a value of 1 is assigned when the subject consumes more than the median of the population. A 0 value is assigned to individuals whose consumption is below the median. Concomitantly, for food categories considered detrimental (meat, especially red and processed meats, and full fat dairy products), a value of 1 is assigned to subjects whose consumption is below the median, and a 0 value is given to individuals whose consumption is above the median. The resulting score gives an indication of each individual’s adherence to the Mediterranean diet, which ranges from 0 (non-adherent) to 7-9 points (high adherence).

In your clinic, similar logic can be applied to help patients’ evaluate their adherence to a Mediterranean-type diet; however, since fewer than 1 in 10 Americans meet their calorie-specific MyPyramid fruit or vegetable recommendations (only 2.2% of men ≥19 years and 3.5% of women ≥19 years meet recommendations for fruit and vegetable consumption—a paltry 2.5 cups of vegetables and 2 cups of fruit in a 2,000 Kcal daily diet,{re45}  being above the median will not suffice for prevention of AD. Thus following chart is an adaptation of the adherence score used in the research, and is provided as a simple aid in assisting patient awareness of the extent to which they are following a Mediterranean-type diet.

Mediterranean Diet Reduces Risk of Alzheimer’s Disease: Epidemiological Research

The possible association between the Mediterranean diet and reduced risk of AD is supported by a number of recent studies conducted by Scarmeas et al.{re44}  In 2006, this group first published the results of their research on the effects of the Mediterranean diet and risk of AD in a community-based study involving 2,258 non-demented elders in New York, New York. Subjects were prospectively evaluated then followed for an average of 4 years. Higher adherence to the Mediterranean diet, i.e., being in upper third tertile of the adherence score, was associated with a 40% lower risk for AD.⁴⁶

This result was confirmed by a further analysis of the data, a case-control study nested within the original cohort that investigated whether vascular risk factors (previous stroke, previous heart disease, diabetes, hypertension, lipids) lessened the protective effect of adherence to a Mediterranean diet.  The positive association was found to be even stronger—a 68% reduction in AD risk among those in the highest tertile of adherence.⁴⁷

In 2007, Scarmeas et al. published another paper in which they examined the association between adherence to the Mediterranean diet and mortality among the 192 subjects identified as having AD at the onset of the original study. Of these AD patients, 82 died during the following 4.4 years; however, patients with AD in the highest tertile of adherence to Mediterranean diet were 73% more likely to survive compared to those in the lowest tertile—a finding that strongly suggests adherence to a Mediterranean diet affects not only the risk of developing AD, but also the subsequent course of the disease.⁴⁸

Additive Effect of Physical Activity and the Mediterranean Diet

The benefits of physical activity in reducing risk of cognitive decline (28% reduction in risk) and AD (45% reduction in risk) have been recently confirmed by a meta-analysis that included 16 prospective studies.⁴⁹  But results are even better when physical activity is combined with adherence to the Mediterranean diet.  Analyzing data gained from their prospective study of community-dwelling elders in New York, Scarmeas et al. found that physical activity plus higher adherence to Mediterranean diet have an additive effect. The combination results in a hazard ratio (HR) for AD of 35% versus an HR of 40% in those following a Mediterranean diet but engaging in little physical activity.⁵⁰

Adherence to the Mediterranean diet has also been found to lessen risk of mild cognitive impairment—an early symptom of neurological dysfunction whose eventual outcome, if untreated, is likely to be AD, just as insulin resistance is an early warning sign of impending type 2 diabetes. Another study, again conducted by Scarmeas et al., identified an association between higher adherence to a Mediterranean diet and mild cognitive impairment in a population of 1,393 cognitively normal participants, 275 of whom developed mild cognitive impairment during 4.5 years of follow-up. Being in the upper third tertile of adherence to a Mediterranean diet was highly protective against developing mild cognitive impairment (and by implication AD), reducing risk by 48%.⁵¹

In their most recent paper, entitled “Food Combination and Alzheimer Disease Risk: A Protective Diet,” Scarmeas et al. evaluated dietary patterns in terms of their content of 7 potentially AD-related nutrients: saturated fatty acids, monounsaturated fatty acids, omega-3 polyunsaturated fatty acids, omega-6 polyunsaturated fatty acids, vitamin E, vitamin B12, and folate. What emerged was a dietary pattern strongly associated with lower AD risk. This dietary pattern was characterized by higher intakes of cruciferous vegetables, dark and green leafy vegetables, tomatoes, fruits, salad dressing, nuts, fish and poultry, and a lower intake of high-fat dairy products, red meat, organ meat, and butter. Sound familiar? Compared with subjects in the lowest tertile of adherence to this dietary pattern, the AD hazard ratio for subjects in the highest tertile was 0.62—i.e., a 38% reduction in risk for AD.⁵²

Research presented at the Experimental Biology Meeting, held in Anaheim, Calif., April 26, 2010, also showed adherence to a Mediterranean diet slowed age-associated mental decline. Christy Tangney, PhD, and colleagues at Rush University Medical Center, followed 3,790 men and women in the Chicago Health and Aging Project (average age 75) for 7 years. Subjects answered a food-frequency questionnaire, spelling out in detail which components of the Mediterranean diet they ate and how often, and took a battery of tests every 3 years to evaluate mental functions, such as short- and long-term recall. Those with high adherence to the Mediterranean diet scored younger—the equivalent of 2 years younger than their age--in mental function. "The beauty of the finding,” Tangney is quoted as telling WebMD, “is that following the diet perfectly isn't necessary to get a brain-protective effect. When someone incorporates a diet rich in fruits and vegetables and non-refined grains, such as cereals and breads, and breaks it up with a little wine, there appears to be at least some protection against cognitive aging."⁵³   Not too onerous a prescription.

The Alzheimer's Interstate   (Right side of chart above)

(X) Environmental factors:

Standard American Diet: three problems—too much refined carbohydrate, saturated and trans fats AND pesticides, AND not enough micronutrients (e.g. insufficiencies of virtually all micronutrients, including vitamin A, vitamin D, vitamin K, omega-3s, B vitamins, & all the minerals including magnesium, calcium, zinc, selenium, potassium, etc.)

Couch Potato Lifestyle: combined with SAD exponentially promotes accumulation of visceral adipose tissue (VAT – belly fat), which is highly pro-inflammatory

Environmental toxins, e.g., pesticides (atrazine), BPA, POPs—all dysregulate endocrine function, promoting obesity, VAT, inflammation

Food intolerance, e.g., wheat, dairy, soy—provokes defensive immune response à inflammation

Genetic susceptibility, e.g., ApoE4

The above result in Cardiovascular and Endocrine Dysfunction as indicated by:

Unhealthy lipid profile: High Peripheral LDL Cholesterol / Low HDL Cholesterol levels

High Triglycerides

Hypertension (endothelial dysfunction)

Insulin resistance

Metabolic Syndrome

Diabetes

Obesity

(Y) Cardiovascular and Endocrine Dysfunctions promote:

Any and all of the above diseases/conditions promote Pro-inflamatory Metabolism, which causes à Leaky blood brain barrier à which opens up 2 pathways to AD:

1. (Z1) Insulin resistance in periphery à hypometabolism in brainà increased production of ROS, RNS in periphery and brainà promotion beta-secretase activity à promotion Abeta formation àAD
2. (Z2) High LDL/low HDL cholesterol in periphery à increased production of ROS, RNS in periphery à influx of Abeta-carrying LDL into the brain à dysregulation cholesterol homeostatis in brain à promotion beta-secretase activity à promotion Abeta formation àAD

End result: ↑ Alzheimer’s disease

Getting Off the AD Interstate (Left side of chart above)

Healthy Brain

(X) Mediterranean Dietàlow glycemic, low sat and trans-fat, high fiber, high omega-3, high micronutrients, e.g. B vitamins, myriad protective phenols (eg, resveratrol in red wine, vitamin E and hydroxytyrosol, tyrosol, and oleuropein in extra virgin olive oil)àall of which promote healthy lipid profile and good insulin sensitivityà a non-inflammatory metabolism

If avoid environmental toxicants (organic, use glass or stainless steel not  BPA containing plastics) à less inflammation, less likelihood of endocrine dysruption

 (Cautions—supply of vitamin A may not be adequate  if one cannot convert carotenoids to retinol; supply of vitamin D may not be adequate depending on latitude and skin color)

(Y) Supplemental nutrientsà needed because: (1) a variety of SNPs result in increased need: SNPs have been identified for vitamin A, vitamin D (or, in the case of vitamin D, if a person has dark skin or lives in areas located at higher latitude), vitamin K, B vitamins. (2) because foods, especially conventionally grown foods, supply fewer micronutrients than they did 50 years ago, and (3) To restore mitochondrial efficiency in aging individuals, eg., CoQ10, N-acetyl cysteine, lipoic acid AND lastly, to help restore mitochondrial function in those experiencing brain hypometabolism -- Medium chain triglycerides/ketones.

(Z) Daily exercise improves endothelial and mitochondrial function

The above support Optimal Cardiovascular and Endocrine Function as indicated by:

Healthy lipid profile: Total Cholesterol >200 / LDL > 130 / HDL ≤ 60

Triglycerides >150 mg/dL

Blood pressure >120/80 mm Hg

Insulin sensitive fasting insulin > 10 microU/mL

BMI within in range of 18.5 – 24.9

Normal cardiovascular and endocrine function promotes:

Anti-inflammatory metabolism à minimally permeable blood brain barrier

Insulin sensitivity and good endothelial function à normal glucose delivery and metabolism in the brain

Optimal cholesterol recycling / homeostasis in the brain

↑Alpha secreatase activity à ↑ neuroprotective APPsα

↓ Beta-secretase activity à ↓ Abeta

End Result: ↓ Alzheimer’s disease

The Spanish Ketogenic Mediterranean Diet

For those willing to go the extra dietary mile (or those at increased risk of AD, e.g., ApoE4 carriers), what is being called the Spanish Ketogenic Mediterranean Diet is likely to provide even more anti-AD bang for the dietary buck.⁵⁴

Mildly ketogenic diets help preserve muscle mass, reduce appetite, favor increased fat loss, promote a non-atherogenic lipid profile, lower blood pressure and decrease insulin resistance. In contrast, it now appears that high carbohydrate diets, particularly when composed largely of refined carbohydrates (e.g., the SAD), are associated with: low levels of HDL; high levels of triglycerides, LDL and total cholesterol; MetS, type 2 diabetes, essential hypertension and cancer.⁵⁴

In Spain, fish is an important protein source, thus the "Spanish Ketogenic Mediterranean Diet" whose effects were studied in 31 obese subjects, relied upon fish as the main source of protein, while using virgin olive oil as the principal source of fat and green vegetables and salads as the main source of carbohydrates, along with moderate red wine intake. Calories were unlimited, but subjects were encouraged to consume per day:

• A maximum of 30 grams of carbohydrates in the form of green vegetables and salads (2 portions of salad vegetables [such as alfalfa sprouts, lettuce, escarole, endive, mushrooms, radicchio, radishes, parsley, peppers, chicory, spinach, cucumber, chard and celery]), and 1 portion of low-carbohydrate vegetables (such as broccoli, cauliflower, cabbage, artichoke, eggplant, squash, tomato and onion). Allowable salad dressings were olive oil, vinegar, lemon juice, garlic, salt, herbs and spices.
• A minimum of 30 ml (=1 ounce or 2 tablespoons) of virgin olive oil distributed as 10 ml (2 teaspoons) at breakfast, lunch and dinner.
•  200–400 ml (6-12 ounces) of red wine distributed as 100-200 ml (6-8 ounces) at lunch and dinner.
• Unlimited protein in the form of fish 4 days each week (all types except longer-lived predators, swordfish and shark due to concerns re mercury), and meat, fowl, eggs, shellfish and cheese on the remaining 3 days.
• Trans-fats and processed meats with added sugar were not allowed.
• No more than two cups of coffee or tea and at least 3 litres of water (~12 8-oz glasses) were recommended daily.

While for the average American patient, the Spanish ketogenic diet borders on the draconian, at least some might be willing if given the data on this dietary regime’s highly impressive beneficial impacts on numerous risk factors for AD. These included significant reductions in body weight (108.62 kg--> 94.48 kg), BMI (36.46 kg/m(2)-->31.76 kg/m(2), systolic blood pressure (125.71 mmHg-->109.05 mmHg), diastolic blood pressure (84.52 mmHg--> 75.24 mmHg), total cholesterol (208.24 mg/dl-->186.62 mg/dl), triglycerides (218.67 mg/dl-->113.90 mg/dl—a 47.91% reduction), glucose (109.81 mg/dl--> 93.33 mg/dl), and LDL (114.52 mg/dl-->105.95 mg/dl), and a significant increase in HDL (50.10 mg/dl-->54.57 mg/dl).

Toning Up the AD Brain with Ketones

Exceptional results, such as those seen in the preceding study, are the driving force behind growing research interest in ketones’ potential as protective agents against AD, particularly in carriers of ApoE4.

As noted in Part I of this review, a decline in brain glucose metabolism is seen long before significant amounts of Abeta are present, years before the onset of frank AD (even as early as age 30 in ApoE4 carriers), and brain hypometabolism is a consistent pathological feature of AD. 

Unlike other tissues in the body, the brain does not efficiently metabolize fats, instead relying almost exclusively on glucose as its energy substrate. Thus, chronic cerebral glucose hypometabolism can profoundly affect brain function, and some researchers believe dysfunctional energy metabolism not only precedes but is key to alterations in APP processing that favor Abeta production.⁵⁵⁵⁶

Dysfunctional alterations in lipid/glucose metabolism may be triggered by:

•  Environmental factors, e.g., exposure to the commonly used herbicide, atrazine. Recently published animal research demonstrated that chronic exposure to extremely low levels of atrazine decreased basal metabolic rate, and increased body weight, intra-abdominal fat and insulin resistance without changing food intake or physical activity level. (Reason to suggest your patients eat organic!)⁵⁷
•  The standard American diet. High in refined carbohydrates, the SAD inhibits the use of fatty acids, increases triglyceride levels,⁵⁸ and is also very low in omega-3 fatty acids. The latter results in low levels of DHA in the brain. Insufficient brain levels of DHA promote beta-secretase cleavage of APP, and the presence of less flexible fats in neural plasma membranes may also result in impaired functioning of other proteins key for glucose metabolism, such as glucose transporters.⁵⁹
• Genetic predisposition, e.g., possession of an APOE4 allele. The ApoE4 protein increases triglyceride levels by binding triglycerides much more readily than ApoE2 or 3, while also inhibiting lipolysis.⁵⁸

Supplementing  the normal glucose supply of the brain with ketone bodies (i.e., acetoacetate, beta-hydroxybutyrate, and acetone), which are normally produced from fat stores when glucose supplies are limited, e.g., during prolonged fasting or a ketogenic diet, has demonstrated significant benefit in both animal models of neurodegenerative disorders and in human clinical trials, including trials involving patients with AD.⁶⁰

However, although the ketogenic diet has demonstrated beneficial effects on dyslipidemia and insulin resistance, and specifically in AD, research on ketones and epilepsy indicates that to produce therapeutic levels of ketone bodies, 90% of calories must come from fat. Obviously, such a diet is impractical for chronic use because a regimen this high in fat and low in carbohydrate is unpalatable; compliance will be poor. For this reason, a way in which to obtain high ketone levels, while allowing the patient to eat a relatively normal diet, has been under investigation. Medium chain triglycerides may offer a solution.⁶⁰

Reversing AD-Promoting Brain Hypometabolism with Medium Chain Triglycerides

To date, only one study has been published evaluating the effectiveness of ketone bodies in treating human AD subjects. This study utilized medium chain triglycerides (MCT), unique triglycerides comprised of fatty acid chains between 5 to 12 carbons. Because of their short fatty acid chain lengths, MCT are not treated like long chain fatty acids, but undergo obligate oxidation. If sufficient MCT are ingested, the excess acetyl-CoA produced will generate ketone bodies. Also, importantly, the oxidation of MCT occurs regardless of other macronutrients consumed; thus, MCT use escapes the carbohydrate restrictions necessary for ketone production in a ketogenic diet.

A crossover study examined the effects of acute elevation of serum ketone bodies on cognitive performance in 20 mild to moderate probable AD subjects. A single, 40 gram dose of MCT induced a 10-fold elevation in ketone bodies (specifically, beta-hydroxybutyrate) after 2 hours. Ninety minutes after dosing, subjects were tested for changes in cognitive performance. The single administration of MCT led to a significant correlation between performance on the Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog), a paragraph recall test, and serum beta-hydroxybutyrate concentration, with those subjects presenting the highest beta-hydroxybutyrate levels showing the most improvement. The rapid (90 minute) improvement suggests that the effect is a result of improved neuronal metabolism.⁶¹

Also, in this study, subjects who were not carriers of ApoE4 showed greater improvement in ADAS-Cog scores than those who were ApoE4 carriers, and this outcome has been replicated in a longer, 90-day dosing study.⁶²

Why would MCT work preferentially in subjects who are not carriers of APOE4? Suzanne Craft, PhD, whose research is discussed in Part I of this review, has suggested that differences in insulin sensitivity resulting from Apo E4 carriage status are responsible. Non-carriers of ApoE4 have been shown to have higher fasting insulin levels and lower glucose disposal rates than normal controls, suggesting insulin resistance.⁶³

The insulin resistance of non-ApoE4 AD subjects may also explain their responsiveness to ketone bodies, which are transported into the brain by the monocarboxylate transporter (MCTs) carrier proteins, one of which (MCT1) is widely expressed and found in endothelial cells of the blood brain barrier (BBB). BBB Levels of monocarboxylate transporters are elevated in diabetes and in other conditions in which insulin resistance occurs, such as in the hypometabolic state that precedes and accompanies AD in the brain.

A clinically relevant point: the serum level of ketone bodies reached in the above study is easily achievable, requiring only a single 40 gram dose of MCT, and thus individuals at risk or suffering from AD may gain the benefits of ketosis without draconian dietary changes. However, the research clearly indicates that one cannot remain on the SAD, but must move to the Mediterranean diet to prevent or mitigate progression of AD.

MCTs are naturally present in coconut oil and red palm kernel oil. MCT oil derived from these sources is also available.

From A to Zinc—Optimal Nutrition, the Best Hedge vs. Alzheimer’s?

As triage theory, the latest hypothesis generated by legendary researcher Bruce Ames, PhD, suggests, nutrients are triaged to meet the body’s most pressing survival needs. For example, under scarcity conditions, vitamin K is preferentially shunted to its use in the clotting cascade as K1, rather than converted to K2 in the intestines and used to activate the matrix Gla protein that prevents arterial calcification or carboxylate osteocalcin to build bone.⁶⁴⁶⁵

Even from this necessarily truncated review, it should be apparent that sufficiency of virtually all nutrients needed for optimal lipid and glucose metabolism, and mitochondrial function, plays a role in AD prevention—how important a role being related to an individual’s genetic inheritance of single nucleotide polymorphisms (SNPs). For example, several B vitamin SNPs have been identified that result in higher than RDI need for B6, B12 and folate, all of which are necessary for methylation / homocysteine metabolism, and for choline, which is an essential component of acetylcholine. What this means is that a significant number of individuals are functionally deficient even if consuming the RDI levels of these nutrients—and we know that few Americans are getting RDI levels from the SAD.⁶⁶

Oxygen, although considered free currency, has also been found to be a conditionally essential nutrient in many brain disorders.⁶⁷⁶⁸ Chronic stress causes transient cerebral hypoxia,⁶⁹ which leads to expression of iNOS in the brain, causing microglial activation and increased risk for AD. Stress reduction via physical exercise and meditation may thus provide significant protective benefit against AD. Hypoxia is also associated with hypertension, implying the necessity of healthy endothelial function, which is supported by L-arginine. For a review of L-arginine’s effects on endothelial function, please see: “Longevity Medicine Strategies for Cardiovascular Disease: Closing the Statin Gap in Endothelial Dysfunction and Insulin Resistance Naturally, with L-Arginine and Citrulline: Part I and Part II”

Heavy metal toxicity promotes AD. Both aluminum and mercury bind to thyroid hormone receptors and prevent proper metabolism. In addition to chelation, iodine is needed, both to displace heavy metals and to produce thyroid hormone, which also necessitates selenium. Thyroid hormone regulates APP gene expression. Hypothyroidism is common in the elderly, and the reduced action of thyroid hormone on the APP gene contributes to AD pathology by increasing APP expression*, brain hypometabolism, ROS, RNS, and thus increased levels of Abeta. (*Specifically, T3 represses APP promoter activity by binding to the nuclear T3 receptor (TR). The unliganded receptor increases promoter activity; the effect of T3 binding is to return promoter activity to basal levels.⁷¹)

While one could argue that insufficiency of virtually any nutrient contributes to AD pathology, the most recent research identifies a couple with major impact: vitamins A and D.

Two Vital Anti-Alzheimer’s Micronutrients

“A” Does Not Stand for Alzheimer’s

Vitamin A plays key roles in alpha-secretase production, acetylcholine transmission, and the regulation/inhibition of excessive microglial activation.

Two binding sites for the retinoic acid receptor (RXR) are found just 203 and 302 nucleotides upstream of the gene for the most prevalent alpha-secretase, ADAM-10, and, not coincidentally, vitamin A, specifically retinoic acid (RA), has been shown to significantly upregulate ADAM-10 mRNA levels.⁷²⁷³In cell cultures, direct application of RA to hippocampal slices from vitamin A deficient mice reverses impairment of ADAM-10 transcription and upregulates production of both ADAM-10 and APP, resulting in reduced Abeta formation and increased formation of APPsα, which has neurotrophic and neuroprotective properties. In adult rats, dietary deficiency of vitamin A results in Abeta deposition in cerebral blood vessels.⁷⁴⁷⁵

In humans, vitamin A insufficiency has been identified as a contributing factor in late-onset AD (LOAD), the form of the disease that afflicts 98% of those affected.⁷⁶⁷⁷⁷⁸A number of potential mechanisms, not simply RA’s effect on alpha-secretase production, are likely responsible.

In addition to its impact on alpha-secretase, RA insufficiency downregulates production of acetylcholine transferase, thus inhibiting neurotransmission of acetylcholine, whose impaired release is another hallmark of AD.⁷⁹

Retinoic acid may also help prevent AD via numerous anti-inflammatory effects, which combine to inhibit microglial activation. RA strongly suppresses production of IL-6, a key pro-inflammatory cytokine, and also inhibits amyloid-beta-induced production of TNF-alpha, and thus the expression of inducible NO synthase (iNOS), in microglia—effects that are thought to be mediated via retinoids’ inhibition of NF-kappaB nuclear translocation.⁷⁹

As discussed in the LMR review Vitamin A – Tolerance Extends Longevity, RA is involved in numerous anti-inflammatory activities that promote the development of regulatory T cells, which tune down the inflammatory TH1/TH17 immune response and increase the production of anti-inflammatory cytokines, e.g. 1L-10.

Lastly, it is has just recently been recognized that the vitamin A metabolite, retinol, not only functions as the precursor for retinaldehyde (the universal chromophore in the vertebrate and invertebrate eye) and RA, but itself plays a fundamental role in the mitochondria. Retinol is an essential cofactor for protein kinase C delta, and the PKC-delta/retinol complex signals the pyruvate dehydrogenase complex to increase influx of pyruvate into the Krebs cycle. When cells are deprived of retinol, respiration and ATP synthesis defaults to basal levels. Thus, vitamin A serves as a nutritional sensor with a key role in regulating mitochondrial energy homeostasis.⁸⁰

Vitamin A insufficiency may be a key contributing factor, to the hypometabolism seen in the AD brain. Since reduced cerebro-glucose metabolism can be seen years before AD diagnosis (as noted by AD-researcher Suzanne Craft and discussed in Part I of this review), vitamin A supplementation may help correct this underlying dysfunction and prevent progression to AD.

The recognition of even one of the above facets of vitamin A activity would identify impaired retinoid metabolism as significant in AD. Fortunately, it is one that can be easily resolved by ensuring vitamin A sufficiency. The potentially good news is that, since vitamin A insufficiency is much more common than has been previously thought, it may be a key contributing – and rectifiable -- factor in a large percentage of those afflicted with AD.

Vitamin A insufficiency can result not only from insufficient consumption of foods rich in pre-formed vitamin A (liver is the best source of vitamin A; egg yolk, cow’s milk, butter, cheese and fatty fish provide some) or pro-vitamin A (aka beta-carotene, which is concentrated in carrots, sweet potato, kale, spinach, winter squash, collard greens, cantaloupe, mango), but to recently identified genetic risk factors. Two common SNPs (present in ~25-40% of the population) result in very poor conversion of carotenoids to retinoic acid. For full discussion of these issues plus additional factors that can result in retinoic acid insufficiency, please see our LMR article: Common Genetic Variants and Other Host-related Factors Greatly Increase Susceptibility to Vitamin A Deficiency. For an in-depth discussion of vitamin A’s effects on immunity, relationship to vitamin D, and recommendations regarding assessment of patients’ vitamin A status, dosage and safety issues, please see our review: Vitamin A -- Tolerance Extends Longevity.

Vitamin D: Illuminating the Brain’s Golden Years with Supplemental Sunshine

Vitamin D insufficiency correlates with increased risk for AD. In a study of 318 elders receiving home care from 2003 to 2007, vitamin D [25(OH)D3, aka calcidiol] insufficiency (≤20 ng/mL) was associated with more than twice the odds of all-cause dementia (odds ratio [OR] = 2.3), Alzheimer disease (OR = 2.5) and stroke (with and without dementia symptoms) (OR = 2.0).⁸¹

Examination of a cross-sectional group of 80 participants, 40 with mild AD and 40 non-demented persons selected from a longitudinal study of memory and aging, found vitamin D deficiency (<20 ng/mL) was associated an 11.69 odds ratio for an active mood disorder and significantly impaired cognitive performance.⁸²

A number of studies have also revealed comorbidity of AD and osteoporosis, and a recent paper discussed the finding that blood biomarkers of osteoporosis (C-terminal collagen fragments and osteocalcin) are significantly increased in AD patients.⁸³⁸⁴

Vitamin D’s anti-inflammatory effects, which in the brain include down-regulating microglial activation, may provide one mechanism for this connection. Inflammation activates osteoclasts, and thus plays an important role in osteoporosis. The AD brain exhibits significant inflammation, with microglial activation as the driving force provoking its inflammatory cascade.⁸⁵⁸⁶⁸⁷

Abeta protein activates microglia, which produce ample quantities of peroxynitrite and other oxidants, cytokines, and pro-inflammatory prostaglandins that hit neurons with a double destructive whammy: they boost Abeta production and induce apoptosis. Peroxynitrite also increases neural sensitivity to excitotoxicity, and potentiates the direct toxicity of Abeta to neurons.

Activated microglia may also adversely impact neuron function and survival less directly by impairing astrocyte function. Healthy astrocytes sequester glutamate, thus protecting neurons by alleviating excitotoxicity. Peroxynitrite and microglial-derived cytokines have been shown to impair glutamate uptake by astrocytes. Furthermore, peroxynitrite and PGE2 (a prostaglandin derived from the omega-6 fatty acids rampant in the standard American diet) increase neurons’ susceptibility to excitotoxic cell death.⁸⁸

Microglial activation also induces expression of the 1-alpha-hydroxylase that converts 25(OH)D3 to its active hormonal form. Microglial cells express the vitamin D receptor, and the binding of 1,25(OH)D3 (aka calcitriol) suppresses the expression of iNOS by microglial cells exposed to inflammatory activators. (The research has used lipopolysaccharide (LPS), an endotoxin that induces a strong immune response and to which humans are particularly sensitive.)

In addition, calcitriol boosts astrocyte production of a protective neural growth factor called glial-derived neurotrophic factor (GDNF), which protects dopaminergic neurons of the substantia nigra. 1α-Hydroxylase, the enzyme that converts 25(OH)D3 (calcidiol) to the active hormone, 1,25(OH)D3 (calcitriol), is only expressed by activated, but not quiescent, microglia, and activated microglia generate calcitriol when incubated with 25(OH)D3, suggesting invocation of a self-regulatory inhibition of the inflammatory cascade—if sufficient vitamin D is present.

Conclusion

While the research evidence rarely, if ever, reaches a “the sun is likely to rise tomorrow” level, surely the evidence presented in this review provides rational grounds to argue that the fear, pessimism and victim mentality espoused by the NIH consensus panel on AD prevention is unfounded, and worse, may exert a nocebo effect on patient health outcomes. We now have a level of insight into the underlying causes of AD pathology that reveals late-onset AD is largely due to diet and lifestyle practices, which are modifiable and highly responsive to dietary and supplement interventions.⁸⁹

Please read Part I: Alzheimer’s and Atherosclerosis - Siblings in a Dysfunctional Family: Part I

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Amyloid precursor protein (APP) is the transmembrane protein that, if cleaved by the enzyme beta-secretase, produces amyloid beta (the histopathological hallmark of Alzheimer’s disease [AD]). If acted upon by the enzyme alpha-secretase, however, APP cleavage produces neuroprotective compounds.  What determines which enzyme will gain access to APP? The answers to this question reveal connections among cardiovascular disease, neurological dysfunction and nutrient insufficiencies that provide the rationale for new therapeutic strategies to prevent AD.

Part I of this review discusses recent evidence that Alzheimer’s disease and atherosclerosis are siblings. Amyloid plaque and atherosclerotic plaque have much in common. Both are damage control responses to high cholesterol in a pro-inflammatory environment, the result, in many individuals, of insulin resistance.  Amyloid beta (Abeta) plays an apolipoprotein-like role in helping to maintain cerebral cholesterol homeostasis, and Abeta peptides are found in atherosclerotic plaque. In both brain and arteries, soluble Abeta peptides upregulate inducible nitric oxide synthase (iNOS), leading to increased production of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Exacerbated ROS/RNS production results in macrophage activation in the arteries and glial activation in the brain, both of which promote beta-secretase processing of APP and Abeta formation—a potentially vicious cycle. In contrast, alpha-secretase and the metabolic conditions that promote its activation and access to APP—insulin sensitivity, good mitochondrial and endothelial function; a healthy lipid profile, and key micronutrients, including the lead actors necessary for oxidative phosphorylation, vitamin D and retinoic acid—are highly neuroprotective.

Part I of this review summarizes the latest research, which reveals that AD is the ultimate outcome in the brain of systemic vascular and glycemic dysregulation. Awareness of what goes awry promotes optimism along with new understanding of the mechanisms underlying dietary and micronutrient modulation, the topic of Part II of our review. The causes of AD are neither mysterious nor untreatable: Alzheimer’s disease is not the inescapable fate of the aging brain.

Amyloid Precursor Protein Processing: Promoting or Preventing Alzheimer’s Disease

Amyloid precursor protein (APP) is a transmembrane protein concentrated in the plasma (outer) membrane of brain cells, including neurons, glial cells, the cells lining the perivascular channels that drain into the CNS, and the endothelial cells of the blood brain barrier. Not only is APP one of the most abundant proteins present in the CNS, it is also ubiquitously expressed in peripheral tissues including muscles, epithelial tissue, and circulating cells. Platelets represent the most important peripheral source of APP and contain APP concentrations equivalent to those found in brain.¹

APP can be cleaved and processed by either alpha- or beta-secretase enzymes. In both cases, the initial cleavage is followed by an additional cleavage by gamma-secretase. All tissues and cells containing APP, including platelets, also contain alpha, beta-secretase and gamma-secretases. Thus, Abeta can be formed and stored within platelets, then released upon platelet activation. (Not only are platelets essential for primary hemostasis and repair of the endothelium, they also play a key role in the development of atherosclerotic plaques; platelets are a source of inflammatory mediators, and platelet activation by inflammatory triggers is a significant contributing factor in cerebrovascular disease.²

Alpha-secretase is protective not only because it cleaves APP inside the amylolid beta (Abeta) sequence, splitting this sequence apart and thus preventing the formation of Abeta peptides, but also because it releases a soluble fragment called APPsα that is neuroprotective. Beta-secretase cleavage frees the entire Abeta sequence, producing Abeta peptides which, if not quickly cleared when produced in or delivered to the brain, become potent neurotoxins that ultimately self-aggregate and deposit as amyloid plaque.³

A substantial amount of research has been conducted on ADAM-10, a member of the ADAM family of proteins (ADAM =a disintegrin and metalloproteinase) that, among other functions, acts as an alpha-secretase and has been shown to prevent amyloid plaque formation in an Alzheimer’s disease mouse model.⁴ In mice bred to over-express APP in neurons, concomitant over-expression of ADAM-10 causes increased production of APPsα, reduced formation of amyloid plaque, and a reduction in cognitive deficits. In contrast, in mice bred to possess an inactive ADAM-10, its lack results in Alzheimer-like pathology.⁵

Levels of alpha-secretase (ADAM-10) and APPsα are reduced in the cerebral spinal fluid (CSF) of Alzheimer’s disease (AD) patients compared to controls. Thus, researchers have begun to ask, “What lessens alpha-secretase production or activity, and what might increase it?” Surprising discoveries appearing in the evolving research on APP and Abeta provide potentially therapeutic answers.

Be forewarned: many new pieces of the puzzle are emerging. What may, in the following discussion, initially appear to be disparate topics will meld together by this article’s end to present a deeper understanding of Alzheimer’s pathophysiology that enables proactive, effective prevention.

Abeta: Good Intentions, Gone Awry

Abeta, a readily self-aggregating peptide that produces severe neurotoxicity, particularly in its initial form as a soluble monomer or oligomer*, is eventually deposited in brain tissue interstices in plaques, which, recently styled “the cobwebs of the brain,” are the histopathological hallmarks of AD.⁶ (*It is now generally recognized that mature amyloid fibrils/plaques are virtually devoid of toxicity to cells; it is their unstable precursors, the oligomers or prefibrillar aggregates of Abeta peptides, that are extremely toxic.)⁷ For a detailed discussion of this topic, please see the LMR review titled: Alzheimer’s Disease: a 21st Cenury Epidemic.

Although Abeta is widely known only for its infamous role as the driving pathological persona in AD, seemingly invented by Nature in order to ruin the aging brain, the impetus for its increased cerebral production is cholesterol homeostasis. New research shows that Abeta’s physiological raison d’etre is to help recycle cholesterol within the brain as well as promote its efflux to the systemic circulation. Increased Abeta production is part of a protective response through which the brain attempts to lower its cellular cholesterol levels.

As noted above, Abeta is produced from amyloid precursor protein (APP) in a sequential process in which APP is cleaved first by beta- and then gamma-secretases, enzymes whose activity is upregulated by high levels of cholesterol – one of many reasons why high cholesterol (particularly in individuals carrying the ApoE4 genotype, which binds more avidly to Abeta and is also pro-inflammatory⁸) is a risk factor for Alzheimer's disease.

 Abeta increases cholesterol efflux from cells, assembling high-density lipoprotein-like particles during its secretion. In animal studies, lipoproteins with Abeta are excreted into peripheral tissues much more efficiently than those without Abeta.⁹ Thus, Abeta is not simply a neuro-terrorist. Abeta plays an apolipoprotein-like role in the maintenance of cerebral cholesterol homeostasis.

Increasing cholesterol in the plasma membrane of brain cells increases cerebral Abeta production

Cholesterol-rich lipid rafts are platforms where APP clusters in the plasma membrane (the outer cell membrane) of neurons, astrocytes and microglia. When cholesterol concentration decreases in the plasma membrane, so does Abeta production from APP, while neuroprotective alpha-secretase activity goes up. When cholesterol concentrations increase in the plasma membrane, alpha-secretase activity drops, and beta-secretase activity and Abeta production increase.

Recently, researchers unexpectedly found that increasing plasma membrane cholesterol caused endosomes in hippocampal neuronal cells to enlarge by 31%.¹⁰ (Endosomes are organelles in which molecules are sorted on their way to lysosomes for degradation.) Enlarged endosomes are seen in affected neurons in the brains of AD patients and develop years before either of the neuropathological hallmarks of AD, Abeta plaque and tau hyperphosphorylation, become evident.

The appearance of enlarged endosomes coincides with increased production of Abeta peptides, suggesting that increased plasma membrane cholesterol causes endosomal pathology that increases APP processing by beta-secretase, resulting in Abeta overproduction. This pathology tends to become self-perpetuating since endosome enlargement is also induced by the C-terminal fragment of APP, which is the first item produced en route to Abeta formation when APP is cleaved by beta-secretase.¹¹

Therapeutic news to use: the amyloidogenic pathway is initiated to lower brain cholesterol levels

A large body of evidence now indicates that increased levels of plasma membrane cholesterol promote amyloidogenic processing of APP. Cholesterol normally concentrates in the plasma membrane of neurons and glial cells. The immediate precursor to Abeta, the C-terminal domain of APP (called C99 because it consists of a domain containing 99 residues), is the product of beta-secretase cleavage of APP and the substrate of gamma-secretase cleavage, from which Abeta issues. It now appears that this section of APP has a propensity to bind with cholesterol.

Such binding favors the amyloidogenic pathway because it promotes localization of APP/C99 to lipid rafts in the plasma and endosome membranes, where beta and gamma-secretase are concentrated.  On the other hand, non-amyloidogenic alpha-secretase is believed to reside primarily in the bulk membrane (the non-raft/ free cholesterol sections of the plasma membrane) and to be inactivated when forced to associate with lipid rafts.¹²

Cholesterol within the plasma and endosome membranes is distributed between the two pools: free cholesterol in the bulk membrane and cholesterol associated with lipid rafts. At low plasma membrane/endosomal cholesterol concentrations, free cholesterol and free APP/C99 will predominate, and APP will primarily be subject to alpha-secretase-initiated processing. However, as cholesterol levels in the membrane rise, lipid rafts will predominate. APP/C99 will complex with cholesterol in these rafts, and will be processed by beta- and gamma-secretase.

Research suggests that the second and third products in the amyloidogenic pathway—Abeta and the intracellular domain of the APP (AICD)—act to lower cellular cholesterol levels. It has been shown that Abeta stimulates the release of cholesterol and some other lipids from cells in the form of lipoproteins, and also that Abeta fibrills down-regulate cholesterol biosynthesis.

Other studies suggest that Abeta reduces biosynthesis of cholesterol and other lipids under conditions of ischemia, and that cytosolic Abeta acts as an inhibitor of HMG-CoA reductase, the rate-limiting enzyme in the biosynthetic pathway for cholesterol whose activity is blocked by statins. (Statins, however, are not the answer to the cholesterol issues in AD. The reasons why are discussed in Part II of this review.)

In addition, the AICD, which is released when gamma-secretase cleaves C99 to produce Abeta, translocates to the cell nucleus, where it acts as a transcriptional suppressor to the gene that encodes the LRP1 protein. LRP1 is a major apoE receptor in the brain that mediates cellular cholesterol uptake via endocytosis (the process by which cells absorb molecules from outside the cell by engulfing them with their cell membrane). Thus AICD may also help down-regulate cellular cholesterol uptake.

In sum, Abeta can down-regulate cerebral cholesterol content. All these observations suggest that the amyloidogenic pathway’s true function is to reduce total cellular cholesterol levels. Further, the fact that APP now appears to be a cholesterol binding protein suggests that one of its raisons d’etre may be to act as a cellular cholesterol sensor/receptor. When membrane cholesterol levels are elevated, APP forms a complex with cholesterol, which promotes the amyloidogenic pathway, whose products then reduce both cholesterol uptake and biosynthesis, completing a negative-feedback loop.¹²

Amyloidogenic and Non-Amyloidogenic APP Processing

Flowchart by John Morgenthaler

Amyloid Beta and the Cholesterol Connection to Alzheimer’s Disease

In the brain, persistently elevated cholesterol promotes excessive Abeta production, the formation of neurotoxic souble Abeta oligomers, and the deposition of amyloid plaque. When cholesterol levels are within optimal range, alpha-secretase is the constitutive enzyme, APP is channeled to non-amyloidogenic processing, and Abeta is produced in lesser amounts and assists in cholesterol recycling and efflux, helping to maintain cholesterol homeostasis.⁴ When cholesterol levels remain high, however, Abeta quickly metamorphoses from Dr. Jekyll to Mr. Hyde.

Location, location, location

The alpha- and beta-secretases concentrate in different areas of the cell. Not surprisingly, given beta-secretase’s cholesterol efflux function, this enzyme is primarily found in the Golgi apparatus and lysosomes. The Golgi is an organelle in most eukaryotic cells that serves as the processing, packaging center for macromolecules, e.g., proteins and lipids, which pass through the Golgi soon after their synthesis before making their way to their destination. When brain metabolism is functioning well, Abeta’s next destination is the endosome, a further sorting compartment from which Abeta is sent on to the lysosomes, the disposal unit of the cell, for degradation. Also not surprisingly, beta-secretase has optimal activity at the acidic pH found within the endosomes. As noted above, beta-secretase is also active in “lipid rafts” in the plasma membrane.¹³¹⁴

In contrast, alpha-secretase activity is optimized in cell surface membrane regions with few  lipid rafts, especially areas with high fluidity where this secretase is primarily localized, another reason for the importance of the most fluid of all fats, the omega-3 fatty acid, DHA, in promoting a-secretase activity and cognitive function.¹⁵ For a detailed discussion of this topic, please see the LMR review titled Omega-3s, ApoE Genotype and Cognitive Decline.

Thus, if APP accumulates at the cell surface, it has a much better chance of being cleaved by alpha-secretase. If APP is shifted to inside the cell to the Golgi, endosomes or lysosome compartments where beta-secretase is the key player, then processing to Abeta will be the likely outcome. Emerging evidence indicates that cholesterol and the family of low-density lipoprotein receptors (LDLR) impact where APP will accumulate.¹⁶ And so does insulin, which significantly accelerates APP trafficking from the Golgi to the plasma membrane (more on insulin’s connections to Abeta and AD below in the section titled Insulin Resistance: the Dysglycemic Route to Atherosclerosis & Alzheimer’s below).¹⁷

APP Location Chaperones – the Low-Density Lipoprotein Receptors

At least four members of the low-density lipoprotein receptor (LDLR) family regulate APP location: LRP (LDLR-related protein), LRP1B, SorLA/R11 (Sorting protein related receptor containing LDLR class A repeats) and apoER2 (apoE receptor 2). LRP and apoER2 promote delivery of APP to areas rich in beta-secretase; LRP1B and SorLA/R11 deliver APP to alpha-secretase-rich areas.16  (More on these chaperones, which work with ApoE, below.)

Cholesterol dysregulation in the brain: key cause of Abeta overload and AD

Although it accounts for only 2% of the body mass, the brain contains 25% of the total body amount of unesterified (free) cholesterol; over 99% of the cholesterol in the brain is unesterified. Brain tissue is separated from blood circulation and other tissues by the blood–brain barrier, (although this barrier is turning out to not be nearly as impermeable as formerly thought)⁷ and its cholesterol is almost completely synthesized de novo by glial cells. Brain lipids, including cholesterol, are primarily located in cell plasma membranes and are constantly being replaced.¹⁸

Brain cholesterol is essential for a wide variety of key brain functions, and when released from degenerating nerve terminals, is actively recycled by glial cells and astrocytes, esterified by cholesterol esterase, then cleared from the extracellular matrix by binding, primarily to ApoE, which delivers it to neurons. A negative feedback loop between free cholesterol, Abeta and HMG-CoA reductase maintains intracellular cholesterol equilibrium in the brain. In the AD brain, however, this orderly process of cholesterol turnover is impaired.

Although the brain synthesizes its own cholesterol and does not depend on the circulation for its cholesterol supply, it has recently been shown that, contrary to previously accepted opinion, plasma lipoproteins do cross the blood brain barrier (BBB). In individuals in whom cholesterol levels are persistently (or even frequently post-prandially) elevated, plasma lipoproteins can deliver significant amounts of cholesterol (and the Abeta it contains) to the CNS.⁷

A key role for ApoE

AD can be classified broadly into two groups, early onset AD (EOAD, occurring < 65 years) and late-onset AD (LOAD, occurring > 65 years). EOAD, although the most severe form, accounts for only a small percentage of AD cases (<5%), and the majority of these are caused by mutations in one of three genes: those for APP, presenilin (PS) 1 and PS2.

LOAD accounts for the vast majority of AD cases (>95%). Age and possession of the ApoE4 single nucleotide polymorphism (SNP) are considered the major risk factors for LOAD. ApoE4 is also a major risk factor for CVD, due not only to its association with increased low density lipoprotein levels, but also higher oxidative stress, and a more pro-inflammatory / immunoreactive phenotype.¹⁹ For more on this topic, please see our LMR article: Omega-3s, ApoE Genotype and Cognitive Decline

In the adult brain, astrocytes not only synthesize, but also internalize and recycle the cholesterol released from degenerating nerve terminals. This recycled cholesterol is then delivered back to neurons, an activity that requires cholesterol’s binding to one of the variants of apolipoprotein E (ApoE), which serves as the major lipoprotein in the CNS. ApoE is a ligand for cell surface lipoprotein receptors (including the APP chaperones noted above); is responsible for regulating lipid transport among brain cells; and clears cholesterol from the extracellular space, delivering it to the lysosomes for recycling.

Abeta is internalized in ApoE-containing particles, which then move either to endosomal compartments in the plasma membrane, from which they are sent to lysosomes for recycling, or efflux from the brain into perivascular channels that drain into the CNS and the systemic circulation. Once in the systemic circulation, they are sent to and processed by the liver, where Abeta peptides are catabolized and excreted into the bile.⁸

The ApoE4 isoform has the highest affinity for Abeta binding, but is the least effective ApoE isoform in promoting cholesterol efflux (from macrophages as well as from neurons and astrocytes); the most immunoreactive isoform; and has the least antioxidant capacity – all reasons ApoE4 increases risk for AD).¹³ If these ApoE4 traits were not problematic enough, recent papers indicate that the ApoE4 variant also promotes Abeta-induced apoptosis in neuronal cells. After actively binding Abeta, ApoE4 forms a reactive molecular intermediate with the potential to insert into the lysosomal membrane, destabilize it, and cause lysosomal leakage, provoking apoptosis.²⁰²¹

Arterial Abeta Leads to Leaky Brain and Dysregulation of Cerebral Cholesterol Homeostasis

While the intact BBB normally largely prevents cholesterol import into the CNS from the peripheral circulation, vascular injury can enable cholesterol-carrying lipoproteins to gain relatively easy access to the CNS. This results in increased brain membrane cholesterol, increased beta-secretase cleavage of APP, and increased generation of Abeta peptides. In addition, oxidized cholesterol derivatives found in the circulating blood can also cross the BBB. Hence, any vascular damage (e.g., the endothelial damage seen in atherosclerosis, hypertension, MetS, diabetes) can result in increased importation of damaged cholesterol into the brain.²⁰²¹ This is where systemic, not just cerebral, endothelial dysfunction enters the AD causality chain.

Plus, Abeta is also found in platelets inside human atherosclerotic plaques. In vitro studies have shown that when perivascular macrophages phagocytose platelets within atherosclerotic plaques, APP is released and processed to Abeta-like peptides, which then promote the upregulation of inducible NO-synthase (iNOS). iNOS activation results in increased production of toxic amounts of NO, causing additional RNS/ROS production, additional macrophage activation, endothelial dysfunction, and the onset of a vicious cycle.²²²³

Recent data indicate that by enhancing iNOS production, Abeta peptides in atherosclerotic plaque promote vasoconstriction that ultimately reduces cerebral blood flow and increases the production of superoxide by NADPH oxidase, which is located in both vascular and brain cell membranes. The outcomes: damage to the vascular endothelium, reduced brain perfusion and cerebral ischemia, increased production of iNOS, glial activation, mitochondrial dysfunction – a brain-destructive Pachinko game.

Arterial and Amyloid Plaque: Different Location, Same Theme

Altered cholesterol metabolism results in failure to clear Abeta and a chronic inflammatory response by the microglia, the brain’s version of macrophages, which are surprisingly similar to those found in the intima of atherosclerotic lesions. Brain microglia produce virtually all the same cytokines, chemokines, growth factors, enzymes, complement and coagulation factors, and ROS, as their vascular counterparts, plus the microglia express many of the same surface receptors that mediate local immune reactions. In addition to microglia, both astrocytes and neurons, also swing into action, producing inflammatory mediators, including C-reactive protein, amyloid P, and complement factors.¹³ All of this contributes to increased production of ROS and RNS, endothelial dysfunction, mitochondrial dysfunction, hypoxia—sound familiar?—many roads to Rome, all converging on Alzheimer’s disease.

In what other research also suggests is the brain’s variation on atherosclerosis, Abeta plays a role similar in many respects to that undertaken by marcophages in the vasculature. Macrophages ingest and attempt to sequester oxidized LDL, becoming foam cells and furthering an inflammatory process whose end result is the formation of arterial plaque. Similarly, Abeta, failing to sufficiently lower cholesterol in the brain (where highly active neuronal mitochondria greatly increase the potential for damage by ROS/RNS), sequesters excess cholesterol in amyloid plaque. Different location, same theme.¹³

Convergent Pathways to Alzheimer’s Disease: High Systemic Cholesterol &/or Insulin Resistance

Flowchart by John Morgenthaler

Insulin Resistance: the Dysglycemic Route to Atherosclerosis & Alzheimer’s

Although the human brain represents only 2% of the body weight, it utilizes 15% of the cardiac output, 20% of total body oxygen consumption, and 25% of total body glucose.²⁴ Because the energy-hungry brain relies on glucose as its only source of fuel for ATP production, (except during fasting or significant carbohydrate restriction when a switch is made to ketone metabolism), insulin plays a pivotal role in cognition and other aspects of normal brain function.*

Insulin resistance results in lowered insulin activity and chronically elevated levels of insulin in the periphery, but a reduction in insulin levels in the brain. Knowing this, it is not surprising that dysfunctions related to systemic insulin resistance -- metabolic syndrome (MetS, aka insulin resistance syndrome) and related conditions such as type 2 diabetes mellitus, hypertension and dyslipidemia -- are all also associated with age-related cognitive decline and AD.²⁶

In addition to depriving the brain of insulin (and therefore its glucose fix), insulin resistance promotes an increase in the production of inflammatory agents, including Abeta. When plasma insulin is experimentally increased in healthy humans to the high levels typically seen in patients with insulin resistance, levels of inflammatory agents and Abeta increase in the brain.²⁷

Insulin and Abeta have a reciprocal relationship

Insulin regulates Abeta in several ways:

By increasing its trafficking from within to outside the cell. When high levels of insulin are given, Abeta levels go up in spinal fluid. Insulin crosses the BBB, enters the CNS, and promotes trafficking of Abeta out into extracellular space in the brain, which then drains into the spinal fluid. While this is a positive effect of insulin, it’s also a double-edged sword. Abeta needs to be outside the cell to get degraded, but if not degraded quickly, soluble Abeta peptides are highly neurotoxic. Enter “insulin-degrading enzyme” to the rescue.²⁸

By increasing the expression and therefore availability of insulin-degrading enzyme. As its name implies, insulin-degrading enzyme (IDG) degrades insulin, but it is also responsible in the brain for degrading Abeta. Insulin thus impacts Abeta levels both by competing for IDG, so supplies become insufficient to clear Abeta, and by regulating levels of IDG in the brain since insulin increases the expression and therefore availability of IDG.²⁸

Abeta that manages to bind to dendrites fights back by causing the neurons’ insulin receptors to move off the neuronal surface and into the cell body, where they are no longer accessible to insulin.²⁸²⁹ On the positive flip side, restoration of insulin sensitivity down regulates the availability of Abeta binding sites, thus preventing pathogenic Abeta oligomers from binding.³⁰

The Insulin Connection to Alzheimer’s Disease

Flowchart by John Morgenthaler

Many tributaries, two main streams, one pathological outcome

It has recently been proposed by Suzanne Craft and her research team at the University of Washington School of Medicine, Seattle, WA, that there are two main pathways to AD, one of which is driven by physiological processes associated with the ApoE4 allele, while the second is driven by insulin resistance.³¹

According to Dr. Craft, if you take a group of patients with AD, about 50% will have the ApoE4 allele, a much higher percentage of ApoE4 carriers than in the general population. The other 50% have AD but do not have an identified genetic risk factor. Interestingly, these patients are much more likely to be insulin resistant. Although the mechanisms differ through which ApoE4 and insulin resistance initiate pathology, both promote dyslipidemia, endothelial dysfunction and damage, and oxidative stress—effects that interact with Abeta to produce the final common pathway that results in AD.

Furthermore, we now know that the CNS and the periphery are closely interrelated, and each is capable of driving AD pathology. Dietary insults provoke insulin resistance, endothelial dysfunction, and dyslipidemia—and affect the CNS. In the brain, Abeta causes insulin resistance that may drive compensatory insulin resistance in the periphery.

As Craft noted in her January 2010 Functional Medicine Update interview with Jeff Bland, PhD, over the last 10 years, a rapidly increasing number of papers have reported on co-morbid relationships in which insulin resistance appears to act as a mechanistic fulcrum, connecting hypertension, dyslipidemia, type 2 diabetes and dementia /AD.²⁸

Insulin resistance promotes endothelial dysfunction/hypertension and dyslipidemia/atherosclerosis (and food sensitivities/allergies/gut dysbiosis – but that’s another topic necessitating its own review). Any one or any combination of these can engender a leaky blood brain barrier that impacts Abeta transport (both efflux and influx) between the brain and the periphery. The end result is increased levels of Abeta in the brain, increased Abeta-induced inflammation and damage to the endothelium, increased deposition of Abeta plaque--and AD.

The bad news is that is has become abundantly clear that no single molecular target or silver bullet will prevent or cure AD. The good news is that the latest research is moving to a model in which therapies with pleiotropic effects on metabolic function combine to decrease the risk of developing AD. The best news is that all identified risk factors for AD are modifiable. There is only one genetic risk factor, ApoE4, and actualization of its potential negative effects involves interaction with environmental factors—the incremental insults that result from poor diet, micronutrient insufficiencies and an inactive lifestyle.

Although the complexity of what has been called “the morass of intermediary metabolism” appears daunting, Dr. Crarft remains optimistic. The inter-relationships among the myriad modulators of system and cellular function offer the happy prospect that intervention at one level of the metabolic web may have beneficial effects across many levels. For example, treatment that restores insulin sensitivity also typically improves endothelial function and lipid profiles. Part II of this review will discuss what the research is revealing to be the most promising means of getting off the side roads that funnel into the Alzheimer’s Interstate.

(*Although the liver is the primary source of ketones, and a ketogenic diet increases BBB permeability to ketones from the periphery, ketones are also produced from fats within the brain by astrocytes—more on this in Part II]).²⁵

EDITORS NOTE: We are scheduled to post part II of this article on May 14, 2010 or a bit sooner.

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Introduction

As primary prevention for a myocardial infarction or stroke, you’ve prescribed a statin, possibly in conjunction with a fibrate, for a 50 year old male patient with one or more of the following: metabolic syndrome, type 2 diabetes, hypercholesterolemia, coronary artery disease or peripheral artery disease. In a follow-up visit, his PSA is elevated, and he complains of awakening several times each night to urinate. You prescribe finasteride. He returns several months later, worried the recession will result in his being laid off, feeling very anxious, and experiencing insomnia. After further evaluation, it’s clear he is depressed. You prescribe an SSRI.

What’s the likelihood that this patient will experience erectile dysfunction (ED) as a result of one or more of these therapies and will simply stop taking the medication(s) rather than discuss this problem with you? Unfortunately, risk for this scenario is surprisingly high.

ED remains a greatly under-reported adverse drug reaction (ADR) because male patients often are not forthcoming about, and doctors usually do not raise the issue of, sexual function. The Massachusetts Male Aging Study data showed an annual incidence of ED in men 40 – 69 years of age of 26 per 1000 men (an incidence of 12 in the 40–49-year age group and 46 in the 60–69-year age group); however, these statistics may seriously underestimate the problem.¹  Research presented at the Second Princeton Consensus Conference on Sexual Dysfunction and Cardiac Risk indicates that ED affects more than half of men >60 years.²

Since the major comorbidities of ED –diabetes, hypertension, hyperlipidemia and depression—are also key risk factors for cardiovascular disease, the question of whether the drugs used to treat these conditions might also be contributing to ED has not received much consideration, despite the fact that many of the drugs used to manage the underlying risk factors of atherosclerosis (e.g. ß-blockers, thiazide diuretics, ACE inhibitors) are well known to be risk factors for ED.³⁴⁵⁶⁷

A recent French Pharmacovigilance System Database assessment of the association between drugs and risk of ED not only confirmed a significant association for finasteride (adjusted relative odds ratio [aROR] = 14.5), fibrates (aROR = 3.6), ß-blockers (aROR = 1.5) and tricyclic antidepressants (aROR = 2.0), but revealed a surprising statistically significant association between all statins (aROR = 2.4) and ED.^{8 9} 

Statin-associated ED

Given statins’ efficacy in lowering cholesterol and therefore lessening cardiovascular disease risk, an association between these drugs and increased risk for ED seems counterintuitive. However, a number of recent studies indicate the statin-to-ED connection is real.

When a group of 76 men with stable coronary artery disease, average age 64 years (range 40 to 82), were evaluated using the validated Sexual Health Inventory for Men (SHIM), 57 of the 76 (75%) had ED.  Of those with ED, 28% were on diuretics, 47% on β-blockers, but almost all, 92%, were on statins.¹⁰ 

As noted above, a significant association was found in the French Pharmacovigilance System Database between exposure to statins and ED. Among the total of case reports of ADRs, exposure to statins was identified in 4471 cases (4%), of which 51 reports (1.1%) concerned ED, in comparison to 431 (0.4%) cases of ED without exposure to statins. The mean delay of onset of ED after starting statins, known for 19 cases, was 62 days (median 29 days). In 56.9% of cases, recovery occurred after withdrawal of statin, and rechallenge was positive in five cases. The association with ED was statistically significant for all statins (adjusted ROR = 2.4) with adjusted relative risks for simvastatin of 2.6;  atorvastatin, 3.4; and rosuvastatin, 7.1.⁸

An earlier study involved 38 cases of impotence associated with statins in the database of the Spanish pharmacovigilance system plus 37 statin-associated cases in French pharmacovigilance system. Among the Spanish men, ED disappeared after drug withdrawal in 93% of the cases. In France, recovery was observed in 85% of cases after drug withdrawal, and in 5 cases, a positive rechallenge confirmed the ADR and recovery after cessation of statin use.⁹

How might statins promote ED?

The statin-induced decrease in cholesterol levels could negatively impact testosterone synthesis.¹¹   In vitro assays have shown that in concentrations “probably [italics added] exceeding those achieved in vivo,” simvastatin inhibits not only HMG-CoA reductase, but the 17-ketosteroid-oxidoreductase catalyzed conversion of dehydroepiandrosterone and androstenedione to androstenediol and testosterone, respectively.¹²   In support of this hypothesis, a significant decrease in free testosterone has been noted in men treated with simvastatin for hypercholesterolemia. In a study of 8 hypercholesterolemic men, free testosterone levels were determined at baseline in basal conditions and after stimulation by human Chorionic Gonadotropin, and again after 3, 6 and 12 months of treatment with simvastatin (20 mg/day). Significant reductions in free testosterone, both basal and hCG-stimulated, were observed at 6 and 12 months.¹³ 

In a larger study, involving 81 men, the pooled total testosterone level at baseline was 541 and 513 ng/dL in the placebo and simvastatin-treated groups, respectively, and declined to 536 ng/dL (-1.5%) and 474 ng/dL (-13.6%) after simvastatin treatment. The pooled free testosterone declined by 6.3% in the simvastatin group, while increasing 4.9% in the placebo group. Pooled bioavailable testosterone declined 10.2% in the simvastatin group, while increasing 1.4% in the placebo group.¹⁴ 

In addition, in familial hypercholesterolemia, the low-density lipoprotein receptor is dysfunctional, which renders the Leydig cells more dependent on 'de novo' synthesis of cholesterol (Leydig cells are interstitial cells in the testes that secrete testosterone when stimulated by lutenizing hormone. [Lutenizing hormone increases the activity of cholesterol desmolase, which converts cholesterol to pregnenolone, the prohormone precursor of progesterone, mineralocorticoids, glucocorticoids, androgens, including testosterone, and estrogens].) Statins have been found in small quantities in the testes, where they could inhibit this de novo synthesis of cholesterol.^{8 15}

Most recently (February 2010), the association between statin therapy and hormonal parameters was evaluated in a consecutive series of 3,484 patients (mean age 51.6 +/- 13.1 years) seeking medical care for ED at the Andrology Unit, University of Florence, Florence, Italy.  Among the patients studied, 244 (7%) patients were being treated with statins. Both total and calculated free testosterone levels were significantly lower in subjects taking statins. The use of statins was also associated with a reduced testis volume and a higher prevalence of hypogonadism-related symptoms and signs. The lower levels of total and calculated free T observed in subjects treated with statins were confirmed by comparing them with age-waist circumference and CV risk score matched controls. The researchers concluded that statin therapy might induce an overt primary hypogonadism and should be considered as a possible confounding factor for the evaluation of testosterone levels in patients with ED.¹⁶ 

Although ED appears, statistically, to be a rare ADR of statin therapy, the high rate of under-reporting of ED by male patients is likely to mitigate against an accurate estimate of the true prevalence of statin-related ED. Despite this, a significant literature from case reports, review articles, clinical trials and regulatory agencies has now identified statins, as well as fibrates, as a likely cause of ED.  The topic of a possible association between sexual disturbances and statins is being currently discussed at the European Medicine Agency.⁸

Exposure to statins may act as the final straw, lowering testosterone levels and producing frank ED in patients with endothelial dysfunction for whom cholesterol-lowering is neither the key—nor sufficient—means of resolving the underlying factors contributing to their risk for cardiovascular disease. (For a more complete review of this issue and discussion of agents that effectively improve cardiovascular function without increasing risk of ED, please see our earlier LMR articles “Managing Erectile Dysfunction—When Viagra Doesn’t” and the series on “Closing the Statin Gap”).

5-alpha reductase inhibitors (e.g., Finasteride) and ED

5-alpha reductase inhibitors (finasteride and dutasteride), the most commonly used drugs to treat benign prostatic hyperplasia, are associated with adverse sexual outcomes, including ED, at rates ranging from 2.1% to 38% in clinical trials. These drugs inhibit type II 5-alpha reductase, the enzyme that converts testosterone to dihydrotestosterone (DHT). Of the two, dutasteride is more likely to cause ED since unlike finasteride, it inhibits both isoenzymes of 5-alpha reductase and results in near-complete suppression of serum dihydrotestosterone.¹⁸  The proposed mechanism via which the 5-alpha reductase inhibitors promote ED is decreased nitric oxide synthase activity as a result of decreased dihydrotestosterone.¹⁹

 Natural agents, shown in systematic reviews to improve overall urologic symptoms of BPH including nocturia, without any increase in risk of ED, include cernilton, which is prepared from the rye-grass pollen Secale cereal,²⁰   Pygeum africanum, the extract of the African prune tree,²⁰  and Serenoa repens, the extract of the berry of the American saw palmetto, although the two most recent Cochrane Database Systematic Reviews have produced inconsistent results regarding its efficacy. Research included in the 2002 review, which included 21 randomized trials involving 3139 men, found that Serenoa repens provides mild to moderate improvement in urinary symptoms and flow measures.²¹  The 2009 review, which included 9 new trials involving 2053 additional men, found saw palmetto no more effective than placebo for treatment of the urinary symptoms of BPH.²²

SSRIs, highly likely to cause ED

Sexual dysfunction secondary to the use of antidepressants, especially SSRIs, is an adverse effect that is often underestimated, and one that, according to a number of studies, may affect from 58% to 73% of patients.^23242526 Approximately 42% of men discontinue antidepressant treatment due to its adverse sexual effects.   These present as a decrease in libido, alterations in the ability to reach orgasm/ejaculation, and ED, which appears to be related to the resulting increase in serotonin. Increased availability of serotonin results in its increased binding to and activation of the serotonin (aka 5-hydroxytryptamine) receptors 2 and 3 [the 5-HT2 and 5-HT3 receptors], which inhibit sexual desire, ejaculation, and orgasm. Thus increasing serotonin levels   promotes ED, whereas dopamine release enhances sexual function. Amineptine, a drug with an increased dopamine transmission and scant serotonin transmission, may have a more benign impact on the sexual function of depressed patients.²⁸  After substituting amineptine for SSRIs in patients with sexual dysfunction, the incidence of any type of sexual dysfunction decreased significantly from 100% (baseline) to 55.3% after 6 months. Incidence of delayed orgasm dropped to 15.8%, anorgasmia to 17.4%, and impotence to 15.8%, while the antidepressant effect that had already been achieved with the SSRI was maintained.²⁹ 

The best researched alternative to SSRIs, which does not increase risk of ED, is Hypericum perforatum (St. John’s Wort). Numerous randomized clinical trials, including those covered in the most recent Cochrane Database Systematic Review, confirm that Hypericum extracts are superior to placebo in patients with major depression, are similarly effective as standard antidepressants, and have fewer side effects than standard antidepressants.³⁰³¹

Conclusion

Considering the widespread use of the above drugs, especially statins, and the under-reporting of ED as an ADR, drug-associated ED may affect a large number of male patients. Fortunately, ED resulting from treatment with ß-blockers, thiazide diuretics, ACE inhibitors, fibrates, statins, finasteride, tricyclic antidepressants or SSRIs seems to be reversible in most of cases after drug withdrawal. Doctors should be aware of this potential adverse reaction when prescribing any of these drugs to their patients.

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Phosphodiasterase-5 (PDE-5) inhibitors, e.g., sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis), are ineffective in 30-40% of men diagnosed with erectile dysfunction (ED).  New studies show that erectile function can be restored in these men, without resorting to intracavernous injections or a penile prosthesis, by using natural agents (including NO-producing substrates such as N-hydroxyarginine [OH-arginine]) in combination with the PDE-5 inhibitors.¹

In ~70% of impotent men, ED is due to cardiovascular disease, MetS, diabetes mellitus, hypogonadism, and/or use of prescription and non-prescription drugs (e.g., antihypertensive agents including diuretics and betablockers; psychotropic drugs including neuroleptics, and SSRIs and other antidepressants; anti-arrhythmics, anti-androgens, steroids, nicotine [smoking] and alcohol).^{2 3 4} Identifying and treating the individual’s underlying cause(s) of ED will not only rejuvenate his sexual performance but significantly enhance his overall health and longevity.⁵

Just say NO to ED

In the vast majority of individuals with ED, the dysfunction is nitric oxide-related. In the healthy male, in response to sexual stimulation, nitric oxide (NO) is released in the corpus cavernosum, the cylinder of erectile tissue forming the dorsal part of the body of the penis, and binds to receptors on the enzyme guanylate cyclase, which results in production of cyclic guanosine monophosphate (cGMP), leading to smooth muscle relaxation (vasodilation) of the intimal cushions of the helicine arteries, increased inflow of blood and an erection.

By selectively blocking the normal hydrolysis of cGMP, the PDE-5 inhibitors promote cGMP accumulation, thus partially reversing deficiencies in the NO/cGMP pathway. The key word here, however, is “partially,” since decreased expression or activity of endothelial NO synthase (eNOS), impaired NO release, and/or rapid NO destruction will preclude sufficient cGMP formation to enable benefit from a PDE-5 inhibitor. Thus, to make possible PDE-5 efficacy in men unresponsive to these drugs, the amount of available NO must be increased in penile tissue.¹ This can be accomplished safely and without the risk of inducing adverse side effects with the use of natural agents that have been shown in the peer-reviewed research to enhance NO synthesis, facilitate NO release, and/or inhibit NO breakdown.

Enhancing NO Synthesis

L-arginine: NO is generated via the activity of NOS, which catalyzes the oxidation of L-arginine with NADPH and molecular oxygen to yield L-citrulline and NO. L-arginine, as substrate for this reaction, is its key limiting factor. (For a full discussion of L-arginine metabolism and NO production, please see “Longevity Medicine Strategies for Cardiovascular Disease: Closing the Statin Gap in Endothelial Dysfunction and Insulin Resistance Naturally, with L-Arginine and Citrulline”.

Supplemental L-arginine is readily absorbed and well tolerated in single doses of 3-6 grams.⁶   When larger doses are desirable, adverse gastrointestinal effects can be avoided with multiple daily doses of <6 grams.⁷   However, optimizing L-arginine delivery for NO production is not as straightforward as this since L-arginine may be co-opted for a number of other uses in the body.

~50% of ingested L-arginine is rapidly converted in the body to ornithine, primarily by the enzyme arginase, whose expression and activity is upregulated in the presence of oxidized LDL and conditions associated with endothelial dysfunction, including—in addition to ED—hypertension, heart failure, atherosclerosis, and diabetic vascular disease—co-morbidities  so frequently seen in men with ED that a growing number of studies recognize ED as a reliable marker not only for overt cardiovascular disease, but also one that serves as an early indicator of subclinical systemic vascular disease.^{8 9}   In the man with ED, arginase is highly likely to be upregulated, which translates to a need for higher than typical doses of supplemental L-arginine to produce benefit.

Furthermore, L-arginine, in addition to its roles as substrate for NOS and arginase, is also a precursor for the synthesis of proteins, urea, creatine, vasopressin, and agmatine. In addition to NOS, L-arginine that escapes metabolism by arginase is targeted by three other enzymes: arginine:glycine amidinotransferase (to become creatine); arginine decarboxylase (to become agmatine); and arginyl-tRNA synthetase (to become arginyl-tRNA, a precursor to protein synthesis).¹²

Sustained-release preparations of L-arginine have been suggested as a means of helping to maintain blood levels over time, yet arginase’s substantial intestinal and hepatic metabolism of L-arginine to ornithine and urea, combined with L-arginine’s use for other functions in the body, lessen the likelihood of optimal improvement in NO production from oral supplements of L-arginine.¹⁰   L-citrulline or hydroxyl-arginine (OH-arginine) may provide a better option.

L-citrulline is readily absorbed and efficiently converted to L-arginine, but since the conversion occurs in the kidneys, vascular endothelium and other tissues, and not in the liver and intestine where arginase is concentrated, the formation of L-arginine from supplemental L-citrulline does not induce arginase activity, increasing the amount of L-arginine available for NO production.  Published data also indicates that L-citrulline has relatively better absorption and systemic bioavailability than L-arginine.^{12 13}

N-hydroxy-arginine (OH-arginine) is a stable intermediate product formed during the synthesis of NO. Arginine is first oxidized into OH-arginine, which carries an atom of oxygen bonded to the guanidine group of L-arginine, and then further oxidized to citrulline concomitant with the release of NO. Thus, when OH-arginine is provided as substrate, NOS requires one less step and a lesser amount of oxygen and NADPH to produce NO. As a result, OH-arginine is a highly potent competitive inhibitor of arginase.^{14 15}   In rabbit corpus cavernosum, OH-arginine, but not L-arginine, was found to promote NO-mediated relaxation and cGMP accumulation, and OH-arginine has also been shown to improve NO generation in hypoxic and aged tissues.^{1 16}   During the last decade, a great number of compounds have been synthesized and studied as possible NO-producing substrates for NOS. OH-arginine, although not yet available as a supplement, is one of the very few found capable of promoting significant formation of NO. ¹⁷

OH-arginine – a more reliable indicator of NOS activity. Levels of OH-arginine have been found to be significantly reduced in patients with MetS compared to healthy controls, while these men showed no significant differences in concentrations of the NO precursor, L-arginine, or the end product of NO synthesis, L-citrulline. OH-arginine may therefore also serve as a more reliable marker of reduced NO formation and cardiovascular risk in men with MetS, ED and its other co-morbidities than L-citrulline, nitrite and nitrate, the compounds that have been commonly used to evaluate CVD risk. Because NO is very unstable, techniques to measure its formation in vivo have concentrated on measuring NO decomposition products, i.e., citrulline and the electron oxidation products of NO, nitrite and nitrate. But these compounds are also produced by other enzymes, while only NOS produces OH-arginine. For this reason, plasma OH-arginine concentration has been suggested as a more accurate indicator of NOS activity.¹⁸

When lab testing reveals low plasma levels of OH-arginine, this must either be due to decreased eNOS activity or increased breakdown of OH-arginine by oxidants, e.g., superoxide and peroxynitrite. Both oxidants indicate increased need for the cofactors in NO metabolism, i.e., tetrahydrobiopterin (BH4) [and its recycling partner, vitamin C], and the methylating factors, B6, B12 and folate. (For a full discussion of these issues, please see “Longevity Medicine Strategies for Cardiovascular Disease: Closing the Statin Gap in Endothelial Dysfunction and Insulin Resistance Naturally, with L-Arginine and Citrulline, Part II”).

Ginseng has also demonstrated efficacy in enhancing NO synthesis and improving erectile function in men with ED.¹⁹  Ginseng, specifically an extract of Panax ginseng containing the ginsenoside Rg1, has been shown to increase phosphorylation of eNOS, leading to enhanced production of NO and of vascular endothelial growth factor, which also increases production of eNOS, and thus, NO synthesis.^{20 21 22} In a double-blind, placebo-controlled 8-week study involving 143 men with ED, scores on the International Index of Erectile Function (IIEF) questionnaire were significantly higher, and erectile function significantly improved, in subjects given extract from Panax ginseng extract (1,000 mg b.i.d.), while no improvement was experienced by those receiving placebo.²³   At least one mechanism through which ginseng exerts its beneficial effects was identified in a recently published rat study in which Rg1 increased serum testosterone concentration, and enhanced NO release and cGMP accumulation in the corpus cavernosum both in vivo and in vitro. Within 16 days, Rg1 (10 mg/kg) significantly increased mounting, pelvic thrusting frequency and intromission by male mice.²⁴

Facilitating NO Release

Yohimbine, an α-2 antagonist, inhibits activation of the nitrergic nerves in the penile arteries and corpus cavernosum tissue, thus promoting nitrergic relaxation and NO release.^{1 2 16} Although controlled, randomized human studies involving yohimbine are few, meta-analyses of the available research have consistently shown an advantage of yohimbine over placebo.²⁵   The most recent was a double-blind, placebo-controlled, three-way crossover, randomized clinical trial, conducted by the Urology Department at the Hopital Foch, Suresnes, France, that involved 45 men with mild to moderate ED and compared the efficacy of a combination of 6g of L-arginine glutamate and 6 mg of yohimbine hydrochloride with that of 6 mg of yohimbine hydrochloride alone or placebo. Capsules were administered orally one to two hours before intended sexual intercourse. At the end of each treatment period, scores on the IIEF were 17.2+/-7.17, 15.4+/-6.49 and 14.1+/-6.56, for the combination of arginine and yohimbine, yohimbine alone, and placebo respectively, with the difference between the arginine plus yohimbine combination achieving statistical significance. According to both investigator and participant assessments, erectile function was significantly improved by yohimbine, which the paper describes as a promising addition to first-line therapy for ED.²⁶

Further recent evidence of yohimbine’s potential benefit for men with ED was noted in a study conducted in 18 non-smoking men (since tobacco has been thought to affect yohimbine’s efficacy more than other risk factors for ED); 50% of subjects were successful in completing intercourse in more than 75% of attempts. Yohimbine responders did have less severe ED (as manifested by improved increased rigidity on RigiScan testing, higher Florida Sexual Health Questionnaire scores, and slightly higher levels of testosterone).²⁷   A review of the clinical, pharmacological and therapeutic profiles of yohimbine concluded that, as monotherapy, yohimbine exerts only modest efficacy in ED patients, but increasing evidence indicates concomitant administration of yohimbine with other agents that augment NO release or action in the corpus cavernosum significantly improves erectile function.²⁸

Thermal therapy. Systemic thermal therapy, such as taking a warm-water bath or sauna (60 degrees C / 140 degrees F for 15 minutes, followed by bed rest with a blanket for 30 minutes), induces systemic vasodilation and has been shown to improve vascular endothelial dysfunction within 2 weeks in patients with congestive heart failure, hypertension, hyperlipidemia, diabetes mellitus, obesity, and compromised endothelial function due to smoking.^{29 30} In experiments with cardiomyopathic hamsters with heart failure, sauna therapy increased expression of eNOS mRNA and aortic eNOS protein expression. Far-infared sauna therapy has been shown to significantly increase hindlimb eNOS  expression and capillary density in apoE-deficient mice with hindlimb ischemia. No improvement was seen in animals given a NOS inhibitor [N(G)-nitro-L-arginine methyl ester], confirming benefit was due to eNOS/NO activity.³¹

In obese patients, body weight and fat—contributing factors to all the lifestyle-related diseases typically present in men with ED—significantly decreased after 2 weeks of sauna therapy with no increase in plasma ghrelin concentrations.³⁰³² 

Not only does regular thermal therapy, using saunas or hot baths, boost expression of eNOS, but it may promote insulin sensitivity, whose importance, given the number of men with ED for whom MetS or type 2 diabetes is a contributing factor, should not be underestimated. And thermal therapy’s beneficial effects on eNOS expression are not slight, but comparable to those of regular aerobic exercise.³³  Several recent animal studies provide evidence that thermal induction of heat shock protein 72 (Hsp72) can counter insulin resistance induced by a high-fat diet by suppressing activation of Nterminal-Jun kinase (JNK) in skeletal muscle. Hsp72 is clearly an inhibitor of the fat-mediated activation of JNK, which leads to phosphorylation of Ser307. Serine 307 is a major site of JNK phosphorylation in insulin receptor substrate-1 [IRS-1] whose phosphorylation causes inhibition of IRS-1’s function in insulin signaling.³⁴³⁵  IRS-1 is now believed to be a key mediator of fat-induced insulin resistance in skeletal muscle. This may be the mechanism through which glycemic control improves in type 2 diabetic patients receiving regular hot tub treatments. Skeletal muscle expression of hsp72 mRNA tends to be decreased in patients with type 2 diabetes as compared to healthy age-matched controls, and a polymorphism of hsp72 has been linked to increased diabetes risk.

Thus, thermal therapy may be of special benefit for diabetic and/or obese men with ED who find it difficult to participate in significant aerobic activity. However, thermal therapy does produce physiological stress and is therefore contraindicated in men with unstable angina, recent myocardial infarction, decompensated heart failure, cardiac arrhythmias, uncontrolled hypertension, and severe aortic stenosis.³³

Testosterone. Approximately one-third of men with ED have low serum testosterone levels.³⁶³⁷ A number of human clinical trials have demonstrated that testosterone replacement therapy improves erectile function and the response to PDE-5 inhibitors in hypogonadal patients with ED—even in hypogonadal men who previously failed to respond to PDE-5 inhibitors alone.³⁷  Testosterone acts as a vasodilator in the penis via its potent stimulation of NOS protein expression and activity. In one study of castrated rats, testosterone fully restored penile NOS activity, which had been reduced by 45%.³⁸  In humans, bioavailable testosterone’s direct relationship with cavernous vasodilation was demonstrated in a retrospective, double-blind correlation analysis of 52 impotent men who had no confounding risk factors for ED. Low free testosterone correlated independently of age with impaired relaxation of cavernous endothelial and corporeal smooth muscle cells in response to a vasoactive challenge, confirming the importance of androgens in regulating smooth muscle function in the penis.³⁹ 

Testosterone – the Structure-Function Connection

In addition to stimulating NOS expression and activity, testosterone also plays a key role in maintaining penile structural integrity. Recent studies indicate that androgens are necessary for the maintenance of the anatomical and physiological substrate of erections, and that the beneficial effects of PDE-5 inhibitors can only be optimally expressed in the eugonadal male.⁴⁰  Experimental animal models have demonstrated that androgens beneficially affect structural components in the corpus cavernosum, including the smooth muscle fibers and connective tissue necessary for the structural and functional integrity of penile erection. Androgen deprivation promotes differentiation of progenitor stroma cells in the corpus cavernosum into adipocytes; clearly, replacement of muscle and connective tissue with fat cells will impair erectile function.⁴¹  The diameter of the dorsal nerve and nerve fibers in the rat corpus cavernosum are also dependent on androgens, evidenced by their shrinking in diameter in castrated rats.⁴²⁴³  In both animals and humans, the penile corpus cavernosum is a vascular bed; androgen insufficiency-related alterations to its structure will produce vascular dysfunction.⁴⁴  In recognition of these factors, European guidelines now recommend that testosterone concentrations be measured in all men presenting with ED.³⁷

Inhibiting NO breakdown

As discussed in “Longevity Medicine Strategies for Cardiovascular Disease: Closing the Statin Gap in Endothelial Dysfunction and Insulin Resistance Naturally, with L-Arginine and Citrulline: Part II,” oxidative inactivation of NO caused by the increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the vasculature that accompany cardiovascular disease, MetS and diabetes, are a significant contributing factor to NO insufficiency. As noted in a review titled, Oxygen free radicals and the penis, “evidence from basic scientific studies indicates that oxidative stress mediated through the superoxide radical and other reactive oxygen species (ROS) may be central to impaired cavernosal function in erectile dysfunction.” ⁴⁵

Tetrahydrobiopterin (BH4). RNS are of special concern since peroxynitrites oxidize tetrahydrobiopterin (BH4), the cofactor for eNOS, and once uncoupled, eNOS itself will produce superoxide radical by reducing oxygen. This promotes a vicious cycle in which levels of reactive species build, causing further eNOS uncoupling, insufficient NO production, and vascular oxidative damage. BH4 levels can be preserved by supplementation with ascorbate and 5-methyltetrahydrofolate (5-MTHF), the circulating form of folate. Ascorbate does not fully protect BH4 from oxidation by perxoynitrite, but effectively recycles BH3 radical back to BH4.⁴⁶⁴⁷⁴⁸  5-MTHF is a strong peroxynitrite scavenger, which has been shown to increase vascular BH4 and the BH4/total biopterin ratio.⁴⁹ 

Methylating Factors. Approximately 10-fold more L-arginine is metabolized to creatine than is used for NO synthesis,⁵⁰  and an intermediary compound formed during creatine synthesis is homocysteine. Thus, in addition to 5-MTHF, the other key nutrients needed for the metabolism of homocysteine, namely the other B vitamins needed for methylation, B6 and B12, are also required to prevent the accumulation of homocysteine, which lessens NO production both by promoting increased ROS production, and also by inhibiting dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that metabolizes the potent NOS inhibitor, asymmetric dimethylarginine (ADMA) to L-citrulline.⁵¹⁵²

Alpha-lipoic acid (ALA). A potent water- and fat-soluble antioxidant, ALA has also been shown to improve endothelial function by ameliorating the age-related decrease in the phosphorylation of NOS.⁵³  When given to diabetic rats exhibiting a 41% reduction in endothelium-dependent NO-mediated relaxation in the corpus cavernosum, ALA reversed this reduction by 65%.¹

In research using rats with diabetes-induced ED, ALA was found to decrease inducible macrophage-type NOS (iNOS), while increasing eNOS and neuronal NOS (nNOS).⁵⁴   nNOS is expressed in penile neurons innervating the corpus cavernosum, while eNOS protein expression has been identified primarily in both cavernosal smooth muscle and endothelium.⁵⁵   Vasodilatation of penile arteries and large veins during erection is mediated by nNOS. The subsequent increased arterial inflow to the cavernosal sinoids and shear stress on the endothelium lining the penile arteries triggers Akt phosphorylation of eNOS and NO production.¹⁶  iNOS induction during aging has been hypothesized to cause neurotoxicity in critical related regions of the hypothalamus during senescence and to be a major cause in the net loss of trabecular smooth muscle in the corpus cavernosum through apoptosis.⁵⁶

Ascorbic acid.  In addition to recycling BH3 radical back to BH4, L-ascorbic acid’s ability to enhance impaired endothelium-dependent vasodilation is likely attributable to its antioxidant actions, including scavenging intracellular superoxide, direct reduction of nitrite to NO, and recycling of vitamin E.⁵⁷⁵⁸

Alpha tocopherol. In studies of hypertensive rats, alpha tocopherol decreased oxidant levels, while increasing the activity of superoxide dismutase and corpus cavernosum relaxation, and improving erectile function.⁵⁹   When given concurrently with PDE-5 inhibitors to 89 men with ED (average age 61.6) who had failed to respond to PDE-5 inhibitors, administration of alpha-tocopherol (300 mg/day for 1 month) resulted in an increase in the average IIEF score from 13.8 to 17.1. ⁶⁰

Thermal Therapy. In addition to increasing expression of eNOS mRNA (discussed above), sauna treatments have been shown to increase production of BH4, thus also protecting eNOS from ROS and increasing NO availability by lessening eNOS uncoupling. Sauna therapy increases BH4 availability via two distinct pathways. Increased blood flow in heated surface tissues results in increased vascular shear stress, thus inducing increased activity of GTP cyclohydrolase I (GTPCH-I, the rate-limiting enzyme involved in de novo biosynthesis of BH4) in those vascular tissues. Even modest heating of mammalian tissues also induces heat shock protein 90 (Hsp90), which interacts with GTPCH-I, increasing its activity by lowering its degradation. Increased BH4 synthesis in surface tissues of the body will result in increased circulating BH4, providing BH4 to other body tissues, e.g., penile tissue, which may be BH4 deficient.⁶¹

Conclusion

ED, like diabetes, should be considered a “cardiovascular equivalent.”⁶²  ED unresponsive to PDE-5 inhibitors should definitely be a red flag for risk of serious endothelial dysfunction. As the relationship among cardiovascular disease, MetS/type 2 diabetes, and ED clearly indicates, first line therapy in the treatment of ED should be promotion of the diet and lifestyle choices now shown in hundreds of studies to improve cardiovascular health and restore insulin sensitivity: a Mediterranean-style diet including frequent consumption of omega-3-rich fish, regular aerobic and resistance exercise, and 6-8 hours nightly sleep.

Aging itself is already associated with increased oxidative stress, but the process is greatly accelerated by MetS, type 2 diabetes and obesity, all of which promote CVD and reduced production of NO, which in turn suppresses the NO-cGMP pathway, resulting in ED unresponsive to PDE-5 inhibitors. Addressing the underlying causes of endothelial dysfunction is a necessary prerequisite for PDE-5 efficacy.

NO production lessens slightly even in the healthiest of aging males, and PDE-5 inhibitors can help extend the years in which to enjoy an active sex life. Even in healthy, younger men, these cGMP-conserving drugs can bolster sexual function, enabling a stronger erection and enhanced ability to engage in sexual intercourse. The ideal outcome for the aging male is for PDE-5 inhibitors to be optional—to be able to function with—or without—these performance enhancing drugs.  The natural therapies discussed in this article provide this option, increasing not only a man’s potential for healthy erectile function but his overall health and longevity.
 

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Women are at risk of vitamin A deficiency.

In his presentation at the 2nd Hohenheim Nutrition Conference in Stuttgart, Germany, November 2009, Dr. Georg Lietz of England's Newcastle University, the senior investigator in research published April 2009 in the FASEB Journal (and summarized in our June 2009 LMR review, “Vitamin A – Tolerance Extends Longevity”), reported that a high percentage of women in the UK are at risk of vitamin A deficiency. Two common genetic variants greatly lessen the body’s ability to convert beta-carotene into vitamin A.

As noted in LMR’s Vitamin A review, concerns regarding potential toxicity from hypervitaminosis A have led to the recommendation that much, if not all, of the vitamin A requirement be met by consuming pro-vitamin A carotenoids, which have been thought to be readily converted to retinoic acid as needed. In fact, due to concerns about the teratogenic potential of hypervitaminosis A, beta-carotene has been considered the preferred source of vitamin A for women of reproductive age, and due to their dietary restrictions, is the primary source of the nutrient for vegetarians and its only source for vegans. The ability to convert beta-carotene into vitamin A has, however, recently been found to vary significantly in up to 45% of healthy individuals.¹

When Lietz and colleagues examined the gene that encodes the enzyme beta-carotene 15,15'-monoxygenase (BCMO1) the enzyme responsible for conversion of beta-carotene to retinoic acid in 62 female volunteers in Great Britain, 47% of the women were found to have at least one of two SNPs that greatly reduce activity of BCMO1.

Furthermore, these two SNPs (R267S and A379V) are common in the UK population at large, with variant allele frequencies of 42 and 24%, respectively. In vitro analysis by Lietz et al. revealed that the 267S + 379V double mutant produced a 57% reduction in catalytic activity of BCMO1. Female volunteers carrying both variant alleles and given a pharmacological dose of β-carotene were shown to have seriously reduced ability to convert β-carotene, indicated by reduced retinyl palmitate: beta-carotene ratios in the triglyceride-rich lipoprotein fraction of -32% and -69% , respectively, and increased fasting beta-carotene concentrations of +160% and +240% , respectively.

Interviewed at the 2nd Hohenheim Nutrition Conference, Leitz emphasized, "Vitamin A is incredibly important—particularly at this time of year when we are all trying to fight off the winter colds and flu. It boosts our immune system and reduces the risk of inflammation such as that associated with chest infections. What our research shows is that many women are simply not getting enough of this vital nutrient because their bodies are not able to convert the beta-carotene."

"Worryingly, younger women are at particular risk," Dr Lietz added. "The older generations tend to eat more eggs, milk and liver which are naturally rich in vitamin A whereas the health-conscious youngsters on low-fat diets are relying heavily on the beta-carotene form of the nutrient." Many health conscious elders also strive to follow a low-fat diet and thus avoid vitamin A rich foods.

In addition to the significant number of individuals whose genetic inheritance renders them “low-responders,” unable to absorb and/or convert provitamin A carotenoids to vitamin A, a number of food and other host-related factors can significantly impact carotenoid bioavailability, absorption and conversion to retinol.²

The presence of virtually any of the following factors can inhibit the conversion or render the amount of carotenoids absorbed insufficient to produce or maintain adequate levels of vitamin A, even in individuals who are not carriers of the SNPs R267S and/or A379V and thus should be able to metabolize provitamin A to vitamin A:

• Normal absorption of carotenoids is minimal: About 70-90% of ingested retinol is absorbed, but even under optimal circumstances, only 3% or less of carotenoids.³
• Vitamin A stores are depleted by at least 5% each day: Despite its being a fat-soluble nutrient, under normal conditions, 5% of vitamin A stores are lost daily.
• Large variations in the concentration of provitamin A carotenoids in foodstuffs are typical—even in the same type of food—due to varietal differences, stage of maturity, climatic conditions, processing and/or cooking.
• Food matrix and preparation: Efficiency of carotenoid absorption is affected not only by the amount of carotenoid ingested, but by processing and/ or cooking of the food, other dietary ingredients that stimulate absorption (e.g., the type and amount of dietary fat [carotenoids must be incorporated into mixed micelles and then into chylomicrons for absorption] or inhibit it [fiber, especially pectins], food matrix effects, and interactions between carotenoids [lutein and β-carotene appear to inhibit one another’s absorption]).⁴
• Availability of bile acids, iron, riboflavin, niacin, and zinc: The enzyme beta-carotene 15,15'-monoxygenase, which converts the provitamin A carotenoids to retinal mainly in the intestinal mucosa (and, to a lesser extent, in testes, liver and kidneys), requires bile acids and iron for its three-step activity (epoxidation at the 15,15'-double bond, hydration to the diol, and oxidative cleavage). Retinal resulting from carotenoid cleavage is metabolized, mainly to retinol, by several tissue specific enzymes that require riboflavin, niacin, and zinc as cofactors.
• Bile flow is compromised by acute and chronic liver diseases, including non-alcoholic steatohepatitis (NASH) related to insulin resistance / metabolic syndrome, which is now estimated to affect ~30% of the US population⁵, and alcoholism.
• Alcoholism promotes vitamin A deficiency via two mechanisms. Not only is bile flow compromised, but alcohol dehydrogenase—the enzyme that catalyzes conversion of retinol to retinaldehyde, which is then oxidixed to retinoic acid—has a stronger affinity for ethanol.⁶
• Drug induced depletion: Antacids containing aluminum hydroxide can impair vitamin A absorption. Cholesterol-lowering agents, e.g., bile acid sequesterants such as cholestyramine, cholesipol, colestipol, reduce fat absorption and thus absorption of vitamin A and other fat-soluble nutrients. Colchine impairs vitamin A absorption by blocking the release of retinol-retinol binding protein. Neomycin, if high doses over extended period, may impair vitamin A absorption. Sucralfate decreases absorption of fat-soluble nutrients.⁷
• Impaired digestion: Absorption of β-carotene and other carotenoids from vegetables is only 5-30% of that absorbed from synthetic supplements, due to the food matrix of fiber and/or protein that must first be broken down by mastication, gastric acid and bile acids.⁸
• Imbalanced gut ecology: due, for example, to parasitic infestation, food allergies such as gluten intolerance, celiac disease (U.S. incidence 1 in 133)⁹, lipid malabsorption, hydrochloric acid insufficiency (30% of the US population >60 is estimated to have atrophic gastritis)⁸, infection with H.pylori, (prevalence of H.pylori infection is 50% worldwide, 40% in the US¹⁰) or Clostridium difficile (annual incidence of C.difficile infection increased from 49.2 to 101.6 per 100,000 population from 2001-2005¹¹)
• Exposure to heptatoxicants. Numerous hepatotoxicants, e.g., carbon tetrachloride, reduce retinol storage and or alter retinoid metabolism. In 2008, a study of common cleaning products found the presence of carbon tetrachloride in "very high concentrations" (up to 101 mg m−3) as a result of manufacturers' mixing of surfactants or soap with sodium hypochlorite, i.e. chlorine bleach.¹²
• Supplementation with increasingly high levels of vitamin D. A plethora of studies published within the last 5 years have ensured clinicians awareness of the pandemic of vitamin D deficiency with the result that many are testing patients’ vitamin D levels and prescribing supplementation with 1,000 to up to 10,000 IU vitamin D daily. Retinoic acid and 1,25(OH)2D3 compete for the same nuclear receptor partners; both the retinoic acid receptor (RAR) and the vitamin D receptor (VDR) must form heterodimers with retinoic X receptors (RXRs) to be able to bind to response elements and initiate transcription. For this reason, 1,25(OH)2D3 and retinoic acid naturally mitigate against each other’s uncontrolled effects. High doses of vitamin D may upset the balance between it and vitamin A, which, as discussed in “Vitamin A -- Tolerance Extends Longevity,” is a key regulator of self and oral tolerance.¹³

Taken together, the genetic, food and host-related factors affecting carotenoid conversion to vitamin A clearly indicate that while the RDIs for vitamin A were intended to include a reasonable safety margin, they are based on overly optimistic assumptions regarding the amount of β-carotene the majority of individuals actually absorb and convert to retinol.

The spotlight Leitz has focused on the SNPs likely to induce vitamin A insufficiency is timely, especially given current concerns re the potential for an epidemic of H1N1 Swine Flu¹⁴, a public health threat that should underscore the importance of vitamin A sufficiency for immune function. Above and beyond this current challenge, vitamin A will remain critical for overall health and longevity because retinoic acid affects the expression of at least 532 genes, and the study by Leitz et al. both explains and confirms research conducted in a number of other geographical areas (including Indonesia¹⁵, France¹⁶, Germany¹⁷¹⁸and the U.S. (specifically University of California, Davis)¹⁹, suggesting widespread penetrance of the R267S and A379V SNPs in the human species.

For an in-depth discussion of vitamin A’s effects on immunity, relationship to vitamin D, and recommendations regarding assessment of patients’ vitamin A status, please see our review, “Vitamin A -- Tolerance Extends Longevity.”.Dr Lietz and his associates plan to assess whether body composition, i.e., BMI, also affects the ability to convert beta-carotene into vitamin A. Given the rapidly rising incidence of obesity in the western world, a negative impact of excessive adipose tissue on carotenoid conversion to retinoic acid would have serious implications. We will follow Lietz’s progress and keep you updated.

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Despite their efficacy in lowering cholesterol, statins remain ineffective for the primary or secondary prevention of myocardial infarction (MI) in two-thirds of patients, and cardiovascular disease (CVD) remains the #1 cause of death in the U.S. A key reason for statins’ failure to reduce MI incidence is that they ameliorate neither endothelial dysfunction, nor its corollary, hypertension. Additionally, statins do not impact insulin resistance, a major contributing factor to CVD as well as diabetes, which itself quadruples risk for CVD. Statins’ lack of efficacy against these risk factors constitutes a treatment gap that results in high risk of morbidity notwithstanding low cholesterol levels—a gap that has now been connected to elevated levels of a recently identified CVD risk factor, asymmetric dimethylarginine, whose deleterious effects can be overcome by treatment with L-arginine, which has been shown to significantly improve endothelial function both alone and when added to statin therapy, particularly if accompanied by necessary co-factors, including tetrahydrobiopterin and the methylating factors, B6, B12 and folate. Part I of this review summarizes the latest research on L-arginine’s beneficial effects on nitric oxide production, endothelial function and insulin resistance. Part II reviews the research on the co-factors without which L-arginine supplementation may not only lack efficacy, but could promote CVD; improvement in CVD outcomes from combined statin and L-arginine therapy; and why L-citrulline may offer an even better option.

Part II: Key Considerations When Prescribing L-arginine: Maximizing Efficacy, Preventing Harm

Co-factors, without which, L-arginine may contribute to CVD

Tetrahydrobiopterin (BH4) — key to L-arginine’s efficacy in established atherosclerosis^{1 6 2}

BH4 is a necessary cofactor for endothelial nitric oxide synthase (eNOS) to produce NO. Furthermore, in conditions of deficient BH4, eNOS generates superoxide (O₂-) instead of NO; therefore, it is particularly important to use BH4 in conjunction with L-arginine. 

NO is synthesized by the family of oxidoreductase enzymes, the NO synthases, which utilize L-arginine as their principal substrate, oxidizing it to L-citrulline and NO, in a process involving the cofactors reduced nicotinamide adenine dinucleotide phosphate (NADPH) [which requires niacin], flavin adenine dinucleotide  (FAD) [which requires riboflavin], and tetrahydrobiopterin (BH4). 

Insufficiency of endothelial NO can occur as a result of decreased synthesis due to decreased expression of eNOS and/or insufficient substrate (L-arginine) and/or co-factors (tetrahydrobiopterin, niacin, riboflavin); or an increase in NO’s oxidative inactivation to nitrite, nitrate, and peroxynitrites due to the presence of increased ROS in the vasculature, including O₂-, H2O2, derivative hydroxyl radical (OH-); lipid peroxides (e.g., malondialdehyde) and derivative peroxyl radicals; or both. Under normal circumstances, these highly reactive derivatives of oxidative metabolism are neutralized by antioxidant enzymes (e.g., superoxide dismutases and glutathione peroxidases) and antioxidants (e.g., -tocopherol and ascorbate neutralize ROS; γ-tocopherol neutralizes reactive nitrogen species [RNS]) (Vitamin A – Tolerance Extends Longevity); however, virtually all risk factors for CVD, as well as frank CVD, increase the load of ROS and RNS in the vasculature. eNOS itself will produce O₂- by reducing oxygen when eNOS becomes "uncoupled" by limited availability of its cofactor tetrahydrobiopterin or of its substrate, L-arginine.

In established atherosclerosis, expression of the inducible form of NOS ( iNOS) is strongly upregulated. iNOS is present in activated macrophages, is found within atherosclerotic lesions, and is proatherogenic. iNOS produces greater amounts of NO compared to eNOS—a big problem in an atheromatous phenotype in which ROS are abundant because the large increase in NO production results in NO reacting with O₂- and lipid peroxyl radicals to yield the RNS, peroxynitrite and lipid peroxynitrites.

These RNS are potent oxidants and inactivate NO because peroxynitrites oxidize tetrahydrobiopterin (BH4), which uncouples eNOS. The uncoupling of eNOS not only decreases NO production, but the uncoupled enzyme itself increases production of O₂-.  This promotes a vicious cycle in which levels of reactive species build, causing further eNOS uncoupling, insufficient NO production, and oxidative damage to proteins in the vessel wall—a recipe for CVD progression.³

Even if ROS are controlled by antioxidants, in the absence of sufficient BH4, the oxidation of L-arginine is no longer coupled to NADPH consumption, so NOS isoforms catalyze the formation of O₂- at the oxygenase domain. In fact, BH4 levels appear to regulate the ratio of O₂- and NO made by NOS enzymes. When BH4 levels are adequate, eNOS produces NO; when BH4 levels are limiting, eNOS becomes enzymatically uncoupled and generates O₂-, contributing to vascular oxidative stress and endothelial dysfunction. Thus, insufficient BH4 can be a key cause of NO-related endothelial dysfunction.

BH4 levels can be preserved by supplementation with ascorbate and 5-methyltetrahydrofolate (5-MTHF), the circulating form of folate. Ascorbate does not fully protect BH4 from oxidation by perxoynitrite (ONOO-), but effectively recycles BH3 radical back to BH4.⁴ 5-MTHF is a strong peroxynitrite scavenger, which has been shown to increase vascular BH4 and the BH4/total biopterin ratio.⁵

Methylating Factors⁶

Since, as noted above, iNOS promotes CVD progression, researchers hypothesized that providing supplemental L-arginine to apoE null mice [animals bred to quickly develop atherosclerosis] also lacking iNOS would decrease plaque burden. It didn’t; in fact, it increased atheromatous burden to levels observed in the apoE null mice, completely offsetting the benefit of eliminating iNOS.⁷ 

The most likely explanation for this outcome is related to the fact that approximately 10-fold more L-arginine is metabolized to creatine than is used for NO synthesis,^{8 9} and creatine synthesis requires the methylation of guanidinoacetate by S-adenosyl-methionine in the liver, which yields S-adenosyl-homocysteine. S-adenosyl-homocysteine is then hydrolyzed to adenosine and homocysteine by S-adenosyl-homocysteine hydrolase. 

Homocysteine can either be metabolized by methylation to methionine or undergo transsulfuration to cysteine, which can be utilized to form glutathione. However, converting homocysteine to methionine requires adequate methylation support, which may be in short supply because -- although the source of methyl groups is in a separate, although metabolically linked cellular process -- as much as 70% of accessible methyl groups may be used up for creatine synthesis.¹⁰ 

In addition, vascular cells are unable to transsulfurate homocysteine to cysteine (and thence to glutathione), so their only option for neutralizing homocysteine is remethylating it to methionine.7  Thus, if nutrients necessary for methylation (B6, B12, folate) are in short supply, local concentrations of homocysteine, a highly atherogenic amino acid, will increase in the vasculature. To cap it all off, homocysteine increases plasma concentrations of ADMA by inhibiting dimethylarginine dimethylaminohydrolase, the enzyme that metabolizes this NOS inhibitor to L-citrulline.¹¹

The clinical takeaway here is to ensure adequacy of both tetrahydrobiopterin and methylating factors when supplementing L-arginine, not only to promote L-arginine’s efficacy in improving endothelial function, but because inadequacy of these factors may result in increased CVD risk.

L-arginine Dosage Considerations

A recent study evaluating the responses of healthy individuals to increasing doses of L-arginine suggests that supplementation with 3 grams bid is optimal. This level, which is about twice the amount of L-arginine present in a typical Western diet, was associated with no adverse side effects.¹²

Study participants were instructed to take L-arginine for 1-week periods at daily doses of 3, 9, 21, and 30 grams. Ten of the 12 subjects reported adverse gastrointestinal side effects at higher dosages, five subjects at 21 grams/day, and five subjects at 30 grams/day. It was hypothesized that a large bolus of L-arginine may disturb the acid–base balance of the stomach and GI tract, thus provoking gastrointestinal symptoms.

Mean L-arginine concentrations peaked at 9 grams/day (169 μmol/L), were found to be significantly higher than values at baseline (101 μmol/L) or 3 grams/day (110 μmol/L), and also slightly higher than L-arginine levels at 21 g/day (164 μmol/L). 

Availability of L-arginine, relative to that of ADMA, increased significantly at both 9 grams/day and 21 grams/day. As ADMA is produced by proteolysis of proteins containing methylated arginine residues, its levels would not be expected to be altered by L-arginine supplementation and, in fact, were not. Indeed, lack of effect on ADMA levels is an aspect of the practical utility of L-arginine supplementation, which increases the substrate available to NOS without raising the level of inhibitors. Any increase in arginine accessibility when ADMA levels remain constant or decline slightly will favor NO production. 

Potential side-effects of L-arginine supplementation

L-arginine induces water and electrolyte secretion that is mediated by NO, which acts as an absorbagogue at low levels and as a secretagogue at high levels. The action of many laxatives is NO mediated, and diarrhea following oral administration of arginine in single doses >9 grams has been reported. 

The clinical data cover a wide span of arginine intakes from 3 grams/day to>100 grams/day. Single doses of 3-6 grams rarely provoke side effects. Single doses >9 grams are more likely to provoke gastrointestinal symptoms in healthy athletes than diabetic patients, which may relate to the effects of disease on gastrointestinal motility and pharmacokinetics. Most side effects have occurred at single doses of >9 grams in adults, often when part of a daily regime of >30 grams/day. Adverse effects seemed dependent on the dosage regime and disappeared if divided doses were ingested.¹³

L-arginine may promote replication of herpes simplex virus 1 (HSV-1) in already infected individuals.  HSV-1 multifunctional regulatory protein ICP27 shuttles between the nucleus and cytoplasm in its role as a viral mRNA export factor. Arginine methylation has been shown to regulate protein ICP27 export and is thus required for efficient HSV-1 replication. Therefore, individuals infected with oral and/or genital herpes may need to reduce L-arginine dosage or avoid foods high in L-arginine when under high stress, fighting off a viral infection, or otherwise immune-challenged.¹⁴

On the other hand, arginine has recently been investigated as an antiherpetic agent against HSV-1. Arginine suppressed the growth of HSV-1 concentration-dependently and was particularly effective when added within 6 hours post-infection. When administered in the early stages of the infection, the latent period was significantly extended; the rate of viral replication decreased, and the final yield of viral progeny decreased to 1%. However, when arginine was added at 8 hours post infection after the completion of viral DNA replication, the amount of viral progeny produced in the subsequent 4 hours reached normally expected levels.¹⁵ 

Citrulline – a Preferential Source of L-arginine?

Supplemental L-arginine is readily absorbed; however, ~50% of ingested L-arginine is rapidly converted in the body to ornithine, primarily by the enzyme arginase.¹⁶ As noted above, in the presence of oxidized LDL, arginase activity is increased, and arginase competes with NOS for L-arginine in its role as a substrate for NOS, which results in impaired production of NO and increased production of ROS by NOS. 

In addition to supplemental L-arginine, arginase expression and activity is enhanced by diseases associated with endothelial dysfunction, including hypertension, heart failure, atherosclerosis, diabetic vascular disease and ischemia-reperfusion injury, thus further reducing the effectiveness of L-arginine therapy in those who need it most.¹⁷

Furthermore, L-arginine, in addition to its role as a substrate for NOS, is also a precursor for the synthesis of proteins, urea, creatine, vasopressin, and agmatine. Thus, L-arginine that escapes metabolism by arginase is targeted for processing by four other enzymes: NOS (to become NO); arginine:glycine amidinotransferase (to become creatine); arginine decarboxylase (to become agmatine); and arginyl-tRNA synthetase (to become arginyl-tRNA, a precursor to protein synthesis).¹⁷

Because of L-arginine’s fast turnover, sustained-release preparations are thought to be a better way to maintain blood levels over time, yet arginase’s substantial intestinal and hepatic metabolism of L-arginine to ornithine and urea, and L-arginine’s use for other functions in the body, impede the likelihood of optimal efficacy from oral supplements of L-arginine.¹⁸

L-citrulline may be a better option. L-citrulline is readily absorbed and efficiently converted to L-arginine. But its conversion does not take place in the intestine or liver, and therefore not only does not induce tissue arginase, but inhibits its activity. Upon entering the kidney, vascular endothelium and other tissues, L-citrulline is readily converted to L-arginine, raising plasma and tissue levels of L-arginine and enhancing NO production.14 Published data indicates that L-citrulline has relatively better absorption and systemic bioavailability than L-arginine.¹⁹

In a double-blind, randomized, placebo-controlled cross-over study, 20 healthy volunteers were given 6 different dosing regimens of placebo, citrulline, and arginine. After one week of oral supplementation, the plasma L-arginine/ADMA ratio was measured. L-citrulline dose-dependently increased plasma L-arginine concentration more effectively than L-arginine. The highest dose of citrulline (3 grams bid) improved the L-arginine/ADMA ratio from 186 +/- 8 (baseline) to 278 +/- 14, a 49% increase.²⁰

In another recent study, L-citrulline supplementation was shown to  attenuate brachial blood pressure and aortic hemodynamic responses to stress induced by the cold pressor test (CPT) in 17 young (average age 21.6 years) normotensive men. Subjects were randomly assigned to 4 weeks of oral L-citrulline (6 grams/day) or placebo in a crossover design. Hemodynamic responses to CPT were evaluated after each treatment. Compared to placebo, oral L-citrulline decreased brachial systolic blood pressure (-6 +/- 11 mm Hg), aortic systolic blood pressure (-4 +/- 10 mm Hg), and aortic pulse pressure (-3 +/- 6 mm Hg) during CPT but not at rest, suggesting improved induction of NO under stress.²¹

L-citrulline’s beneficial effects on NO were also demonstrated in a group of 17 male professional cyclists in whom oral L-citrulline administration prior to a cycling race increased plasma arginine availability for NO synthesis and immune cells’ (polymorphonuclear neutrophils [PMNs]) priming for oxidative burst without oxidative damage. Cyclists were randomly assigned receive 6 grams L-citrulline-malate or placebo, after which they participated in a race. Blood samples were taken in basal conditions, immediately after and 3 hours post-race. Citrulline supplementation significantly increased plasma concentration of both arginine and citrulline post-race. In controls, PMNs responded to exercise with a progressive decrease in immune-defensive ROS production, while PMNs in those supplemented with L-citrulline significantly increased ROS production after exercise compared to basal values and then diminished to values lower than basal at recovery.²² L-citrulline’s enhancement of immunity may benefit endurance athletes as it has long been noted that immune defenses drop post-event, increasing susceptibility to infection.^{23 24 25 26 27}

Citrulline’s potential benefit to endothelial function is also evidenced by the fact that impairment in the activity or expression of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that metabolizes ADMA to L-citrulline and dimethylamine, results in elevated ADMA concentrations and reduced NO synthesis, and promotes the onset and progression of atherosclerosis in experimental models. AMDA elevation may be a marker for insufficient availability of citrulline due to DDAH activity and/or expression, which then contributes to the pathogenesis of endothelial dysfunction in various diseases.^{18 28} 

Adding L-Arginine Closes the Statin Gap

Despite the fact that statins increase eNOS expression (by stabilizing eNOS mRNA) and enhance eNOS activity (by decreasing caveolin, which otherwise forms an inhibitory complex with eNOS and impairs NO release), statins have failed to improve endothelial function in the majority of studies in which their effects on the endothelium have been evaluated.^{2 29 30}

A key reason for statins’ lack of efficacy in this regard is ADMA, which inhibits eNOS by a mechanism unaffected by statins, but reversible by L-arginine.³¹ It was therefore hypothesized, and has now been demonstrated in several studies, that providing both a statin and L-arginine to individuals with high levels of AMDA can close the statin gap. 

In a study involving 98 clinically asymptomatic elderly subjects, those in the highest and lowest quartiles of the ADMA distribution were given, in a randomized order, simvastatin (40 mg/day), L-arginine (3 grams/day), or a combination of both, each for 3 weeks. Endothelium-dependent vasodilation (EDD) was assessed by brachial artery ultrasound. While simvastatin alone had no effect on EDD in subjects with high ADMA (6.2 at baseline vs. 6.1 after simvastatin treatment), simvastatin plus L-arginine significantly improved EDD (9.8 at baseline vs. 5.3 after treatment). In subjects with low ADMA, L-arginine alone, simvastatin alone, or the two combined improved EDD. When given alone, only L-arginine, but not simvastatin, improved endothelial function in both groups.³⁰

L-arginine has also been shown to enhance the triglyceride-lowering effect of simvastatin in a 2-arm, randomized, double-blind study of 33 hypertriglyceridemic patients that consisted of a 6-week run-in phase, 6 weeks of treatment with L-arginine (1.5 grams bid) or placebo, and a 6-week extension period in which simvastatin (20 mg q.d.) was added. The combination of L-arginine with simvastatin led to a significantly stronger reduction in triglycerides compared to placebo plus simvastatin (-140.5 +/- 149.2 mg/dL vs. -56.1 +/- 85.0 mg/dL). L-arginine also attenuated simvastatin-induced increases in aspartate transaminase (elevated levels of this enzyme indicate acute liver, cardiac and skeletal muscle, blood cell, kidney and/or brain tissue damage) and also in fibrinogen (the protein in plasma from which fibrin is generated in the process of clot formation), but had no triglyceride-lowering effects when given alone.³²

Assessing Endothelial Function

Although FMD is the “gold standard” to evaluate endothelial function, this technique requires specialist imaging equipment and great attention to detail in order to obtain reproducible results. An alternative approach, suitable for assessing pre-clinical atherosclerosis and evaluating the effect of interventions on endothelial function, e.g., L-arginine supplementation, has recently become available. Vascular tone of the small arteries can now be easily and inexpensively measured in-office by checking the digital pulse wave reflection index, which has been shown to correlate with FMD and has high sensitivity and specificity in detecting arterial distensibility and stiffness, and abnormal endothelial function as defined by FMD.^{33 34 35} In recently published studies using pulse wave methodology, L-citrulline has been shown to lower aortic pulse pressure and improve hemodynamic responses to stress in normotensive men, and L-arginine to improve carotid-femoral pulse wave velocity, an index of aortic stiffness, in healthy smokers at rest and after acute smoking.^{36 37}

Conclusion

Lifetime risk for CVD is currently 49% for men and 32% for women ≥age 40—statistics that are likely to increase exponentially given the fact that individuals with diabetes are two to four times more likely to develop CVD, and, as of 2006, 66.7% of the adult population in the United States was overweight or obese, 34.6% had MetS, and 5.9% had been diagnosed with type 2 diabetes.

Despite their efficacy in lowering cholesterol, statins are unable to improve endothelial function, a risk factor for CVD-related mortality on a par with hypercholesterolemia. Nor do statins impact insulin resistance, a key cause of endothelial dysfunction. Recent studies indicate that endothelial dysfunction, as reflected by FMD (or pulse wave reflection index), is a more powerful prognosticator of future cardiac events than carotid artery plaque burden, and patients with high FMD have low cardiovascular event rates irrespective of their degree of carotid atheroma. Statins’ lack of efficacy against endothelial dysfunction constitutes a gap in the treatment of CVD that results in high risk of morbidity notwithstanding low cholesterol levels—a gap that is largely due to elevated levels of ADMA and may be overcome by supplementation with L-arginine and/or L-citrulline.

Read Part I: L-Arginine Closes the Statin Gap by Overcoming ADMA’s Promotion of Endothelial Dysfunction and Insulin Resistance

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Despite their efficacy in lowering cholesterol, statins remain ineffective for the primary or secondary prevention of myocardial infarction (MI) in two-thirds of patients, and cardiovascular disease (CVD) remains the #1 cause of death in the U.S. A key reason for statins’ failure to reduce MI incidence is that they ameliorate neither endothelial dysfunction, nor its corollary, hypertension. Additionally, statins do not impact insulin resistance, a major contributing factor to CVD as well as diabetes, which itself quadruples risk for CVD. Statins’ lack of efficacy against these risk factors constitutes a treatment gap that results in high risk of morbidity notwithstanding low cholesterol levels—a gap that has now been connected to elevated levels of a recently identified CVD risk factor, asymmetric dimethylarginine, whose deleterious effects can be overcome by treatment with L-arginine, which has been shown to significantly improve endothelial function both alone and when added to statin therapy, particularly if accompanied by necessary co-factors, including tetrahydrobiopterin and the methylating factors, B6, B12 and folate. Part I of this review summarizes the latest research on L-arginine’s beneficial effects on nitric oxide production, endothelial function and insulin resistance. Part II reviews the research on the co-factors without which L-arginine supplementation may not only lack efficacy, but could promote CVD; improvement in CVD outcomes from combined statin and L-arginine therapy; and why L-citrulline may offer an even better option.

Part I: L-Arginine Closes the Statin Gap by Overcoming ADMA’s Promotion of Endothelial Dysfunction and Insulin Resistance

Introduction

Despite widespread use of statins, cardiovascular disease remains the #1 cause of death in the U.S., afflicting 36.3% of the American population, including more than 38 million individuals ≥ 60.¹ Albeit the most successful pharmacotherapy agents used to treat atherosclerosis, statins remain ineffective for the primary or secondary prevention of myocardial infarction (MI) in two-thirds of patients.²

A key reason for statins’ failure to reduce MI incidence is the fact that, in the majority of studies examining their impact, statins, although highly effective in lowering cholesterol, have failed to mitigate endothelial dysfunction, a risk factor for CVD-related mortality on a par with hypercholesterolemia.³ Recent studies indicate that brachial endothelial function, as reflected by flow-mediated vasodilation (FMD), actually has more powerful prognostic value for predicting future cardiac events than carotid artery plaque burden,⁴ and patients with high FMD have low cardiovascular event rates irrespective of their degree of carotid atheroma.⁵

Statins’ lack of efficacy against endothelial dysfunction and its corollary, hypertension, constitutes a gap in the treatment of CVD that results in high risk of morbidity notwithstanding low cholesterol levels – despite the fact that statins have been shown to upregulate gene expression of endothelial nitric oxide synthase (eNOS) [the enzyme responsible for the production of vasodilating endothelial nitric oxide (NO)].³ In a significant number of patients, a primary reason for the statin gap is elevated levels of a recently identified cardiovascular risk factor, asymmetric dimethylarginine (ADMA), an inhibitor of eNOS whose deleterious effects may be overcome by supplementation with an inexpensive, natural agent, L-arginine.

A second key reason for statins’ failure to prevent MI is that statins also have no impact on insulin resistance, which is most often related to obesity, especially abdominal obesity, and has been recognized as a major contributing factor to hypertension. In the Framingham study, each 10% gain in weight was associated with a 6.5 mm Hg increase in systolic blood pressure.⁶ The connection can be partly understood by noting that L-arginine levels are significantly lower, and ADMA levels higher, in individuals with impaired glucose tolerance/metabolic syndrome (MetS) and type 2 diabetes. 

It is important to note that this relationship between body fat and blood pressure is not restricted to the obese patient, but progressively worsens throughout the entire range of above normal body weight. A direct association between hypertension and body mass index (BMI) has been observed in cross-sectional and longitudinal population studies from early childhood to old age.⁷ A BMI of <25 is considered normal or healthy; a BMI of 26 to 28 increases risk of high blood pressure by 180%, and risk of insulin resistance by >1000%.⁸

In 2006, 66.7% of the adult population in the United States was overweight or obese, 34.6% had MetS (aka insulin resistance syndrome), and 5.9% had been diagnosed with type 2 diabetes.^{9 10} Since individuals with diabetes are two to four times more likely to develop CVD, recent increases in Americans’ BMI and the prevalence of MetS and type 2 diabetes are likely to sharply increase lifetime risk for CVD, which according to current statistics, is already 49% for men and 32% for women ≥age 40.¹¹

Given this scenario, it is important to note that L-arginine levels are significantly lower in individuals with impaired glucose tolerance and type 2 diabetes,¹² while levels of ADMA are not only increased in these populations,¹³ but correlate with other predictors of MetS, even in supposedly “healthy” young adults.¹⁴ This article reviews current research on L-arginine, a conditionally essential amino acid that has been shown to not only lower levels of AMDA and alleviate insulin resistance, two key CVD risk factors unaffected by statins, but to significantly improve functional medicine outcomes when added to statin therapy. 

Endothelial Function, L-arginine and the Nitric Oxide Pathway

The vascular endothelium plays a key role in cardiovascular physiology and pathophysiology, largely via processes dependent upon nitric oxide (NO).  An endothelium-derived vasoactive mediator, NO is formed from L-arginine by the constitutively expressed enzyme endothelial nitric oxide synthase (eNOS), which is activated by shear-stress of the flowing blood or agonists such as acetylcholine and bradykinin.^{15 3}

In the vasculature, NO plays vital protective roles in a wide variety of regulatory mechanisms affecting vascular tone (NO aka endothelium-derived relaxing factor [EDRF] is the major mediator of endothelium-dependent vasodilation), vascular structure (NO inhibits proliferation of smooth muscle cells), and cell-to-cell interactions in blood vessels (NO protects blood vessels from thrombosis by inhibiting platelet aggregation and adhesion; prevents leukocyte adhesion to the vascular endothelium and leukocyte migration into the vascular wall; decreases endothelial permeability; inhibits LDL oxidation;  and reduces lipoprotein influx into the vascular wall).³

Impairment of the endothelial L-arginine/NO pathway is a common underlying mechanism through which major cardiovascular risk factors--including hypercholesterolemia, hypertension, smoking, diabetes mellitus, homocysteine, and vascular inflammation—mediate their deleterious effects on the vascular wall.^{3 16}

Impairment of the Endothelial L-arginine/NO Pathway

L-arginine Supplementation Significantly Improves Endothelial Function

In individuals with essential hypertension

It is estimated that 43 million people in the United States – ~24% of the adult population – have hypertension with essential or idiopathic hypertension accounting for 95% of all cases of hypertension.8 Oral L-arginine has been shown to improve endothelial dysfunction in patients with essential hypertension within 1.5 hours.¹⁷

In a prospective, randomized, double-blind trial, 35 patients (ranging in age from 57-69) with essential hypertension received either 6 grams L-arginine (18 subjects) or placebo (17 subjects). Patients were examined for flow-mediated endothelium-dependent dilatation of the brachial artery before and 1.5 hours after administration of L-arginine or placebo. L-Arginine resulted in significant improvement in FMD (median FMD increased from 1.7% to 5.9%), while placebo had virtually no effect (median FMD 3.0% vs. 3.1%).¹⁷

In individuals with compromised flow-mediated dilation

L-arginine benefits those most in need (those with the lowest FMD), which is not surprising since it restores NO production, thus normalizing endothelial function. A recent meta-analysis of 12 randomized, placebo-controlled trials involving 492 participants evaluated the effect of short-term (3 days to 6 months) L-arginine supplementation (3 to 24 grams/day) on endothelial function. L-arginine supplementation significantly increased FMD when baseline FMD levels were <7% but had no effect on FMD when baseline FMD was >7%.¹⁸

In individuals with chronic heart failure

L-arginine has been shown to induce beneficial effects on endothelial function in patients with chronic heart failure. Forty patients with severe chronic heart failure (left ventricular ejection fraction 19 +/- 9%) were randomized to an L-arginine group (8 grams/day), a training group with daily handgrip training (T), an L-arginine and T group, or an inactive control group (C). After four weeks, in response to administration of acetylcholine [an eNOS agonist] (30 microg/min), internal radial artery diameter increased 8.8 +/- 0.9% in the L-arginine group, 8.6 +/- 0.9% in the T group, and 12.0 +/- 0.3% in L-arginine and T group, compared to  group C (controls).¹⁹

In patients with impaired glucose tolerance (MetS) and type 2 diabetes

Patients with impaired glucose tolerance show a lessening in NO bioavailability that correlates with the degree of insulin resistance and is associated with increased endothelin-1 activity.  (Endothelin-1 is a vasoconstrictive peptide whose effects include activation of smooth muscle cell mitogenesis, leukocyte adhesion, and monocyte chemotaxis, all of which contribute to the initiation and progression of the atherosclerotic process.^{20 21} ) L-arginine supplementation, most likely due to its effect of restoring the balance between NO and endothelin-1, has been shown to improve insulin sensitivity and endothelial function in lean and in obese individuals with insulin-resistant type 2 diabetes mellitus.²²

In a study involving obese type 2 diabetic patients, 33 individuals were placed on a hypocaloric diet along with an exercise training program for 21 days. In addition, they were randomly divided into two groups, the first of which also received L-arginine (8.3 grams/day), while the second group was given placebo.²² L-Arginine treatment not only caused a more rapid improvement in fasting glucose levels, which were almost normalized within 3 weeks, but also a normalization of postprandial glucose levels, a result of special interest in relation to CVD since recent studies have found that management of postprandial blood glucose levels may influence microvascular and possibly cardiovascular risk in patients with type 2 diabetes.²²

L-arginine reverses the age-associated impairment in FMD and endothelial function

Of particular interest for anti-aging and longevity medicine, L-arginine reverses the normally-observed age-associated impairment of endothelial function. A negative correlation has been noted between aging and peak coronary blood flow response to the endothelium-dependent vasodilator acetylcholine—a negative correlation that is lost after L-arginine infusion, suggesting that aging selectively impairs endothelium-dependent coronary microvascular function and that this impairment can be restored by L-arginine administration.²³

Aging also correlates with impairment of FMD of the brachial artery and a reduction in vascular NO bioavailability, particularly in elderly individuals with cardiovascular disease. However, even in healthy elders, aging is associated with progressive endothelial dysfunction. In a prospective, double-blind, randomized crossover trial, 12 healthy older subjects (age 73.8 +/- 2.7 years) took L-arginine (8 grams/bid) or placebo for 14 days each, separated by a wash-out period of 14 days. L-Arginine significantly improved FMD (from 3.88 =/- 0.18 at base line to 5.7 +/- 1.2%), whereas placebo had no effect.²⁴

L-arginine may benefit men with erectile dysfunction

Although little is known about how effective L-arginine will be for men with erectile dysfunction or which subset of men would most likely be helped, preliminary research suggests that some men may benefit. Elevated ADMA is commonly seen in erectile dysfunction, (ED), and ED is commonly associated with other conditions affecting the vasculature in aging men, including ischemic heart disease, peripheral vascular disease, hypertension, atherosclerosis, hyperlipidemia, stroke, and diabetes mellitus.²⁹

In a controlled clinical trial, 50 patients with ED were given 5 grams L-arginine daily or placebo for 6 weeks. Nine of 29 patients taking L-arginine (31%), but only two of 17 patients taking placebo (11.7%), reported significant subjective improvement of sexual function, although all objective variables (complete physical examination including an assessment of bulbocavernosus reflex and penile haemodynamics) remained unchanged. All 9 patients who experienced a subjective improvement in sexual performance had initially had a low urinary nitrate and nitrate (NOx) level, which had doubled at the end of the study, indicating improved NO production secondary to L-arginine treatment.²⁵

L-arginine may further facilitate erections in men with severe ED using sildenafil (Viagra) or its analogues tadalafil (Cialis), and vardenafil (Levitra). In a study involving 40 men between 50 and 60 years old with insulin-dependent diabetes and ED, those given L-arginine, propionyl-L-carnitine, and nicotinic acid daily, along with vardenafil 20 mg twice weekly, for 12 weeks, experienced better FMD and erectile function (estimated with the International Index of Erectile Function questionnaire) than those receiving only vardenafil.²⁶

Sildenafil and its analogues improve ED via a different, albeit NO-related, mechanism of action than L-arginine. These drugs are potent and selective inhibitors of cGMP specific phosphodiesterase type 5 (PDE-5), which is responsible for degradation of cGMP in the corpus cavernosum. Since their molecular structure is similar to that of cGMP, sildenafil and analogs act as a competitive binding agent of PDE-5 in the corpus cavernosum, which raises cGMP levels when the NO/cGMP system is activated in the penis, resulting in improved erections. Sildenafil (under the name Revatio) has also been approved since 2005 for the treatment of pulmonary arterial hypertension. Sildenafil relaxes the arterial wall, decreasing pulmonary arterial resistance and pressure, which lessens the workload on the right ventricle of the heart, improving symptoms of right-sided heart failure. Because PDE-5 is primarily distributed within the arterial wall smooth muscle of the lungs and penis, however, the PDE-5 inhibitors act selectively in both these areas without inducing vasodilation in other areas of the body. L-arginine promotes vasodilation systemically.

Impairment of the Endothelial L-arginine/NO Pathway

Primary Mechanisms through which L-arginine Improves Endothelial Function²⁷

Antagonizes ADMA 

Most likely, the key mechanism behind both the occurrence of endothelial dysfunction and the beneficial effects of supplemental L-arginine in restoring healthy endothelial function is that L-arginine antagonizes asymmetric dimethylarginine (ADMA), a naturally occurring amino acid found in plasma and various tissues that is an endogenous inhibitor of NO synthase (NOS). By blocking endothelial NOS (eNOS), and therefore NO production from L-arginine in the vasculature, ADMA induces endothelial dysfunction, which contributes to the initiation and progression of CVD.²⁸ Concentrations of ADMA normally seen in pathophysiological conditions (3-15 micromol/L)inhibit NO production.²⁹

Elevated ADMA levels may explain the "L-arginine paradox": the observation that L-arginine supplementation improves NO-mediated vascular functions in vivo, although the enzyme kinetics of eNOS have been determined in vitro, and the data show that physiological plasma L-arginine concentrations are in a range about 25-fold higher than the Michaelis-Menten constant (KM) of endothelial NO synthase in vitro – a range that should enable full activity of the enzyme in the presence of physiological, low ADMA levels.

In the presence of elevated levels of ADMA, however, NOS is inhibited and the conversion of L-arginine to NO is impaired, resulting in decreased biological actions of NO. Under such circumstances, boosting the concentration of L-arginine, NOS’ natural substrate, by dietary supplementation may normalizes the L-arginine/AMDA ratio,28  and restore NO production to near-normal levels.30,31 Normally, the L-arginine/ADMA ratio is in the range of 50:1 to 100:1, given a range of L-arginine levels between 50 and 100 μmol/L, and ADMA concentrations between 0.3 and 0.7 μmol/L.³

Circulating levels of ADMA are elevated in association with virtually all traditional CVD risk factors and indicators of established CVD. Elevated AMDA levels are associated with low brachial FMD; insulin resistance/the MetS, diabetes; elevated CRP, VCCAM-1, elevated coronary artery calcium score; hypercholesterolemia, hypertriglyceridemia, hyperhomocystinemia; essential hypertension, unstable angina, peripheral arterial disease, congestive heart failure, renal failure, and aging. Evidence for a causal relationship between increased ADMA levels and endothelial dysfunction has been demonstrated in many of these conditions.^{28 32 33}

In normotensive insulin resistant subjects, ADMA plasma concentrations correlate with insulin resistance independently of other CVD risk factors, being higher in obese, insulin-resistant women than in obese, insulin-sensitive women, and decreasing when weight loss results in improved insulin sensitivity.²⁸

In numerous prospective clinical trials, plasma ADMA has been found to be a significant, independent predictor of CV events and mortality, even after controlling for CVD risk factors. Elevated ADMA is associated with a three-fold increased risk of future severe cardiovascular events and mortality in patients undergoing hemodialysis; a four-fold increased risk for acute coronary events in clinically healthy, nonsmoking men; and in humans with no underlying cardiovascular disease who are undergoing intensive care unit treatment, ADMA is a marker of mortality risk.34 As these trials have also revealed that ADMA levels vary in patients previously regarded as having a similar CVD risk profile, they suggest that plasma ADMA levels may be used to identify individuals at increased risk for a major cardiovascular event, e.g., individuals in the “statin gap.”²⁸

Counteracts the negative effects of oxidized LDL cholesterol on endothelial-mediated vasodilation:

Oxidized LDL impairs endothelium dependent vasodilatation via numerous mechanisms including decreasing transport of L-arginine into cells, increasing superoxide (O2-) production, and inhibiting eNOS and NO activity. Specifically, in regards to eNOS, the presence of oxidized LDL leads to the activation and upregulation of the enzyme arginase II, which competes with NOS for L-arginine as a substrate. Not only does this result in impaired NO production, but it also causes increased production of reactive oxygen species (ROS) by NOS. Furthermore, arginase activation contributes to aging-related vascular changes by mechanisms unrelated to NO production, including polyamine-dependent vascular smooth muscle proliferation and collagen synthesis. Provision of supplemental L-arginine and antioxidants reverses all these effects.^{16 35 36}

Increases insulin secretion

Insulin secretion promotes not only vasodilation, but decreased platelet aggregation and blood viscosity.^{37 38} Since the vasodilation produced by L-arginine can be prevented by octreotide, a somatostatin analogue that inhibits insulin release, it has been proposed that L-arginine’s stimulation of insulin release, rather than its enhancement of NO production, is responsible for its cardiovascular benefits.³⁹  It has also recently been proposed that insulin resistance is primarily due to ADMA’s inhibition of the neuronal isoform of NOS (nNOS), while the simultaneously observed atherosclerosis is a consequence of ADMA’s inhibition of endothelial NOS (eNOS); thus ADMA—whose effects can be greatly ameliorated by L-arginine supplementation—is thought to be the molecule responsible for the coexistence of these two conditions.^{40 41}

Maintains muscle mass while decreasing visceral obesity and inflammation in type 2 diabetics

Normally, a hypocaloric diet results in a loss of an equivalent amount of fat mass and fat-free (muscle) mass, although exercise helps preserve fat-free mass. In the study discussed immediately above, the addition of L-arginine to exercise caused a further muscle-saving effect, nearly abolishing the loss in fat-free mass and inducing greater reduction in fat mass. Furthermore, a twofold decrement in waist circumference occurred when L-arginine was added to the hypocaloric diet and exercise, suggesting L-arginine specifically decreased visceral obesity. These findings confirmed previous results in studies of Zucker diabetic fatty rats in which L-arginine therapy was found to increase expression of key genes responsible for fatty acid and glucose oxidation in adipose tissue.⁴² 

L-arginine also lowered levels of adipokines (pro-inflammatory cytokines released by adipose tissue), while enhancing levels of adiponectin (a hormone secreted by adipose tissue that improves insulin sensitivity and triglyceride clearance, and protects against endothelial dysfunction).⁴³ 

A similar earlier study of 33 middle-aged patients with chronic heart failure, visceral obesity and MetS-associated type 2 diabetes also produced highly beneficial results. Subjects were not receiving any medication other than diet for their diabetes; statins were withdrawn one week before trial onset. Standard treatments for hypertension (angiotensin-converting enzyme inhibitors and β-blockers) were matched in L-arginine and placebo groups. Subjects were put on a hypocaloric diet (1,000 kcal/day) and a 3-week exercise program (a 45-minute twice daily exercise session 5 days/week). L-arginine plasma levels increased significantly in L-arginine group (from 81.8 ± 12.3 to 131.8 ± 16.5 µmol/l); no increase was seen in the placebo group.

After 21 days, both L-arginine and placebo therapy caused significant loss in whole body weight and fat mass; however, in the L-arginine group, fat mass accounted for 100% of the total weight loss, whereas in the placebo group, fat mass comprised 57% while fat-free mass accounted for 43% of total weight loss. The same research group had previously demonstrated that 3 weeks of a similar hypocaloric diet treatment without exercise training resulted in a 51% decrease of fat mass and a 49% decrease of fat-free mass. These data collectively indicate that L-arginine promotes VAT loss while sparing lean body mass.⁴⁴

Conclusion

Statins alone are ineffective for the primary or secondary prevention of MI in two-thirds of patients because they do not improve endothelial dysfunction, its corollary hypertension, or insulin resistance. Particularly in individuals in whom the ratio of ADMA: L-arginine is elevated, supplementation with L-arginine restores NO production, improving endothelial function, while also improving insulin sensitivity.  Not only does L-arginine improve insulin sensitivity, but it specifically promotes loss of visceral adipose tissue while saving muscle mass—effects that greatly lessen adipokine-related inflammation, a key factor in the downward spiral to CVD associated with type 2 diabetes. Despite these benefits, L-arginine may actually promote CVD in individuals with an atheromatous or highly inflammatory phenotype. Part II of this review explains why and what to do to optimize the benefits, while avoiding the potential dangers, of L-arginine therapy. 

Read Part II: Key Considerations When Prescribing L-arginine: Maximizing Efficacy, Preventing Harm

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Cardiovascular disease, which now afflicts more than 80 million Americans – 36.3% of the U.S. population, including 38,100,000 individuals ≥ 60 – continues to be the No. 1 cause of death in the U.S., despite widespread use of statins, which, in 2002,  were being taken by >30% of Medicare patients ≥ 65.¹ Statins, the most successful pharmacotherapy agents used to treat atherosclerosis, remain ineffective for the primary or secondary prevention of myocardial infarction in about two-thirds of patients.²

Heart disease and stroke are still the first and third most common causes of death, accounting for 35.3% of all deaths each year (1 of every 2.8 deaths).   Although those ≥65 years of age are most at risk, as this age group accounts for nearly three-quarters of all strokes and 82% of deaths due to coronary heart disease (CHD), cardiovascular disease begins much earlier. According to current statistics, after age 40, lifetime risk of CHD is 49% for men and 32% for women. Deaths from coronary disease decreased during the period between 1980 and 2000, but recent increases in Americans’ body mass index and the prevalence of metabolic syndrome and type 2 diabetes have offset any reductions since individuals with diabetes are two to four times more likely to develop cardiovascular disease.³ In 2006, 66.7% of the adult population was overweight or obese, 34.6% had MetS, and 5.9% had been diagnosed with type 2 diabetes.^{4 5}

Given this scenario, the following four recently published studies may offer significant “news to use” to promote the health and longevity of patients with, or at risk for, cardiovascular disease – in other words, virtually everyone.  

Anthocyanin supplementation significantly improves cholesterol profiles in dyslipidemic subjects in 12 weeks

Anthocyanins – the water-soluble plant pigments that impart colors ranging from violet to blue to most shades of red – are highly concentrated in bilberries, black currants and wild blueberries. In this double-blind, placebo-controlled clinical trial, 120 dyslipidemic subjects (mean total cholesterol 225 mg/dL, LDL 159 mg/dL, and HDL 46 mg/dL) aged 40-65 years, were given 160 mg anthocyanins extracted from bilberry and black currant, or placebo, twice daily (after breakfast and supper) for 12 weeks.⁶ By the end of the trial, in those receiving anthocyanins, LDL had decreased 13.6%, and HDL had increased 13.7%, in contrast to those in the placebo group in which LDL increased 0.6% and HDL increased 2.8%. Cellular cholesterol efflux to serum—the first and most critical step of reverse cholesterol transport—increased 20% in the anthocyanin group compared to 0.2% in the placebo group. Study authors noted that the changes in lipid profile seen in subjects receiving anthocyanins would result in a nearly 27.3% reduction in their risk of coronary heart disease.

For comparison, simvastatin (20 mg/day) reduced LDL-C by 35% in 12 weeks and has also been shown to reduce hsCRp.⁷ However, statins have no effect on cellular cholesterol efflux and little effect on HDL, and thus, as noted in a recent paper reporting on the outcomes of the REVERSAL (Reversal of Atherosclerosis with Aggressive Lipid Lowering) Trial, a double-blind, randomized 18 month trial at 34 centers in the U.S. that involved 654 patients and compared the effect of two different statins on atherosclerotic burden:

“Even intensive LDL cholesterol and CRP reduction did not reverse coronary atherosclerosis. Perhaps, emerging therapies designed to enhance HDL cholesterol can produce clinically meaningful regression of coronary atherosclerotic disease burden.” [italics added] 

As demonstrated by the above study, anthocyanins significantly increase concentrations of HDL cholesterol.

Diet is another therapy that has repeatedly been shown to affect lipid profiles – for better and for worse. Specifically, it is well known that the Standard American Diet, high in processed foods, meat and saturated fat, promotes an atherogenic lipid profile. In contrast, a Mediterranean diet reduces total and LDL-cholesterol, and concomitantly increases HDL-cholesterol and reduces the total cholesterol/HDL-cholesterol ratio.⁸ Expecting patients hooked on the Standard American Diet (aptly given the acronym “SAD”) to suddenly switch from the convenience of their processed, prepared, pop-in-the-microwave foods to a diet based on largely unprocessed plant foods seasoned with olive oil, may be a bit of a stretch, but most people enjoy berries. Berries demand virtually no prep time, provide significant health benefits even when frozen, and are easily accessible year round. Explaining anthocyanins’ cardioprotective health benefits may encourage patients to begin selecting a more heart-healthy diet. According to the USDA database⁹, 100 grams (3.5 oz) of raw blueberries contains ~160 mg of anthocyanins, and even frozen, blueberries deliver ~90 mg per 100 grams. Raspberries and strawberries are not as effective a source, containing ~40 mg of anthocyanins per 100 grams if fresh, but only ~20 mg if frozen. Raw bilberries provide 430 mg anthocyanins per 100 grams and, for this reason, are often used as a source for these phytonutrients in high quality supplements – obviously, the easiest way for patients to receive their cardioprotective benefits.

EPA (eicosapentaenoic acid) significantly reduces incidence of cardiovascular events in dysglycemic as well as normoglycemic patients

Further analysis of data from the Japan EPA Lipid Intervention Study (JELIS), a large clinical trial involving 18,645 hypercholesterolemic patients followed over 4.6 years, divided the subjects into two groups—those with impaired glucose metabolism (IGM), i.e., diabetic patients and those with a fasting plasma glucose of ≥110 mg/dL (n=4565), and those with normal glucose levels (NG, n=14,080). Not surprisingly, IGM patients had a significantly higher risk of coronary artery disease (CAD) (1.71 in the group that did not receive EPA) than NG patients. However, treatment with EPA (two 300 mg capsules t.i.d. for a total of 1,800 mg/day) resulted in a 22% decrease in CAD incidence in IGM patients, and an 18% decrease in NG patients. 

Of special interest is that EPA supplementation was shown to be highly effective in decreasing CAD incidence among IGM and NG Japanese, a population in which fish intake, and therefore consumption of EPA/DHA (docosahexaenoic acid) is already high. The Japanese consume 0.8 to 1.5 g/day of EPA/DHA, which is between 8 and 15 times more EPA/DHA than is consumed by typical Westerners.^{10 11} In the JELIS study, the EPA concentration among Japanese individuals, given as the EPA concentration in the non-EPA-treated IGM patients, was 2.9 mol%, which is approximately 10-fold higher than that of white Americans – yet EPA supplementation in this cohort substantially decreased CAD risk.^{12 13}

Another compelling fact is that all patients in the JELIS were prescribed a statin. As a result, during the course of the study, LDL cholesterol levels decreased from a baseline of 180mg/dL to 137–138 mg/dL in NG patients, and from a baseline of 180mg/dL to 133–134 mg/dL in IGM patients. Despite this statin-related decrease in LDL cholesterol, IGM patients still had a significantly increased risk for CAD. Another large scale study performed in Japan, the Japan Lipid Intervention Trial (J-LIT), has also reported that CAD risk is significantly higher in diabetic than non-diabetic patients, even if they receive statin treatment.¹⁴ Both the JELIS and J-LIT studies found increased CAD risk in diabetic patients despite statin therapy’s reduction of LDL cholesterol levels by ~28%. The results of the present study concur, clarifying that abnormal glucose metabolism is a CAD risk factor independent of the level of LDL cholesterol, and further showing that EPA significantly decreases CAD risk via mechanisms independent of decreasing hemoglobin A1C (HbA1C) or fasting plasma glucose. 

In other recent studies in type 2 diabetic patients, EPA has been found to reduce carotid intima-media thickness (an indicator of arteriosclerosis), and, while not lowering total LDL concentrations, to significantly reduce levels of small dense LDL.^{15 16} EPA is also known to have anti-inflammatory effects, to reduce platelet aggregation, inhibit cell proliferation and stabilize plaque – beneficial effects all of which contribute to lessening risk of major coronary events in IGM as well as NG patients.^{17 18 19 20 21 22 23 24}

If EPA supplementation is so effective in reducing CAD risk in fish-loving Japanese dysglycemic patients taking statins, shouldn’t supplementation with this essential fatty acid be first line therapy for Americans at risk of cardiovascular disease, especially those with metabolic syndrome or type 2 diabetes?

DHA – determining the optimal dosage for its effects on platelet reactivity and redox status

Some studies suggest that DHA may be even more cardioprotective than EPA. DHA appears to be more effective than EPA in suppressing arrhythmia and is the principal omega-3 responsible for the vasorelaxant and hypotensive effects of fish oils. DHA is a much more potent inducer of nitric oxide (NO) production, and NO, in addition to its vasodilating effect as endothelium-derived relaxing factor (EDRF), has inhibitory effects on platelet aggregation and adhesion, and vascular smooth muscle cell proliferation and migration.²⁵

Research published in The FASEB Journal, September 2009, looked at the effects of varying dosages of DHA on both platelet activity and redox status in 12 healthy men, aged 53-65 years.²⁶ Over 4 successive 2-week periods, the men consumed 200, 400, 800 and 1600 mg of DHA per day. Blood and urine samples were collected before and after each dose of DHA and at 8 weeks, after supplementation was stopped.

DHA was incorporated in a dose-response fashion in platelet phospholipids. After supplementation with 400 and 800 mg/day, but not with 200 mg/day of DHA, platelet reactivity was significantly decreased. On the other hand, levels of urinary isoprostane, a marker of lipid peroxidation/oxidative injury, were significantly lowered after 200 mg/day of DHA, but increased after 1600 mg/day, leading the researchers to suggest daily intake of 200 mg of DHA/day as an effective way to protect healthy men from platelet-related cardiovascular events. 

We disagree. This study shows that higher doses of DHA – in the range of 400 – 800 mg/day – are needed to decrease platelet reactivity. The findings show, not that higher doses of DHA should not be used, but that higher doses of DHA must be complemented with increased levels of vitamin E. The reason for this is that DHA – and EPA – are highly susceptible to oxidation, thus higher amounts of these omega-3 fatty acids require additional lipid-soluble antioxidant protection, e.g., vitamin E. 

A further consideration is that vitamin E should be supplied in the form of mixed tocopherols rather than alpha tocopherol alone since gamma tocopherol is significantly more effective in preventing platelet aggregation and lipid peroxidation from reactive nitrogen species (RNS) than alpha tocopherol, which is more effective in neutralizing reactive oxygen species (ROS). For a full discussion of these issues and a review of the current research, please see Beyond α-Tocopherol: A Review of Natural Vitamin E’s Potential in Human Health and Disease, Part I: In Defense of Vitamin E and Part II: Vitamin E in Action.

Lowering the burden of cardiovascular disease

Although slight differences in biological activity exist between EPA and DHA, both exert numerous actions protective against cardiovascular disease. Both EPA and DHA inhibit platelet aggregability, reduce serum triglycerides, reduce production of pro-atherogenic cytokines, improve endothelial function, reduce vascular occlusion, reduce heart rate, increase heart rate variability and prevent arrhythmia. Most importantly, a number of large studies have shown reductions in clinical endpoints like sudden cardiac death or major adverse cardiac events. As a consequence, European and American Cardiac Societies have incorporated EPA and DHA into recent treatment guidelines, recommending 1 gram/day for cardiovascular prevention, after a myocardial infarction and for prevention of sudden cardiac death.^{27 28}

Advocating the use of anthocyanins (160 mg b.i.d) -- which significantly increase HDL and may therefore help fill the treatment gap left by statins – as well as EPA/DHA, may greatly improve the likelihood of clinically meaningful regression in coronary atherosclerotic disease.

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Part III: Restoring Mitophagy – the Key To Mitochondrial Rejuvenation

Introduction

Mitochondrial decay resulting from oxidative damage accumulates with age and is universally recognized as a major contributing factor to the whole range of functional decline and tissue deterioration associated with aging.^{1 2 3 4 5 6 7} Part I of this review discussed Bruce Ames’ application of the Michaelis constant (KM) concept to the ramifications of age-associated oxidative damage to proteins. Aging-associated increases in oxidative damage to key enzymes results in their structural deformation and decreased binding affinity for the co-enzyme, causing a decrease in enzyme funtion.⁸ Ames’ research has demonstrated that increasing the availability of acetyl L-carnitine and α-lipoic acid, two nutrients that serve as mitochondrial enzyme co-factors, restores the velocity of the reactions (KM) in the related enzymes, and thus restores aging mitochondria’s ability to produce youthful levels of ATP.^{9 10 11 12 13} Part II focused on the inter-relationships among the folate, methylation and transsulfuration pathways, whose dysfunction results in increased free radical production coupled with disruption of glutathione (GSH) synthesis, thus accelerating mitochondrial decay and aging. Ensuring adequacy of all the nutrient co-factors necessary to restore KM in these pathways, as well as in mitochondrial oxidative phosphorylation, is first line therapy for promoting healthy aging. 

However, a protocol whose end goal is restoration of KM, while certainly helpful in delaying mitochondrial decay, does not address the more fundamental issue – why does human physiology shift from a homeostasic state that repairs and balances itself to one that allows decay to accumulate? Researcher Wulf Dröge has called this shift “the first cause of death,” and his insight into its likely causes may both explain why calorie restriction increases longevity and provide a much less onerous way to opt out of the vicious cycle responsible for the age-associated transition from a state of youthful homeostatic repair to one that promotes mitochondrial decay. This is the topic of Part III of this review.

“Biological aging is no longer an unsolved problem"

In press releases for a symposium that took place at the International Association of Gerontology and Geriatrics 19th World Congress, held in Paris, July 5-9, 2009, the father of the oxidative theory of aging, Dr. Leonard Hayflick, is quoted as saying,

Aging occurs because the complex biological molecules of which we are all composed become dysfunctional over time as the energy necessary to keep them structurally sound diminishes, thus our molecules must be repaired or replaced frequently by our own extensive repair systems. These repair systems, which are also composed of complex molecules, eventually suffer the same molecular dysfunction. The time when the balance shifts in favor of the accumulation of dysfunctional molecules is determined by natural selection — and leads to the manifestation of age changes that we recognize are characteristic of an old person or animal. It must occur after both reach reproductive maturity, otherwise the species would vanish. These fundamental molecular dysfunctional events lead to an increase in vulnerability to age-associated disease therefore, the study, and even the resolution of age-associated diseases, will tell us little about the fundamental processes of aging.

Perhaps the key point made by Hayflick, both above and in a recent paper titled, “Biological aging is no longer an unsolved problem,” (and generally accepted among leading gerontology researchers) is that the body's repair and maintenance systems are the primary determinants of longevity.¹⁴ The question then becomes, “What causes these systems to lose their ability to outpace the damage of daily life?” Hayflick asserts the shift is determined by natural selection, suggesting little can be done to extend lifespan. Wulf Dröge has proposed an alternate, much more optimistic, explanation: the shift from a homeostasis of repair to one of snowballing damage is due to the age-related onset of aberrant insulin signaling, which prevents autophagic recycling.^{15 16 17 18}

Autophagic mitochondrial recycling – key to longevity

Aging is a waste-full process. In youth, autophagy—controlled self-destruction, literally “self-eating”—removes and recycles cellular waste that would otherwise compromise cellular function and regeneration of replacement structures. In the mitochondria, which are easily damaged due to their role as the energy production factories in cells and can themselves become a key source of cellular damage, adequate autophagic waste recycling is critical for rejuvenation but diminishes in aging cells. Since several longevity mutants (such as long-lived C. elegans or Drosophilia mutants^{19 20}) manifest defects in a signaling mechanism that normally slows the rate of autophagic waste recycling, Dröge and other researchers have postulated that the aging-associated decline of mitochondrial autophagy or “mitophagy” may be the first most limiting mechanism that determines maximum life span in most animal species, including man.^{21 22}

Insulin signaling inhibits autophagy

In humans, the signaling mechanism that slows autophagic recycling is identical to the signaling mechanisms that control our response to insulin and insulin-like growth factor, thus it can also be called the insulin mechanism. Bottomline: aberrant insulin signaling that inhibits autophagy, particularly mitophagy, is the first cause of death.

In addition to its role in clearing glucose from the bloodstream, insulin stimulates protein synthesis in skeletal muscle and other tissues (provided sufficient amounts of free amino acids are available). Thus, most protein synthesis takes place during the day when food consumption raises blood levels of amino acids and insulin. Through the same signaling mechanism, the increase in amino acid and insulin concentrations triggers suppression of autophagic activity in the fed state, i.e., for most of the day.

Autophagic waste recycling happens mainly in the fasted state—in man, mainly at night—when amino acid and insulin concentrations in the blood drop off, protein synthesis largely shuts down, and autophagy is allowed to proceed. In young individuals, the resulting nightly autophagic protein breakdown is normally so strong that it causes skeletal muscle tissues to release substantial amounts of free amino acids, including the cysteine needed for glutathione synthesis and defense against oxidative stress. In the fed state during the daytime, glutathione biosynthesis is maintained by cysteine from dietary protein.

Excessive autophagic destruction is avoided at night through a feedback mechanism that halts autophagic clearance when its products, free amino acids, reach a sufficient concentration. Autophagy converts proteins from damaged mitochondria and other forms of cellular waste into free amino acids. The resulting increase in free amino acid concentrations activates the serine/threonine kinases, Akt1 and Akt2, which mobilize the signaling protein mTOR (the mammalian target of rapamycin), which then suppresses autophagic activity. The same mechanism operates during the day when dietary protein and carbohydrate are consumed; the resulting increase in plasma amino acid and insulin concentrations results in activation of mTOR, which stimulates protein synthesis while suppressing autophagic activity.

Oxidative stress causes aberrant insulin signaling in older individuals

The weak point in the homeostatic control of cysteine supplies for glutathione synthesis is that autophagic activity is regulated not only by the free amino acid levels, but also by the insulin receptor signaling cascade, which even in the absence of insulin in the post-absorptive (fasting) state, can be abnormally triggered by oxidative stress.¹⁶

Activation of the insulin receptor involves its autophosphorylation, which is followed by phosphorylation of several target proteins in the signaling cascade. The insulin signaling cascade is inhibited by several phosphatases, including protein tyrosine phosphatase 1B (PTB 1B), phosphatase, tensin homologue on chromosome 10 (PTEN), and SH2-domain-containing inositol phosphatase (SHIP2), all of which are inactivated under moderately oxidative conditions. Thus the age-related increase in oxidative stress sets up a vicious cycle in which the mechanisms that maintain adequate levels of cysteine in the fasted state are overcome, leaving older adults (and specifically, aging mitochondria) increasingly susceptible to oxidative damage and less able to remove damaged cellular components via autophagy.

Aberrant insulin signaling drains glutathione reserves. PTP 1B, a key redox-sensitive signaling phosphatase in the insulin receptor signaling pathway, is inhibited by hydrogen peroxide (H2O2), which converts PTB 1B’s catalytically necessary cysteine moiety into cysteine sulfenic acid (Cys-SOH), which then interacts spontaneously with glutathione to form glutathione disulfide (GSSH), the inactive form of glutathione. GSSH can also convert PTP 1B’s redox-sensitive cysteine residue into the inactive disulfide. Changes in the thiol/disulfide redox status (discussed in Part II of this review in relation to the connections among homocysteine, methylation and transsulfuration) can also significantly inhibit the activity of the redox-sensitive phosphatases that regulate insulin signaling.

In addition to causing the functional inactivation of these phosphatases, low concentrations of H2O2 or an oxidative shift in the GSH:GSSH redox status strongly increases the activity of the basic insulin receptor tyrosine kinase in the absence of insulin. Since the activity of the insulin receptor signaling pathway is determined by a balance between kinase and phosphatase activities, oxidative activation of the kinase combined with the simultaneous inactivation of phosphatases synergistically upregulates the insulin signaling pathway. The end result is an age-associated decrease in autophagy and the body’s ability to maintain adequate post-absorptive (night time or fasting) cysteine levels. The resulting drop in glutathione production and intracellular glutathione concentrations compromises the mitochondria’s ability to scavenge reactive oxygen species (ROS), producing a vicious cycle that drives the progressive increase in ROS-mediated structural damage and its corollary, the progressive decline in energy production and repair that accompanies aging.

Aberrant insulin signaling inhibits key recycling and repair proteins

If this were not harmful enough, aberrant insulin signaling also results in inhibition of the forkhead transcription factor, FOXO 1, and peroxisome proliferator-activated receptor-coactivator 1 (PGC-1α).15

Activation of the insulin receptor leads to sequential activation of a number of protein and lipid kinases, including the serine/threonine kinases Akt1 and Akt2, which not only stimulate mTOR and thus downregulate autophagic protein catabolism (and thus cysteine supplies), but elicit phosphorylation (inhibition) of FOXO1, a transcription factor that induces expression of proteins involved in both of the proteolysis recycling pathways: the autophagic/lysosomal pathway and the ubiquitin-proteasomal pathway. (In contrast to the role of autophagy in removal of defective organelles, the ubiquitin-proteasomal pathway is responsible for recycling damaged, long-lived muscular proteins that are not degraded through the autophagic/lysosomal pathway.) FOXO genes also interact with SIRT1, a sirtuin protein that is the human homologue of the yeast sirtuin, silent information regulator 2 (SIR2), which assists in DNA repair and regulates genes that undergo alteration with age. Sirtuin activators, notably resveratrol (which activates both SIRT1 and PGC-1α, and improves the functioning of the mitochondria) ^{23 24} have been shown to inhibit gene expression profiles associated with muscle aging and age-related cardiac dysfunction in mice and may extend lifespan.^{25 26}

Akt2 also phosphorylates and inhibits the transcriptional coactivator PGC-1α, a regulator of post-absorptive hepatic metabolism that works in close association with FOXO1. In mice, PGC-1α is required for the expression of several mitochondrial genes in the liver, skeletal muscle, heart, brain, and brown fat. Strongly induced in the liver upon fasting, PGC-1α promotes hepatic fatty acid oxidation and shifts fuel usage from glucose to fat in the post-absorptive period. Thus, ROS-related aberrant activation of Akt2 may contribute to the development of hyperlipidemia and obesity. This may be the mechanism behind the association noted between low plasma cysteine concentrations and both hyperlipidemia and obesity. In support of this hypothesis, the redox sensitivity of the basic insulin receptor signaling activity in vivo has been confirmed in a placebo-controlled clinical study of non-diabetic obese persons in whom basic (i.e., post-absorptive) insulin receptor signaling decreased after supplementation with relatively small doses of N-acetyl cysteine (200 mg NAC t.i.d. for a total daily dose of 0.6 g for 8 weeks).²⁷

In young healthy individuals, the role played by oxygen radicals and H2O2 in enhancing insulin signaling is helpful, and so important that almost all cells and tissues contain special enzymes that produce H2O2, and O2- at a well-regulated rate. But in older individuals in whom basal levels of oxidative stress are already increased, aberrant insulin signaling during the fasting state that militates against mitophagy and further decreases glutathione stores, becomes the final straw. A vicious cycle is initiated. Lessened mitophagy results in a decrease in plasma cysteine concentrations, which causes a decrease in intracellular glutathione, which causes a corresponding increase in H2O2 and O2-, which leads to further insulin-independent upregulation of the basic insulin signaling mechanism, which further represses mitophagy, continuing the cycle.

Calorie restriction extends lifespan by promoting autophagy

The relationship between FOXO1 and the sirtuins, both of which are inhibited by insulin receptor signaling, may be the key mechanism behind the life-extending effects of calorie restriction with adequate nutrition (CRAN). CRAN reduces mitochondrial ROS production, lessening aberrant insulin signaling (and thus inhibition of FOXO1 and SIRT1), and promotes mitochondrial renewal via autophagy.²⁸ Preliminary research indicates that even moderate CRAN (a reduction in caloric intake of just 8% rather than the traditional 30-40%) may promote muscle mitochondrial biogenesis in middle-aged human subjects and may therefore both delay onset and mitigate progression of sacropenia in older adults.^{29 30 31}

The insulin receptor signaling cascade is inhibited by several phosphatases, including protein tyrosine phosphatase 1B (PTB 1B), phosphatase and tensin homolog on chromosome 10 (PTEN) and SH2-domain-containing inositol phosphatase (SHIP2), all of which are inactivated by ROS. In addition, activity of the basic insulin receptor tyrosine kinase is strongly increased by low concentrations of H2O2 or by an oxidative shift in the GSH redox status. 

In the presence of H2O2 and ATP, the insulin receptor kinase domain is phosphorylated at its catalytic site and thereby rendered catalytically active in the absence of insulin. The phosphatases, in turn, contain a redox-sensitive cysteine moiety in their catalytic site, which is converted by H2O2 into a sulfenic acid moiety, which renders the phosphatase inactive. The balance between kinase and phosphatase activities determines the rate of phosphorylation of the insulin receptor kinase domain and several downstream targets including the phosphatidylinositol phosphates, the serine/ threonine kinase Akt1 and the mTOR. Ultimately, this balance controls the aging-related functions of autophagy and sirtuin proteins.

The insulin-independent oxidative upregulation of insulin receptor signaling activity (basic IRS) results in inhibition of autophagic removal of damaged cell structures (Autophagy) and ability to maintain post-absorptive plasma cysteine concentrations. This causes a decrease in intracellular glutathione (GSH) and a corresponding increase in ROS (H2O2, O2-) concentrations that further upregulates the insulin-independent insulin receptor signaling, perpetuating the cycle.

These changes occur in primarily during the night and early morning hours, i.e., in the post-absorptive/fasting condition. In the postprandial (fed) state, the insulin receptor plays a positive role as a key regulator of glucose clearance, protein synthesis, etc. Its negative effects are related to aberrant (insulin-independent) insulin signaling in the post-absorptive (fasting) condition.

Mitochondria – key players in initiating the first cause of death

Mitochondria are widely believed to play the pivotal role in aging because they are a major source of oxygen radicals and arguably their most important targets. Thus stressed mitochondria are a preferred target for autophagy. However, it is a common finding in old age that structurally abnormal mitochondria accumulate in post mitotic cells, mitochondrial oxidative phosphorylation decreases, and mitochondrial H2O2 production increases—the latter indicating that at least some of the abnormal mitochondria have defective electron transport chains and produce abnormally high amounts of O2-, H2O2, and other ROS.

This happens because mitochondrial DNA is particularly vulnerable to oxidative damage and shows a more than ten-fold greater mutation rate than nuclear DNA. O2- and other ROS induce mitochondrial DNA deletion mutants, which have been hypothesized to out replicate the wild-type (normal) mitochondrial DNA genome.³² The DNA of these mutated mitochondria may then code for abnormal electron transport chain complex enzymes, resulting in increased electron loss, increased production of H2O2 and O2-, and the initiation of the vicious cycle of progressively increasing oxidative stress coupled with progressively decreasing autophagic recycling. ROS may also cause lipofuscin deposits that further compromise autophagic removal of defective mitochondria.

Breaking the vicious cycle with N-acetyl cysteine

A pivotal factor in the shift from damage repair to damage accumulation in the aging adult is that during the night and early morning hours, older individuals are producing less glutathione (GSH). Fortunately, this can be ameliorated or even reversed. Supplementation with oral N-acetylcysteine (in doses ranging from 0.6 to 8 grams/day) has been shown to replenish intracellular GSH levels even in a wide range of infections, genetic defects and metabolic disorders, including HIV infection, cystic fibrosis, COPD, cardiac dysfunction, diabetes, colon cancer and Alzheimer’s disease.³³ (Since cysteine is easily oxidized, most clinical studies have used either the relatively stable synthetic cysteine derivative, N-acetylcysteine (NAC), or a naturally derived cysteine-rich undenatured whey protein isolate, both of which have been shown to decrease insulin responsiveness in the fasted state.)¹⁵

Furthermore, in vitro, in vivo and clinical studies have all confirmed that the insulin-independent basal activity of the insulin receptor, while increased by ROS, can be downregulated by supplementation with NAC. In clinical trials, NAC supplementation has also been shown to improve a number of parameters associated with aging including an increase in skeletal muscle wasting and the ratio of body fat/lean body mass (sarcopenia), an increase in plasma levels of the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and decreases in immune function and plasma albumin* levels.¹⁵

[ *Hypoalbuminaemia has been strongly correlated with functional decline and increased mortality risk in older individuals. Elderly subjects and patients with practically all types of catabolic conditions typically show a conspicuous decrease in the plasma albumin level. Albumin normally constitutes about 60% of human plasma protein and plays an important role in regulating blood volume by maintaining the oncoosmotic pressure of blood needed to avoid edema, and by serving as the carrier for hydrophobic molecules, including lipid soluble hormones, bile salts, unconjugated bilirubin, free fatty acids (apoprotein), calcium, ions (transferrin), and some drugs (e.g., warfarin, phenobutazone, clofibrate & phenytoin). Lowered albumin levels (normal range is 3.5 to 5 g/dL) may also increase risk of ADRs since competition among drugs for albumin binding sites can result in increased levels of the free fraction of one or more of the drugs, thereby affecting potency.]^{34 35 36 37 38}

In a placebo controlled double-blind study of frail elderly patients, treatment with NAC doubled the increase in knee extensor strength during a 6-week program of physical exercise and slowed the subsequent decline during a 6-week follow-up period. On average, the exercise program was completely ineffective in persons who had low plasma concentrations of cysteine under fasting conditions. Conversely, persons with low fasting cysteine levels showed the greatest benefits from supplementation. While the plasma level of TNF-α increased significantly during the exercise program (and an increase in TNF-α is typical following exercise, even in healthy, young individuals), cysteine supplementation completely prevented this increase.^{17 39}

Studies of both elderly subjects and cancer patients have found that the aging and disease related loss of skeletal muscle mass, or sarcopenia, is significantly related to the oxidative shift in the plasma thiol/disulfide redox status. Treatment with NAC has been shown to ameliorate muscle wasting both in senescence and in cancer patients.^{39 40}

As Dröge noted in a paper titled, “Oxidative stress and ageing: is ageing a cysteine deficiency syndrome?,” all of this suggests that loss of youth, health and quality of life may be partly explained by a age-related deficit in the body’s cysteine and glutathione reservoirs, and that aging may be postponed by supplementation with NAC.⁴¹

Optimizing NAC Supplementation

The goal is NAC supplementation sufficient to increase glutathione levels, prevent aberrant insulin signaling, and restore night-time mitophagic activity and amino acid homeostasis to a youthful level.

The typical dosage of NAC given as an oral supplement in clinical trials is 600 mg b.i.d., although 200 mg t.i.d. has been shown to provide benefit in just 8 weeks.²⁷ On the other hand, doses as high as 1 gram t.i.d. have been used in cystic fibrosis patients for 4-12 weeks with no adverse effects, and in AIDs patients, oral NAC doses up to 8 grams/day did not cause clinically significant adverse reactions.^{42 43 44 45 46 47}

For anti-aging purposes, 600 mg b.i.d. of NAC is likely to be adequate. Furthermore, unnecessarily high cysteine supplementation should be avoided. Glutathione biosynthesis proceeds up to a certain concentration, which in the liver is approximately 10 millimolar (mM), after which any excess cysteine is converted into cystine or catabolized into sulfate and protons (ie., metabolic acid).Metabolic acidification leads to dedgradation of skeletal muscle protein and loss of lean body mass. Ideally, the diet should include 75-85% alkali-producing foods (grains, vegetables, most fruits) and 15-25% acid-producing foods (meat, fish, dairy, eggs [specifically egg whites]). The Standard American Diet renders most people in the U. S. slightly acidic. The cost of buffering excess acid is initially levied on bones but compromises function in all cells and tissues. Excessive intake of cysteine may increase formation of metabolic acid in tissues. Urine pH can be checked to evaluate the patient’s metabolic pH. The urine should be approximately neutral, i.e., with a pH of 6.5-6.8.15

In addition, consumption of an excessively large amount of cysteine as one dose will result in a potentially significant portion not being quickly cleared from the oxidative environment of the blood for use in biosynthesis of proteins and glutathione. Cysteine remaining in the bloodstream will be oxidized to disulfide cystine, causing an undesirable shift in the thiol/disulfide status of the blood plasma, which in turn, will alter the set points of redox-sensitive signaling pathways. Excessive cysteine may also lower basal insulin receptor signaling activity to the point that glucose clearance after food intake is compromised to the level of mild diabetes—an effect that can be easily monitored by a routine test of glucose clearance.¹⁵

Since glutathione and cysteine concentrations are relatively low in the post-absorptive state, especially in older individuals, NAC is best taken early in the morning and before retiring for the night -- several hours after consumption of the evening meal to ensure a postabsorptive state (i.e., that normal insulin signaling in response to food intake is not occurring).

Effectiveness of NAC dosage may be evaluated by measuring the early morning fasting plasma cysteine levels. Do not request total cysteine (tCys); this test reports only cystine levels since labs often oxidize the specimen, so all cysteine is converted to cystine.⁴⁸ Request cyst(e)ine; this terminology is used to indicate a combination of reduced (cysteine) and oxidized (cystine) forms. These will be in dynamic equilibrium. Normal reference range is 4.9 – 8.0 mmol/dL.⁴⁹

A functional assessment of glutathione production and cysteine adequacy to meet the need for GSH synthesis can be provided by a urinary organic acids test evaluating α-hydroxybutyrate and sulfate. Since α-hydroxybutyrate is a byproduct of GSH production, high levels indicate increased hepatic GSH synthesis due to chronic oxidative stress. Urinary sulfate levels reflect the adequacy of sulfur-containing amino acids (e.g., cysteine) and also GSH adequacy (low sulfate levels indicate low total body GSH and thus increased need for sulfur-containing amino acids, e.g., cysteine). Taken together, urinary α-hydroxybutyrate and sulfate provide a picture of the dynamics of GSH synthesis and whether the body’s GSH needs are being met.^{48 50}

Immediately before or after a bout of physical exercise, consumption of 10-30 grams of cysteine-rich whey protein (depending on body weight and exercise intensity) is a better option since this delivers amino acids preferentially to skeletal muscle while also providing cysteine to lessen oxidative stress.¹⁵

Night-time autophagic activity can be increased by drinking 3.5 to 16 ounces of water about 2-3 hours before breakfast. This will temporarily dilute blood plasma concentrations of leucine and other regulatory amino acids, triggering autophagy to the point where the original equilibrium of regulatory amino acids is restored. Since the cysteine and leucine content of muscle protein is comparable, an autophagy-mediated increase in plasma leucine will be accompanied by a similar increase in cysteine, thus facilitating glutathione biosynthesis. While for younger adults, waking at 4-5 a.m. to drink water will be problematic, it is less likely to be onerous for older individuals, who often wake at least once during the night and frequently have a reduced sensation of thirst, so do not consume enough water to optimally support cellular functions. Optimal daily water intake is estimated to be 3.7 liters (3.9 quarts) for men and 2.7 liters (2.8 quarts) for women.¹⁵

Conclusion

Autophagy, the body’s mechanism for the removal and recycling of cellular waste, is key to rejuvenation, especially in the mitochondria, and is allowed to proceed only in the postabsorptive state, in which insulin signaling ebbs to its lowest (basal) level. With age, ROS-induced aberrant triggering of the insulin signaling mechanism in the postabsorptive state inhibits autophagy, preventing maintenance of plasma cysteine and intracellular glutathione levels throughout the night and early morning hours, and initiating a vicious cycle of progressively increasing oxidative stress and mitochondrial dysfunction. Since accumulation of cellular waste, decreased cellular glutathione concentrations and increased oxidative stress are common hallmarks of aging in a broad range of species, while lowered insulin signaling that promotes autophagy, whether resulting from genetic mutation or caloric restriction (with adequate nutrition), has been shown to extend lifespan in virtually all species tested to date, it seems likely that human life- and health-span may be extended by a protocol designed to re-establish youthful levels of mitophagy and postabsorptive cysteine concentrations, such as the one proposed by Dröge and outlined in this article.

Read Part I: Delaying the Mitochondrial Decay of Aging

Read Part II: The Methylation – Transsulfuration Connection to Mitochondrial Decay

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Part II: The Methylation – Transsulfuration Connection to Mitochond

Introduction

Mitochondrial decay resulting from oxidative damage accumulates with age and is universally recognized as a major contributing factor to the whole range of functional decline and tissue deterioration associated with aging.^{1 2 3 4 5 6 7} Part I of this review discussed Bruce Ames’ application of the Michaelis constant (KM) concept to the ramifications of age-associated oxidative damage to proteins. Specifically, with age, increased oxidative damage to key enzymes produces deformation in their structure, resulting in an increased Michaelis constant (KM), i.e., a decreased binding affinity for the co-enzyme (the nutrient co-factor for the enzyme), thus causing a decrease in enzyme function.⁸ Ames’ research has demonstrated that increasing the availability of acetyl L-carnitine, the substrate for the enzyme acetyl-L-carnitine transferase, (which plays a key role in transporting long-chain fatty acids into the mitochondria for β oxidation and ATP production), along with α-lipoic acid, (a mitochondrial antioxidant that also serves as cofactor for two key enzymes in the Krebs cycle, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase), restores the velocity of the reactions (KM) for these enzymes, and thus restores aging mitochondria’s ability to regain youthful levels of energy production.^{9 10 11 12 13}

Single Nucleotide Polymorphisms plus KM equals Double Jeopardy

In addition to the aging-associated accumulation of oxidative damage, single nucleotide polymorphisms (SNPs) are another factor affecting KM, and one that, since the activity of the enzymes affected is down-regulated from birth, may promote premature aging. As many as one-third of all mutations in a gene result in the corresponding enzyme having an increased KM (decreased binding affinity) for a coenzyme, and therefore a lower rate of reaction.

One well-researched and pivotal example in terms of its impact on mitochondrial function is the 677CàT SNP of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in the methylation cycle. MTHFR is responsible for the conversion of folic acid into methionine synthase, which, along with B12, recycles homocysteine to methionine.

In the fairly common 677CàT variant (~30-40% incidence), thymine is substituted for cytosine in the gene, resulting in production of a slow variant of the MTHFR enzyme in which valine is substituted for alanine at position 222 (for which reason this SNP is also labeled Ala222Val). In individuals in whom both alleles have this SNP (i.e., individuals homozygous for the TT genotype of MTHFR, (~10% of the North American population¹⁴), the resulting enzyme expressed has only 50% of the activity of the wild (more common) CC type.¹⁵

Similar to the way in which providing supplemental acetyl-L-carnitine restores the KM for acetyl-L-carnitine transferase, providing supplemental folate, the substrate for MTHFR, may help compensate for the drop in MTHFR activity that will otherwise occur in carriers of this SNP. Another way of looking at this is that carriers of the MTHFR 677CàT SNP have an increased requirement for folate to optimize the activity of this enzyme, and thus require more folate for normal methylation.

Whether impaired MTHFR activity is the result of age-associated oxidative damage or genetic inheritance, restoring normal levels of MTHFR activity is essential for optimal function in aging mitochondria. When MTHFR is compromised, not only is methylation impaired, but unmetabolized homocysteine accumulates in the cell and effluxes into the bloodstream. In both, homocysteine will react with reactive oxygen intermediates, greatly increasing the rate of antioxidant consumption, including that of GSH, an antioxidant critical for neutralizing mitochondrial reactive oxygen species (ROS). 

High levels of homocysteine have been shown to increase ROS and lipid peroxidation in the endothelium, liver, kidney and brain.^{16 17 18 19} In addition to decreasing antioxidant defenses and total thiol content (Thiols are compounds that contain the functional group [–SH], aka a sulfhydryl or thiol group. [–SH] is the sulfur analogue of an alcohol group [–OH]. Key compounds in mitochondrial energy production, coenzyme A, cysteine, GSH, and α-lipoic acid are all thiols), hyperhomocystenemia has been shown to disrupt the activity of all three key mitochondrial antioxidant enzymes (superoxide dismutase [which neutralizes superoxide radical (O2−)], catalase [which neutralizes hydrogen peroxide (H2O2)] and glutathione peroxidase (GPx) [which reduces H2O2 and lipid peroxides]).¹⁷

Homocysteine Metabolism - Linking Methylation, Transsulfuration and Mitochondrial Function

Decreased MTHFR activity hamstrings the methylation cycle, resulting in increasing levels of homocysteine, increased ROS production, decreased S-adenosylmethionine (SAM) production, and increased oxidative stress, which greatly increases the rate of glutathione (GSH) depletion. 

In addition to its role in the methylation cycle, homocysteine is involved in transsulfuration as the precursor to cysteine, the rate-limiting factor for the production of GSH, the premier endogenous antioxidant. Thus homocysteine serves as an essential factor in the body’s major antioxidant system—when other processes utilizing homocysteine, i.e., methylation, are functioning normally. However, when the interactive balance among these pathways is compromised, alteration in the level any of the thiols affects the others.²⁰

Compromised methylation results in increased homocysteine efflux into plasma, where it is highly susceptible to being oxidized, increasing the formation of superoxide radical (O2−) and hydrogen peroxide (H2O2), thus increasing systemic demand for GSH. When methylation is chronically compromised (as in an individual homozygous for the 677CàT SNP or one with insufficient intake of nutrient co-factors essential for the activity of enzymes involved in methylation, e.g., vitamins B6, B12, folate and riboflavin), plasma levels of homocysteine are chronically elevated, resulting in chronically increased need for GSH. In turn, low GSH concentrations dramatically accelerate homocysteine oxidation, perpetuating the vicious cycle.²⁰ 

Clinical and epidemiological evidence suggests that a plasma level of homocysteine >15 μmol/l, although only modestly elevated, is associated with vascular disease, a finding that may be linked to homocysteine’s effects on thiol balance. In recent years, several studies have shown that moderate hyperhomocysteinemia is associated with premature cerebrovascular, peripheral and coronary artery disease.20 Disruption of the interactive balance among the roles played by thiols, resulting in GSH depletion and mitochondrial dysfunction, may prove to be an underlying factor as important as high cholesterol. 

The effects of thiol disequilibrium have also been noted in a recent study of autistic children. In these children, the ratio of plasma S-adenosylmethionine (SAMe) to S-adenosylhomocysteine (SAH) was significantly reduced; the mean concentration of reduced GSH, the major intracellular antioxidant and mechanism for detoxification, was significantly decreased; and the oxidized disulfide form of GSH (GSSG) was significantly increased, resulting in a 2-fold reduction in the mean GSH:GSSG redox ratio. Several metabolic precursors for GSH synthesis were found to be lower in autistic children, suggesting insufficient GSH synthesis. These combined findings indicate a significant decrease in methylation capacity (↓SAM:SAH), resulting in a decrease in antioxidant/detoxification capacity (↓GSH:GSSG) and an increase in oxidative stress (↑GSSG) in autistic children.²¹ 

In this study, 40 autistic children were treated with 75 µg/kg methylcobalamin (2 times/week) and 400 µg folinic acid (2 times/day) for 3 months. 

While the intervention did not alter methionine, SAM, and SAH concentrations significantly (despite the fact that methylcobalamin and folinic acid provide methyl groups for the methylation cycle), concentrations of the transsulfuration metabolites, cysteine, cysteinylglycine, and GSH, increased significantly, and oxidized GSSG levels were reduced to the point that after the intervention, these metabolites were no longer statistically different compared to those in healthy control children. Measures of autistic behavior were assessed by a trained study nurse pre- and post-treatment using the Vineland Adaptive Behavior Scales, and significant improvement was observed after treatment, although scores remained below normal. 

The finding that plasma concentrations of methionine and SAM were unaffected (and remained significantly below those in the control subjects) was unexpected because methylcobalamin and folinic acid directly provide methyl groups for synthesis of methionine and SAM, and only secondarily provide metabolic precursors for transsulfuration reactions.

The explanation appears to lie in the fact that the enzymes within the methylation cycle, (methionine synthase, betaine-homocysteine methyltransferase, and methionine adenosyltransferase) are redox-sensitive and are down-regulated by oxidative stress. Under pro-oxidant conditions, this serves to promote preferential utilization of homocysteine for GSH synthesis at the expense of methylation. 

Similarly, in addition, cystathionine beta synthase (CBS) contains a heme component, which serves as a redox sensor that upregulates CBS activity under oxidative conditions. This adaptive up-regulation of CBS activity further promotes cysteine and GSH synthesis by irreversibly diverting homocysteine away from methionine remethylation and down the transsulfuration pathway. 

Short term, the response of these enzymes to oxidative stress is protective, allocating metabolic resources to quickly restore GSH concentrations and maintain intracellular redox status in response to oxidative challenge.

Chronically compromised methylation, however, leads to increased plasma homocysteine levels, promoting chronic oxidative stress. In a state of chronic oxidative stress, synthesis of methionine and its product, SAM, progressively decline with a key factor being oxidative inactivation of the cobalamin cofactor of methionine synthase. In this way, unresolved oxidative stress can promote precursor depletion and a progressive decrease in cysteine and GSH synthesis. 

Ames’ KM concept provides an explanation for the positive outcome in this study of children with autism, a condition characterized by unremitting oxidative stress. By restoring enzyme activity, treatment with methylcobalamin and folinic acid lowers homocysteine and rescues GSH synthesis. 

The Methylation – Transsulfuration Connection to Mitochondrial Decay

As noted in Part I of this review, GSH is the body’s most critical intracellular antioxidant and a key conjugating agent in Phase II detoxification. High levels of homocysteine, which lead to chronic oxidative stress and GSH depletion, gravely impact not only mitochondrial function, but that of every cell and system in the body.

Following the logic chain outlined in this article, it is not surprising that folate insufficiency, and the resultant increase in homocysteine levels, which, if chronic, can lead to a decrease in SAM and GSH, has also recently been recognized to be a significant risk factor for Alzheimer's disease and other dementias, and is strongly associated with depression, with approximately one-third of depressed individuals having frank folate deficiency.^{15 22} SAM is a key donor of methyl groups, which are necessary for the formation of neurotransmitters (e.g., serotonin) and phospholipids that are a component of neuronal myelin sheaths, and cell receptors.²³

{image_1}

Methylation pathway: (1)Methionine is converted by methionine adenosyltransferase into the methyl donor S-adenosylmethionine (SAM), which is acted upon by methyltransferase and gives up a methyl group, becoming S-adenosyl homocysteine (SAH). SAH hydrolase converts SAH to homocysteine. (2)Homocysteine can either be remethylated through the folate cycle or go down the transsulfuration pathway. (3) The folate cycle requires the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) and folic acid (which enters the cycle as tetrahydrofolate [THF]), plus the enzyme methionine synthase (MS) and vitamin B12, which is the cofactor [in the form of methylcobalamin] for methionine synthase and uses the substrates 5-methyl-tetrahydrofolate and homocysteine. (4) In liver and kidney, homocysteine is also remethylated by the enzyme betaine homocysteine methyltransferase (BHMT), which transfers a methyl group to homocysteine via demethylation of betaine to dimethylglycine (DMG).

Transsulfuration pathway: (1)Homocysteine is converted to cystathionine by the enzyme cystathionine beta-synthase (CBS), which requires the cofactor vitamin B6 (pyridoxyl phosphate). Once formed from cystathionine via the action of cystathionine gamma-lyase, cysteine can be utilized in a number of cellular functions, including glutathione (GSH) production and protein synthesis. 

Methylation / Transsulfuration / Homocysteine Connection to GSH Depletion: When methylation is compromised, plasma homocysteine levels rise, increasing oxidative stress and the rate at which glutathione stores are depleted. When homocysteine elevation is chronic, redox sensitive enzymes in the methylation cycle (methionine synthase and the methyl transferase enzymes, betaine-homocysteine methyltransferase and methionine adenosyltransferase) are down-regulated, which promotes preferential utilization of homocysteine for GSH synthesis at the expense of methylation. In addition, cystathionine beta synthase (CBS) contains a heme component that is thought to serve as a redox sensor that upregulates CBS activity under oxidative conditions. This adaptive up-regulation of CBS activity promotes cysteine and GSH synthesis by irreversibly diverting homocysteine away from methionine remethylation and down the transsulfuration pathway. Short term, this serves as a protective mechanism, quickly restoring GSH concentrations to maintain intracellular redox status during oxidative stress.Chronically compromised methylation, however, leads to increased homocysteine levels and chronic oxidative stress. This results in oxidative inactivation of the cobalamin cofactor of methionine synthase causing progressive decline in synthesis of methionine and SAM, which ultimately promotes precursor depletion and a progressive decrease in cysteine and GSH synthesis.

Optimal Nutrition for Mitochondrial Function

While it has commonly been thought that Americans’ intake of essential micronutrients is adequate, evidence indicates that damage occurs at nutrient levels higher than those known to cause acute deficiency disease and, in some individuals (e.g., those with SNPs resulting in lessened KM), it is obvious that higher than DRI levels of the associated enzyme co-factors are necessary for optimal function. 

In addition to folate, MTHFR contains a bound flavin cofactor (derived from riboflavin, which as noted in Part I is a nutrient cofactor also required in the Krebs cycle and electron transport chain) and uses NADPH (which requires niacin as its co-factor and also plays a central role in mitochondrial oxidative phosphorylation) as the reducing agent. SNPs resulting in an increased need for both riboflavin and niacin have also been identified. 

The key clinical take-away, first proposed by Bruce Ames in his seminal paper on SNPs, decreased coenzyme binding affinity, and related nutrient needs, and recently re-emphasized by leading nutrigenomics researcher Michael Fenech, is that the activity of the reaction catalyzed by the MTHFR gene—and therefore the risks associated with the lessened MTHFR activity seen in carriers of 677CàT—can be markedly modified by the providing higher than "average" amounts of not just folate, the substrate for MTHFR, but also riboflavin and niacin, its cofactors.^{25 8}

Basic biochemistry and other studies suggest that not only higher than DRI amounts of riboflavin and niacin, but of vitamin B6, cobalamin (vitamin B12) and choline, may also be needed to optimize MTHFR-related methylation capacity in carriers of MTHFR SNPs. Homocysteine is metabolized through three vitamin B–dependent pathways. It can be either (1) remethylated and recycled as methionine, a reaction catalyzed by either the enzyme betaine homocysteine methyltransferase (in which the co-factor betaine is derived from choline) or (2) by the vitamin B12-dependent enzyme methionine synthase, or (3) homocysteine can be removed from the remethylation cycle by undergoing irreversible B6-dependent transsulfuration via the enzyme cystathionine beta synthase to form cysteine.^{26 27}

Conclusion

The folate, methylation and transsulfuration pathways are not separate entities but comprise an interconnected cellular network whose disruption, at any point, may have far-reaching deleterious effects, one of which is increased free radical production coupled with disruption of glutathione (GSH) production—a surefire recipe for accelerated mitochondrial decay and aging. 

Ensuring adequacy of all the nutrient co-factors necessary to restore KM in all these pathways, as well as for mitochondrial oxidative phosphorylation (discussed in Part I of this review), is first line therapy for promoting healthy aging. However, a protocol whose end goal is restoration of KM, while certainly helpful in delaying mitochondrial decay, does not address a more fundamental issue – why does human physiology shift from a homeostasic state that repairs and balances itself to one that allows decay to accumulate? Researcher Wulf Dröge has called this shift “the first cause of death,” and his insights into its likely causes may provide the means to opt out of the vicious cycle responsible for the age-associated move from a state of youthful homeostatic repair to a one that promotes mitochondrial decay. This will be the topic of Part III of this review: Reversing the Age-related Metabolic Shift towards Mitochondrial Decay. 

Read Part I: Delaying the Mitochondrial Decay of Aging

Read Part III: Restoring Mitophagy – the Key To Mitochondrial Rejuvenation

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Part I: Delaying the Mitochondrial Decay of Aging

Introduction

Mitochondrial decay resulting from oxidative damage is known to accumulate with age and is universally recognized as a major contributing factor to the whole range of functional decline and tissue deterioration associated with aging.^{1 2 3 4 5 6 7} Age-associated changes in mitochondria are characterized by increased generation of oxidants during oxidative phosphorylation and a decline in energy production, due in part to impaired enzyme activity and also to a decrease in cardiolipin, a phospholipid concentrated in the inner mitochondrial membrane that is essential for the function of key enzymes in the electron transport chain (see Glossary for more complete definition of italicized terms).⁸

Image Source: http://en.wikipedia.org/wiki/Mitochondria

During the phase of ATP production in which oxidative phosphorylation takes place within the mitochondria, electrons from NADH or FADH2 are transferred through the electron transport chain (Complexes I through IV), with the end result that ATP is generated and molecular oxygen (O2) is reduced to water. However, in this process, even in youth, ~ 2% of the electrons escape the electron transport chain (ETC) and reduce O2 to the highly reactive oxidant species, superoxide radical (O2−) and hydrogen peroxide (H2O2). This basal leakage of oxidants from the ETC not only appears to be unavoidable, but increases with age, rendering mitochondria the main endogenous center of superoxide radical formation.⁸

A side effect of being the primary source of these toxic oxidants is that mitochondria become their immediate targets. Mitochondria’s proximity to the oxidants they produce (O2− and H2O2), combined with their intricate structure, and the age-associated decline in antioxidant capacity (whose underlying mechanisms are discussed in Parts II and III of this review), render these organelles especially vulnerable to oxidative damage. In addition, mitochondria lack catalase, which catalyzes the decomposition of H2O2 to water and oxygen; the ability to synthesize glutathione (GSH), the body’s premier intracellular antioxidant; the ability to transport glutathione disulfide (GSSG), the oxidized form of GSH, out of the matrix; and chelators for heavy metals – all of which serve as key mechanisms through which cells decrease oxidant production.⁸

Tuning Up Mitochondrial Function with Acetyl L-Carnitine and A-Lipoic Acid

For more than thirty years, the legendary Bruce Ames, PhD, whose more than 450 scientific publications have resulted in his being among the few hundred most-cited scientists in any field—he certainly qualifies for, and has, his own entry on Wikipedia⁹—has been conducting research focused on tuning up mitochondrial metabolism to optimize health and longevity.

In recent studies, Ames and his team at the University of California, Berkeley, have made significant progress, reversing indicators of age-associated mitochondrial dysfunction in old rats by feeding them high doses of two crucial mitochondrial metabolites, acetyl L-carnitine and α-lipoic acid. Specifically, in old compared to young rats, mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O2 uptake are lower, while levels of oxidants, and mutagenic aldehydes from lipid peroxidation are higher. Yet when old rats are given a diet containing high levels of acetyl-L-carnitine and α-lipoic acid, within just a few weeks, these signs of mitochondrial decay are reversed, restoring their mitochondrial function to a much more youthful level.^{10 11 12 13 14}

While their mechanisms of action differ, acetyl L-carnitine and α-lipoic acid complement one another, in some cases synergistically.

Acetyl L-carnitine has been described as a conditionally essential nutrient for humans. In addition to glycolysis, acetyl-CoA is produced from the oxidation of fatty acids by acyl-CoA synthetase enzymes in the outer mitochondrial membrane, and then transported into the inner mitochondrial matrix by acetyl L-carnitine for β oxidation and ATP production. Acetyl L-carnitine also facilitates removal from the mitochondria of the excess short- and medium-chain fatty acids that accumulate during fat metabolism. Acetyl-L-carnitine is the preferred form of the nutrient in aging or conditions of disease since it is better absorbed and more efficiently crosses the blood–brain barrier compared to L-carnitine.¹⁵

Feeding old rats an acetyl-L-carnitine-supplemented diet restores tissue levels of free and acyl carnitines to those found in plasma and brain tissues of younger animals and, when combined with α-lipoic acid, significantly increases carnitine acetyltransferase (CAT) activity. More than 70% of CAT, an enzyme present in all mammalian tissues, is located in the mitochondrial matrix, where it catalyses the reversible conversion of acetyl-CoA and carnitine to acetyl carnitine and CoA. Thus CAT regenerates CoA, which allows peroxisomal β-oxidation to proceed, and facilitates transport of acetyl moieties to the mitochondria for oxidation. (β-oxidation of fatty acids generates acetyl-CoA, the entry molecule for the Krebs or citric acid cycle.)¹³

Alpha lipoic acid is a cofactor for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, two key enzymes involved in the Krebs cycle. Not only does α-lipoic acid function as a mitochondrial coenzyme, but it also is reduced in the mitochondria to a powerful antioxidant, dihydrolipoic acid, which recycles other antioxidants, including vitamins C and E.¹³

In addition, α-lipoic acid is a potent inducer of the ~200 Phase II enzymes with antioxidant activity, including those required for the synthesis of GSH, the body’s most important intracellular antioxidant.

Thus supplementation with α-lipoic acid also increases intracellular GSH levels, which is critical for neuronal function. As a result, in aged rodents, α-lipoic acid supplementation restores long-term potentiation, a synaptic analogue of learning and memory, and partially restores ambulatory activity and memory lost during aging.^{13 16 17 18}

As noted above, when old rats receive high levels of both acetyl L-carnitine and α-lipoic acid, within several weeks, the combination greatly improves mitochondrial function, resulting in restoration of ambulatory activity and cognition (as assayed with the Skinner box and Morris water maze), heart, and immune function to levels seen in young rats.

The KM Concept: Key to Mitochondrial Decay?

Ames’ rationale for the mechanism behind the rejuvenating effects of acetyl-L-carnitine and α-lipoic acid is that, with age, increased oxidative damage to key proteins, causes deformation in their structure, resulting in an increased Michaelis constant/(KM) or decreased binding affinity for a co-enzyme (i.e., the nutrient co-factor for the enzyme), and thus a decrease in enzyme function.¹⁹

Ames’ research has demonstrated that this age-associated effect on enzyme-binding affinity can be mimicked by exposing enzymes to malondialdehyde, a lipid-peroxidation product, levels of which also increase with age. Increasing availability of the substrate acetyl L-carnitine along with α-lipoic acid, a mitochondrial antioxidant, restores the velocity of the reaction (KM) for the enzyme acetyl-L-carnitine transferase and thus improves mitochondrial function.⁸

Common Nutrient Deficiencies Accelerate Mitochondrial Decay

Ames also targets iron and vitamin B6 as two common nutrient deficiencies that accelerate mitochondrial decay.²⁰ Heme biosynthesis occurs predominantly in the mitochondria. Iron insufficiency disrupts heme synthesis resulting in loss of Complex IV and, as a result, significantly increased release of oxidants. Iron deficiency is fairly common among menstruating women in the U.S., 25% of whom ingest ≤50% of the DRI. Vitamin B6 deficiency, also not uncommon (10% of Americans ingest ≤50% of the DRI), can also cause heme deficiency since heme biosynthesis requires B6. A further consideration is that menstruating women may be using oral contraceptives, which have repeatedly been found to be associated with significantly lowered levels of vitamins B6 and B12.²¹ Ames notes that insufficiencies of iron or B6 are likely to result in accelerated aging and neural decay.

Conclusion

While it has commonly been thought that Americans’ intake of essential micronutrients is adequate, evidence indicates that damage occurs at levels higher than those that cause acute deficiency disease. In addition, as many as one-third of all single nucleotide polymorphisms (SNPs) in a gene result in the corresponding enzyme having an increased KM (decreased binding affinity) for its coenzyme, and therefore a lower rate of reaction. Given that some of these SNPs (to be discussed in Part II of this review: The Methylation Transsulfuration Connection to Mitochondrial Function) are found in ~30-40% of the population,²⁹ it is obvious that higher than DRI levels are necessary for optimal function in significant numbers of people.

Dr. Ames’ KM concept applies both to aging-associated mitochondrial dysfunction, as discussed above in regards to acetyl-L-carnitine and lipoic acid, and to inborn (genetic) weaknesses in metabolism. In both cases, the functional capacity of defective enzymes can be ameliorated by administration of high doses of the corresponding cofactors, which raises coenzyme levels and at least partially restores enzymatic activity and mitochondrial function.

However, a protocol focused on restoration of KM, while certainly helpful in delaying mitochondrial decay, does not address a more fundamental issue – why does human physiology shift from a homeostasic state that repairs and balances itself to one that allows decay to accumulate? A new theory concerning what researcher Wulf Dröge has called “the first cause of death” may provide insight into a vicious cycle responsible for the shift from a state of youthful homeostatic repair to a homeostatic state that promotes mitochondrial decay. This will be the topic of Part III of this review: Reversing the Age-related Metabolic Shift towards Mitochondrial Decay.

Image Source: http://en.wikipedia.org/wiki/Mitochondria

Glossary

α-ketoglutarate dehydrogenase (aka xoglutarate dehydrogenase)—enzyme involved in the citric acid or Krebs cycle in which it catalyzes the reaction: α-ketoglutarate + NAD+ + CoA → Succinyl CoA + CO2 + NADH

β oxidation—the process by which fatty acids, in the form of Acyl-CoA molecules, are broken down in mitochondria and/or in peroxisomes to generate Acetyl-CoA, the entry molecule for the Krebs cycle.

betaine—in chemistry, any neutral compound with both a positively- and a negatively-charged functional group, so that it carries a total net charge of 0, while carrying formal positive and negative charges, also called a zwitterion. In biological systems, betaines, which are polar and water-soluble, permit water to remain in cells, thus serving as intracellular protectors against osmotic stress. Betaines also serve as methyl donors; trimethylglycine is a betaine.

cardiolipin—an important component accounting for ~20% of the total lipid content of the inner mitochondrial membrane, cardiolipin (so named since it was first identified in beef heart in the 1940s) is essential for the optimal function of numerous enzymes involved in mitochondrial energy production including cytochrome bc1 (Complex III) and cytochrome c oxidase (Complex IV) in the electron transport chain. Decreased cardiolipin synthesis is thought to be associated with mitochondrial dysfunction in Parkinson’s disease, non-alcoholic fatty liver disease, heart failure and diabetes.^{30 31 32 33} Increased mitochondrial ROS production promotes oxidation and depletion of cardiolipin, as well as inhibition of cytochrome c oxidase activity. Peroxidation of cardiolipin has been suggested to impair the barrier function of the inner membrane and facilitate the detachment of cytochrome c from the electron transport chain.²⁴

catalase—a common enzyme found in virtually all organisms exposed to oxygen, catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. Catalase has one of the highest turnover numbers of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second.³⁴³

Glutathione—GSH is a tripeptide composed of cysteine, glycine, and glutamate that is synthesized de novo in all cells and serves as the major intracellular antioxidant and redox buffer.

heme—the major functional form of iron, synthesized in the mitochondria.³⁶

pyruvate dehydrogenase—enzyme involved in transforming pyruvate from glycolysis into acetyl-CoA, which is then used in the citric acid or Krebs cycle. Thus, pyruvate dehydrogenase serves as a link from glycolysis to the citric acid or Krebs cycle, which is followed by oxidative phosphorylation in the electron transport chain of the mitochondria.

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Vitamin A is a family of essential fat-soluble dietary compounds, three of which—retinol, retinal and retinoic acid—play significant roles in the human body, with each compound performing functions the others cannot. Retinol is the major transport and storage form of vitamin A; retinal is essential for vision, and retinoic acid acts like a hormone, binding to nuclear-hormone receptors and affecting the expression of more than 500 genes. Vitamin A is required for vision, immune function, skin health, regulation of cell growth and bone metabolism, thus symptoms of vitamin A insufficiency include night blindness; increased susceptibility to infection and loss of immune tolerance (environmental and food allergies, autoimmune diseases); hyperkeratosis, dry skin, brittle nails, hair loss; epithelial cancers and bone loss.

Due to the perceived risk of hypervitaminosis A, provitamin A carotenoids have been recommended as the preferred source of this nutrient for women of reproductive age and are its primary source for vegetarians and its only source for vegans. While it has long been recognized that vitamin A and provitamin A differ significantly in bioavailability and therefore biologic activity, recently published research reveals that a significant number of individuals are “low-responders” unable to absorb and/or convert provitamin A carotenoids to vitamin A, and that even in individuals who would normally be able to metabolize provitamin A to vitamin A, numerous factors can effectively impede this conversion. Research showing large individual variations in the actual absorption of pro-vitamin A from foods and in its conversion to retinol has demonstrated that current data on retinol activity equivalents (RAEs) for vitamin A sufficiency is highly misleading, and that a significant percentage of the American population may be vitamin A insufficient.

In addition to the aforementioned research, this article reviews recent discoveries regarding the key roles played by vitamin A in promoting the development of regulatory T cells that favor oral and self- tolerance, and in both potentiating and balancing the effects of vitamin D. Symptom and lab assessment of vitamin A status is discussed. 

Healthy centenarians are consistently found to have vitamin A levels comparable to those in healthy young adults, in contrast to lower levels of vitamin A seen in typical older control subjects.

Aging is characterized by a peculiar chronic inflammatory status for which researchers have recently coined the term, “inflammaging,” a key aspect of which is the age-dependent expansion of effector T cells with pro-inflammatory cytokine production potential. Particularly in light of vitamin A’s emergence as a pivotal inducer of oral and self-tolerant immune function, vitamin A sufficiency should be recognized as a key contributing factor to longevity.

Introduction to Vitamin A

Vitamin A is a family of essential fat-soluble dietary compounds that contain a retinyl group. Three different forms of vitamin A, collectively referred to as retinoids, are active in the body: retinol (alcohol), retinal (aldehyde) and retinoic acid (acid). All natural retinoids have a least one β-ionone ring linked to a side chain of conjugated carbon-carbon double bonds that may exist in trans or cis conformation.^{1 2 3}

In animal-derived foods, the major form of vitamin A is the retinyl ester (primarily as retinyl palmitate, but also as retinyl stearate), which is hydrolyzed in the small intestine to yield an alcohol (retinol) and the corresponding fatty acid (palmitate or stearate). The provitamin A carotenoids (β-carotene, α-carotene and β-cryptoxanthin) can be converted into retinal and then to retinol. β-carotene, which contains β-rings at both ends, is cleaved to yield two retinol molecules, while α-carotene and β-cryptoxanthin, which contain one β-ring, yield one.

Conversion of retinol to retinal is reversible, but the further conversion of retinal to retinoic acid is not—a significant fact since each form of vitamin A performs functions the others cannot. (Please see Conversion of Vitamin A Compounds chart above.) 

Most (80%) of the body’s vitamin A reserves (around 450 mg in healthy vitamin A-replete adults) are stored in the liver (500 µg/g wet tissue), an amount that can cover vitamin A requirements for several months. High alcohol intake, however, can rapidly deplete vitamin A stores.

Retinoic acid and other vitamin A metabolites can be conjugated to glucuronide by CYP26 and excreted with bile; however, the high efficiency of intestinal absorption for most retinoids minimizes losses and maintains extensive enterohepatic recycling. In addition, megalin, a particularly large member of the LDL-receptor family, binds retinol binding protein 4 (RBP4) and mediates its uptake by endocytosis. Despite the considerable size of the retinol carrying RBP4-transthyretin complex, it has been shown to proceed through the epithelial cell and return intact into circulation. Oxidation of retinoic acid is the only known inactivating catabolic pathway; all retinoids, however, are highly susceptible to oxidation.⁴

Retinol

Retinol is the major transport and storage form of vitamin A. Retinol is absorbed from the proximal small intestine in a process that requires concurrent fat absorption and the formation of mixed micelles, which are transferred into chylomicrons and then exit enterocytes to be exported into portal blood and taken up by the liver. Retinol is released from the liver within retinol-binding protein 4 (RBP4), which engulfs the retinol molecule, shielding it from the hydrophilic environment in circulation or inside cells. After its release from the liver, RBP4 combines with transthyretin, forming a complex that transports retinol in the blood. A surface receptor on many peripheral cells binds RBP4, mediating retinol uptake. In vitamin A deficiency, the liver’s production and release of RBP4 increases. About 70-90% of ingested retinol is absorbed, but only 3% or less of carotenoids. Carotenoids, which also must be incorporated into mixed micelles and then into chylomicrons for absorption, are exported into lymph, appearing in the blood about 8 hours after a challenge meal. Chylomicrons rapidly lose triglycerides, but not carotenoids, and the depleted chylomicron remnants are taken up into heptaocytes and extrahepatic cells via diverse lipoprotein receptors.

Retinal

Retinal serves as the intermediate in the conversion of retinol to retinoic acid and is involved in vision as an integral component of the pigment molecules in the retina, rhodopsin, which are composed of one molecule of opsin bonded to one of retinal. When light passes through the cornea and strikes the retina, the retinal in rhodopsin shifts from a cis to a trans configuration, which cannot remain bonded to opsin. Opsin changes shape, generating an electrical impulse that transmits visual information to a nerve cell and thence to the brain.

While most of the retinal converts back to its cis isoform and recombines with opsin to regenerate rhodopsin, some is oxidized to retinoic acid. Thus visual activity results in continuous small losses of retinal, which must be constantly replenished from retinol stores, food or supplements.

Retinoic acid

Vitamins A and D are notably distinct from other vitamins in that their respective bioactive metabolites, retinoic acid and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), have hormone-like properties. Both 1,25(OH)2D3 and retinoic acid are synthesized from their vitamin precursors by different tissues and cells in the body and exert their effects on remote target cells by binding to nuclear-hormone receptors.⁵ Retinoic acid regulates normal endodermal differentiation, morphogenesis, embryonic and childhood development; and sustains balanced cell proliferation, differentiation, and apoptosis in adulthood. Many of the underlying events involve the binding of retinoid receptors to specific binding elements in nuclear DNA and control of the expression of associated genes. More than 532 genes are recognized as either directly or indirectly regulated by retinoic acid.^{4 6 7}

Two groups of retinoid receptors have been identified. The first includes the retinoic acid receptors (RAR) α, β, and γ, which bind both the trans and cis isoforms of retinoic acid. The second group is comprised of the retinoic X receptors (RXR) α, β, and γ, with the cis isoform of retinoic acid as the main activating ligand. The RXR group is of special interest since its binding is necessary for the actions of numerous other nuclear receptors. The list of RXR-dependent nuclear binding proteins includes the RAR, vitamin D receptor (VDR), thyroid receptors (TR), peroxisome proliferation activating receptors (PPAR), pregnane X receptor (steroid and xenobiotic receptor, SXR/PXR), liver X receptors (LXR), famesoid X-activated receptor (FXR), and benzoate X receptor (BXR). The importance and diversity of retinoic acid’s gene regulating effects are evidenced by the wide ranging diversity of these receptors.⁸

Functions of Vitamin A and Related Symptoms of Deficiency

Since vitamin A is essential for vision, skin health, immune function/oral tolerance (discussed below), regulation of cell growth, and bone metabolism, symptoms of vitamin A insufficiency include:

• Impaired vision—initially most evident in reduced light (e.g., night blindness, due to lack of vitamin A at the back of the eye, the retina)
• Xerophthalmia—total blindness resulting from lack of vitamin A at the front of the eye, the cornea
• Xerosis—abnormal drying of the skin and mucous membranes
• Dry skin
• Dry hair
• Pruritus
• Easily broken or ridged fingernails
• Hyperkeratosis—goose bump-like appearance of the skin caused by excessive production of keratin, which blocks hair follicles. Skin becomes dry, scaly and rough, initially on the forearms and thighs. In advanced stages, the entire body is affected, causing hair loss.
• Skin lesions—actinic keratoses, increased susceptibility to basal and squamous skin cancers
• Epithelial cancers (lung, colon, breast, prostate cancers).
• Viral infections, most notably measles, chicken pox, pneumonia, and respiratory syncytial virus (RSV—the most common cause of bronchiolitis and pneumonia in children under 1 year of age and now being recognized as an important cause of respiratory illness in adults).
• Infections of the respiratory, GI, and urinary tracts, the vagina, and possibly the inner ear.
• Fungal infections, e.g., nail fungal infections, athlete’s foot
• Environmental and food allergies
• Inflammatory bowel disease
• Autoimmune disease
• Bone loss

Vitamin A Toxicity – a Question of Balance?

Excessive retinol intake (UL = 3,000 µg/day [10,000 IU/day]) may increase bone fracture risk in older people; however, recent studies indicate retinol, which promotes osteoblast differentiation and activation, helps prevent bone loss in combination with vitamin D,^{9 10 11 12 13} suggesting imbalance between vitamins A and D is the real issue. Two recently published studies in which the relationship between vitamin A intake and fracture risk was evaluated support this hypothesis.

A study involving 3,113 postmenopausal women living at higher latitudes in the UK found that retinol derived from cod liver oil showed no detrimental associations with markers of bone health. Retinol from other vitamin supplements was initially associated with lower BMD; however, a trend for lower bone resorption was noted at the follow-up visit, and less bone loss occurred over 6 years. Retinol from food was associated with increased bone resorption, leading study authors to note that it appears “retinol from supplements and food have different effects, which may in part be due to whether the source of retinol also provides vitamin D.¹⁴

The second study, which involved 75,747 women from the Women’s Health Initiative Observational Study, found a modest risk in total fractures associated with increased total vitamin A or retinol intakes; however, this association was observed only among women with low vitamin D intakes. In addition, no association between vitamin A intake and the risk of hip fracture was observed. Researchers speculated, “A deleterious effect of vitamin A on bone may operate through its antagonistic relation with vitamin D.¹⁵

We would characterize the relationship between vitamin A and vitamin D as one of maintaining balance rather than antagonism. 

Retinoic acid and 1,25(OH)2D3 compete for the same nuclear receptor partners; both the RAR and the VDR must form heterodimers with RXRs to be able to bind to response elements and initiate transcription. For this reason, 1,25(OH)2D3 and retinoic acid naturally mitigate against each other’s uncontrolled effects.¹⁶

In addition, vitamin A optimizes the body’s use of vitamin D. Recent research has shown that 9-cis-retinoic acid, a derivative of vitamin A, increases the affinity of VDR/RXR to its DNA recognition site, induces recruitment of coactivators by the DNA-bound heterodimer, and potentiates vitamin D-dependent transcriptional responses—all of which suggests that vitamin A, when properly balanced with vitamin D, promotes bone health.^{17 18}

Vitamin A intake >3,000 µg/day [>10,000 IU supplemental pre-formed vitamin A] during early pregnancy may increase risk of birth defects.^{4 3 1} Since such negative outcomes may be related to vitamin D insufficiency, maintaining balance between vitamins A and D (indicated by lab values in normal range for both nutrients) is especially important for women who might conceive.

Food Sources of Vitamin A

Only animal-derived foods contain retinol. Highest concentrations of retinol are found in liver (106µg/g), butter (6.84µg/g), hard and cream cheeses, e.g., Swiss cheese (2.5 µg/g), cheddar (2.8µg/g) and regular (not low fat) cream cheese (3.8µg/g). Eggs contain 1.9µg/g, and cow’s milk 0.6 µg/g. 

Good sources of provitamin A include carrots (24.6 µg RAE [retinal activity equivalent]/g), sweet potatoes (16.4µg RAE/g), spinach (8.2µg RAE/g), kale (7.4 µg RAE/g), and broccoli (1.4µg RAE/g). Cantaloupe (1.69µg/g), mangos (.38 μg/g), and many other dark green or orange-yellow colored fruits and vegetables are also good sources of provitamin A. In the case of mangoes, however, up to 64% of the total β-carotene is present as the cis-isomer, which is not taken up or transported as efficiently as the trans-isomer.¹

Many at Risk of Vitamin A Insufficiency Due to Increased Intake of Vitamin D and High Variability in β-carotene Absorption and Conversion to Retinol

Bioavailability of pre-formed vs. pro-vitamin A

Vitamin A and provitamin A differ significantly in bioavailability and therefore biologic activity. The retinol activity equivalent (RAE) was adopted in 2001 to account for the much lower biological activity of provitamin A carotenoids compared to pre-formed vitamin A. In RAE units, 1 µg RAE is equivalent to 1µg of pure all-trans-retinol, 2 µg of all-trans-β-carotene in oil (a highly absorbable form), 12µg of food-based all-trans-β-carotene, or 24 µg of other, food based, all-trans provitamin A carotenoids. Of the 600 carotenoids discovered so far, only β-carotene, α-carotene and β-cryptoxanthin possess vitamin A activity.¹

The RDAs for vitamin A are extrapolated from the estimated average requirement (EAR), which is equal to the intake that meets the estimated nutrient needs of half of the individuals in a group. To this amount is factored in the considerations that 0.5% of vitamin A stores are lost daily, and minimum acceptable liver vitamin A storage reserves are 20µg. Thus, the EAR for men >18 is 625 and the RDA is 900, while for women the EAR is 500 and RDA is 700 µg RAE/d.1

While these amounts appear to include a reasonable safety margin, the RDAs are based on assumptions regarding the amount of β-carotene absorbed and converted to retinol that do not take into account a number of food and host-related factors that significantly impact carotenoid bioavailability, absorption and conversion to retinol.¹⁹

Factors affecting carotenoid absorption and metabolism to retinol

Current evidence indicates that large variations in the concentration of provitamin A carotenoids in foodstuffs occur—even in the same type of food—due to varietal differences, stage of maturity, climatic conditions, processing and/or cooking. 

Efficiency of carotenoid absorption is affected not only by the amount of carotenoid ingested, but by processing and/ or cooking of the food, other dietary ingredients that stimulate absorption (e.g., the type and amount of dietary fat [carotenoids must be incorporated into mixed micelles and then into chylomicrons for absorption] or inhibit it [fiber, especially pectins], food matrix effects, and interactions between carotenoids [lutein and β-carotene appear to inhibit one another’s absorption]).²⁰

Other factors affecting the individual’s ability to utilize carotenoids as a source of retinol include:

• Current nutritional status: protein and nutrient-cofactors are necessary for function of the enzymes involved in carotenoid metabolism to retinal, including 15,15΄monoxygenase, the brush border retinal ester hydrolases, and retinal dehydrogenase [RALDH]1
• Inadequate bile flow: affected by acute and chronic liver diseases, including non-alcoholic steatohepatitis related to insulin resistance / metabolic syndrome, which is estimated to affect ~30% of the US population²¹, and by alcoholism. Alcohol dehydrogenase—the enzyme that catalyzes conversion of retinol to retinaldehyde, which is then oxidixed to retinoic acid—has an affinity for ethanol.
• Imbalanced gut ecology: due, for example, to parasitic infestation, food allergies such as gluten intolerance, celiac disease (U.S. incidence 1 in 133)²², lipid malabsorption, hydrochloric acid insufficiency (30% of the US population >60 is estimated to have atrophic gastritis)20, infection with H.pylori, (prevalence of H.pylori infection is 50% worldwide, 40% in the US²³ ) or Clostridium difficile (annual incidence of C.difficile infection increased from 49.2 to 101.6 per 100,000 population from 2001-2005²⁴)
• Genetic inheritance: discussed immediately below

Single nucleotide polymorphisms (SNPs) have recently been identified that significantly lessen the activity of 15,15'-monoxygenase, the key enzyme responsible for the conversion of β-carotene to retinal, and can therefore greatly impact an individual’s ability to derive adequate levels of vitamin A from carotenoid-rich foods. These SNPS (R267S and A379V) are common, with variant allele frequencies in the population of 42 and 24%, respectively. In vitro biochemical characterization of the 267S + 379V double mutant revealed a 57% reduction in catalytic activity of 15,15'-monoxygenase. Assessment of the responsiveness of female volunteers to a pharmacological dose of β-carotene confirmed that carriers of both the 379V and 267S + 379V variant alleles have a greatly reduced ability to convert β-carotene, indicated by reduced retinyl palmitate: beta-carotene ratios in the triglyceride-rich lipoprotein fraction of -32% and -69% , respectively, and increased fasting beta-carotene concentrations of +160% and +240% , respectively.²⁵

Beta-carotene 15,15'-monoxygenase, which converts the provitamin A carotenoids to retinal mainly in the intestinal mucosa (and, to a lesser extent, in testes, liver and kidneys), also requires bile acids and iron for its three-step activity (epoxidation at the 15,15'-double bond, hydration to the diol, and oxidative cleavage). Retinal resulting from carotenoid cleavage is metabolized, mainly to retinol, by several tissue specific enzymes. In testes, a specific isoform of lactate dehydrogenase is closely associated with beta-carotene I5, 15'-dioxygenase, and the reduction of newly generated retinal is thought to be its main physiological function. In most other tissues, class III alcohol dehydrogenase may be more important. Co-factors required by these enzymes are riboflavin, niacin, and zinc.⁴

The activity of each of these dehydrogenase enzymes is also dependent on sufficient protein in the diet and is surely affected by SNPs as well. Although the research directly connecting various dehydrogenase SNPs with vitamin A metabolism is still forthcoming, researchers have recently identified six alcohol dehydrogenase SNPs, several of which appear to confer protection against aerodigestive cancers, suggesting a connection to retinol metabolism.²⁶ While these SNPs appear to promote enzyme activity, it seems only reasonable to assume that other SNPs result in its decrease.

Environmental toxicants affect retinol storage and metabolism

Numerous hepatotoxicants, e.g., carbon tetrachloride, reduce retinol storage and/or alter retinoid metabolism. In 2008, a study of common cleaning products found the presence of carbon tetrachloride in "very high concentrations" (up to 101 mcg per cubic meter) as a result of manufacturers' mixing of surfactants or soap with sodium hypochlorite, i.e. chlorine bleach.²⁷

So, while on the basis of central enzymatic cleavage alone, one molecule of β-carotene provides two molecules of retinol (so 1 mg β-carotene in a food is theoretically equal to 1 mg retinol), in vivo, the above factors affecting carotenoid absorption greatly impact how much of the provitamin ingested in the diet is actually presented to and absorbed by an individual’s intestinal mucosa. 

Research confirms provitamin A may not supply adequate retinol

Several studies have demonstrated the impact of the above-noted factors on carotenoid absorption and metabolism to retinol. In one study in which stir fried vegetables or supplements of β-carotene enriched wafers were given to Indonesian breast feeding women with low hemoglobin, no significant changes were seen in the vegetable group in serum retinol, β-carotene or other serum carotenoids, or breast milk retinol. In the enriched wafer group, serum retinol, serum β-carotene, and breast milk retinol all increased 0.32 nmol/L (38%), 0.73 nmol/L (390%) and 0.59 nmol/L (67%), respectively. The authors concluded that the assumption that provitamin A-containing vegetables can be relied upon to prevent or combat vitamin A deficiency should be re-examined.²⁸

Although the processing and/or cooking of vegetables may cause a change from the all-trans to the cis isomer of carotenoids¹⁹, which results in less efficient production of retinol²⁹, partially because up to 95% of the cis isomer is first converted back to trans-β-carotene before absorption³⁰, studies indicate that up to 50% of carotenoids may be absorbed from cooked vegetables in comparison to less than 10% from raw vegetables. Other research shows that absorption of β-carotene and other carotenoids from vegetables is only 5-30% of that absorbed from synthetic supplements, due to the food matrix of fiber and/or protein that must first be broken down by mastication, gastric acid and bile acids.²⁰

In another study, a prepared meal containing a pharmacological dose of 120 mg of β-carotene in oil was fed to 79 healthy male volunteers. Response was highly variable (coefficient of variation [CV] =61%) due to inter-individual differences in the efficiency of intestinal absorption of β-carotene and in chylomicron metabolism. The inter-individual variability of the apparent vitamin A activity of β-carotene ingested was also high, although the higher the amount of β-carotene absorbed, the higher the amount of retinol palmitate secreted into chylomicrons. The authors concluded that the dietary equivalence of β-carotene and retinol varies greatly among individuals.³¹

Variability in the conversion of beta-carotene to vitamin A has also been shown in a recent study at UC Davis in which 11 healthy women were given 30 micromol retinyl acetate orally, followed one week later by an approximately equimolar dose of 37 micromol β-carotene. Although mean absorption of β-carotene was 3.3%, only 6 of the 11 subjects had measurable plasma β-carotene and retinol concentrations. Mean absorption of β-carotene in the 6 women with measureable levels was 6.1%, and their conversion ratio was 1.47 mol for retinol to 1 mol β-carotene. The remaining 5 subjects were “low responders” with

In Germany, pregnant women or those considering becoming pregnant are generally advised to avoid the intake of liver due to concerns about the teratogenic potential of hypervitaminosis A, a concern that may be misguided (as noted above and in the following section, vitamins A and D mitigate and balance one another’s effects). As a result, β-carotene is their primary source of vitamin A. A clinical study in pregnant women with short birth intervals or multiple births showed that, regardless of a high to moderate socio-economic background, 27.6% of the women had plasma retinol levels below 1.4 micromol/l, corresponding to a borderline deficiency (or worse.) Despite high total carotenoid intake of 6.9 +/- 3.6 mg/d, 20.7% of mothers showed plasma levels <0.5 micromol/l β-carotene, indicating frank deficiency. Vitamin A is recognized by the American Pediatric Society as one of the most critical vitamins during pregnancy and the breastfeeding period, especially in terms of lung function and maturation. If the vitamin A supply of the mother is inadequate, her supply to the fetus will also be inadequate, as will be the supply of vitamin A in her breast milk. The developmental effects of these inadequacies cannot be reversed by postnatal supplementation and may be a contributing factor to the escalation in the incidence of allergic diseases, including asthma, and autoimmune diseases, including type 1 diabetes, seen in the western world.^{33 34 35 36 37}

Vitamin A – dancing with vitamin D to promote balanced immunity

Both the retinoic acid receptor (RAR) and the vitamin D receptor (VDR) must form heterodimers with RXR to signal, and 9-cis-retinoic acid, a derivative of vitamin A, increases the affinity of VDR/RXR to its DNA recognition site, induces recruitment of coactivators by the DNA-bound heterodimer, and potentiates vitamin D-dependent transcriptional responses. 

Since not only is 1,25(OH)2D3 recognized to play a protective role in dampening or limiting potential autoimmune responses at the cellular level (vitamin D regulates TGF-β signaling molecules called Smads that affect gene expression, decreasing induction of pro-inflammatory TH1 [type 1 helper T cell] cytokines^{38 39}), but vitamin A, specifically all-trans retinoic acid, has recently been shown to decrease effector T-cell function, while also increasing regulatory T-cell populations and activities (further discussed below), one might predict that insufficiency of either vitamin D or vitamin A could predispose to hypersensitivity or autoimmunity. 

Consistent with this hypothesis, both vitamin A and vitamin D have been found to be relatively deficient in adults as well as children with type 1 diabetes. Serum levels of 1,25(OH)2D3 are also often decreased in patients with systemic lupus erythematosus and are inversely correlated with disease activity in patients with rheumatoid arthritis. Insufficiency of both vitamins A and D is found in children with rickets (typically due to insufficient vitamin D), who also have a higher incidence of diabetes than vitamin D-sufficient children, and are more susceptible to infection} ⁴⁰ suggesting a lack of vitamin A for protective immune responses.⁴¹

Research reported in the January 2009 issue of Diabetes provides evidence that all-trans retinoic acid inhibits the development of type 1 diabetes in NOD mice. Spleen cells from diabetic mice were transferred into NOD.scid mice normally resistant to type 1 diabetes development, a protocol through which diabetes is consistently transferred to recipient mice. NOD.scid mice are naturally type 1 diabetes resistant since, although they have the same genetic background as NOD mice, they also carry a mutation rendering them immunodeficient (i.e., no T- or B-lymphocytes). 

Treating NOD.scid recipients with all-trans retinoic acid markedly suppressed the transfer of diabetes by diabetogenic splenocytes. All-trans retinoic acid also significantly delayed progression to type 1diabetes in a “late prevention” model in which it was given by intraperitoneal injection to treat 10-week-old NOD mice. These beneficial therapeutic outcomes were found to result from the immunoregulatory effects of all-trans retinoic acid (discussed below).⁴² In an article also published in the January 2009 issue of Diabetes and entitled “Taking a daily vitamin to prevent type 1 diabetes?” Wasserfall and Atkinson note that both vitamin A and D are fat-soluble and found in fish oil supplements, which epidemiological evidence suggests are associated with reduced type 1 diabetes-associated autoantibody conversion, raising “an intriguing possibility that a combination of vitamins A and D, in safe pharmacologically formulated doses rather than the usual daily recommended dose, might be of benefit in the treatment of those at increased risk for type 1 diabetes.⁴¹

Vitamin A’s Effects on Immunity

T Lymphocytes

T lymphocytes (T cells), which activate and direct other immune cells, account for 70-80% of peripheral lymphocytes in the blood and express the surface protein CD4, so are often referred to as CD4+ T cells. Naive T cells originate in bone marrow and mature primarily in the thymus where, after activation by unique cytokines, they differentiate into lineages of effector/memory (TH) and regulatory T (Treg) cells, each of which are characterized by distinct developmental pathways and unique biologic functions. 

Meet the new subset of T cells

Until recently, helper T cells that develop into effector T cells were thought to be a binary system, including only TH1 and TH2 effector cell types, but the TH family is now known to include an additional lineage, the TH17 cell. TH1 cells produce IFN-γ and TNF-β, activate marcophages, and are involved in cell-mediated immunity. TH1 cells elicit delayed-type hypersensitivity responses and effectively clear intracellular pathogens. TH2 cells produce IL-4, IL-5, IL-6 and IL-10, partner with B cells, and contribute to humoral immunity. TH2 cells are involved in IgE production, eosinophilic inflammation, and the clearance of helminthic parasite infections. 

The recently recognized T cells, TH17 cells, which are induced to differentiate from naïve CD4+ T cells by the transcription factor receptor-related orphan receptor-γt (RORγt) in the presence of TGF-β and IL-6, generate IL-17 cytokines that initiate activation of NFκB, which leads to the transcription of multiple target genes involved in innate immunity. TH17 cells appear to be critical for enhancement of host protection against extracellular bacteria and fungi, which are not efficiently cleared by TH1 and TH2 responses, and also have emerged as potent mediators of autoimmune disease. 

T regulatory cells

In counterbalance, Treg cells maintain immune tolerance and protect against excessive TH effector activity and autoimmune pathology. Treg cells are characterized by the expression of a DNA binding protein, the forkhead–winged helix transcription factor family member, FOXP3+, which is induced by TGF-β in the presence of IL-2. This process is significantly upregulated by retinoic acid (RA), which enforces the generation of Treg cells and inhibits the differentiation of TH17 cells. FOXP3+ Treg cells secrete IL-10, which suppresses activation of aggressive effector T cells. 

So, we now have four functionally unique populations of CD4+ T cells that are directly involved in the regulation of immune responses to pathogens, allergens, and self-antigens: TH1, TH2, TH17 and Treg cells.⁴³

Vitamin A Key to Self and Oral Tolerance

This emerging picture has revealed vitamin A as an important regulator of the TH1/TH17 versus TH2 balance in the immune system. Vitamin A deficiency skews balance in a TH1/TH17 direction, while adequate levels of vitamin A, by promoting the development of Treg cells responsible for self-tolerance and the prevention of autoimmune diseases, favors immune responses characterized by production of anti-inflammatory IL-10 cytokines.^{44 45}

Regulatory T cells develop both constitutively in the thymus (referred to as natural Tregs [nTregs]) and peripherally, as a result of induction by FOXP+3 (referred to as induced Tregs [iTregs]).8 Retinoic acid [RA] promotes induction of FOXP3+ Tregs in the presence of TGF-β by downregulating RORγt, thus also blocking the differentiation of TH17 cells. 

Transforming growth factor-β (TGF-β), a family of cytokines with numerous signaling effects, (members of the TGF- β superfamily are involved in cell growth and differentiation and influence immune and endocrine functions⁴⁶) is required for both constitutive nTreg development in the thymus and peripheral induction of iTreg, but TGF-β plays a dual role since, in the presence of inflammatory cytokines such as IL-6, it is also able to promote development of pro-inflammatory TH17 cells. RA thus emerges as a key regulatory factor, modulating immunity by inhibiting the TGF-β/IL-6-driven induction of pro-inflammatory TH17 cells and simultaneously promoting the TGF-β-dependent peripheral differentiation of anti-inflammatoryFOXP3+ iTregs, which are indispensable to prevent excessive and self-destructive immune responses.⁸

In addition to enhancing the TGF-β-driven generation of iTregs in peripheral tissues, RA enhances the production of iTregs in response to antigen-presenting dendritic cells in the gut‑associated lymphoid tissue (GALT) and small intestinal lamina propria, and upregulates gut-homing receptors on iTreg cells and B lymphocytes, targeting both cell types to the gut mucosa. Effector and memory T cells exhibit plasticity in their homing commitment: skin-homing T cells can become gut-homing T cells and vice versa if they are restimulated either with or without RA, respectively. Like T cells, B cells also exhibit plasticity in their homing commitment and can either acquire or lose gut-homing potential when reactivated with or without RA.⁴⁰

Both TGF- β and RA are actively produced by the intestinal epithelium and play important roles in mucosal epithelial cell differentiation and in maintaining the integrity of its barrier function. For example, by actively promoting IgA class switching, TGF-β and RA positively regulate secretory IgA production, important in mucosal barrier function and antibacterial protection.

Vitamin A’s effects on B cells

During the primary immune response, naïve B cells proliferate and differentiate either into IgM-producing plasma cells, followed by hypermutation into plasma cells producing high affinity IgG antibodies, or into long-lived memory B cells, which are characterized by a lower threshold for activation and differentiation. Memory B cells also have a higher density of toll like receptor 9 (TLR9) than naïve B cells. In vitro, TLR 9 binds unmethylated CpG oligonucleotides mimicking the unmethylated DNA typically found in bacteria and viruses. RA has been shown to markedly increase proliferation of memory B cells while inhibiting proliferation of naïve B cells. Thus, RA plays a role in maintaining long-lived humoral memory.⁴⁴

nTregs represent 5–10% of peripheral CD4+ T cells in naive mice and humans. Chronic ablation of Treg cells in adult healthy mice results in their death within 3 weeks, demonstrating how important Treg cell-mediated suppression is for preventing immune pathology throughout the lifespan.⁴⁷ In the intestine, where an improper balance between inflammatory and suppressive immunity can jeopardize mucosal homeostasis and destroy the integrity of the mucosal barrier, FOXP3+ Tregs, induced by RA, play a key role in maintaining the steady-state of tolerance towards innocuous antigens, thus preventing excessive, self-destructive immune responses and the development of inflammatory bowel disease and autoimmunity. 

In a recent paper entitled “Vitamin A rewrites the ABCs of oral tolerance,” Strober sums up the situation saying, “Thus, we arrive at the somewhat surprising realization that mucosal unresponsiveness [self and oral tolerance] relies upon the bioavailability of a factor in the food stream.”⁴⁸

Plasma Vitamin A Correlates with Healthy Aging and Longevity

Healthy centenarians are consistently found to have vitamin A levels comparable to those in healthy young adults, in contrast to lower levels of vitamin A seen in typical older control subjects.^{49 50 51 52} Vitamin A’s effects on immune regulation provide a likely explanation for this correlation. 

Recent observations indicate that immunosenescence is not accompanied by an unavoidable and progressive deterioration of immunity, but results from a remodeling in which some immune functions are reduced, while others remain unchanged or even increase. Specifically, major age-related changes in immunity include a progressive, age-dependent decline in virgin T cells combined with a progressive age-dependent increase in effector T cells that produce pro-inflammatory cytokines (e.g., IL-2, IFN-γ, TNF-α).^{53 54} These changes support the hypothesis of “inflame-aging,” which suggests that immunosenescence is driven by an increasing lack of oral and self-tolerance that results in a chronically elevated perceived antigenic load, which not only induces enormous expansion of effector T cells, but increases their production of pro-inflammatory cytokines. In older subjects, these cells appear to be equipped with a greater capability to produce IFN-γ and TNF-α, two cytokines, while they amplify, via IFN-γ, the immune response against internal or external pathogens, also, promote an unremitting inflammatory and/or autoimmune processes that negatively correlate with human longevity.⁵⁵

Vitamin A Assessment

The most commonly used indicator of vitamin A status is the serum retinol concentration. If measuring serum retinol, ensure the sample is protected from light and heat. Normal range using HPLC methodology is 24 to 90 µg/dL.^{56 57}

Since retinol is unstable when exposed to heat or light, and HPLC requires costly, complicated lab equipment, making it impractical for field studies, retinol-binding protein has been proposed as a surrogate measure. RBP is significantly more light-and-heat stable than retinol, with which it is released into the circulation from the liver in a 1-1 complex. Despite the fact that RBP concentrations may fall under circumstances of protein malnutrition or the acute phase response (which accompanies inflammatory states), equimolar RBP cutoffs have been shown to predict vitamin A deficiency with high sensitivity and specificity, even in the context of significant infection (HIV-1) and protein malnutrition. RBP cutoffs of 1.05 µmol/L and 0.70 µmol/L identify marginal vitamin A status (retinol < 1.05 µmol/L) and vitamin A deficiency (retinol < 0.70 µmol/L), respectively. Finally, the techniques used to quantify serum RBP are easier and less expensive.⁵⁸

Conclusion

Large individual variations in the actual absorption of β-carotene from foods and its conversion to retinol demonstrate that reliance on current data on retinol equivalents for vitamin A sufficiency is highly misleading and that a significant percentage of the American population may be vitamin A insufficient, despite the fact that foods containing pre-formed vitamin A and β-carotene are generally available. Given recent discoveries regarding the importance of vitamin in balancing the activities of vitamin D and its pivotal role in promoting oral and self- tolerance, thus counterbalancing the tendency to “inflammaging,” vitamin A sufficiency should be considered a key factor in healthy aging.

Aging is characterized by a peculiar chronic inflammatory status for which researchers have recently coined the term, “inflammaging,” a key aspect of which is the age-dependent expansion of effector T cells with pro-inflammatory cytokine production potential. Particularly in light of vitamin A’s emergence as a pivotal inducer of oral and self-tolerant immune function, vitamin A sufficiency should be recognized as a key factor in healthy aging.

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Strong correlations have been noted between cardiovascular diseases and low bone density / osteoporosis—connections so strong that the presence of one is considered a likely predictor of the other. This relationship has led to the hypothesis that these conditions share core pathophysiological mechanisms. Recent advances in our understanding of the complimentary roles played by vitamin D3 and vitamin K2 in vascular and bone health provide support for this hypothesis, along with insight into key metabolic dysfunctions underlying cardiovascular disease and osteoporosis.

Part I of this review, Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease -- summarized current research linking vitamin D deficiency to cardiovascular disease, the physiological mechanisms underlying vitamin D's cardiovascular effects, and leading vitamin D researchers' recommendations for significantly higher supplemental doses of the pro-hormone. Read Part I: Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease

Part II, The Vitamin K Connection to Cardiovascular Health, reviews the ways in which vitamin K regulates calcium utlization, preventing vascular and soft tissue calcification while complimenting the bone-building actions of vitamin D, and also discusses vitamin K safety and dosage issues, and the necessity of providing vitamin K and vitamin A along with vitamin D to preclude adverse effects associated with hypervitaminosis D.

Part II: The Vitamin K Connection to Cardiovascular Health

Introduction

First recognized by German researchers as a nutrient required for normal blood “koagulation,” vitamin K is actually a family of structurally similar, fat-soluble compounds, some of which (the K2 forms) play essential roles in cardiovascular health, primarily through regulating the body’s use of calcium – both promoting its integration into bone and preventing of its deposition within blood vessels -- and also by exerting anti-inflammatory and insulin-sensitizing actions.¹

In nature, vitamin K appears primarily in two forms: K1 (phylloquinone [phyllo – relating to a leaf] and K2 (the menaquinones [mena – in reference to their methylated napthoquinone ring structure]). While all forms of vitamin K share 2-methyl-1,4-naphthoqinone as their common ring structure, individual forms differ in the length and degree of saturation of a variable aliphatic side chain attached to the 3-position.

K1, a single compound that contains a monounsaturated side chain of four isoprenoid residues, is found primarily in plants and algae in association with chlorophyll. Dietary sources of K1 include green leafy vegetables, such as broccoli, kale and Swiss chard, and unhydrogenated plant oils, including canola and soybean oil.

K2, the menaquinones (MKs) are classified based on the length of their unsaturated side chains into 15 different types denominated as MK-n, with “n” denoting the number of isoprenyl residues in the side chain. The most common MKs in humans are the short-chain menaquinone, MK-4, which is now thought to be primarily produced via the systemic conversion of K1 to K2 in the body} ^{2 3 4} and the long-chain menaquinones, MK-7 through MK-10, which are exclusively synthesized by bacteria and gut microflora in all mammals, including humans. K2 (primarily its long-chain forms, MK-7, MK-8 and MK-9) is found in fermented foods, notably cheese and natto (fermented soybean); the latter is the richest dietary source of vitamin K presently known, almost all of which occurs in the form of MK-7.⁴⁵

Vitamin K1, MK-4 and MK-7 are available as supplements: MK-4 as a synthetic version called menatetrenone, and MK-7, as the natural compound extracted from natto. MK-7 has a much longer half-life than either K1 or MK-4, which share similar molecular structures (both contain 4 isoprenoid residues, 3 of which are saturated in K1 but contain a double bond in MK-4) and therefore similar physiokinetics. In contrast, the longer-chain menaquinones, including MK-7, are much more hydrophobic and are handled differently by the body. In vivo, they have longer half-lives and are incorporated into low-density lipoproteins in the circulation, resulting in much more stable serum levels and accumulation to 7- to 8-fold higher levels during prolonged intake.⁵

K3 (menadione), a third, much simpler form of the vitamin, is considered a synthetic analogue, although intestinal bacteria can produce minute amounts from K1.⁶ K3 has been utilized in research on vitamin K's anti-cancer effects because it potentiates the cytotoxic activity of chemotherapeutic agents and vitamin C (when acting as an antioxidant, vitamin C is oxidized to dehydroascorbate, a potent free radical that is spontaneously reduced by glutathione as well as in reactions using glutathione or NADPH⁷; however, because of its toxicity, the FDA has banned its use in nutritional supplements.⁸

Although, following intestinal absorption, both K1 and K2 are taken up in the triglyceride fraction from which they are rapidly cleared by the liver, only the K2 forms are also taken up and systemically redistributed by low-density lipoproteins.⁹¹⁰ Compared to K1, whose primary activity is the carboxylation of blood coagulation factors (II [prothrombin], VII, IX, and X, the anticoagulant proteins C, S and Z), which are synthesized in the liver, K2 has a much wider range of action, playing a significant role in bone formation and protection against bone loss, arterial calcification, and oxidation of LDL cholesterol.^{11 12} In addition, K2 is a 15-fold more powerful antioxidant than K1 and is the predominant form of vitamin K in all tissues, except the liver.¹³ Finally, K2 is better absorbed than K1 and remains biologically active far longer; K1 is cleared by the liver within 8 hours, while measurable levels of the MK-7 form of K2 have been detected up to 72 hours after ingestion.¹⁴

Underlying Mechanism of Action: Gamma-carboxylation

Vitamin K is the cofactor for the enzyme, γ-glutamyl carboxylase, which converts specific glutamic acid residues in a number of substrate proteins to γ-carboxyglutamic acid (Gla) residues, which then serve to form calcium-binding groups in these proteins and are essential for their biologic activity.

Carboxylation thus activates this family of Gla-proteins, which are involved in numerous essential activities throughout the body, including blood coagulation, bone metabolism, vascular repair, prevention of vascular calcification, regulation of cell proliferation, and signal transduction.^{15 16}

K1 is preferentially utilized in the carboxylation of clotting factors in the liver. K2 is preferentially used in the rest of the body to carboxylate the other vitamin K-dependent Gla-proteins, including osteocalcin (which is essential for bone health and primarily synthesized in bone), and matrix-Gla protein (MGP) (which prevents calcification of soft tissue, [e.g., the vasculature, myocardium, breasts and kidneys], and is primarily synthesized in cartilage and the vessel wall. Vitamin K2 is also found in high concentrations in the brain, where it contributes to the production of myelin and other important compounds.^{17 18}

Vitamin K Recycling

The body very efficiently utilizes vitamin K by recycling this nutrient in a cyclic interconversion called the vitamin K cycle.^{19 20} In this cycle, the vitamin K quinone form is reduced by the FAD-containing enzyme DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase ) into the vitamin K hydroquinone (KH2), which then serves as the cofactor for vitamin K carboxylation of Gla-proteins and, in so doing, is oxidized to vitamin K epoxide. Vitamin K epoxide is then recycled back to the quinone form by the enzyme vitamin K epoxide reductase (VKOR), completing the cycle. On a molecular level, vitamin K expoxide is reduced in two steps: first to the quinone form by VKOR, then to vitamin K hydroquinone (KH2) by DT-diaphorase.

Besides being a cofactor in the vitamin K-dependent carboxylation, KH2 also possesses antioxidant activity and is highly sensitive to free radicals, which may oxidize (and thus inactivate) KH2 before it can take part in the carboxylation reaction. KH2’s reactivity to free radicals may increase need for K2 in arteries burdened by atherosclerotic plaque, where high levels of oxidized LDL can contribute to a local vitamin K deficiency, further exacerbating the atherosclerotic process.²¹

As noted, VKOR is a crucial enzyme in vitamin K metabolism, enabling its re-utilization after it has been oxidized in the carboxylase reaction through which it activates Gla-proteins. Because of VKOR recycling, the human requirement for vitamin K is extremely low—just 45 mcg/day is suggested to be all that is needed of its most potent form, MK-7. VKOR is also the target for warfarin and related coumarin derivatives, which block the recycling of vitamin K by inhibiting this enzyme, thereby decreasing vitamin K available for the activation of Gla-proteins. The gene for VKOR has recently been identified, and it appears that most of the variability observed in patients’ response to warfarin is attributable to variability in the VKOR gene.^{21 22 23}

Click here for a PDF version of the chart

K2 Regulates Calcium Deposition: Mineralizing Bone, Preventing Vascular Calcification Mineralizing Bone

Mechanisms

Only after its carboxylation by vitamin K is osteocalcin, the major non-collagenous protein responsible for inducing bone mineralization in human osteoblasts, able to attract calcium ions and incorporate them into hydroxyapatite crystals forming the bone matrix. When vitamin K2 levels are insufficient, osteocalcin remains uncarboxylated with the result that bone mineralization is impaired.²⁴

Not only is vitamin K2 a key inducer of bone mineralization in human osteoblasts, but this form of vitamin K also inhibits osteoclast differentiation and is necessary to bring to fruition the bone-building effects of vitamin D3's upregulation of osteoblast's expression of osteocalcin.^{24 25 26 27 28}

Research Evidence

Numerous epidemiologic and intervention studies have shown that vitamin K insufficiency, with associated high levels of undercarboxylated osteocalcin, causes reductions in bone mineral density (BMD) and increases fracture risk. Conversely, supplementation with vitamin K2 has been shown to increase osteocalcin activation(carboxylation), promote bone mineralization and lessen risk of fracture.^{1 29 30 31 32}

A further consideration is that a number of clinical trials have demonstrated that the combination of K2 and vitamin D3 is more effective in preventing bone loss than either nutrient alone.^{33 34} In a study of 173 osteoporotic/osteopenic women, those given both K2 and D3 experienced an average 4.92% increase in bone mineral density (BMD), while average BMD increase was just 0.13 in those receiving K2 alone.35 In another study evaluating the effects of vitamin D or K singly or in combination, 92 postmenopausal women were assigned to one of four groups: K2 (45 mg/day), D3 (0.75 mcg/day [1 mcg D3 = 40 IU, so this was a 3,000 IU dose), both K2 and D3 at the aforementioned dosages, or calcium lactate (2 g/day). In the women receiving only calcium, lumbar BMD decreased. Those given either D3 or K2 experienced a slight increase in BMD. In those taking both, K2 and D3, lumbar BMD increased an average of 1.35%.³⁵

K2 has also been shown to work with D3 to lessen the risk of osteoporosis in Parkinson's disease, which is thought to be related in part to immobilization as well as a deficiency of vitamin D caused, not by a lack of vitamin D, but rather to suppression of D3 by the high blood levels of calcium seen in Parkinson's. When K2 (MK-4) (45 mg/day for 12 months), was given to 54 female Parkinson's patients with osteoporosis, only one hip fracture occurred, compared to 10 fractures in a control group of 54 women with Parkinson's who were not treated with K2. Average bone loss in the untreated group was 4.3% compared to 1.3% in those given K2.⁸

Preventing Arterial Calcification

Mechanisms

Matrix Gla-protein (MGP) is the strongest inhibitor of tissue calcification presently known. Its importance for vascular health was first demonstrated in animals bred to be MGP-deficient, all of which died of massive arterial calcification within 6–8 weeks after birth.⁷¹

MGP is produced by small muscle cells in the vasculature where—once carboxylated by vitamin K2—it protects against calcification through several mechanisms, including inhibiting bone morphological protein-2 (BMP-2), upregulating the gene for DT-diaphorase, and downregulating the gene for osteoprotegerin:

Bone Morphological Protein-2 (BMP-2)

MGP inhibits calcification by binding to and inhibiting the activity of BMP-2, a potent bone morphogen whose expression triggers the induction of an osteogenic gene expression profile in vascular smooth muscle cells (VSMC), which causes them to transform into osteoblast-like cells, a transformation known to precede arterial calcification. BMP-2 is expressed by cells in atherosclerotic lesions, and its expression can be induced by oxidative stress, inflammation or hyperglycemia.^{36 37 67} Overexpression of non γ-carboxylated MGP, as is seen in calcified lesions in the aorta, results in unopposed BMP-2 activity, which promotes osteoblastic differentiation of VSMC and the laying down of a calcified matrix.³⁸

DT-diaphorase

DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase) is a FAD-containing enzyme (i.e., incorporates riboflavin as its cofactor) that plays a key role in vitamin K recycling by reducing vitamin K to vitamin K hydroquinone, which then serves as the cofactor for vitamin K carboxylation of Gla-proteins. Specifically, K2’s effects on the gene expression of DT-diaphorase increase the enzyme’s activity in the vasculature 4.8-fold, greatly increasing levels of activated MGP.^{19 38}

Osteoprotegerin

Osteoclast-like cells have been identified in calcified human aortic plaques.38 Their activation is inhibited by osteoprotegerin but upregulated by activated MGP. Specifically, osteoprotegerin promotes vascular calcification by acting as a RANKL (RANK/receptor activator of NF-kappa B ligand) decoy receptor, thus preventing RANKL from binding to the transmembrane receptor RANK on osteoclast precursors, where it induces the differentiation and activation of osteoclasts.^{39 40} By lessening the production of osteoprotegerin in the vessel wall, activated MGP increases RANKL concentrations, thus increasing osteoclastic activity and the removal of calcified areas from the vasculature.

Safeguarding Elasticity

While oxidized cholesterol's contribution to atherosclerosis has been treated as the primary issue in cardiovascular disease, arteriosclerosis, the calcification of the arterial intima, is just as lethal. The elasticity characteristic of a healthy artery is what enables it to accommodate increases in blood flow. Enough calcium deposition and that pliability is lost: blood pressure rises, damaging the vasculature and contributing to atherosclerosis. The two pathologies—arteriosclerosis and atherosclerosis—are synergistic. By preventing arterial calcification, vitamin K2 also provides protection against atherosclerosis.

In addition, K2 directly promotes blood vessel elasticity by safeguarding elastin, the core protein in the muscle fibers primarily responsible for the elasticity of the arterial wall.  Calcium deposition not only damages existing elastin, but inhibits new elastin production.⁴¹

Clinical Evidence

The question of whether high vitamin K-intake is protective against arterial calcification was first addressed in the Rotterdam Study, a massive European clinical trial following 4,807 subjects aged ≥55 over a 7-10 year period. Dietary intake of vitamin K2 (but not K1) was inversely correlated with cardiovascular calcification and cardiovascular death. Elderly people in the highest tertile of vitamin K2 intake had 52% reduction in severe aortic calcification, a 57% reduced risk of cardiovascular disease, and a 26% decreased risk for all-cause mortality. K1 intake correlated with none of these beneficial outcomes.⁴¹

Sudden death from heart attack is even more highly correlated with calcification of the aorta than cholesterol. In Framingham study research, aortic calcification was associated with double the risk of death from cardiovascular disease in men and women younger than 65, even after other risk factors (e.g., cholesterol) were taken into account. In men younger than 35, aortic calcification increased risk of sudden coronary death 7-fold.^{42 43}

Coronary Artery Calcification – A Key Biomarker of Functional Age

Research involving more than 100,000 men and women in California revealed that aortic calcification increased risk of coronary heart disease 127% in men and 122% in women. Among women, risk of stroke also increased concurrently by 146%.⁴⁴

A high coronary artery calcium score on electron beam tomography has been found to be a better predictor of mortality than age. A calcium score of less than 10 confers a reduction in functional age by 10 years in subjects older than 70, while a calcium score of >400 adds as much as 30 years of functional aging to younger patients.^{45 46 47}

Vitamin K-dependent Gla-proteins have been shown to inhibit calcification in the heart and arteries; in the kidneys, where K2 prevents the calcification that typically accompanies dialysis and diabetes; and in the breast. Women whose diets provide the most vitamin K2 have significantly less breast calcification compared to those whose diets provide the least.⁴⁸ In women, calcification of breast tissue (which several studies have correlated with vitamin K2 insufficiency^{49 50}) is associated with a 132% increased risk of cardiovascular disease, a 141% increased risk of stroke, and a 152% increased risk of heart failure.⁵¹

Uncarboxylated MGP has also been identified as a key player in the increased calcification seen in the development of varicosis, as well as in other vascular diseases. Researchers compared healthy veins from 36 male patients (aged 30 to 83) and varicose veins from 50 male patients (aged 40 to 81). In the men with varicose veins, levels of uncarboxylated MGP, were high, indicating the local vascular vitamin K status in varicose veins is insufficient to mediate full carboxylation of all newly formed MGP. Vitamin K supplementation inhibited the mineralization process in varicose small muscle cell cultures, suggesting that in vitro, carboxylation of MGP could be induced and that its inhibitory effect on varicosis could be restored.⁵²

In a clinical intervention study in which 78 women between 55 and 65 years of age received either vitamin K2 (1 mg/day) or placebo for three years, vascular characteristics were assessed (elasticity and distensibility). In subjects in the placebo group, vascular elasticity had decreased by 10–13%, which is consistent with what has been considered a “normal” decrease during a three year time period for women in this age group; in the vitamin K group, however, vascular characteristics remained unchanged, suggesting that the process of vascular aging can be retarded by increased vitamin K intake.⁵³

Intra-cranial atherosclerosis, a newly identified risk factor for ischemic stroke⁵⁴, has been shown to be an age-independent risk factor for cerebral atrophy.⁵⁵ Given the protective effects of carboxylated MGP against calcification the heart, vasculature, kidneys, and breasts, and the fact that K2 is concentrated in the brain where it has been shown to completely block free radical accumulation and cell death in cell cultures of developing fetal cortical neurons} ⁵⁶ it does not seem unreasonable to hypothesize that K2 may also play a protective role against calcification in the brain.

Vitamin K: Key to the Osteoporosis – Atherosclerosis Connection

Osteoporosis and arterial calcification have been thought to be unrelated conditions, but a number of recent studies suggest a connection.31 In the U.S., ~75–95% of men and women have some degree of coronary artery calcification on autopsy; 54% of postmenopausal women have osteopenia, and 30% have osteoporosis.31 It has been noted that patients with low bone mass, osteopenia or osteoporosis frequently also exhibit vascular calcification, which has been shown to predict both cardiovascular morbidity/mortality and osteoporotic fractures.⁵⁷

Aortic calcifications, specifically, have been positively associated with osteoporotic fractures, and the progression of aortic calcification has been positively associated with the rate of decline in lumbar spine BMD.⁵⁸ In a study of 195 postmenopausal women, the association between echogenic carotid artery plaques, low bone mass and vertebral fractures was so strong that researchers suggested it could partly explain why osteoporotic vertebral fractures are linked to increased mortality.⁵⁹ Similar associations have been found in men. In a 10 year prospective study of 781 men ≥ 50, calcifications in the abdominal aorta increased fracture risk 2 to 3-fold, regardless of subjects’ BMI, comorbidities and medications.⁶⁰

An explanation for this correlation between osteoporosis and atherosclerosis is being developed in studies analyzing the two conditions’ underlying pathophysiological mechanisms, which appear to coincide in one common factor: vitamin K deficiency.

It is becoming apparent that the development of arterial calcification resembles the process of osteogenesis.⁶¹

• Both involve the same cell types, proteins and cytokines that lead to tissue mineralization.
• More than 90% of atherosclerotic plaques undergo calcification. Ectopic bone tissue has been identified in calcified plaques, and bone-specific cells have been found in the arterial wall, with evidence that endothelial cells have transdifferentiated into osteoblasts.
• Calcified arteries have also been shown to contain osteoclast-like cells.
• Local and serum lymphocytes, monocytes and macrophages are involved in both osteoporosis and vascular calcification.
• Chemical mediators of bone metabolism including osteocalcin, bone morphogenetic protein (BMP), osteopontin (OPN), osteonectin, osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and inflammatory cytokines are also present in atherosclerotic arteries.
• The vitamin K-dependent Gla proteins, osteocalcin and MGP, are mainly expressed in bone and vascular cells, and are mediators and inhibitors of osteoid formation.
• Although osteocalcin does not appear to play a significant role in the process of vascular calcification, MGP (if carboxylated) is a key protective factor. Not only do MGP-knockout mice form extensive and lethal arterial calcifications, they also present with osteopenia, fractures, short stature, and erratic mineralization of the growth plates.
• As noted earlier in this review, carboxylated MGP protein inhibits mesenchymal differentiation into osteogenic cell lines by blocking the action of bone morphogenic protein (BMP), a potent factor of bone maturation that initiates the differentiation of vascular mesenchyme into bone cells, thus increasing calcification.
• MGP isolated from calcified atherosclerotic plaques of mice shows incomplete carboxylation.

The Calcification Paradox – Another Iteration of the Same Theme

Upon entering menopause, women simultaneously lose calcium from bone and increase its deposition in arteries—a negative double whammy called the "calcification paradox," which greatly increases their risk of both osteoporosis and cardiovascular disease.³¹ The drop in estrogen causes both problems; vitamin K2 can help rectify them.

Estrogen impacts calcium regulation metabolism through several different pathways. Estrogen is involved in the conversion of vitamin D to its active bone-building form (1,25-dihydroxycholecalciferol [1,25(OH)2D] or calcitriol). When estrogen levels drop, osteoclasts become more sensitive to parathyroid hormone, which signals them to increase their activity. Plus, the decline in estrogen allows production of the cytokine, interleukin-6, to increase, and IL-6 stimulates the production of even more osteoclasts.^{27 62}

Among postmenopausal women not using estrogen replacement, low levels of vitamin K or high levels of uncarboxylated osteocalcin are associated with low spine BMD.⁶³ Supplementation with vitamin K2, however, has been shown to prevent bone loss associated with estrogen decline. In a 3-year study, 325 postmenopausal women were given either K2 (in the form of MK-4 or menatetrenone, for which the dosage is 45 mg/day, specifically 15 mg/tid) or placebo. In those receiving K2, bone mineral content increased, and hip and bone strength remained unchanged, whereas in the placebo group, both bone mineral content and bone strength decreased significantly.⁶⁴

Estrogen also protects premenopausal women from cardiovascular disease by increasing endothelial production of prostacyclin, PG12, which inhibits platelet aggregation and promotes vasodilation. When estrogen levels drop in menopause, these protective effects are lost.⁶⁵

Fortunately, MGP (if carboxylated) both inhibits vascular calcification and, as noted above, helps maintain blood vessel elasticity. In a 3-year study of 181 postmenopausal women, one-third were given a supplement containing vitamin D, one-third got a supplement providing both vitamin K and D, and one-third were given a placebo. In both the vitamin D and the placebo group, elasticity of the common carotid artery decreased; in those receiving K along with D, elasticity was maintained.⁶⁹

Vitamins K and A: Essential for the Prevention of Vitamin D Toxicity

Vitamin D upregulates the expression of Gla-proteins, whose activation depends on vitamin K-mediated carboxylation. Vitamin D thus increases both the demand for vitamin K and the potential for benefit from K-dependent proteins, including osteocalcin in bone and MGP in blood vessels.²⁵

Another way of looking at this, however, is that by increasing the need for vitamin K2, increased levels of vitamin D may actually induce a functional vitamin K2 deficiency, with the result that levels of uncarboxylated osteocalcin and matrix-Gla protein rise in the circulation and vasculature. In this case, not only is calcium not delivered to the bones, which become porous, but it is deposited in the arteries, which become calcified.^{31 66 11 67 68 69 70 71}

It has recently been proposed that vitamin D toxicity is the result of precisely such induction of vitamin K2 deficiency.²⁵. As vitamin D induces levels of Gla proteins to rise, the pool of available vitamin K available to carboxylate them becomes depleted, so vitamin K-dependent processes that retain minerals in the bone matrix, protect the soft tissues from calcification, and support the nervous system can no longer be performed.

In support of this hypothesis, warfarin, a coumadin derivative that induces a functional vitamin K deficiency by inhibiting the recycling of the vitamin, has definitively been shown to produce extensive hypervitaminosis D-like calcification of the soft tissues and to exert toxicity synergistically with vitamin D when the two are combined.^{72 73 74 75}

Uncarboxylated MGP is abundant in calcified arterial plaque, where its presence is thought to reflect a reactive attempt by the local tissue to protect itself from calcification—an attempt rendered futile by inadequate supplies of vitamin K2. In addition, vitamin K alone has been shown to fully reverse the calcification induced by warfarin} ⁷⁶ both confirming that the drug’s inhibition of vitamin K is directly responsible for its induction of calcification, and also adding to the likelihood that vitamin D toxicity is due to the same or a similar mechanism.²⁵³¹

Vitamin A — Balancing the Actions of Vitamin D

The hypothesis further proposes, and a number of recent studies suggest, that vitamin A protects against possible vitamin D toxicity by downregulating the expression of MGP, thus exerting a vitamin K-sparing effect, which counteracts the depletion of vitamin K potentially induced by increased levels of vitamin D.^{77 78 79}

A number of animal experiments have shown that high doses of vitamin A protect against the growth retardation, soft tissue calcification and bone resorption induced in rats by dietary vitamin D3, and that vitamin A completely protects against renal calcification induced by dietary vitamin D3 in turkeys. Vitamin A has also been shown to decrease MGP expression in human cells.25 Retinoic acid and 1,25(OH)2D3 compete for the same nuclear partners; both the retinoic acid receptor and the VDR must form heterodimers with retinoid X receptors (RXRs) to binding to response elements and initiate transcription. For this reason, 1,25(OH)2D3 and retinoic acid naturally balance one another’s effects.⁸⁰

Also, in relation to the efficacy of vitamin D at potentially lower doses or in individuals carrying VDR SNPs with impaired binding efficacy, recent research has shown that 9-cis-retinoic acid, a derivative of vitamin A, increases the affinity of VDR/RXR to its DNA recognition site, induces recruitment of coactivators by the DNA-bound heterodimer and potentiates vitamin D-dependent transcriptional responses.⁸¹

Thus, the proposed model suggests vitamin D toxicity is actually due, not to higher supplemental doses of vitamin D, but results from an imbalance among vitamins D, A and K. Proper consideration of the synergistic relationship among these vitamins could allow vitamin D to be therapeutically effective at lower doses or to be administered in higher therapeutic doses without incurring the risks associated with hypervitaminosis D.

As noted in Part I of this review, the body's ability to utilize cholecalciferol in the numerous roles played by the vitamin D endocrine system is not optimized until blood levels of 25(OH)D are ≥40 ng/ml (98 nmol/L). Not until this level is the Vmax, of the 25-hydroxylase enzyme achieved (i.e., are all enzyme sites saturated). Below this level, chronic substrate deficiency prevents full actualization of the myriad benefits of vitamin D.⁸² For some individuals, supplementation of vitamin D3 in the range of 5,000 – 10,000 IU/day may be necessary to reach and maintain these blood levels, which underscores the concomitant need for adequate supplies of vitamin A as well as vitamin K. The National Institutes of Health has set the RDI for vitamin A at 3,000 IU for males ≥ 14 years and 2,310 IU for females ≥ 14 years, and the tolerable upper limits for retinols in both men and women at 10,000 IU.⁸³

Factors Affecting Vitamin K Deficiency

Assuming that normal, healthy levels of beneficial bacteria are present in the intestines, these bacteria produce about 75% of the vitamin K2 the body absorbs each day. Thus, even a diet quite rich in leafy greens when consumed by an individual with healthy gut flora supplies less than half the vitamin K2 needed for this nutrient's calcium-regulating activities.

Unlike the other fat-soluble nutrients (vitamins A, D and E), vitamin K1 is cleared from the body within 8 hours, and even the MK-7 form of vitamin K2 is not stored in the body for more than 72 hours, thus this nutrient is best provided daily. Despite the production of vitamin K2 (specifically MK-4) by healthy intestinal bacteria, humans can develop a deficiency of the vitamin in as few as 7 days on a vitamin K-deficient diet.⁸⁴

Absorption of vitamin K, like that of other fat-soluble nutrients (A, D and E), is dependent upon healthy liver and gallbladder function. Digestive health is also a factor. Deficiency is more likely in people with digestive problems such as celiac disease, irritable bowel disease, or who have had intestinal bypass surgery, all of which increase the likelihood of fat malabsorption.

Vitamin K recycling is dependent upon DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase), a FAD-containing enzyme that reduces vitamin K to vitamin K hydroquinone, which then serves as the cofactor for vitamin K carboxylation of Gla-proteins. FAD is derived from riboflavin (B2), thus vitamin K recycling is dependent upon adequate supplies of riboflavin.

Vitamin K needs increase with age. Older individuals (>70) require higher levels of vitamin K1 or K2 to maintain low levels of uncarboxylated vitamin-K dependent proteins.⁸⁵

Bile acid sequestrants (e.g., Cholestyramine, Colestipol), which bind to bile acids, forming large compounds that are poorly reabsorbed from the gut and eliminated in the feces, also bind and remove fat-soluble vitamins, including vitamin K.

Canola and soybean oils are the primary source of vitamin K in the American diet. Hydrogenation changes the vitamin K1 (phylloquinone) in these oils into dihydrophylloquinone, a form that does not carboxylate osteocalcin and other vitamin-K dependent proteins. In 2,544 men and women (average age 58.5) who participated in the Framingham Offspring Study, those with the highest intake of vitamin K from hydrogenated oils had the lowest BMD at the neck, hip and spine.⁸⁶ If your patient eats a fair amount of processed or fast foods that contain hydrogenated oils, risk of functional vitamin K deficiency is greatly increased.⁸⁷

While levels of vitamin K (K1, specifically) are rarely insufficient to meet clotting needs, levels of vitamin K necessary for clotting are much lower than those needed (in the form of K2) for bone and arterial protection. Studies of healthy adults have found high levels of uncarboxylated osteocalcin and matrix Gla-protein (MGP) in all subjects tested.⁷¹

Laboratory Assessment of Vitamin K Status

A normal prothrombin time is not an indication that sufficient vitamin K is present to maintain carboxylation of osteocalcin or MGP.^{24 68 71}

To check vitamin K levels, request an osteocalcin test; this measures how much uncarboxylated osteocalcin is present in the blood. High levels of uncarboxylated osteocalin (ucOC) indicate insufficient vitamin K for bone health and indirectly indicate that MGP is insufficiently carboxylated.⁷¹

Safety and Efficacy

Even in high doses, neither K1 nor K2 has produced adverse effects in individuals not on coumadin derivatives. For this reason, the Institute of Medicine at the National Academy of Sciences chose not to set a Tolerable Upper Limit (UL) for vitamin K when it revised its public health recommendations for this vitamin in 2000.

Drug Interactions

Anticoagulant Medications

In patients on warfarin or other coumadin derivatives, vitamin K1 can interfere with these drugs’ anti-clotting activity in amounts as small as 1 mg. 

As noted above, oral anticoagulant medications, e.g., warfarin and other coumadin derivatives, promote arterial calcification by preventing vitamin K from activating matrix Gla-protein.^{15 88}

These medications decrease clotting by blocking vitamin K epoxide reductase (VKOR), thus preventing vitamin K recycling and greatly increasing risk of vitamin K deficiency, and have also been shown to block the conversion of K1 to K2.⁸⁹

A case report recommended physicians prescribing warfarin consider arterial calcification as a potential consequence after routine examination of a healthy man on long-term warfarin treatment found his coronary arteries were highly calcified.⁹⁰ Other case reports have noted pathologic tracheobronchial calcification with long-term warfarin therapy in children, an 18-year-old male, and an elderly woman.^{91 92 93} Two recent studies involving more than 100 subjects have shown that patients treated with oral anticoagulants have double the calcification of patients not on these vitamin K-blocking drugs.⁸⁸

When improving vitamin K status, however, patients on these medications must be closely monitored. A dose of just 1-2.5 mg of oral vitamin K1 reduces the range of the international normalized ratio (INR) from 5.0-9.0 to 2.0-5.0 within 24-48 hours; even eating a vitamin K-rich diet can make anticoagulant medications less effective.⁹⁴

On the other hand, recent studies have shown that the INR is more sensitive to vitamin K changes in patients with a low vitamin K status than in those with a normal or high vitamin K status and that dietary vitamin K intake in unstable patients is considerably lower than in stable patients.^{95 96 97}

Research conducted by Schurgers et al., sugggests that MK-7 supplements supplying <50 mcg/day are not likely to affect the INR value; however, doses of >50 mcg/day may interfere with oral anticoagulant treatment in a clinically relevant way. A 50 mcg dose is comparable to the menaquinone content of 75 to 100 grams (2.6 to 3.5 ounces) of cheese, an amount that should lead to a disturbance of the INR value of no more than 10%. In addition, the long half-life of MK-7 suggests that regular intake in combination with properly adapted coumarin doses may result in more stable INR values.5

Other Interactions

K3, the synthetic form of vitamin K, promotes ROS production and glutathione depletion. High doses of K3 have been used in cancer research precisely for its ability to promote oxidative stress and cell death. Even in lower doses, K3 has produced jaundice and hemolytic anemia in human infants. For these reasons, the U.S. Food and Drug Administration banned the use of K3 in nutritional supplements.

Considerations when Choosing a Vitamin K Supplement

In animal studies, at very high intakes of K1, (200-fold the daily requirement of the liver), vitamin K1 is converted to K2 (MK-4) in amounts that may be sufficient to help decrease arterial calcification.⁹⁸

It is important to differentiate between the two commercially available forms of K2 (the MK-4 and MK-7 menaquinones) since they differ in clinically significant ways.^{5 99 100} MK-4 is a short-chain menaquinone available as a synthetic compound (menatetrenone), while MK-7, a long chain menaquinone, is a natural menaquinone derived from natto fermentation.

The vast majority of studies evaluating the effectiveness of vitamin K for the prevention of both osteoporosis and arterial calcification have used K2 (MK-4) at a dosage of 45 mg/day (specifically, 15 mg/tid). Not only has the majority of the research been done using MK-4, but MK-4 is the predominant form of K2 into which the body converts K1. MK-4 appears quickly in the blood but has a half-life of only 1-2 hours, for which reason, high pharmacological doses (typically 45 mg/day given as 15 mg tid) are necessary. Such large doses necessitate medical supervision in patients on blood-thinning medications (e.g., warfarin).

MK-7 is not only highly bioavailable and bioactive—45 mcg/day was sufficient to activate osteocalcin in the Rotterdam study—but has a much longer serum half life of 3 days, which enables the body to build up a buffer that can supply vitamin K2 to all tissues 24 hours a day. At 45 mcg/day (a dose 1,000 times less than that typically used in the research for MK-4), natto-derived MK-7 is less likely to interact negatively with blood-thinning medications.

Conclusion

As research documenting the widespread and significant beneficial actions of vitamin D continues to appear in the peer-reviewed medical literature accompanied by reports that the majority of the U.S. population is deficient in this nutrient, more clinicians are evaluating their patients’ vitamin D levels and prescribing supplementation, often in amounts as high as 5,000 to 10,000 IU/day, without awareness of the risk of provoking an imbalance among vitamins D, K and A. Consideration of the synergistic relationship among these vitamins could allow vitamin D to be administered in doses of greater therapeutic value without incurring the risks of osteoporosis and vascular calcification associated with hypervitaminosis D.

Read Part I: Vitamin D and Vitamin K Team Up to Lower CVD Risk:
Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Strong correlations have been noted between cardiovascular diseases and low bone density / osteoporosis—connections so strong that the presence of one type of pathology is considered a likely predictor of the other. This potentially causal relationship has led to the hypothesis that these conditions share core mechanisms. Recent advances in our understanding of the complimentary roles played by vitamin D3 and vitamin K2 in vascular and bone health provide support for this hypothesis, along with insight into key metabolic dysfunctions underlying cardiovascular disease and osteoporosis.

Part I of this review summarizes current research linking vitamin D deficiency to cardiovascular disease, the physiological mechanisms underlying vitamin D's cardiovascular effects, and leading vitamin D researchers' recommendations for significantly higher supplemental doses of the pro-hormone. Part II reviews the vitamin K connection to cardiovascular disease; the ways in which vitamin D and vitamin K pair up to prevent inflammation, vascular calcification and osteoporosis; and the necessity of providing vitamin K along with vitamin D to preclude adverse effects associated with hypervitaminosis D, which include vascular and other soft tissue calcification.

Part I : Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease

Introduction

Risk for both cardiovascular disease and osteoporosis significantly increases with age. Even after adjustment for age, strong correlations have been noted between cardiovascular diseases (including atherosclerosis, coronary heart disease, congestive heart failure, hypertension, myocardial infarction and peripheral artery disease) and low bone density and osteoporosis—connections so strong that the presence of one type of pathology is considered a likely predictor of the other. This apparently causal relationship has led to the hypothesis that these conditions share core mechanisms. Recent advances in our understanding of the complimentary roles played by vitamin D3 and vitamin K2 in vascular and bone health provide support for this hypothesis, along with insight into key metabolic dysfunctions underlying cardiovascular disease and osteoporosis.^{1 2 3 4 5 6} This article focuses on the effects of vitamin D deficiency on cardiovascular disease and the mechanisms through which vitamin D sufficiency promotes cardiovascular health.

Vitamin D and Cardiovascular Disease

Technically, vitamin D is not a "vitamin". Its metabolic product, calcitriol, is a secosteroid hormone that affects more than 2,000 genes in the body (about 10% of the human genome). Current research implicates vitamin D deficiency as a major factor in the pathology of not only cardiovascular disease (CVD)—the focus of this review—but at least 17 varieties of cancer, diabetes, autoimmune diseases, osteoporosis, osteoarthritis, chronic pain, periodontal disease, sarcopenia, depression and more.⁷ A rapidly growing number of recently published studies link vitamin D deficiency with virtually all forms of CVD, including arteriosclerosis, atherosclerosis, hypertension, coronary artery disease, congestive heart failure, peripheral artery disease, myocardial infarction, and stroke.^{8 9 10 11 12 13 14 15 16 17 18}

Myocardial infarction

Serum 25(OH)D (25-hydroxyvitamin D or calcidiol, the pre-hormone produced by hydroxylation of cholecalciferol in the liver and the form measured in blood to assess vitamin D status) has recently been shown to be an independent predictor of CVD morbidity and mortality. A prospective study involving 18,225 men, aged 40-75 years at baseline (April 1993) and followed for 10 years, found that men deficient in 25(OH)D (serum levels ≤15 ng/mL or 37.5 nmol/L [to convert ng/mL to nmol/L, multiply by 2.496]) were at significantly increased risk for myocardial infarction (relative risk 2.09) compared with those considered to be sufficient in 25(OH)D (≥30 ng/mL [75 nmol/L]). Even men with intermediate 25(OH)D levels (22.6-29.9 ng/mL [or 56.4-74.6 nmol/L]) were at elevated risk (RR, 1.60) compared to those with sufficient 25(OH)D levels.¹⁴

Peripheral arterial disease

Other NHANES III-related research indicates an inverse relationship between 25(OH)D serum levels and prevalence of peripheral arterial disease (PAD), which is associated with a 2-fold increase in incidence of heart failure.¹⁹ In this analysis of nationally representative data from 4,839 participants including white, black and Hispanic ethnicities, the prevalence ratio of PAD for the lowest, compared to the highest 25(OH)D quartile (<17.8 and ≥29.2 ng/mL [44.4 and 72.8 nmol/L], respectively) was 8.1% compared to 3.7%.²⁰

Hypertension and cardiac hypertrophy

Vitamin D deficiency is associated with cardiac hypertrophy and hypertension in animal and human studies. Vitamin D receptor expression is increased in the myocytes, fibroblasts and intact ventricular myocardium of the hypertrophic heart, and 1-α hydroxylase and 24-hydroxylase, the two enzymes involved in the synthesis and metabolism of 1,25 dihydroxyvitamin D (calcitriol, the active form of vitamin D), are also present in the heart, allowing for local production of bioactive D3 from 25(OH)D.¹³

In animal studies, vitamin D deficiency leads to both hypertension and cardiac hypertrophy, while treatment with the vitamin D analogue, paricalcitol, reverses cardiac hypertrophy. The VDR knockout mouse exhibits hypertension and cardiac hypertrophy. In humans, low circulating levels of vitamin D3 in patients with chronic renal failure on dialysis are associated with ventricular hypertrophy, and treatment with supplemental vitamin D3 results in amelioration of the hypertrophy. All of the above suggests that the key components required for a functional 1,25 dihydroxyvitamin D-dependent signaling system are present in the human heart and that it provides an anti-hypertrophic system, which is protectively amplified in cardiac hypertrophy.¹³

Association with other CVD risk factors

Low vitamin D levels have also been associated with prevalence of a number of key risk factors for CVD. Analysis of data collected by the Third National Health and Nutrition Examination Survey (NHANES III) found that individuals in the lowest quartile of serum 25(OH)D levels had a significantly higher odds ratio for obesity (OR 2.29), diabetes (OR, 1.98), and high serum triglyceride levels (OR 1.47), as well as hypertension (OR 1.30).²¹

Another cross-sectional analysis of NHANES III data involving a large sample representative of the U.S. adult population (16,603 men and women, ≥18 years) found a strong correlation between low levels of 25(OH)D and CVD that was independent of traditional CVD risk factors.. Even after adjustment for age, gender, race/ethnicity, season of measurement, physical activity, body mass index, smoking status, hypertension, diabetes, elevated LDL cholesterol, low HDL, hypertriglyceridemia, and chronic kidney disease, vitamin D3-deficient participants had a 1.20 increased risk of CVD.¹⁰

Low 25(OH)D concentrations (<15 ng/mL [37.5 nmol/L]) have been clearly associated with a steep increase in incidence of cardiovascular disease in the Framingham Offspring Study22; with heart failure, sudden cardiac death, and fatal stroke in patients routinely referred for coronary angiography23,24; and with increased risk for all-cause and cardiovascular mortality.²⁵ A recent meta-analysis of studies reviewing 18 independent randomized controlled trials (n= 57,311 participants) suggests that low vitamin D status is associated with higher risk of mortality (from cardiovascular disease, diabetes mellitus and cancer) that accounts for 60% to 70% of the total mortality in high-income countries!²⁶

Obesity

Obesity is a well recognized contributing factor to CVD via its associations with risk factors such as hypertension, dyslipidemia, decreased glucose tolerance, metabolic syndrome and diabetes, and elevations in levels of markers of inflammation—all of which contribute to CVD pathology. Prevalence of overweight (BMI ≥ 25) and obesity (BMI ≥ 30) has dramatically increased during the last several decades. According to World Health Organization estimates, 1.6 billion adults were overweight, and 400 million adults were obese worldwide in 2006.²⁷ In the U.S., 2 in 3 adults have a BMI >25.^{28 29}

A recent double-blind, placebo-controlled clinical trial involving 200 healthy overweight subjects with mean 25(OH)D concentrations of 30 nmol/L (12 ng/mL), who received vitamin D (83 microg [3,320 IU]/day) or placebo for 12 months while in a weight-reduction program, revealed several beneficial effects of vitamin D supplementation at this dosage. Pronounced decreases in parathyroid hormone levels (-26.5%), triglycerides (-13.5%), and the inflammation marker tumor necrosis factor alpha [TNFα ] (-10.2%) were seen in the vitamin D group compared to the placebo group, in which results were -18.7%, +3.0%, and -3.2%, respectively.³⁰

Parathyroid hormone

As noted in the discussion of the results of this clinical trial, elevated levels of parathyroid hormone (PTH) are a non-traditional risk factor for CVD.30 Primary hyperparathyroidism is associated with hypertension, coronary atherosclerosis and other cardiovascular diseases. Elevated PTH concentrations due to hyperparathyroidism have been correlated with significantly higher risk of cardiovascular morbidity and mortality.³¹ Elevated serum PTH has been found to be an independent predictor of all-cause mortality in a study of frail elderly subjects, among whom 50% of deaths were cardiovascular-related³². And in the Tromsø Study, in subjects with calcium levels within the normal reference range, the rate of coronary heart disease (CHD) was higher in subjects with serum PTH > 6.8 pmol/l than in those with normal or low serum PTH levels (relative risk 1.67). The highest PTH quartile (> 3.50 pmol/l in men and > 3.30 pmol/l in women) predicted CHD, with mean odds ratios of 1.70 for men and 1.73 for women.³³

TNFα

As mentioned above, concentrations of TNFα were also significantly lessened (-10.2%) in overweight subjects supplemented with vitamin D3, confirming the suppressive effects of vitamin D3 on TNFα concentrations in an earlier study conducted by the same researchers in patients with congestive heart failure.³⁴

High TNFα concentrations (> 2.8 pg/mL) are recognized as a risk factor for cardiovascular disease³⁵. Studies have confirmed a significant association between high levels of TNFα, congestive heart failure and coronary heart disease mortality.^{36 37} Accumulating evidence suggests that TNFα plays a pivotal role in disruption of endothelial function and macrovascular and microvascular circulation, thus contributing to atherosclerosis.³⁸

As noted by Zitterman A et al.8, high levels of TNFα have been shown to suppress circulating calcitriol concentrations by inhibiting 1α-hydroxylation of 25(OH)D (calcidiol) to 1,25-(OH2)D3 (calcitriol).³⁹ Concentrations of vitamin 25 (OH)D of at least 30 ng/mL (75 nmol/L)—which were not achieved in all patients in the above study even with daily vitamin D supplementation of 3,320 IU—may be necessary to achieve calcitriol concentrations sufficient to prevent a vicious cycle of low calcitriol and high TNFα in the majority of patients. Individuals with genetic polymorphisms associated with increased TNFα production or impaired VDR binding activity (e.g., the BsmI BB SNP) may need considerably more (4,600 IU – 5,000 IU/day).^{40 41}

Natriuretic peptide

The natriuretic peptide family is a group of peptide hormones that play a major role in cardiovascular, endocrine, and renal homoeostasis. B-type natriuretic peptide and its N-terminal counterpart (N-terminal pro-brain natriuretic peptide or NT-proBNP) are secreted from cardiomyocytes in response to increased wall tension. Both natriuretic peptides have been comprehensively studied in heart failure and are considered major prognostic factors for high risk of death or serious complications across the whole spectrum of acute coronary syndromes and beyond traditional risk markers.⁴² In studies of patients with renal disease, circulating levels of 25(OH)D have been shown to correlate inversely with both BNP and NP-proBNP.^{43 44} In patients with congestive heart failure, levels of 25 (OH)D and calcitriol have also been found to correlate inversely with another natriuretic peptide, N-terminal pro-atrial natriuretic peptide (NT-proANP), which is a strong predictor of congestive heart failure severity.⁴⁵

Mechanisms: How Vitamin D Supports Cardiovascular Health

Endogenous production of vitamin D begins when the sun’s ultraviolet light acts upon 7-dehydrocholesterol (a precursor of cholesterol) in the skin, converting it to pre-vitamin D3, which then spontaneously isomerizes to vitamin D3 (cholecalciferol). Whether made in the skin or ingested, cholecalciferol is then hydroxylated in the liver by the enzyme 25-hydroxylase, to form 25-hydroxycholecalciferol [25(OH)D]or calcidiol, the metabolite that reflects bodily vitamin D stores. Calcidiol is further hydroxylated in the kidneys by 1α-hydroxylase into 1,25-dihydroxycholecalciferol [1,25(OH)2D] or calcitriol—the biologically active form of D3. Although calcitriol is primarily produced in the kidneys, a number of other tissues, including vascular smooth muscle cells and cardiac myocytes, also express 1α-hydroxylase and can locally produce the activated form of vitamin D3 [1,25(OH)2D].1

Decreases cell proliferation and cardiac hypertrophy

Local 1α-hydroxylase activity and consequent production of 1,25(OH)2D within endothelial cells and the myocytes of the heart provides an autocrine/paracrine link for control of hypertrophic activity within the myocardial wall.⁴⁶ Specifically, 1,25(OH)2D increases expression of myotrophin (a factor that stimulates myocyte growth), but balances this action by decreasing expression of atrial natriuretic peptide (a powerful vasodilator) and c-myc (a transcription factor that upregulates expression of numerous genes that drive cell proliferation). In addition, 1,25(OH)2D increases expression of the VDR in cardiac myocytes. The combined effect of these actions is a decrease in cell proliferation and prevention of cardiac hypertrophy.⁴⁷

Vitamin D3 has been shown to control genes affecting coagulation at the level of transcription, leading one group of researchers to propose that "vitamin D derivatives may develop as new types of antithrombotic and anti-atherosclerotic agents which change the character of cells."⁴⁸ In human peripheral monocytes, 1,25(OH)2D has been shown to exert anticoagulant effects by upregulating expression of an anticoagulant glycoprotein, thrombomodulin, while also downregulating expression of a critical coagulation factor, tissue factor.⁴⁹

Decreases rennin expression

Vitamin D decreases rennin expression and thus lessens activation of the rennin-angiotensin-aldosterone system (RAAS), which regulates blood pressure and fluid balance. Renin, secreted by the kidneys when blood volume is low, stimulates production of angiotensin, which causes blood vessels to constrict, increasing blood pressure, and also stimulates secretion of aldosterone, which causes the tubules of the kidneys to retain sodium and water, increasing the volume of fluid in the body and further increasing blood pressure. Studies using VDR and 1α-hydroxylase knockout or genetically modified mouse models, in which the vitamin D system has been inactivated, show that its ablation leads to increased renin expression, resulting in increased angiotensin II and aldosterone concentrations, abnormal sodium handling, hypertension, and left-ventricular hypertrophy.^{1 50 51 52} In contrast, high levels of calcitriol have been shown to decrease plasma renin activity, producing a decrease in angiotensin II levels.⁵³

By lessening RAAS activation, vitamin D not only decreases blood pressure, but also decreases the inflammation in the vascular endothelium that results from angiotensin activation, thus lessening progression of atherosclerosis. RAAS inhibition has also been shown to reduce common carotid and femoral artery intima-media thickness.⁵⁴

Decreases C-peptide

Low levels of vitamin D have been correlated with higher levels of C-peptide, a surrogate marker for insulin resistance that has recently been identified as a key mediator of atherosclerotic lesion development in individuals with type 2 diabetes. C-peptide facilitates the recruitment of inflammatory cells into early lesions and promotes lesion progression by inducing smooth muscle cell proliferation.⁵⁵

Data gathered from two large cross-sectional studies among men from the Health Professionals Follow-up Study and women from the Nurses' Health Study revealed that individuals with the highest calcium intake and plasma concentrations of 25(OH)D had significantly lower fasting C-peptide concentrations (35% lower in men, 12% lower in women) than individuals with the lowest levels of the two nutrients. Levels of C-peptide have also been found to be especially high in hypertensive individuals with inadequate calcium intake and low vitamin D levels.⁵⁶

Diabetes is associated with a 3-fold higher CVD mortality risk, and metabolic syndrome is a significant risk factor for type 2 diabetes.^{57 58} Vitamin D's protective effects against the development of metabolic syndrome were apparent in an analysis of data on 10,066 women participating in the Women’s Health Study. Women in the highest, compared to the lowest, quintile of vitamin D intake had an age- and calorie-adjusted relative risk of 0.77 for the metabolic syndrome.⁵⁹

Lowers C-reactive protein and metalloproteinases

Vitamin D supplementation lowers levels of C-reactive protein (CRP), a well-documented marker of inflammation associated with CVD pathology} ⁶⁰ and improves the balance between anti-inflammatory and pro-inflammatory cytokines. In patients with congestive heart failure, supplementation with vitamin D decreases the activity of pro-inflammatory nuclear factor-kappaB (NFκB), thus decreasing levels of pro-inflammatory interleukin-6, interleukin-1, interferon-γ, and TNFα; and also increases production of anti-inflammatory interleukin-10.^{34 61}

Vitamin D levels also correlate inversely with levels of tissue matrix metalloproteinases (MMPs), which control remodeling in the vascular wall, myocardium and other tissues. Plasma levels of MMPs increase in unstable angina and acute infarction. High levels of CRP and MMPs predict atheromatous vulnerability; both are considered indicative of increased risk of acute cardiovascular events. An elevated CRP level is a marker of inflammatory vascular damage to which the body responds by increasing circulating levels of MMPs. Calcitriol (1,25(OH)2D), the activated hormonal form of vitamin D3, modulates tissue MMP expression, plus vitamin D receptors are expressed in the vascular wall and in arterial plaque macrophages, which allows for MMP regulation by both circulating calcitriol and that which is produced locally in vascular tissues. These mechanisms underlying vitamin D's protective effects against CVD also help to explain why vitamin D reduces disease activity in rheumatoid arthritis, another disease in which increased expression of MMP contributes to pathogenesis. ⁶²

Perhaps the most intriguing insight into the mechanisms underlying vitamin D's protective cardiovascular effects is to be found in very recent developments in our understanding of the pharmacology of statin drugs.

Statin's Widespread Benefits Due to Increased Levels of Vitamin D3?

Not only do the benefits of statin drugs extend well beyond their powerful cholesterol-lowering effects, but these agents appear to mimic so many of the actions of vitamin D that they have recently been hypothesized to be vitamin D analogues.⁶³

Statins have been found to not only lower LDL cholesterol concentrations, but to raise concentrations of HDL cholesterol, lower serum triglycerides, reduce rejection of heart and kidney transplants, lessen symptoms in patients with rheumatoid arthritis and multiple sclerosis, improve bone mass, reduce hip fracture, and greatly reduce the risk of several cancers including lung, prostate and, especially, colorectal cancer. The latter two effects seem paradoxical in light of research indicating that high serum cholesterol concentrations are associated with higher bone mineral density in postmenopausal women⁶⁴ and are protective against colon cancer.⁶⁵

These unexpected and apparently contradictory benefits of statins indicate that these drugs are doing more than blocking cholesterol production by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reducatse). So, how can a drug that lowers serum cholesterol levels increase bone density and lower risk of colon cancer, conditions for which high serum cholesterol concentrations have been shown to be protective?63 The explanation may lie with vitamin D.

Insight into the mechanism underlying statin's wonder drug persona has recently been provided by a study that revealed treatment with atorvastatin raised serum 25(OH)D levels in patients with acute ischemic heart disease. After diagnosis, 83 patients (52 men, 31 women) with acute coronary syndrome (75 with acute myocardial infarction and 8 with unstable angina.) were given atorvastatin as secondary prevention. Serum vitamin D was measured at baseline and 12 months later. Atorvastatin treatment significantly decreased cholesterol and triglyceride levels and increased vitamin D levels (41+/-19 vs 47+/-19 nmol/L.). At baseline, 75% of patients had vitamin D deficiency (≤50 nmol/L [≤20 ng/mL]), which was decreased by 75% at 12 months. Results did not differ according to gender or dose of atorvastatin used.⁶⁶ Further analysis of the same patients revealed another benefit of the atorvastatin-induced 33% increase in vitamin D: bone mineral density increased in the spine (1.31%) in male patients whose severely low baseline vitamin D levels had risen to >30 nmol/L (>12 ng/mL).⁶⁷

How do statins increase vitamin D levels? Cholesterol and vitamin D share the same metabolic pathway. Cholesterol is synthesized from 7-dehydrocholesterol, which is also the precursor for vitamin D3. Statins are known to lower cholesterol by inhibiting HMG-CoA reductase, but this enzyme is 10 steps upstream from the production of 7-dehydrocholesterol in the cholesterol synthesis pathway, so blocking HMG Co-A reductase should lessen production of not only cholesterol, but also vitamin D.⁶⁸ Since atorvastatin treatment increases vitamin D levels, the following two statements must be true: (1) statins do not completely block HMG Co-A reductase, but allow some activity of this enzyme, and (2) statins also inhibit the final enzyme in the production of cholesterol, 7-dehydrocholesterol reductase, preventing its conversion of 7-dehydrocholesterol to cholesterol and increasing the amount of 7-dehydrocholesterol in the skin, which can then be energized by ultraviolet (specifically, UV-B) radiation to form 25(OH)D. Thus, it is statins' inhibition of 7-dehydrocholesterol reductase that results in increased levels of 7-dehydrocholesterol, enabling increased synthesis of 25(OH)D, thereby increasing vitamin D levels.^{66 69}

In addition, 25(OH)D has itself been shown in vitro to inhibit HMG-CoA enzyme reductase activity in mouse epithelial and human skin and liver cells.⁷⁰ Thus, a statin-induced increase in vitamin D concentrations could further increase inhibition of HMGCoA. Vitamin D may be acting synergistically with statins to decrease total cholesterol levels.⁶⁶

Click here for pdf version of chart.

Vitamin D3 Dosage Considerations

Using 28 ng/ml (69.8 nmol/L) as a cut-off, it has been estimated that approximately 41% of men and 53% of women in the U.S. have insufficient levels of 25(OH)D.¹

Dose-response curves observed in a recent 6-month, prospective, randomized, double blind, placebo-controlled study of vitamin D3 supplementation suggest that the intake of vitamin D3 needed to raise serum 25(OH)D levels to >30 ng/mL (75 nmol/L) varies depending upon the individual's baseline levels of vitamin D. In one study, a daily dose of 3,800 IU appeared to be adequate for individuals whose baseline vitamin D levels were above 55 nmol/L (22 ng/mL); for individuals whose baseline levels of vitamin D fall below this threshold, the researchers suggest a dose of 5,000 IU/day may be required.⁷¹

However, the optimal serum level of 25(OH)D may be 40 ng/mL or even higher, not 30 ng/mL. A number of other studies reviewed by Heaney suggest 25(OH)D levels of ≥40 ng/ml are needed for full vitamin D efficacy.⁷² These include:

Meta-analyses of controlled trials in which vitamin D supplementation has been shown to reduce osteoporotic fracture show no benefit unless serum 25(OH)D reaches at least 75 nmol/L (30 ng/mL).⁷³

Studies looking at the risk of falling in the elderly, which is also significantly related to serum 25(OH)D and has been shown to lessen all the way up to serum 25(OH)D values of 75 nmol/L (30 ng/mL). The researchers note that risk may be further reduced at higher levels, but there were too few individuals with higher values to assess an association at levels higher than 75 nmol/L.⁷²

Studies analyzing the inverse relationship between serum 25(OH)D and cancers in humans from which the evidence indicates a dose-related, linear decrease in risk as serum 25(OH)D rises to levels of ≥80 nmol/L (32 ng/mL).⁷⁴ One recent 4-year, randomized, controlled trial showed that raising serum 25(OH)D from a mean of 71 nmol/L (28 ng/mL) to one of 96 nmol/L (38 ng/mL) decreased all-cancer risk in 1,169 postmenopausal women by approximately 60%.⁷⁵

NHANES III data on fasting blood sugar and response to a standard glucose challenge that indicates response plateaued at 25(OH)D values between 100 and 120 nmol/L (40 and 48 ng/mL).⁷⁶
Leading vitamin D experts presented a paper at the 2008 American Public Health Association meeting in which they noted, "Observational and randomized controlled trial studies have found that it takes at least 1000-2000 IU of vitamin D3 [per day] and serum levels of 40-60 ng/mL for substantial benefits … 1100 IU/day can raise [vitamin D blood levels] by ~10 ng/mL."⁷⁷

Hollis et al. analyzed the relationship between cholecalciferol (the parent compound) and 25(OH)D (which reflects bodily vitamin D stores). They found that optimal nutritional vitamin D status did not occur until circulating 25(OH)D exceeded 40 ng/mL (100 nmol/L). Not until this level was the Vmax, of the 25-hydroxylase enzyme achieved (i.e., all enzyme sites were saturated). In other words, the body's ability to utilize cholecalciferol in the numerous roles played by the vitamin D endocrine system is not reached until 25(OH)D levels are ≥40 ng/ml. Below this level, chronic substrate deficiency prevents full actualization of the myriad benefits of vitamin D.⁷⁸

In overweight / obese individuals

Body composition may affect vitamin D requirements. Suggested mechanisms include that 1) low vitamin D3, may impair insulin action, glucose metabolism and various other metabolic processes in adipose and lean tissue,⁷⁹ 2) fat soluble-vitamin D3 is sequestered in adipose tissue,⁸⁰ and 3) obese individuals may minimize skin sun exposure due to embarrassment about their body shape.⁸¹

Results of two studies recently conducted on women in sunny Spain, one in Barcelona and the other in Madrid, confirm that overweight/obese persons usually have inadequate vitamin D levels. In the Barcelona study, 51% of the morbidly obese were vitamin D deficient (25(OH)D <38 nmol/L [15 ng/mL]) compared to 22% of the non-obese women.⁸² The women in Madrid were divided into two groups depending on their serum vitamin D concentrations: a low-D group (<90 nmol/L [<36 ng/mL]) and a high-D group (>90 nmol/L [>36 ng/mL]). Although intakes of vitamin D, calcium and supplements were similar in both groups, a BMI of >27.7 kg/m(2) was associated with serum vitamin D concentrations of ≤ 90 nmol/L.⁸³

A study conducted in Auckland, New Zealand, another area of the world in which access to sunshine is not an issue, tested how vitamin D3 levels related to fat mass, markers of metabolic syndrome, and hemoglobin A1c (HbA1c) in 250 overweight and obese adults of different ethnicities. Multivariable regression carried out separately for BMI and waist showed a decrease of 0.74 nmol/L in vitamin D3 per 1 kg/m2 increase in BMI, and a decrease of 0.29 nmol/L per 1 cm increase in waist, with each explaining approximately 3% of the variation in vitamin D3 over and above gender, age, ethnicity and season.⁸¹

In the U.S., a comparison of NHANES data on serum 25(OH)D in the population in 1988-1994 versus 2000-2004 revealed a significant drop (by 5-20 nmol/L [2-8 ng/L]) in 2000-2004 vitamin D levels, which researchers suggest is explained by increases in BMI, decreases in milk intake, and sun protection.⁸⁴

In black individuals

Black individuals, particularly those living in northern latitudes, typically need 5–10 times longer sun exposure to generate vitamin D than Caucasians, and therefore are likely to need higher doses of supplemental vitamin D. Epidemiological studies have shown an inverse association between blood pressure and vitamin D levels, and a direct association between blood pressure and increasing latitude (a surrogate of lower vitamin D levels).^{85 86 87}

Hypertension is more common among blacks living in the U.S. or U.K. than whites. NHANES III data showed mean serum levels of 25(OH)D were lowest in non-Hispanic blacks (49 nmol/L [19.6 ng/mL]), intermediate in Mexican-Americans (68 nmol/L [27.2 ng/mL]), and highest in whites (79 nmol/L [31.6 ng/mL]). When participants were divided into 25(OH)D quintiles, mean systolic BP was 3.0 mm/Hg lower, and diastolic BP was 1.6 mm/Hg lower in subjects in the highest quintile (25(OH)D ≥ 85.7 nmol/L [≥34.3 ng/mL]) compared with the lowest (25(OH)D ≤ 40.4 nmol/L [≤16 ng/mL]). The inverse association between 25(OH)D and systolic BP was strongest in participants aged ≥50 years. Ethnic differences in 25(OH)D explained ~50% of the increased prevalence of hypertension seen in blacks compared to whites.⁸⁸

In older individuals

In the aging patient, an additional factor to consider is that older individuals are more prone to low vitamin D concentrations, both because the skin's capacity to produce vitamin D decreases with age, and because many elders do not get adequate sun exposure.⁸⁹ Worldwide, almost 50% of the elderly are estimated to be vitamin D-deficient. Indication that older individuals are at increased risk for vitamin D deficiency-related CVD is provided by a recent 6.2 year prospective trial, initiated in 2000-2001 that involved 614 older Austrian men and women participating in the Hoorn Study. Risk for all-cause and cardiovascular mortality in individuals in the lowest quartile of serum vitamin D3 levels, compared to those in the upper three quartiles, were 1.97 and 5.38, respectively.⁹⁰

John Cannell, MD, Executive Director and founder in 2003 of the non-profit Vitamin D Council (www.vitamindcouncil.org), a consortium of the world's leading vitamin D scientists, offers the following as "conservative" recommendations for vitamin D supplementation: Healthy adults and adolescents between 80 pounds and 130 pounds should start with 3,000 IU per day; over 130 pounds but less than 170 pounds, 4,000 IU per day; over 170 pounds, 5,000 IU per day. After two months, test serum levels of 25(OH)D3 [calcidiol]. Adjust dose to reach and maintain 25(OH)D [calcidiol] levels between 50 and 70 ng/mL [124.8 nmol/L and 174.72 nmol/L], summer and winter.⁹¹

Vitamin D Safety Issues: Upper Tolerable Limit

A recently published review of the evidence regarding the risk-related consequences of vitamin D supplementation in adults concluded: "Evidence from clinical trials shows, with a wide margin of confidence, that a prolonged intake of 10,000 IU per day of vitamin D3 poses no risk of adverse effects for adults, even if this is added to a rather high physiologic background level [sun exposure, dietary intake] of vitamin D.⁹²

This is not surprising since in an adult with white skin, one minimum erythema dose of total body solar exposure, such as typically achieved in 15 to 20 minutes on a summer day while wearing a bathing suit, generates ~10,000 – 20,000 IU of vitamin D3 in 15 to 20 minutes.⁹²

In humans, the physiologic limit for serum 25(OH)D concentrations is ~220 nmol/L (88 ng/mL), a level that approximates the top of the range of values reported for humans well exposed to ultraviolet light. The fact that humans evolved in a tropical climate with regular skin exposure to sunshine implies that the upper end of the physiological concentration range for 25(OH)D is not only safe, but optimal.⁹²

People with abundant exposure to sunlight can easily exhibit a serum 25(OH)D greater than 150 nmol/L (60 ng/mL). Such 25(OH)D concentrations reflect a pre-supplement oral intake or biosynthesis of vitamin D equivalent to more than 4,000 IU per day. An additional oral intake of 4,000 IU per day of vitamin D would still be far less than the dose of 50,000 IU per day reported to be non-hypercalcemic in clinical trials.⁹²

While the safe upper limit for serum 25(OH)D appears to be 200 nmol/L (80 ng/L),⁹³ the published evidence regarding vitamin D toxicity shows that the lowest 25(OH)D concentration causing hypercalcemia is greater than 500 nmol/L (200 ng/mL). Those patients reported in the literature as exhibiting hypercalcemia with a 25(OH)D concentration less than 500 nmol/L were taking vitamin D in infrequent but extreme doses (e.g., 600,000 IU once weekly). Very strong evidence indicates no hypercalcemia or hypercalcinuria is associated with supplementary vitamin D intakes of at least 10,000 IU per day, in addition to the vitamin D that healthy North American adults acquire in the normal course of modern life.⁹²

Conclusion

Vitamin D exerts numerous protective actions on the cardiovascular system, and a plethora of current studies demonstrate that deficiency of this pro-hormone is a highly significant, if non-traditonal, risk factor for cardiovascular disease. All patients, but particularly those at increased cardiovascular risk, should be screened for vitamin D deficiency. Treatment with physiological doses of vitamin D3 (between 4,000 to as high as 10,000 IU/day from all sources, i.e., intelligent sun exposure, food and supplements) combined with periodic monitoring of serum 25(OH)D calcidiol and calcium levels should be standard medical practice.

Read Part II: Vitamin D and Vitamin K Team Up to Lower CVD Risk
The Vitamin K Connection to Cardiovascular Health

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Despite the widely held assumption that Americans are iodine-sufficient due to the availability of iodized salt, the U.S. population is actually at high risk for iodine insufficiency. Iodine intake has been decreasing in the U.S. since the early 70s as a result of changes in Americans' food and dietary habits, including the facts that iodized salt is infrequently used in restaurant and processed foods, and iodized salt sold for home use may provide far less than the amount of iodine listed on the container's label. The widespread dispersal of perchlorate, nitrate and thiocyanate (competitive inhibitors of iodide uptake) in the environment blocks absorption of the little iodine Americans do consume, further compounding the problem.

In adults, iodine is necessary not only for the production of thyroid hormones, thus affecting systemic metabolism, but is now recognized to play a protective role against fibrocystic breast disease and breast cancer. In addition, a relationship has been hypothesized between iodine deficiency and a number of other health issues including other malignancies, obesity, attention deficit hyperactivity disorder (ADHD), psychiatric disorders, and fibromyalgia.

Analogous to the case of vitamin D, a nutrient for which the 400 IU DRI, although capable of preventing rickets, has been proven inadequate for this pro-hormone's numerous other functions in the body, the iodine DRI for adults of 150 mcg/day (220 mcg/day for pregnant women), while sufficient to prevent goiter (and cretinism), is inadequate for the promotion of optimal health in adults or optimal fetal brain development. Intake of 3-6 mg/day, an amount commonly consumed in Japan without increased incidence of autoimmune thyroiditis or hypothyroidism, may be necessary to support not only thyroid hormone production, but iodine's important antioxidant functions in the breast and other tissues in which this trace mineral is concentrated.

Part I of this article discusses the numerous factors that place Americans at high risk for iodine insufficiency. Part II reviews iodine's roles in the body, the relationship of iodine insufficiency to the above mentioned pathologies, available options in laboratory assessments of iodine levels, optimal intake, preferential forms of supplementation, and cofactors necessary for optimal iodine utilization.

Part II: Not Just for Thyroid

Introduction

Metabolism and Physiological Effects

A trace mineral increasingly recognized as essential for a number of physiologic functions, iodine belongs to the halogen family of elements, a group of highly reactive nonmetals that includes fluorine, chlorine and bromine. Iodine is concentrated by certain seaweeds, but is the least abundant halogen in the Earth’s crust. Although the iodine content of soils varies, most has been leached out since primordial times when much more of the Earth's surface was covered by seas.¹

The second halogen to be identified (after chlorine), iodine was discovered by Bernard Courtois in Paris in 1811, but it took nearly 100 years before its key role in thyroid function was recognized. It was 1927 when Sir Charles Harrington first reported that the major part of the thyroxine (T₄) molecule (65.3% by weight) was made up of iodine.²

While iodine's importance for thyroid function as a primary constituent of thyroid hormones and its use as a topical antibacterial agent^{3 4 5} have long been recognized, only recently has attention been given to this trace mineral's other roles, which include antioxidant and anticancer activity (discussed below).

Usually ingested in the form of an iodate or iodide compound (I¯, inorganic sodium and potassium salts, see Glossary), iodine is also naturally present in seaweeds (e.g., dulse, wakame, kombu) in the form of inorganic diatomic iodine (molecular iodine or I2) and organic monoatomic iodine (C¯I). Iodine is rapidly absorbed into the circulation and actively concentrated within thyroid follicles to 20-40 times its concentration in the blood, a reflection of its critical role in the production of thyroid hormones. Only about 30% of the body's iodine is sequestered in thyroid tissue and hormones; however, the body also concentrates iodide in the salivary glands, breast tissue, gastric mucosa, and choroid plexus, among other sites, indicating that this trace mineral plays vital roles in areas other than the thyroid gland.⁶

As noted in Part I of this review, iodine's concentration at these sites is so critical to physiological function that the body possesses a specific mechanism, the sodium/iodide transporter—a.k.a. the sodium/iodide symporter (NIS) –able to transport iodide from the blood into the thyroid gland and other tissues across a concentration gradient as high as 50-fold.⁷ NIS activity is upregulated by the binding of TSH to receptors on the follicular cells. Inside the follicular thyroid cell, iodide is carried, by a process called iodide efflux, through the apical membrane to the follicular lumen by pendrin, an anion transporter predominantly expressed in the thyroid, kidney and inner ear. (Mutations in the pendrin gene are associated with hypothyroidism and Pendred syndrome, the most common recessive syndromic form of congenital deafness.)⁸ I2 is transported by facilitated diffusion.

Within the thyroid's follicular cells (where the glycoprotein, thyroglobulin, is synthesized), iodide is catalyzed by thyroid peroxidase (TPO) using H2O2, and bound to tyrosine residues in the thyroglobulin molecule to form mono- or diiodotyrosine (MIT or DIT), which in turn combine to produce the thyroid hormones, primarily thyroxine (T₄), which constitutes ~90% of the thyroid hormone secreted from the gland, and triiodothyronine (T₃), which accounts for the remaining 10%. Combining two molecules of DIT produces T₄; combining one molecule of MIT and one particle of DIT produces T₃.{ref⁹ Iodine accounts for 65% of the molecular weight of T₄ and 59% of the molecular weight of T₃.^{10 11}

In addition to its essential involvement in thyroid hormone production, iodine also affects the release of thyroid hormone, which is regulated in two ways: (1) through thyroid releasing hormone (TRH) which stimulates the pituitary gland to secrete thyroid stimulating hormone (TSH), which in turn stimulates the thyroid to release T₃ and T₄, and (2) via autoregulation activated in response to the concentration of iodine in the thyroid (a.k.a. the Wolff-Chaikoff effect, see Glossary). Iodine's rate of uptake into the follicle, the ratio of T₃ to T₄, and their release into the circulation are all affected by the concentration of iodine in the thyroid, such that an increase in iodine intake results in a decrease in its organification (see Glossary) in the follicles, thus preventing excessive hormone production and release, and maintaining stability in hormone secretion despite possibly wide variation in iodine intake.^{1 7}

In an iodine-replete adult, approximately 15-20 mg of iodine (30% of the body's iodine stores) is concentrated in the thyroid; the remaining 70% (~44 mg) is found in a variety of extra-thyroidal tissues, including the breast, eye, gastric mucosa, cervix and salivary glands.¹¹ When the thyroid is stimulated to release its hormones, thyroglobulin is degraded, releasing T₄ and T₃, which, upon entering the circulation, are rapidly bound to transport hormones (~70% to thyroxine binding globulin; the remaining 30% to other proteins, e.g., transthyretinm, albumin and lipoproteins) and delivered to peripheral tissues. Any iodide freed in the degradation of thyroglobulin is for the most part recycled, and the iodinated tyrosine reused for hormone production. T₄, which has a half-life of about one week and serves as a reservoir for conversion to the more active hormone, T₃, whose half-life is only 1 day.¹

Conversion of T₄ to T₃ via deiodination occurs primarily in target organs and is catalyzed by iodothyronine deiodinase (a.k.a. iodide peroxidase) type 2 (DI2).There are two other iodothyronine deiodinases, DI1 and DI3, but all three are selenium-dependent enzymes. DI1, a kinetic enzyme that both activates and inactivates T₄, is the form primarily produced in breast tissue during pregnancy and lactation. DI3 inactivates T₃ producing reverse T₃, and, to a lesser extent, prevents T₄ from being activated. Deiodinase activity has been identified not only in the liver (which contains ~30% of the extra-thyroidal T₄), but also in the breast, kidney, in human skeletal muscle, and in the brain, where DI2 plays a crucial role in deiodinating T₄ to T₃.^{12 13 2}

Approximately 90% of iodine is eventually excreted in the urine. According to the International Council for the Control of Iodine Disorders, WHO and UNICEF, borderline iodine deficiency is indicated by average daily excretion rates of 100 mcg/L per day. As noted in Part I of this review, the World Health Organization has determined 50-99 mcg/L indicates mild deficiency, 20-49 mcg/L indicates moderate deficiency, and less than 20 mcg/L indicates severe deficiency.¹⁴

For comparison, median urinary iodine excretion in the U.S. population was 145 µg/L during the years 1988 through 1994, which was a significant decrease from the 321 µg/L found in a similar survey two decades prior.¹⁰ Among the Japanese, urinary iodine excretion in euthyroid Japanese subjects has been reported to be as high as 9.3 mg per day, and mean urinary iodine levels are approximately twice those reported in the U.S, NHANES 2001-2002 data.^{11 15}

Click here for a PDF version of the chart.

Chart Notes: Normal process: Thyroid hormone levels drop. TSH binds to receptors on the follicular cells, stimulates NIS activity and also ensures H202 will be available as a substrate by inducing NADPH oxidase, which oxidizes NADPH to NADP, liberating superoxide radicals (O2¯), which are then converted to the less potent free radical, H202, by SOD, a selenium-dependent enzyme. NIS transports Iodide (I¯) into the follicular cell where it is catalyzed by heme-dependent TPO using H202 to form I2, which then binds to tyrosine residues in thyroglobulin to form MIT and DIT. DIT+ DIT then produce T₄; DIT + MIT produce T₃. H202 that is not used up in this process is neutralized by selenium-dependent glutathione peroxidase. Iodine deficiency / Selenium deficiency / Iron deficiency: Low iodine stores result in low levels of thyroid hormones, which activates TSH. H202 is produced, but no iodide arrives. If selenium is also insufficient, O2¯, a more potent ROS than H202, is formed and is not converted to H202. If iron is deficient, TPO will not be available to catalyze iodide, so H202 will remain to cause damage to the follicular cell. Iodine repletion: the Wolff-Chaikoff effect will prevent excessive organification of iodide; however, during the formation of thyroid hormones, some ROS will be generated in excess of those used to produce I2, and will cause damage if not reduced by glutathione peroxidase.

Iodine's Effects on Physiological Function

Through its essential inclusion in thyroid hormones, iodine has long been recognized to impact virtually every cell in the body, affecting a wide range of metabolic functions, including basal metabolic rate; protein, fat and carbohydrate metabolism; protein synthesis, and brain development.

Effects on Adult Brain Function

Recently, proper thyroid hormone signaling has been shown to be essential not only for fetal and neonatal brain development, but adult brain function. T₃ is concentrated in the locus coeruleus, a nucleus in the brain stem that is the principal site for synthesis of norepinephrine and is involved in physiological responses to stress. T₃ is also found in the junctions between synapses and regulates the amounts and activity of serotonin, norepinephrine, and gamma-aminobutyric acid (GABA) in the brain. Hence, iodine insufficiency (and/or selenium deficiency since the deiodinases that convert T₄ to T₃ are selenium-dependent) is hypothesized to be a contributing factor in the pathogenesis of a wide range of mental disturbances, from autism to ADHD to post-traumatic stress disorder to depression.²

Recent studies have noted a relationship between subclinical hypothyroidism and decrements in mood and working memory¹⁶, anxiety, psychoses, depression, and dementia (impaired short-term memory, slowed information processing speed, reduced efficiency in executive functions, and poor learning), particularly in women and the elderly.^{17 18 19 20}

Thyroid hormones control several genes in the CNS and are essential for differentiation of somatotrophs (which produce growth hormone) and pituitary lactotrophs (which produce prolactin). A number of thyroid hormone signaling pathways in the hypothalamus are thought to be involved in the adaptation of the thyroid axis, not only to hypo- and hyperthyroidism, but also to inflammation, the stress response, and critical illness (a pattern of decreased pituitary-thyroid function, sometimes referred to as low T₃ syndrome, is known to accompany life-threatening trauma, major surgery and severe illness). Regardless of the challenge, insufficient thyroid hormone leads to defects in hypothalamus-pituitary-thyroid-periphery-feedback regulation.^{21 22}

Other recently published research indicates that thyroid hormones modify genetic expression via their action on nuclear receptors within the large family of receptors that also bind vitamins A and D, and steroids. Most of T₃'s effects are mediated by nuclear receptors, but T₄ itself, and its iodinated metabolites, have also been found to exert direct biological effects in the brain.^{23 24 25 26 21}

Iodine's Antioxidant Actions

For centuries, iodine rich brines or seaweeds have been used as thalassotherapy or balneotherapy (see Glossary) in health spas, treatments that have been repeatedly shown to produce beneficial effects in cardiac and respiratory disease, thyroid function, arteriosclerosis, diabetes mellitus and eye diseases.²⁷ Iodine's antioxidant effects provide one underlying mechanism for these positive clinical results. I¯ itself exerts significant antioxidant effects; NaI levels as low as 15 μM produce equivalent antioxidant effects to those seen with ascorbic acid at levels of 50 μM.²⁸

Iodine's antioxidant effects are a byproduct of the redox reactions that occur during the formation of thyroid hormones, when iodide (I¯) is organified (oxidized) to become iodine (I2).27 The first step in iodide's (I¯) organification to iodine (I2) is accomplished when I¯ is oxidized by thyroid peroxidase (TPO) using hydrogen peroxide (H202). By reducing H202, iodide becomes I2, and binds to tyrosine residues in the thyroglobulin molecule, forming the mono- and diiodotyrosines that are the precursors of triiodothyronine (T₃) and thyroxine (T₄). Since this process decreases available H202, less remains for damaging oxidative activity; thus iodide serves, in effect, as an antioxidant.

Substrate H202 for iodide's organification is provided in the thyroid by TSH-mediated induction of the thyroid oxidases (ThOX1 and ThOX2), which oxidize NADPH to NADP, liberating superoxide radicals (O2¯), which are then converted to the less potent free radical, H202 by superoxide dismutase (a selenium dependent enzyme). H202 that is not utilized for the organification of iodide is, in individuals with adequate selenium, removed by antioxidant enzymes, principally those of the selenium-dependent glutathione peroxidase (GPx) family.²⁷

This is why iodine deficiency, which triggers increased stimulation by TSH resulting in excessive H202 production within the thyroid's follicular cells with little substrate iodide to be oxidized, results in damage to the thyroid. Selenium deficiency exacerbates the risk of oxidative stress since it causes a deficit in the glutathione peroxidases that would normally convert H202 to H2O. Thus, the combination of iodine and selenium deficiencies greatly increases oxidative damage to DNA in thyroid follicular cells and risk for thyroid malignancies. At higher levels of intake, iodide also acts as an antioxidant by a related mechanism—reducing the sensitivity of the thyroid gland to TSH, thus diminishing production of both H202 and T₄.²⁷

Yet another way in which iodine exerts antioxidant effects is through the formation of iodolipids, which are produced when iodine reacts with double bonds on lipids, rendering them less accessible to reactive oxygen species (ROS). This antioxidant effect of iodine provides significant protection in the thyroid where arachidonic acid, a fat that contains four double bonds and is highly susceptible to oxidation, plays a role in intracellular signaling. Iodolipid formation may also play a protective role against lipid peroxidation in other areas of the body that concentrate iodine. Of clinical note, lack of iodolipid formation contributes to the pathological outcomes of hypothyroidism, which results in reduced oxidative metabolism and markedly increased lipid and lipoprotein levels.²⁷

Beyond Thyroid: The Iodine Link to Breast Health

Beyond its thyroid-hormone mediated effects, iodine is required for the normal growth and development of breast tissue, and acts an antioxidant and antiproliferative agent protecting the integrity of the mammary gland. The high level of iodine intake by Japanese women, noted in Part I, has been associated with a low incidence of both benign and cancerous breast disease in this population.^{11 29} In contrast, evidence linking iodine deficiency with an elevated risk of breast, endometrial, and ovarian cancer has been hypothesized since 1976, when it was noted that low dietary iodine intake could result in increased gonadotrophin stimulation, producing a hyperestrogenic state (increased production of estrone and estradiol, and a lower ratio of estriol to estrone + estradiol) that could increase the risk of these cancers.³⁰ Furthermore, estradiol has been shown to increase cell proliferation and down-regulate NIS expression and iodide uptake in vitro.³¹

In support of this hypothesis, in vitro evidence that iodine inhibits cancer promotion through modulation of the estrogen pathway was published in 2008. Researchers looked at the effect of Lugol's iodine solution (5% I2, 10% KI) on gene expression in the estrogen responsive MCF-7 breast cancer cell line. Twenty-nine genes were up-regulated, and 14 genes were down-regulated in response to iodine treatment, including several involved in hormone metabolism as well as others involved in the regulation of cell cycle progression, growth and differentiation.³²

An association between breast cancer and thyroid disease was found in a recent study of 26 breast cancer patients (aged 30-85 years) and 22 age-matched controls. Incidence of thyroid disease was much higher in patients than controls (58% vs. 18%, respectively). Subclinical hyperthyroidism was the most frequent disorder in patients (31%), although hypothyroidism (8%) and positive anti-TPO antibodies (19%) were also seen. The conclusion drawn by the researchers was that subclinical hyperthyroidism was the only statistically significant thyroid alteration found in this breast cancer population. What they failed to mention, except as an afterthought, was that all of the women with breast cancer, but none the healthy controls, came from an area endemic for low iodine intake.³³ It is not surprising that iodine deficiency results in thyroid dysfunction, nor, given iodine's antioxidant, estrogen modulating and gene regulating effects, that a deficiency of this trace mineral increases risk for breast cancer.

Molecular Iodine, the Preferred Form in Breast Tissue

Despite reports of the enhanced expression of NIS in human breast cancer tissue, I2 may be the preferred form of iodine supplementation for the prevention and treatment of breast cancer since non-lactating breast tissue is known to be peroxidase-poor and thus is less capable of iodide organification. The use of I2 bypasses the need for NIS involvement and peroxidase activity. Not surprisingly, the mammary gland more effectively captures and concentrates I2 than the thyroid, and more I2 is concentrated in breast than thyroid tissue.^{34 35}

In vitro studies have found that I2 inhibits proliferation and induces apoptosis in some human breast cancer cell lines by causing loss of selective permeability of the mitochondrial membrane, which leads to the release of apoptogenic proteins normally confined to mitochondrial intermembrane space. Supplementation with I2 has been shown to suppress the development and size of both benign and cancerous neoplasias.^{34 35}

As noted above in the discussion of iodine's antioxidant effects, iodine is also used to produce iodolipids with antioxidant and antiproliferative effects in extra-thyroidal tissues as well as in the thyroid gland. Iodolactones may provide yet another protective mechanism in the breast and other extra-thyroidal tissues since the antiproliferative effects of I2 supplementation are accompanied by a significant reduction in cellular lipoperoxidation.^{36 37}

I2 has also recently been shown to induce formation of an iodolactone derived from arachidonic acid, 6-iodolactone (6-IL), which activates cellular pathways involved in cell cycle arrest and apoptosis. Mammary cancer cells are known to contain high concentrations of arachidonic acid, which may help explain why I2 selectively exerts apoptotic effects at lower concentrations only in mammary tumor cells and not in normal mammary tissue.³⁸

Of clinical note, potassium iodine, the form used in iodized salt, does not have these effects.

Iodine Protective Against Fibrocystic Breast Disease

Benign, fibrocystic breast disease has also been shown to be associated with iodine deficiency. In rat studies, blocking iodine uptake with perchlorate caused histologic changes indicative of fibrocystic breast disease, as well as precancerous lesions in the mammary tissue, with much greater deleterious changes in older animals.^{39 40} Double the risk of fibrocystic breast disease was found among those women with increased blood levels of TSH and a decline in thyroid function in a recent study of 90 women ranging in age from 23 – 50.⁴¹

In a series of three clinical trials, researchers looked at the effect of different forms of iodine-supplementation in women diagnosed with fibrocystic breast disease. In Study 1, an uncontrolled trial in which 233 volunteers received sodium iodide for 2 years, and 588 received protein-bound iodide for 5 years, 70% of the women treated with sodium iodide and 40% of patients treated with protein-bound iodide experienced clinical improvement; however, the rate of side effects (e.g., bad breath, increased salivation, rhinitis, skin eruption) was high.

In Study 2, a prospective, control, crossover study, 1,365 women received I2 (0.8 mg/kg), including 145 patients who were switched over from treatment with protein-bound iodide in Study 1; 74% of these cross-over patients experienced clinical improvement, as did 72% of those receiving I2 initially.

In Study 3, a prospective, control, double-blind study in which I2 was compared to placebo, 65% of those in the treatment group and 33% in the placebo group experienced improvement.⁴² Given that, as noted above, non-lactating breast tissue is peroxidase-poor and less capable of iodide organification, these results add further support for the use of I2 as the preferred form of supplemental iodine for breast tissue.

I2 supplementation has also been shown to ease mastalgia. Supplementation with 3 or 6 mg/day of molecular iodine significantly decreased pain reported by patients, as well as physicians’ assessments of pain, tenderness, and nodularity in benign breast disease, with a dose of 6 mg/day providing significant reduction of pain in more than 50% of patients.⁴³

Iodine—A Protective Role against Cancer?

As noted above, iodine is engaged in a variety of antioxidant activities and has also been shown to induce apoptosis in human breast cancer cells, but not in normal cells, via a mitochondrial-mediated pathway. The data suggests a role for iodine in the prevention and treatment of cancer since, in iodine-deficient individuals, these protective processes are highly likely to be impaired, increasing oxidative damage to DNA, lessening apoptosis, and eventually promoting the development of malignancies.^{34 31}

In rats, chronic dietary iodine deficiency results in thyroid follicular adenomas within 12 months and follicular carcinomas within 18 months. An increased risk of thyroid cancer has been reported in humans with goiter and those living in iodine-deficient areas of the world.⁴⁴

Thyroid cancer incidence increased 2.4 fold from 1973 – 2002.⁴⁵ It has become one of the ten leading cancer types in females. Accounting for 22,590 new cases per year in the United States; thyroid cancer is more frequent than ovarian, urinary bladder or pancreatic cancer. Researchers analyzing the trend in rising thyroid cancer incidence in the U.S., during the period from 1980-2005, concluded that medical surveillance and more sensitive diagnostic procedures cannot account for the observed increases in thyroid cancer and suggest other possible explanations should be explored.⁴⁶ Given that iodine intake has dropped significantly in the U.S. during this same time period, iodine insufficiency seems to be worth considering as a likely contributing factor. Particularly in light of the fact that 4–6% of American adults are goitrous despite what has been considered "adequate" iodine intake.⁴⁷

Another indication of iodine's anti-cancer effects is that iodide uptake is diminished in thyroid cancer compared with normal thyroid tissue, despite the fact that expression of the NIS receptor is increased in malignant cells. When thyroid cancer undergoes total loss of differentiation, and the prognosis is clearly worse, no iodide uptake is observed. In vitro studies have found that the activation of key oncogenes in malignant transformation and tumor progression in thyroid cancer (the BRAF, RAS and RET genes) causes a decrease in NIS mRNA levels among other thyroid-specific genes. Not only does BRAF decrease NIS protein expression, but this oncogene also impairs NIS targeting to the follicular membrane both in vitro and in vivo, a finding consistent with the association between BRAF mutation and the fact that in a high frequency of thyroid cancer recurrences, the gland's ability to concentrate iodide has been wholly lost. Researchers have therefore begun to look into treating thyroid cancer by re-inducing endogenous NIS expression, and therefore iodine uptake. Retinoic acid, a vitamin A derivative that plays a central role in differentiation and cell growth and is known to have tumor-inhibitory effects, has been partially effective in inducing NIS mRNA in thyroid cancer cell lines.^{31 48 49 50}

Thyroid function impacts many organs, including the prostate. A recent prospective analysis of iodine status and prostate cancer risk using data from the NHANES I Epidemiologic Follow-up Study found that men with low urinary iodine had a 1.33 increased age-adjusted risk for prostate cancer. In men with diagnosed thyroid disease, risk was increased 2.34, and a history >10 years of thyroid disease was associated with a 3.38 elevated risk of prostate cancer. Study authors concluded that thyroid disease and/or factors contributing to thyroid disease [e.g., iodine and/or selenium insufficiency] may be risk factors for prostate carcinogenesis.⁵¹

Gastric Disease on the Rise—Iodine Correlation?

Given iodine's antioxidant actions and the fact that the gastric mucosa is one of the areas in which the body concentrates iodine, it is not surprising that iodine deficiency has been linked to an increased risk of gastric carcinoma. One study demonstrated an increased prevalence of gastric cancer and an increased risk of atrophic gastritis in areas with a greater-than-average prevalence of iodine-deficiency related goiter. The researchers also reported that competitive inhibitors of iodine transport by NIS, such as nitrates and thiocyanate, increased the risk of gastric cancer.^{52 53}

In a Chinese cohort of 29,584 adults, self-reported goiter was significantly associated with upper gastrointestinal cancer, specifically, a 2.04 increased risk of gastric non cardia adenocarcinoma, and a 1.45 increased risk for gastric cardia adenocarcinoma (see Glossary).⁵⁴

Another study found a significant correlation between decreased mean urinary iodine levels and prevalence of stomach cancer, as well as a greater frequency of severe iodine deficiency in patients with stomach cancer (49%) than in controls (19.1%).⁵⁵ There is also evidence for lower levels of iodine in cancerous gastric tissue than in surrounding normal tissue.⁵⁶

Iodine Safety Issues

Iodine, per se, is not the Cause of Autoimmune Thyroiditis

Any suggestion of iodine intake at levels above the DRI is always met with concerns about higher levels of iodine causing autoimmune thyroiditis.

Autoimmune thyroiditis, a.k.a, Hashimoto's thyroidits, is characterized by infiltration of the thyroid gland by inflammatory cells and production of autoantibodies to thyroid-specific antigens, thyroglobulin and thyroperoxidase. Autoimmune thyroiditis accompanies and is considered a main cause of hypothyroidisim since it results in destruction and eventual fibrous replacement of thyroid follicle cells.⁵⁷

Although excess iodine intake has been singled out as the cause of autoimmune thyroiditis, current research clearly shows that this condition is multifactorial in etiology. Deficiencies of other key nutrients, genetic susceptibility, and exposure to environmental pollutants are all contributing factors. Iodine repletion without at least one of these other factors, is insufficient to cause autoimmune thyroiditis.

It is well recognized that increased iodine intake results in increased iodination of thyroglobulin, which, since this process also results in increased production of H202, increases thyroglobulin's antigenic potential. In addition, since H2O2 is one of the compounds known to stimulate the intracellular adhesion molecule-1 (ICAM-1) promoter to increase transcription of the ICAM-1 gene, increased iodine intake (which can result in increased levels of unquenched H2O2) can also upregulate expression of ICAM-1. Iodine therefore has significant potential for harm; however, for this potential to be actualized, at least one or more of a number of other contributing factors must be present. These include selenium deficiency, iron deficiency, and/or exposure to environmental pollutants.⁵⁸

Selenium deficiency: Selenium deficiency is widespread. Not only have the selenium contents of surface soils been depleted by erosion and glacial washout, similar to iodine, but the use of nitrate fertilizers (which typically do not replace trace minerals such as selenium in the soil, but do produce perchlorate, an iodide uptake inhibitor), compounds the problem.⁵⁹

Selenium is a necessary component of both superoxide dismutase and glutathione peroxidase, key enzymes for the iodination of iodide and for the neutralization of excess ROS produced during this process, including H2O2 and O2¯. As noted earlier, generation of H202 is the rate limiting step in thyroid hormone synthesis and is regulated by TSH. Thus, the combination of iodine and selenium deficiency, which results in higher levels of TSH and greatly diminished levels of glutathione peroxidase, most severely increases susceptibility of thyroid tissue to free radical damage, upregulated expression of ICAM-1, and activation of antibodies to thyroid peroxidase (TPO) and thyroglobulin.

Iodine repletion coupled with selenium deficiency sets up a situation in which H202 production increases while the balancing factors for its neutralization, selenium-dependent enzymes, are largely absent. Thus programs that rely on iodized salt to restore iodine levels without consideration of selenium sufficiency can promote increased ROS generation, which, particularly in genetically susceptible individuals, may result in enhanced expression of intracellular adhesion molecule-1 (ICAM-1) on thyroidal follicular cells, infiltrating mononuclear cells, and enhanced cytokine production.⁶⁰

In addition to serving as a co-factor for glutathione peroxidase and superoxide dismutase, selenium is an integral component of the thioredoxins, which are key players in a major cellular redox system that maintains cysteine residues in numerous proteins (including glutathione), in the reduced state, thus greatly reducing inflammation. Smoking has been clearly demonstrated to increase risk of thyroiditis. Cigarette smoking increases thiocyanate concentrations to levels that inhibit iodide transport. Plus, selenium concentrations in blood have been found to be significantly lower (and blood cadmium levels significantly higher) in smokers than in nonsmokers, indicating poorly controlled ROS and inflammation. Thioredoxin, which has been shown to inhibit the harmful effects of tobacco smoking in the lungs, is also produced in the thyroid gland—if selenium is present.^{61 62 63 64 58}

Recent clinical studies have documented the suppressive effect of selenium treatment on serum anti-thyroid peroxidase concentrations in patients with Hashimoto's thyroiditis} ⁶⁵ and a number of studies conducted in areas with different iodine and selenium exposures have shown that co-administration of selenium with levothyroxine markedly reduces anti-TPO antibody levels in patients with severe autoimmune thyroiditis, all of which suggests that the problem is not iodine, but the production of thyroid hormone without sufficient selenium for redox control.^{66 67 68 69 70}

Environmental Pollutants: Pollution from car emissions and heavy industry (including particulate emissions of such metals as lead and cadmium, solvents such as benzene and dioxane, as well as polychlorinated biphenyls) increases oxidative stress, increasing need for selenium-dependent antioxidant enzymes. Polychlorinated biphenyls have been shown to interfere with iodide transport.⁷¹ Substantially increased prevalence of anti-TPO antibodies is seen in populations living in areas heavily polluted with polychlorinated biphenyls, and the frequency of various signs of autoimmune thyroiditis (e.g., hypoechogenicity on ultrasound images, increased levels of TSH and the presence of anti- TPO antibodies), has been positively correlated with polychlorinated biphenyl levels.⁵⁸

Genetic susceptibility: Although the molecular mechanisms through which they induce thyroid autoimmunity have yet to be understood, a number of polymorphic genes strongly associated with significantly increased risk for autoimmune thyroiditis have been identified, some of which increase susceptibility to autoimmunity in general (e.g., the human leukocyte antigen gene [HLA], the cytotoxic T lymphocyte antigen-4 gene[CTLA-4], the tumor necrosis factor gene [TNF]), and others thought to be specific to autoimmune thyroid disorders (e.g., the TSH receptor gene [TSHR], and thyroglobulin gene [Tg]).^{72 73 74} Exposure to environmental pollutants combined with insufficient intake of co-factors necessary for normal thyroid metabolism exacerbates the potential for dysfunction significantly increasing risk of autoimmune thyroiditis in genetically susceptible individuals.^{58 59}

Iron—Another Trace Mineral Necessary for Normal Thyroid Hormone Metabolism

The initial steps of thyroid hormone synthesis are catalyzed by heme-dependent TPO. TPO activity is significantly reduced in iron deficiency anemia.⁵⁹

Extensive data from animal studies indicates that iron deficiency, with or without anemia, impairs thyroid metabolism, resulting in blunted TSH responses, lowered hepatic thyroxine- 5'-deiodinase activity, hepatic production of T₃ only 46% that of controls, and 20-60% lower serum levels of T₃ and T₄. In human studies, TSH signals were significantly increased, hepatic thyroxine-5'-deiodinase activity reduced, and serum T₃ and T₄ levels significantly decreased in individuals with moderate-to-severe Fe deficiency.⁵⁹

A series of clinical trials conducted in North and West Africa, in areas of endemic goiter with a high prevalence of iron deficiency anemia, have shown that iron supplementation so significantly improves the efficacy of iodine phrophylaxis that study authors have proposed dual fortification of salt with iodine and iron.⁵⁹

In summary, to claim that iodine per se is the cause of autoimmune thyroiditis and/or hypothyroidism is a gross oversimplification with the potential to cause significant public harm.

Iodine—Time to Consider Changing U.S. Recommendations for Daily Intake?

Although the U.S. Institute of Medicine limit for the tolerable upper intake level for iodine in adults is 1,100 mcg/day, dietary iodine intake in Asia is much higher. High iodine-containing seaweeds are frequently consumed and well tolerated by millions of people in Japan, Korea, and coastal China. In Japan, where seaweed intake averages ~ 4–7 grams/day (with some estimates as high as 10 gram/day), average dietary iodine intake was recently estimated to be 1.2 mg/day.⁷⁵ However, a study of 4,138 apparently healthy, euthyroid Japanese men and women found a mean urinary iodine excretion of 5,100 mcg/day, which translates to a daily intake of 5,500 mcg (~5.5 mg) I/day, and yet other research has estimated daily Japanese iodine consumption ranges as high as 13,800 mcg/day.^{76 11}

Regardless of which estimate of daily iodine intake among the Japanese we accept, their consumption of iodine is magnitudes higher than that in the U.S., where average daily consumption was estimated to be 167 mcg/day11, a number that recent studies suggest may be grossly overestimating actual intake, and certainly NIS uptake, of this trace mineral. Not only does the substantially higher intake of iodine among the Japanese appear to have no harmful effects in this population, but, on the contrary, incidence rates of autoimmune thyroiditis, hypothyroidism, benign and malignant breast disease, and prostate cancer are all dramatically lower among Japanese consuming an iodine-rich diet.^{76 77}

Whether such high levels of iodine intake might cause problems in the U.S., particularly in susceptible individuals (e.g., those with autoimmune thyroiditis), has not yet been the subject of research. However, a number of recent studies suggest iodine intake ranging from 495 mcg/day to as high as 6 mg/day may be beneficial.

A study of the impact of seaweed consumption on thyroid function in American women found that iodine intake of 495 mcg/day, an amount significantly higher than current U.S. DRIs, causes no harm. In this randomized, placebo-controlled crossover trial, 25 healthy postmenopausal women (average age 58 years), 10 of whom had a history of early (Stage I or II) breast cancer but were disease-free at the time of the study, and 15 who had never been diagnosed with breast cancer, were randomized to receive either 6 weeks of supplemental iodine (in the form of 10 seaweed powder capsules providing a total of 475 mcg of iodine/day) or placebo (maltodextrose in 10 identical gelatin capsules).⁷⁷

Since soy, a known goitrogen, is also commonly consumed in Japan, for 1 additional week, the women were also given high-isoflavone powder providing 141.3 mg of isoflavones and 67.5 g of protein/day in addition to seaweed or placebo capsules during the last week of each treatment arm.

Neither 7 weeks of seaweed nor 1 week of soy and seaweed supplementation affected thyroid end points. Seaweed supplementation was associated with a small increase in TSH, but values remained well within normal ranges. The women in this study, (who were already iodine-sufficient with well above average iodine intake for the United States since, during the control period at the beginning of the study, their mean UI was 266 mcg/day), excreted an average of 587 mcg of I/day while ingesting supplements providing 495 mcg of I/day.

As discussed earlier in this review, in the treatment of fibrocystic breast disease, Ghent, Eskin et al (1993), demonstrated the safety of therapeutic doses of molecular iodine (I2) of 3 to 6 mg/day over a period of 2-5 years,42 and a more recent (2004) trial evaluating the effects of varying dosages of I2 on fibrocystic breast disease (1.5, 3 or 6 mg/day) found that the 6 mg dose produced the best results: a 50% reduction in pain in 51.7% of women taking this dose with no adverse effects. No decreases in pain were seen in the groups receiving 1.5 mg or placebo.⁴³

In an editorial in the June 2006 issue of the New England Journal of Medicine, Utiger discusses the evidence, world-wide, for risk of iodine-induced thyroid dysfunction. He concludes that while milligram or higher doses of iodine may cause hypothyroidism in people with damaged thyroid glands, excessive iodine intake for 5 years (defined as urinary iodine >300 mcg by Teng et al., in their investigation of iodine levels and thyroid dysfunction in people living in three regions in China) has only been associated with slightly increased cumulative 5 year incidence of subclinical hypothyroidism and autoimmune thyroiditis, both of which were not sustained in most people. Noting that, "Overall, the small risks of chronic iodine excess are outweighed by the substantial hazards of iodine insufficiency," Utiger recommends that iodine intake be increased to at least 300 to 400 mcg daily.⁷⁸

Supplement Recommendations

A reasonable clinical takeaway from all the data presented in this review overall is that iodine supplementation in amounts ranging from 400 mcg/day to as high as 1 mg/day is likely to be safe and of benefit to most individuals, even those at risk for autoimmune thyroiditis, providing that co-factors of iodine metabolism (e.g., selenium, iron) are not deficient.

Healthy individuals are remarkably tolerant to iodine intakes up to 1 mg per day, as the thyroid is capable of adjusting to a wide range of intakes to regulate the synthesis and release of thyroid hormones. However, in those with damaged thyroid glands, doses of iodine in the mg range may cause hypothyroidism because normal down-regulation of iodine transport is disrupted.⁵⁹

Supplementation using I2 in dosages of 3-6 mg/day or higher may be of significant benefit to women with benign or malignant breast disease. With closely monitored physican care, therapeutic dosages ranging from12.5 to 50 mg/day or sometimes even higher, have been safely and effectively used.⁴²

It should also be noted that intense exercise may increase daily iodine requirements; a study of male university students in Japan found high iodine losses in sweat induced by athletic training.⁷⁶

In all individuals, the critical caveat accompanying iodine supplementation is that co-factors of iodine metabolism must also be present. While selenium, iron, and vitamin A play roles highlighted in current research, a number of other nutrients including zinc, copper, vitamin E, vitamin C and the B vitamins riboflavin (B2), niacin (B3) and pyridoxine (B6) are involved either in the manufacture of thyroid hormone or as cofactors of the deiodinases that convert T₄ to the far more active T₃.⁷⁹ Deficiencies of any of these nutrients can negatively impact the response to prophylactic iodine.⁵⁹

Finally, thyroid function should be carefully monitored in any program of iodine prophylaxis.

Assessment of Iodine Status

Urinary iodine concentration (UI) is a sensitive indicator of recent iodine intake (days) since more than 90% of dietary iodine is excreted in the urine. UI can be expressed as a concentration (mg/L), in relationship to creatinine excretion (mg iodine/g creatinine), or as 24-hour excretion (mg/day). Single random urine sampling is the standard accepted method of measuring iodine body stores in population studies, but multiple spot urine measurements or 24-hour urine collection is recommended for individual measurements as these obviously provide more precise evaluation. As noted earlier, according to WHO standards, 50-99 mcg/L indicates mild deficiency, 20-49 mcg/L indicates moderate deficiency, and <20 mcg/L indicates severe deficiency.⁸⁰

Thyroglobulin (Tg) may be preferable to UI since it is a much more convenient, simple blood test (Tg can also be assayed on dried blood spots taken by a finger prick), and provides a sensitive intermediate assessment (weeks to months) of iodine status. In iodine sufficiency, small amounts of Tg are secreted into the circulation, so serum Tg is normally <10 mcg/L.⁸⁰

TSH. Either UI or Tg is preferable to TSH, which is unreliable because serum TSH values often remain within the normal range in older children and adults when iodine is deficient.⁸⁰ Generally, a normal range for TSH for adults is between 0.4 and 5.0 uIU/mL (equivalent to mIU/L), but values vary slightly among labs. According to the U.S. National Academy of Clinical Biochemistry, the normal range for adults should be 0.4-2.5 uIU/mL since adults whose initial TSH level measures over 2.0 uIU/mL had "an increased odds ratio of developing hypothyroidism over the [following] 20 years."

Thyroid hormone concentrations are poor indicators of iodine status. In iodine-deficiency, serum T₃ can remain unchanged or increase, and serum T₄ usually decreases; however, these changes often remain within the normal range.⁸⁰

Conclusion

Iodine deficiency is of concern not just in Europe and developing nations, but in the U.S. As with vitamin D and the omega-3 fatty acids, two other nutrients recently recognized as deficient in the Western diet, sub-clinical iodine deficiency may soon be found to be a significant contributing factor to declining health in the U.S. Sufficiency of not only iodine, but key co-factors in its metabolism, including selenium, iron and vitamin A, should be ensured in any protocol for healthy aging.

Read Part I: Iodine: the Next Vitamin D? Americans at High Risk for Iodine Insufficiency

Glossary

Balneotherapy: treatment of disease by bathing.

Gastric cardia adenocarcinoma: the gastric cardia is a small anatomical region in the proximal 2-3 cm of the stomach that is especially susceptible to overgrowth by tumors originating from adjacent mucosal sites. Tumors occurring in this region are referred to as gastric cardia adenocarcinomas, gastric tumors arising in the lower esophagus or body of the stomach are labeled noncardia adenocarcinomas. Interestingly, H. pylori is a strong risk factor for noncardia gastric cancer but is inversely associated with the risk of gastric cardia cancer.⁸¹
Iodate: a salt of iodic acid. Iodic acid contains iodine in the oxidation state +5, and is one of the most stable oxo-acids of the halogens in its pure state.

Iodide: A form in which iodine is stored as a single, negatively charged ion. It has recently been shown that iodide, as it is a reducing species that, through the activity of peroxidase enzymes, can detoxify reactive oxygen species such as hydrogen peroxide, functions as an antioxidant.⁸²

Wolff-Chaikoff effect: Named after its discoverers, Wolff and Chaikoff, who reported in 1948 that organic binding of I¯ (i.e., I¯ organification) in the rat thyroid in vivo was blocked when I¯ plasma levels reached a critical high threshold. Since iodide organification resumed when plasma levels fell, Wolff and Chaikoff hypothesized this effect could be the mechanism by which administration of high iodine doses results in remission of Graves’ disease.⁸³

In 1949, Wolff et al. reported that the maximum duration of the inhibitory effect of high concentrations of iodide on its organification was 50 hours in the presence of continued high plasma I¯ concentrations. However, as early as 2 days after onset of the acute effect, an escape or adaptation from the effect occurred, so that the level of organification of I¯ was restored and normal hormone biosynthesis resumed. 

In 1963, Braverman and Ingbar investigated the mechanism underlying the escape from the acute Wolff-Chaikoff effect in rats. They found that the Wolff-Chaikoff effect and the ensuing escape constitute a highly specialized intrinsic autoregulatory system that protects the thyroid from the deleterious effects of I¯ overload, but, at the same time, ensures adequate I¯ uptake for hormone biosynthesis. The level of I¯ capable of inhibiting I¯ organification and concomitantly stopping thyroid hormone synthesis is determined by the ratio of organified to nonorganified intracellular I¯ content, which in turn depends on the previous iodine supply status of the individual.⁷

The mechanism underlying the inhibition of iodide organification by high levels of iodide remains poorly understood, although it has been hypothesized that it is mediated by iodolipids since these inhibit TSH-mediated adenylate cyclase activity.³¹

Organification: The term "organification" refers to the incorporation of iodide (I¯) into organic molecules, as opposed to non-incorporated, inorganic, or free I¯. Organification of iodide (I¯) occurs in a complex reaction in the thyroid follicular cell during which I¯ is oxidized by thyroid peroxidase (TPO, a heme-dependent enzyme) using H2O2 to form I2, which then binds to tyrosine residues in the thyroglobulin molecule to form the iodotyrosine residues, MIT and DIT that are the precursors of thyroid hormones, T₄ and T₃.⁷

Thalassotherapy: treatment of disease with seawater.

Authors

Lara Pizzorno, MDiv, MA, LMT, a member of the American Medical Writers Association with 25+ years of experience writing for physicians and the public, is Managing Editor for Longevity Medicine Review as well as Senior Medical Editor for SaluGenecists, Inc. Recent publications include: contributing author to the Textbook of Functional Medicine, (IFM, 2006), a number of articles for Integrative Medicine: A Clinician's Journal (Innovisions Communications, Inc., 2005 through present), and Textbook of Natural Medicine (Elsevier, 2005, e-dition through present); lead author of Natural Medicine Instructions for Patients (Elsevier, 2002); co-author of The Encyclopedia of Healing Foods (Scribner’s, 2005); and editor, The World's Healthiest Foods Essential Guide for the healthiest way of eating (George Mateljan Foundation, 2006 through present).

Chris D. Meletis, N.D., is an international lecturer and author of over a dozen books. He seeks to empower health care professionals and the public with the latest scientific medical findings as it relates to optimizing true wellness. He has served as Dean and Chief Medical Officer at the National College of Natural Medicine and currently serves as the Executive Director of Healthy Aging, www.TheIHA.org, his personal website is www.DrMeletis.com.

©2010 Smart Publications. All Rights Reserved. www.lmreview.com

Abstract

Despite the widely held assumption that Americans are iodine-sufficient due to the availability of iodized salt, the U.S. population is actually at high risk for iodine insufficiency. Iodine intake has been decreasing in the U.S. since the early 70s as a result of changes in Americans' food and dietary habits, including the facts that iodized salt is infrequently used in restaurant and processed foods, and iodized salt sold for home use may provide far less than the amount of iodine listed on the container's label. The widespread dispersal of perchlorate, nitrate and thiocyanate (competitive inhibitors of iodide uptake) in the environment blocks absorption of the little iodine Americans do consume, further compounding the problem.

In adults, iodine is necessary not only for the production of thyroid hormones, thus affecting systemic metabolism, but is now recognized to play a protective role against fibrocystic breast disease and breast cancer. In addition, a relationship has been hypothesized between iodine deficiency and a number of other health issues including other malignancies, obesity, attention deficit hyperactivity disorder (ADHD), psychiatric disorders, and fibromyalgia.

Analogous to the case of vitamin D, a nutrient for which the 400 IU RDI, although capable of preventing rickets, has been proven inadequate for this pro-hormone's numerous other functions in the body, the iodine RDI for adults of 150 mcg/day (220 mcg/day for pregnant women), while sufficient to prevent goiter (and cretinism), is inadequate for the promotion of optimal fetal brain development or optimal health in adults. Intake of 3-6 mg/day, an amount commonly consumed in Japan without increased incidence of autoimmune thyroiditis or hypothyroidism, may be necessary to support not only thyroid hormone production, but iodine's important antioxidant functions in the breast and other tissues in which this trace mineral is concentrated.

Part I of this article discusses the numerous factors that place Americans at high risk for iodine insufficiency. Part II reviews iodine's roles in the body, the relationship of iodine insufficiency to the above mentioned pathologies, available options in laboratory assessments of iodine levels, optimal intake, preferential forms of supplementation, and cofactors necessary for optimal iodine utilizati