Adrenal Fatigue: The 21st Century Stress Syndrome{title

In Adrenal Fatigue, Dr. James Wilson combines a researcher’s grasp of the latest scientific insights into the causes of adrenal dysfunction with an experienced clinician’s practical understanding of its real life impact on patients. The result is a book that successfully bridges the gap between the therapeutic lifestyle changes the research shows must occur to restore adrenal function and the support clinicians can provide to enable patients to actually make these changes.

Adrenal Fatigue is a condition in which the adrenals, due to chronic over stimulation, begin to fail and produce lower levels of cortisol even when stimulated by higher levels of ACTH (as in an ACTH stimulation test.) Wilson makes a very strong case that adrenal fatigue is an increasingly prevalent yet unrecognized response to the hyper-stress characteristic of the 21st century. Stressors – physical, emotional, psychological, environmental, and infectious – all contribute to adrenal fatigue. The adrenals respond to all types of stress in the same way. And the load is cumulative – if many smaller stresses occur simultaneously, accumulate or become chronic, and the adrenals have no opportunity to recover, adrenal fatigue is all too often the result.

Although adrenal fatigue (aka non-Addison’s or subclinical hypoadrenia) has been described in medical texts since the 1800s, few physicians have recognized the presence of this increasingly widespread problem. Why? Because the “normal” range for adrenal function on standard blood and urine tests includes all but the most severe cases of adrenal malfunction: Addison’s disease (extreme low cortisol and cortisone) and Cushing’s syndrome (extreme high cortisol and cortisone). And patients whose triggering complaint is actually adrenal fatigue typically present with a host of other diseases and syndromes, whose only apparent similarity is that fatigue is a primary symptom.

This diversity is not surprising since the hormones secreted by the adrenals influence all major physiological processes in the body, closely affecting utilization of carbohydrates and fats, conversion of fats and proteins into ATP, fluid and electrolyte balance, the distribution of fat (fat stored as visceral adipose tissue and along the sides of the face is an indication of dysfunctional adrenals), blood sugar regulation, cardiovascular and gastrointestinal function.

In addition, some hormones secreted by the adrenals have anti-inflammatory and anti-oxidant protective activity and also help control allergic and other adverse reactions to alcohol, drugs, foods and environmental allergens. Furthermore, the adrenals become the major source of sex hormones after mid-life (in men as well as women, although andropause occurs a bit later and the decline in hormone production is more gradual.) And, as every athlete knows, muscular strength and stamina are acutely affected by adrenal hormones, i.e., steroids. Lastly, susceptibility to certain types of diseases and ability to respond to chronic illness is significantly influenced by the adrenals. The more chronic the illness, the more critical adequate adrenal response becomes.

The many diverse manifestations of adrenal fatigue

The diverse roles played by adrenal hormones explain why adrenal fatigue can be the underlying cause of such a wide range of symptoms and conditions, including:

•    Fatigue—the hallmark symptom
•    Decreased immunity
•    Functional hypoglycemia
•    Weight gain – the temporary increase in cortisol levels produced by driving the adrenals with fast food and caffeine causes people with chronically low cortisol to put on weight since even a temporary excess of cortisol causes visceral fat deposition
•    Food and environmental sensitivities and allergies
•    Rheumatoid arthritis
•    Premenstrual tension
•    Menopausal symptoms
•    Mental disturbances – increased anxiety, depression, difficulty concentrating, less acute memory recall
•    Insomnia
•    Frequent respiratory infections – chronic and recurrent bronchitis, pneumonia and other chronic lung and bronchial diseases typically have an adrenal fatigue component. Adrenal fatigue is often precipitated by recurring bouts of bronchitis, pneumonia, asthma, sinusitis, or other respiratory infections. The more severe the infection, the more frequently it occurs or the longer it lasts, the more likely the adrenals are involved. If there are other concurrent stresses, like an unhappy marriage, poor diet or stressful job, the downhill slide is steeper and faster.
•    Extended recovery period --longer than normal recovery period with decreased stamina and excess fatigue is a strong indication of adrenal fatigue.
•    Asthma
•    Fibromyalgia & Chronic Fatigue Syndrome -- using recently developed polymerase chain reaction tests, infectious microorganisms have been identified as contributing factors.   These pathogens exert a tremendous drain on adrenal resources.
•    Type 2 Diabetes
•    Auto-immune disorders
•    Alcoholism –the hypoglycemia that results from adrenal fatigue predisposes to a compulsive desire for alcohol

The many diverse causes of adrenal fatigue

Some professions, notably the medical profession, are harder on the adrenal glands. Insurance company actuarial tables show that physicians, on average, die approximately 10 years earlier, have higher rates of alcoholism and several times the drug addiction rates of the “normal” population. If Wilson’s enumeration of key lifestyle factors that promote adrenal fatigue –immediately following – rings too many bells, please read this book!

•    Insufficient sleep
•    Using food and drink stimulants when tired
•    Staying up late when fatigued
•    Constantly driving yourself
•    Trying to be perfect
•    Lack of enjoyable and rejuvenating activities
•    Poor food choices
•    Staying in no-win situations over time

Other professionals at increased risk include the police, secretaries and teachers, and middle executives, all of whom live in the middle, taking the blame when things go wrong but lacking the control to make them go right. Those “in the middle” often present with Syndrome X (glucose intolerance, high triglycerides, low HDL cholesterol, insulin resistance, hypertension, central obesity, accelerated atherosclerosis). All these symptoms reflect the effects of chronic stress that produces elevated cortisol levels. And there are also congenital differences in adrenal resiliency. Children born to mothers suffering from adrenal fatigue or who experience severe stress in the womb typically have lower adrenal function.

The many diverse signs adrenal fatigue

If any of your patients are suffering from a chronic disease and morning fatigue is one of their symptoms, the adrenals are probably involved. If a patient presents with three or more of the following signs and symptoms, adrenal fatigue is highly likely:

•    Difficulty getting up in the morning
•    Continuing fatigue not relieved by sleep
•    Craving for salt or salty foods
•    Lethargy
•    Increased effort to do everyday tasks
•    Decreased sex drive
•    Decreased ability to handle stress
•    Increased time to recover from illness, injury or trauma
•    Light-headed when standing up quickly
•    Mild depression
•    Less enjoyment or happiness with life
•    Increased PMS
•    Symptoms increase if meals are skipped or inadequate
•    Rely on caffeine and sugar to keep going
•    Thoughts less focused, more fuzzy
•    Memory less accurate
•    Decreased tolerance
•    Don’t really wake up until 10am
•    Afternoon low between 3:00 and 4:00 pm
•    Feel better after evening meal
•    Decreased productivity

While Wilson’s focus in Adrenal Fatigue is non-Addison’s or subclinical hypoadrenia, this book could have been marketed as a treasure trove of tools to enable identification of the specific underlying causes of an individual patient’s physiological dysfunctions and the establishment of a targeted curative lifestyle. A self-help manual for the person suffering from adrenal fatigue – both in diagnosing and treating the condition -- the book is arranged sequentially, each part preparing the reader for what follows.

Organizational structure of the book

Part I provides an overview of adrenal fatigue: what it is, what causes it, who is likely to suffer from it, how it progresses and why medicine has not yet recognized it as a syndrome.

Part II contains a questionnaire (available free on-line at http://www.smart-publications.com/take-the-adrenal-fatigue-questionnaire/) and other tactics, such as a Health History Timeline and iris contraction and postural low blood pressure tests, which will help identify whether adrenal fatigue is present, its degree of severity, and what might be causing it. In this section, Wilson also provides an excellent review of laboratory tests for adrenal fatigue discussing not only his preferred option, saliva hormone testing, but problems with the interpretation of standard laboratory tests and ways to still get useful information from these tests.

Part III is an unbelievably comprehensive guide to restoring healthy adrenal function. It identifies virtually every possible hidden source of stress that can drain the adrenals, gives a very detailed description of effective therapeutic options, including a number of stress management techniques; a full dietary program; dietary and botanical supplements; and replacement hormones -- and ends with a Q&A section responding fully to patients’ frequently asked questions. And more – it’s just too much to list here.

Wilson’s discussion of the connection between adrenal fatigue and low blood sugar, exemplifies the thoroughness of his approach. This section includes not just an explanation of why high cortisol levels induce weight gain, what –and what not -- to eat and drink, and why, but also when to eat, key nutrients in foods, the glycemic index, and a handy summary listing patients can print out for a quick reminder.

Part III also includes a section on food allergies and sensitivities that provides the most complete and useful write up of how to track and identify hidden food allergies and sensitivities I have yet to read in my 30+ years as a medical editor. Adrenal Fatigue is well worth a spot on your office reference shelf just for this section.

Part IV is an overview – in patient-friendly English – of the impact of the adrenal glands on physiological function that clearly explains why even mildly impaired adrenal function can have such widespread and dramatic repercussions.

Adrenal Fatigue: The 21st Century Stress Syndrome is a classic. It will serve as an invaluable resource for both patients and clinicians alike.

Abstract

Sufficiency of vitamin K, as K2 specifically, is gaining recognition as a requirement for long term health in many more areas of human physiology than previously realized. The breaking research is revealing a number of roles for vitamin K reaching well beyond coagulation to not only long term cardiovascular and skeletal health, but that of the brain and nervous system, and also for insulin production and sensitivity, and genomic stability / cancer prevention.

An increasing number of aging-associated diseases are now recognized to be related to vitamin K2 insufficiency. Vitamin K1 is not commonly deficient: the vitamin K–dependent (VKD) clotting factors, which are carboxylated (activated) by K1 in the liver, have been found to be fully carboxylated in the healthy population. However, evidence indicates that vitamin K2 is commonly deficient; even in all healthy humans tested, a significant percentage of the extra-hepatic VKD proteins, which are activated by vitamin K2, e.g., osteocalcin and matrix Gla-protein, are only partly carboxylated and therefore inactive. Increasing vitamin K intake has been shown to increase carboxylation of extra-hepatic VKD proteins in apparently healthy adults, as well as those with osteoporosis and atherosclerosis. This indicates that current intake (and RDA) values for vitamin K are too low to support proper function of extra-hepatic Gla-proteins. Foods containing K2 in the Western diet are concentrated sources of calories, cholesterol and saturated fat, thus supplementation is advisable.

As a supplement, vitamin K is available in its K1, MK-4 and MK-7 forms. Until recently, the majority of the research investigating vitamin K’s potential for the prevention and treatment of age-associated diseases has been conducted using pharmacological doses of supplemental K1 or MK-4. However, the latest studies indicate that much smaller doses of MK-7 in combination with K1—both in amounts potentially derived from the diet—may be preferable for a number of reasons. These topics are the primary focus of this article following a brief summary of the mechanism(s) of action through which vitamin K exerts its key protective functions; an examination of why needs for vitamin K (both as K1 and K2), and other micronutrients, increase with age; and a review of the expanding list of age-associated diseases for which K2 insufficiency is being revealed to be an important contributing factor.

Introduction

Similar to “Vitamin E,” “Vitamin K” is not a single substance, but a family of structurally similar, fat-soluble compounds that share a naphthoquinone ring structure with a methyl group at position 2 and an aliphatic side chain at position 3. In nature, vitamin K appears primarily in two forms: phylloquinone (K1), which has an aliphatic side chain of four prenyl residues, the first of which is unsaturated, and the menaquinones (K2), in which the number of prenyl residues may vary from 4 to 13, and all are unsaturated.¹ (K3, the 2-methylnaphthoquinone ring without a side chain, is a synthetic analogue used to potentiate cancer chemotherapy and is banned from use in nutritional supplements. ²

Vitamin K₁ (phylloquinone). Both the K1 and K2 forms of the vitamin contain a functional napthoquinone ring and an aliphatic side chain. Phylloquinone has a phytyl side chain.

Vitamin K₂ (menaquinone). In menaquinone, the side chain is composed of a varying number of isoprenoid residues.

Vitamin K1, a single compound of plant origin found in green leafy vegetables (notably kale, spinach, broccoli, and Brussels sprouts, thus its Latin name “phylloquinone” [phylo = leaf]) and some vegetable oils [e.g., rapeseed, olive, soy, canola oils]), is the primary form of vitamin K in the Western diet.

Vitamin K2, of bacterial origin, refers to the series of menaquinones that share a common methylated napthoquinone ring structure (thus their mena prefix), but differ in the length and degree of saturation of their aliphatic side chain. In humans, the most common menaquinones (MK) are the short-chain MK-4, which is produced endogenously via systemic conversion of K1 to MK-4, and the long-chain MKs, MK-7 through MK-10, which are synthesized by intestinal bacteria in all mammals, including humans. Both MK-4 and the long-chain MKs are present in the diet in minute (mcg) amounts (see Table 1: Micrograms of Vitamin K Present in Foods). Different bacteria synthesize side chains varying from 6 to 11 prenyl units (MK-6 through MK-11); the 4 unit menaquinone (MK-4) is, at most, a minor bacterial product. Nutritionally significant amounts of long-chain MKs also occur in the diet in a few fermented foods. ¹

The richest food source of K2 is the Japanese fermented soy bean food, natto, which is produced with Bacillus natto, a bacterium that converts K1 to MK-7. Outside Japan, cheese, which contains MK-8 and MK-9 and is another food produced with the help of bacteria, is the most important dietary source of K2. Proprioni bacteria, for example, which ferment Swiss Emmental and Norwegian Jarlsberg cheeses, produce MK-9. However, the concentration of K2 in cheese is 20 to 40-fold lower than that in natto. In the U.S., meats and eggs are the most common sources of K2, in the form of MK-4; the concentration of K2 in meat and eggs is >40-fold lower than that in natto. ⁵, ⁶, ⁷

Dietary intake of vitamin K is much higher in the Netherlands than the U.S. (250 mcg versus 80 mcg/day), presumably because of much higher vegetable consumption by the Dutch. In the Rotterdam study, dietary vitamin K intakes corresponding to the highest quartile were 370 mcg/day for K1 and 45 mcg/day of menaquinones (MK-7, MK-8, MK-9), which corresponds to consumption of 100 grams (~3 ounces) green vegetables and 100g (~ 3 oz) of cheese, respectively.

*Adapted from Schurgers LJ, Geleijnse JM, Grobbee DE, et al. Nutritional intake of vitamins K1 (phylloquinone) and K2 (menaquinone) in the Netherlands. J. Nutr. Environ. Med. June 1999;9(2):115–122. DOI: 10.1080/13590849961717

Vitamin K Protects against Degenerative Diseases of Aging by Activating Vitamin K-Dependent Proteins

Vitamin K plays an essential role in the prevention of degenerative age-associated disease due to its being the required cofactor for the enzyme, γ-glutamyl carboxylase, which converts protein-bound glutamate residues (Glu) into γ-carboxyglutamate (Gla) residues in the Gla- (aka vitamin K-dependent [VKD]) proteins. Their Gla-residues form the calcium-binding sites necessary for the biologic activity of these proteins. In the case of vitamin K insufficiency, the carboxylation reaction cannot proceed, and Gla-proteins are released into the circulation in an undercarboxylated form. Undercarboxylated Gla-proteins (also referred to in the literature as Glu-proteins) are inactive.

Fully carboxylated Gla-proteins are involved in numerous critical activities throughout the body, including blood coagulation (K1); bone metabolism, vascular repair, prevention of vascular calcification, cell division and proliferation, myelin development and signal transduction; insulin sensitivity and glucose metabolism (K2). K1’s anti-inflammatory actions also suggest a protective role for this form of vitamin K against age-related chronic diseases including osteoporosis, cardiovascular diseases, type 2 diabetes, osteoarthritis and Alzheimer’s disease. Key recent studies exploring these actions of vitamin K are discussed below under the subheading Vitamin K: A Key Player in the Longevity Game.

Vitamin K Needs in Apparently Healthy Humans Unmet for Extra-Hepatic Vitamin K-Dependent Proteins

Vitamin K1 deficiency, except in newborns, is virtually unknown. The vitamin K–dependent (VKD) clotting factors are all produced in the liver, and all are fully carboxylated (activated by vitamin K1) in the healthy population. ⁹ However, evidence suggests that vitamin K2 is a very common deficiency: in all apparently healthy humans tested, the extra-hepatic VKD (aka Gla-) proteins, osteocalcin and matrix Gla-protein, have been found to be only partly carboxylated (activated by vitamin K2). About 30% of the circulating osteocalcin (OC) occurs in its undercarboxylated form (ucOC) in healthy adults, and this percentage increases with osteopenia / osteoporosis.¹⁰, ¹¹, ¹² An analogous percentage of uncarboxylated matrix-Gla protein (ucMGP) is seen in healthy, and progressively unhealthy arteries. In arteries devoid of calcification and lipid or macrophage infiltration, ~30% of the MGP is uncarboxylated. ¹³ As lipid infiltration and inflammation increase, ucMGP not only accumulates in vesicular structures, but contributes to vessel wall calcification. In Stage III atherosclerosis, expression of ucMGP accelerates in the vessel wall, overwhelming the VKD–dependent carboxylation machinery due to lack of K2 reserves.⁹, ¹⁴ Increasing vitamin K intake has been shown to increase carboxylation of both OC and MGP in apparently healthy adults, as well as in adults with osteoporosis and atherosclerosis. This indicates that current intake (and RDA) values for vitamin K are too low to provide full carboxylation (and thus proper function) of extra-hepatic Gla-proteins.

Since the foods in the Western diet that deliver MKs – dairy, eggs and meat -- are concentrated sources of calories, cholesterol and saturated fat, K2 supplementation is advisable to promote full carboxylation of the VKD proteins required to prevent the increasingly long list of aging-associated diseases now recognized to be related to vitamin K2 insufficiency. ¹³, ¹⁵ As a supplement, vitamin K2 is available in its MK-4 and MK-7 forms: MK-4 as a synthetic version (menatetrenone), and MK-7 as the natural compound extracted from natto. Until recently, the majority of the research investigating vitamin K’s potential for the prevention and treatment of age-associated diseases (specifically, cardiovascular disease and osteoporosis) has been conducted using pharmacological doses of supplemental K1 (1-5 mg/day) or MK-4 (15 mg t.i.d., i.e., 45 mg/day). For a review of the principal studies, please see our earlier article, Vitamin D and Vitamin K Team Up to Lower CVD Risk, Part II: The Vitamin K Connection to Cardiovascular Health. However, the latest studies indicate that much smaller doses of MK-7 in combination with K1—both in amounts potentially derived from the diet—may be preferable for a number of reasons. These will be a primary focus of this article following a brief recap of the mechanism(s) of action through which vitamin K exerts its key protective functions; a discussion of why needs for vitamin K (both as K1 and K2), and other micronutrients, increase with age; and a review of the expanding list of age-associated diseases for which K2 insufficiency is being revealed to be an important contributing factor.

Vitamin K: A Key Player in the Longevity Game

Sufficiency of vitamin K, as K2 specifically, is gaining recognition as a requirement for long term health in many more areas of human physiology than previously realized. The breaking research is revealing a number of roles for vitamin K reaching well beyond coagulation to not only long term cardiovascular and skeletal health, but also that of the brain and nervous system, insulin production and sensitivity, and genomic stability / cancer prevention.

K2’s Regulation of the Body’s Use of Calcium Critical for Bone, Vascular and Kidney Health

Seventeen Gla-proteins have been identified, 7 of which are carboxylated by K1 in the liver, where K1 is preferentially utilized to carboxylate the Gla-proteins involved in regulating blood coagulation. The 10 Gla-proteins not synthesized in the liver (aka, the extra-hepatic VKD or Gla-proteins) are carboxylated by K2, and include two key regulators of calcium usage: osteocalcin (OC), which is primarily synthesized in, and required for calcium deposition in, bone, and plays a key role in the prevention of osteoporosis; and matrix Gla-protein, (MGP), which is primarily synthesized in cartilage and the vessel wall, and prevents calcium deposition in the vasculature and myocardium. Activated MGP, the strongest inhibitor of tissue calcification presently known, is essential for the prevention of coronary artery disease. ¹⁶, ¹⁷

In addition to osteocalcin and MGP, another recently discovered extra-hepatic, calcium-regulatory Gla-protein, the Gla-rich protein (GRP) -- is a circulating protein expressed and accumulated not only in cartilage and bone, but also in soft tissues, particularly the vascular system and skin. Gla-rich protein, which is just being investigated, appears to have the highest content of Gla residues of all the Gla-proteins, and thus an uncommonly high ability to bind calcium. High concentratioins of ucGRP have recently been identified at sites of pathological calcification in the skin (e.g., in pseudoxanthoma elasticum (PXE),an autosomal recessive disorder in which calcification of connective tissue leads to pathology in skin, eye and blood vessels; and dermatomyositis with calcinosis, a rare, often chronic autoimmune disease with onset during childhood that is characterized by weakness in proximal muscles and pathognomonic skin rashes), vasculature (arterial calcification) and kidneys (chronic kidney disease). ¹⁸

Vitamin K Insufficiency – A Risk Factor for Kidney Disease?

It has been repeatedly demonstrated that vascular calcification is present in patients suffering from advanced chronic kidney disease (CKD) to a far greater extent than in individuals with normal renal function; 50% of mortalities in patients with CKD undergoing dialysis treatment are related to vascular calcification. ¹⁹, ²⁰ On the other hand, 20% to 40% of the patients in most CKD cohorts do not develop detectable vascular calcification despite exposure to well-known environmental triggers, such as uremia, diabetes, and hyperphosphatemia. This suggests that naturally occurring vascular calcification inhibitors (i.e., MGP -- after activation by posttranslational γ-glutamyl carboxylation of its five glutamate residues, which requires K2) play an important role in preventing this disease process. ²¹

A recent study evaluating the association between circulating inactive MGP and all-cause mortality in 188 hemodialysis patients, compared with 98 age-matched subjects with normal renal function, supports this hypothesis. Even low levels of inactive MGP were found to increase all-cause mortality risk by 220% and risk of death from cardiovascular disease by 270%. Furthermore, patients with higher vascular calcification scores showed lower levels of inactive MGP. In 17 hemodialysis patients, daily supplementation with vitamin K2 for 6 weeks reduced ucMGP levels by 27%. ²¹

Patients with CKD are also now being recognized to have greatly increased susceptibility to fragility fracture as well as vascular calcifications, both of which are known to be related to vitamin K insufficiency. ²² These findings indicate that vitamin K insufficiency (particularly of K2, the form in which vitamin K activates extra-hepatic Gla-proteins) is an important contributing factor to the dysregulation of calcium usage that contributes to both lack of appropriate calcification in bone and calcium’s pathological deposition in the vasculature, including in the kidneys, where it is a key factor in the development and progression of chronic kidney disease.

Coumarins (vitamin K antagonists used as coagulation inhibitors) are now well known to be a risk factor for vascular calcification. In recently published studies, patients on coumarins have been found to have significantly higher levels of uncarboxylated /inactive MGP (ucMGP); plasma levels of ucMGP have been shown to increase significantly with the use of vitamin K antagonists and, conversely, to decrease after supplementation with vitamin K. ²³

K2 Supplementation Essential in Patients Supplementing with Calcium and Vitamin D

Vitamin K2 is essential to prevent adverse coronary and kidney outcomes in patients supplementing with calcium to prevent osteoporosis, especially if these patients are also being given vitamin D.

Two studies published April 2011 underscore the importance to patient outcomes of an awareness of K2’s role in regulating calcium deposition, and the requirement for K2 sufficient to balance the increased absorption of calcium that occurs when supplemental vitamin D is given. Both studies reported significant increased risk of adverse outcomes in women taking calcium supplements with or without vitamin D.

The first study, widely broadcast in the news after it appeared in the British Medical Journal, was a seven year, randomized, placebo-controlled trial of daily supplementation with calcium (1 gram) and vitamin D (400 IU) in 36,282 postmenopausal women in the Women’s Health Initiative (WHI) study. Meta-analysis of three placebo-controlled trials found calcium and vitamin D increased risk of myocardial infarction 24% and the composite of myocardial infarction or stroke 15%. The conclusion drawn by Bolland et al.: “A reassessment of the role of calcium supplements in osteoporosis management is warranted.” ²⁴

The second paper, published in the American Journal of Clinical Nutrition analyzed data collected on the same 36,282 postmenopausal women participating in the WHI, this time in relation to whether calcium plus vitamin D supplements increased risk of kidney stone formation. Unsurprisingly, a 17% excess in urinary tract stone incidence was noted in the women taking both supplements. ²⁵

Unfortunately, neither in the articles reporting these two studies, nor in the press feeding frenzy that occurred after their publication, was vitamin K’s role in regulating calcium even considered. Had it been, these highly negative outcomes could have easily been predicted – and more importantly, avoided.

It is well known that vitamin D boosts calcium’s absorption from the intestines and its re-absorption from the kidneys, thus greatly enhancing levels of available calcium within the body. Less widely known is that 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. ²⁶

As noted in Part II of our 2009 LMR review, Vitamin D and Vitamin K Team Up to Lower CVD Risk, one potentially adverse repercussion of this, 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 ucOC and ucMGP 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, and also overloads the kidneys, promoting stone formation. ²⁷

Vitamin D toxicity has been proposed to be the result of precisely such induction of vitamin K2 deficiency. ²⁷ 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, and protect the soft tissues from calcification, can no longer be performed. That warfarin, a coumadin derivative that induces a functional vitamin K deficiency, 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, supports this hypothesis. ²⁸ In addition, vitamin K alone has been shown to fully reverse the calcification induced by warfarin, 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. ²⁹

Vitamin K Insufficiency – A Risk Factor for Alzheimer’s Disease?

Recent studies have indicated an association between Alzheimer’s disease (AD) and vitamin K insufficiency. In 2005, a cross-sectional study of 100 Japanese women with AD (mean age, 79.8 years) and 100 age-matched community dwelling controls (mean age, 80.6 years), reported bone mineral density and serum concentrations of K1 to be significantly lower, and serum levels of ucOC significantly higher, in severely demented patients than in those with mild dementia. ³⁰

In 2008, AD patients’ poor K1 status was confirmed with the publication of a study looking at associations between vitamin K status and BMD in AD patients in the Netherlands. AD patients’ dietary intake of K1 was found to be less than 50% of that in age-matched controls, and patients in the early stages of probable AD had significantly lower vitamin K intakes than age- and sex-matched healthy participants, a difference that remained highly significant after data were adjusted for energy intakes. The authors suggested that vitamin K insufficiency could not only be a risk factor for AD, but could also contribute to its accelerated progression. ³¹

Other research has shown that warfarin, a coumarin derivative that prevents vitamin K recycling, leads to increased β-amyloid deposition in brain vessels. ³² (During gamma-glutamate carboxylation, vitamin K is oxidized into its epoxide form (KO), which is reconverted to vitamin K quinone (K) by the enzyme vitamin K epoxide reductase (VKOR). Derivatives of 4-hydroxycoumarin (including warfarin and acenocoumarol) specifically inhibit VKOR, thus preventing the recycling of vitamin K.) ³³

In a paper published in Medical Hypotheses in 2001, Allison proposed that Alzheimer’s disease (AD) is associated with vitamin K status, based on the observation that the circulating concentration of K1 is significantly lower, and incidence of AD significantly higher, in ApoE4 carriers than in those with other ApoE genotypes. ¹

This inverse relationship results from the fact that vitamin K in plasma is bound to chylomicrons and chylomicron remnants, which carry apolipoprotein E. Clearance of chylomicrons and their remnants from the circulation depends on ApoE’s binding to a hepatic receptor, and the rate at which this binding takes place is greatly influenced by ApoE genotype, occurring rapidly in people carrying one or two alleles for E4 and slowly in those whose genes encode E2. Thus ApoE genotype significantly impacts blood levels of vitamin K, which are highest in E2, intermediate in E3 and lowest in E4.

Not surprisingly, carriers of ApoE4 have been found to have significantly higher levels of undercarboxylated osteocalcin (ucOC), indicating extra-hepatic vitamin K deficiency. In confirmation of ApoE4’s deleterious effects on bone, women ≥ 65 years who carry the E4 allele are known to have a significantly increased risk of osteoporotic hip fractures compared with those bearing E2 or E3. ³⁴, ³⁵

ApoE4 carriers are also at greatly increased risk for brain atrophy, and this risk is compounded by cerebrovascular disease. Analysis of quantitative magnetic resonance imaging (MRI) findings on 396 surviving members of the National Heart, Lung and Blood Institute Twin study revealed that carriers of ApoE4 had significantly smaller brain volumes than those with other ApoE genotypes. The presence of both cerebrovascular disease and ApoE4 was associated with significantly greater brain atrophy than was seen with ApoE4 or cerebrovascular disease alone. The authors concluded that ApoE4 enhances the extent of brain degeneration by increasing the brain’s susceptibility to injury and/or impairing brain repair mechanisms.

A possible mechanism for ApoE4’s deleterious effects on the brain is insufficiency of vitamin K, a blood borne factor whose concentration is lowered by ApoE4, and whose actions in the brain improve neuron survival and repair after brain injury.

Vitamin K’s Neuroprotective Activities

That vitamin K, found in high concentrations in the brain as MK-4, is required for normal brain development and function has been demonstrated by that fact that maternal exposure to coumarin derivatives, particularly in the second trimester of pregnancy, causes central nervous system abnormalities and mental retardation in offspring. ³⁶, ³⁷

In the adult brain, vitamin K2 remains involved in sulfation, which is decreased in AD

K2 is required for the activity of brain galactocerebroside sulfotransferase (GST), an enzyme involved in the production of neuronal myelin sulfatides (the development of myelin involves sulfatide accumulation); brain sphingolipid metabolism; and the production of keratin sulfate (KS), levels of which are dramatically reduced in the cerebral cortex of AD patients compared to age-matched AD-free controls.

The KS are sulfated proteoglycans found within several cell types and on their surfaces, where they are components of adhesion molecules. A key KS, SV-2, is a major protein of synaptic vesicles, is structurally similar to neurotransmitter transporters, and is involved in acetylcholine transport. SV-2 is present in synapses in the normal brain, but lacking in cortical neurons in AD, consistent with the loss of synaptic function seen in AD.

Gas-6, another VKD-protein in the brain, is a product of growth arrest specific gene 6, and along with its tyrosine kinase receptor, Ark, is widely distributed throughout the central nervous system. Interaction of Gas-6 with Ark plays a protective role in the brain, both as a growth factor for Schwann cells (non-neuronal cells that maintain homeostasis, wrap around axons of motor and sensory neurons to form the myelin sheath, and provide support and protection for the brain's neurons) and by preventing neurons from entering apoptosis. That the VKD Gas-6-tyrosine kinase receptor can protect neurons against apoptosis is obviously relevant to AD, in which neuronal apoptosis plays a significant role.

Of note, the human body converts K1 to MK-4 in the brain—the form in which vitamin K is involved in brain sulfation. However, humans are unable to convert the hydrogenated K1 found in hydrogenated vegetable oils to MK-4 either in the brain or elsewhere. ³⁸

This deleterious effect of hydrogenation is particularly relevant in the U.S. since, as noted in our earlier article, Vitamin D and Vitamin K Team Up to Lower CVD Risk, Part II: The Vitamin K Connection to Cardiovascular Health, canola and soybean oils are the primary source of vitamin K in the American diet. Hydrogenation changes the K1 (phylloquinone) in these oils into dihydrophylloquinone, a form incapable of carboxylating VKD proteins. Since osteocalcin is also VKD-protein, one would expect bone health in aging adults to be among the long-term adverse outcomes of hydrogenation, and data from the Framingham Offspring Study (n=2,544, average age 58.5) confirm this hypothesis. Subjects with the highest intake of vitamin K1 from hydrogenated oils, the type of oil typically used in processed and fast foods, had the lowest BMD at the neck, hip and spine. ³⁹ The clinical take away: the Standard American Diet, in which processed and fast foods containing hydrogenated oils feature significantly, greatly increases risk of functional vitamin K deficiency. ⁴⁰

Vitamin K2 Insufficiency – A Risk Factor for Type 2 Diabetes?

In addition to a number of animal studies, several recent human studies have suggested an inverse association between vitamin K intake and type-2 diabetes:

Higher dietary K1 intake was associated with greater insulin sensitivity and improved glycemic status in the Framingham Offspring Cohort (~2,000 subjects) ⁴¹, and a post hoc analysis of a placebo-controlled trial of K1 supplementation (initially designed to look at K1’s effects on bone health), revealed that insulin resistance was significantly lower in supplemented older men, after three years of follow-up. ⁴²

To date, only one study has compared the effects of K1 versus K2 on type-2 diabetes, and this research looked at dietary, not supplemental vitamin K. A clear inverse association was found for dietary intake of K2, but not K1, which showed only a weak, non-significant trend in relation to type-2 diabetes. ⁴³

That dietary K2, but not K1, was found to be inversely related to type 2 diabetes is particularly intriguing given the primary dietary sources of K1 (leafy greens) and K2 (cheese, in which K2 is present as the longer chain MKs, MK-8 & MK-9), since diets higher in leafy greens are generally considered protective, while those higher in saturated fat and calories are thought to contribute to MetS and insulin resistance. ⁴⁴ As will be discussed in more detail below, that K2 (in the form of the longer-chain menaquinones, MK-7, MK-8, MK-9), remains systemically available to activate extra-hepatic VKD-proteins for 96 hours (4 days), while K1 is preferentially utilized to carboxylate hepatic clotting proteins, and both K1 and MK-4 are cleared within 8 hours, may help explain this outcome. ⁵

The Osteocalcin Connection to Insulin Sensitivity

The majority of studies have reported that levels of undercarboxylated osteocalcin (ucOC) increase with older age. (Much inactive circulating osteocalcin is partially carboxylated, but since partial carboxylation still results in its being inactive, this distinction is typically not made, and it is referred to as un- rather than under-carboxylated.) Historically, the primary focus of clinical data on osteocalcin (OC) has been its relationship to skeletal health, for which a consistent finding has been an inverse relationship between levels of ucOC and BMD. High levels of ucOC increase fracture risk; postmenopausal women with high ucOC have been found to have a 6-fold increased risk of hip fracture. ⁴⁵, ⁸ (ucOC is known to increase in postmenopausal women and has been negatively correlated with endogenous estradiol levels. Levels of ucOC have been shown to increase after bilateral oophorectomy in premenopausal women, and to decrease with hormone replacement therapy. ⁴⁶) Most recently, researchers have begun looking into OC’s relationship to energy metabolism.

Diabetes, a disease of low insulin secretion and/or insulin resistance and hyperglycemia, has now been shown to be associated with reduced serum OC levels in rodent models and human studies. The rodent model studies of Karsenty’s group jump-started investigation into OC’s effects on energy metabolism with their 2007 publication in Cell of ‘‘Endocrine regulation of energy metabolism by the skeleton.” In this paper, they were the first to suggest that bone, via osteocalcin, acts as an endocrine organ to influence glucose homeostasis. ⁴⁷

Karsenty’s initial “ah-ha” came from an observation that mice deficient in OC were a little fat. Further examination revealed that these mice owed their visceral plumpness to a number of glucose metabolism abnormalities: compared to wild type mice, OC-deficient mice had higher blood glucose, lower serum insulin, impaired glucose-stimulated insulin secretion (GSIS) and poor glucose tolerance. The low serum insulin was found to be the result of a near 50% decrease in pancreatic β-cell mass and insulin content. The OC-knockout mice also had reduced levels of serum adiponectin (an adipocytokine that promotes insulin sensitivity), suggesting a role for OC in insulin sensitivity as well as secretion. Karsenty et al’s research revealed that ex vivo (in rodent cell-based assays), ucOC, but not cOC stimulates CyclinD1 (a molecular marker of cell proliferation), insulin expression in β-cells and adiponectin (levels of which are higher in those with better insulin sensitivity and reduced in diabetes); and in vivo, in rodents, ucOC can improve glucose tolerance.

In humans, however, cOC appears to be the form in which OC improves glucose tolerance. ⁴⁸ In humans, type-1 and type-2 diabetes are associated with lower levels of total (fully carboxylated + partially uncarboxylated) OC. Among those with diabetes, higher total OC is associated with lower fasting glucose and HbA1c (glycosylated hemoglobin). In a study of 50 poorly controlled diabetic patients, Kanazawa et al. reported a decrease in the mean %ucOC with the achievement of improved glycemic control. ⁴⁹

Perhaps even more interesting, total OC also appears to be associated with glucose metabolism and insulin resistance in those without diabetes. A study in which 149 non-diabetic men were assessed, using the frequently sampled intravenous glucose tolerance test, reported a positive correlation between higher levels of total OC and insulin sensitivity. In a study of older Swedish men, higher levels of OC also negatively correlated with fasting glucose, fasting insulin and HOMA-IR in those without diabetes. ⁴⁶ (HOMA, the homeostatic model assessment, is a method used to quantify insulin resistance [HOMA-IR].)

The post hoc analysis of the 3-year trial initially designed to determine the effect of vitamin K supplementation on bone loss (noted above) evaluated the effects of vitamin K’s increasing carboxylation of OC (lowering levels of ucOC) on glucose and lipid metabolism. Levels of ucOC dropped in participants (N = 355) randomized to receive 500 mcg/day of K1, compared to the placebo group. Over the 36-months of the study, mean %ucOC dropped ~19% in supplemented men and women. Measurements of fasting plasma glucose and insulin, and calculated HOMA-IR, were compared at the baseline, 6-month and 36 month visits. At 36 months, men in the vitamin K-treated group, but not the women, showed improved insulin sensitivity based on HOMA-IR, compared with placebo.⁴² Regarding the lack of effect on insulin sensitivity in women, Motyl et al. suggest that these were healthy women, so changes in ucOC with vitamin K supplementation may have been too small to produce significant differences in insulin sensitivity over 3 years. ⁴⁶

In an analysis of the placebo group from this same trial of vitamin K supplementation that reduced ucOC levels, Shea et al. considered whether baseline levels of OC, ucOC or carboxylated (c)OC predicted changes over 3 years of follow-up in HOMA-IR, fasting insulin and fasting glucose. Lower baseline levels of cOC and higher levels of %ucOC were revealed to have accurately predicted greater increases in insulin resistance. ⁵⁰

In sum, human studies are reporting an association between lower total and %cOC and impaired glucose metabolism and insulin resistance. Diabetes (type-1 and type-2) is associated with lower total OC, and total OC is negatively correlated with HbA1c, fasting glucose and insulin resistance. Lower baseline levels of cOC and higher levels of %ucOC predict greater increases in insulin resistance. Improved glycemic control appears to increase total OC, but not ucOC. ⁵¹, ⁵², ⁵³, ⁴⁶

Current thinking is that the acidic conditions (low pH) created by the osteoclastic resorption process activate osteocalcin inside the bone, which is then released from bone to stimulate pancreatic insulin secretion. ⁵⁴ Additionally, it has very recently been discovered that human adipose tissue also produces osteocalcin, adding further complexity to its role in energy metabolism. ⁵⁵ In contrast to the findings reported in regard to OC’s relationship to insulin sensitiivity, Foresta et al. not only found a lower ucOC/cOC ratio in the overweight and obese patients, but also detected expression of osteocalcin mRNA in subcutaneous and omental adipose tissues (aka, visceral adipose tissue), and documented that adipose tissue releases, in vitro, both ucOCN and cOCN. Further studies are needed to clarify the precise regulation of OC carboxylation and release from adipose tissue in obese vs. normal-weight subjects, taking into account factors that influence adipose tissue function, e.g., inflammatory cytokines.

In their paper in Cell, Karsenty et al. raise the teleological questions: “Why would a bone-specific hormone regulate energy metabolism? What is the need for a hormone favoring β-cell proliferation and insulin secretion?” ⁴⁷ They speculate that given the large surface covered by the skeleton, it is an excellent site of hormone synthesis, which suggests that other hormones remain to be identified in osteoblasts. Alternatively, OC (and possibly other hormones) may have been recruited to the skeleton through tinkering during evolution, and the pro-proliferation function of OC may have been required during evolution to maintain the constant size of pancreatic islets in periods of food deprivation.

We would like to suggest that OC’s β-cell proliferation and insulin-sensitizing functions help ensure the availability of ATP for the production of bone and its strengthening in response to muscle contractions, an energy-consuming process.

The next steps will be to determine precisely how osteocalcin -- as cOC and/or ucOC -- interacts with pancreatic β- cells. Are osteocalcin-specific cell surface receptors present on pancreatic β-cells? If so, what are their affinities for cOC and ucOC? Regardless, vitamin K, as both K1 and K2, is highly likely to be found to play an important role.

K1’s Anti-Inflammatory Actions May Promote Insulin Sensitivity

In addition to reducing %ucOC (via its endogenous conversion to MK-4), K1 also reduces inflammation, which may have a separate beneficial effect on insulin sensitivity since inflammation suppresses osteoblast activity and OC expression. ⁵⁶

In cell culture, animal and human studies, respectively, vitamin K has been shown to: reduce lipopolysaccharide-challenged fibroblasts’ secretion of the pro-inflammatory cytokine IL-6 in vitro; suppress expression of genes involved in an acute inflammatory response in an animal model; and lessen inflammatory responses in certain disease states, as evidenced by significantly lower levels of a number of systemic pro-inflammatory biomarkers in subjects in a Framingham Offspring Study cohort with higher vitamin K plasma concentration or K1 intake. ⁵

Interestingly, spaceflight, known to cause bone loss via suppression of osteoblast activity and OC expression, also decreases insulin secretion and increases blood glucose levels. ⁴⁶, ⁵⁷

MK-7 Likely More Effective in Carboxylating Osteocalcin than K1 or MK-4

MK-7 has been shown to have significantly greater efficacy than K1 in carboxylating OC when taken as a daily supplement ( 0.22 μmol/day), and this is thought to be due to MK-7’s much longer residence time and the higher serum concentrations of MK-7 achieved during its prolonged intake. ⁹ For the same reasons, MK-7 is likely to be more effective in carboxylating OC than MK-4, since MK-4 and K1 share comparable molecular structures (both contain 4 isoprenoid residues, 3 of which are saturated in K1 but contain a double bond in MK-4) and clearance rates. The longer-chain menaquinones, including MK-7, are much more hydrophobic, are handled differently, and have much longer half-life times (8 hours for K1 and MK-4 versus 96 hours for MK-7). ⁵⁸

Vitamin K2 Insufficiency – A Risk Factor for Cancer

Other recent studies indicate K2 (as the long chain menaquinones, MK7, MK-8, MK-9; not MK-4) may play a highly protective role against cancer. In 2008, Nimptsch et al. analyzed data from the EPIC-Heidelberg cohort and found that men in the highest quartile of dietary K2 intake (46 mcg/day) had ~50% lower prostate cancer incidence and mortality compared to those in the lowest quartile of K2 intake. No association was found between K1 intake and prostate cancer. As noted above, ⁵, ⁶ cheese, which provides the longer-chain menaquinones (MK-8 and MK-9), is the primary source of dietary K2 in this population. ⁵⁹ Most recently, in a paper published in 2010, Nimptsch et al. extended their analysis of the EPIC-Heidelberg data to other cancers and found all major forms of cancer, except breast cancer, were inversely associated with K2 intake. ⁶⁰

Two VKD-proteins, Tgfbi and periostin, both of which are involved in maintaining genomic stability during cell division, may help explain K2’s cancer-preventive effects. ⁶¹, ⁶²

Earliest Indicator of Vitamin K Insufficiency: Incomplete Carboxylation of Extra-Hepatic VKD-Proteins

As discussed by Ames and McCann in their recent seminal paper proposing nutrient “triage” as a major factor in the development of age-related disease, a key distinction between the hepatic and extra-hepatic Gla-proteins is that the former are fully carboxylated in healthy adults, while the latter are not. “Triage theory” proposes that the reason for this disparity is an evolutionary mechanism via which available nutrients are not equally distributed throughout tissues, but are preempted by those areas in which their use meets the body’s most pressing, immediate survival needs. In relation to vitamin K, triage supports immediate survival by safeguarding against a terminal bleed out, but results in suboptimal vitamin K status in extra-hepatic tissues, which are deprived of the activated VKD proteins required for maintenance of long-term bone and vascular health. ⁶¹

After its absorption in the small intestine, where all vitamin K is incorporated into chylomicrons -- which form in the small intestine in response to dietary lipid intake, are modified into smaller chylomicron remnants, and rapidly shunted to the liver -- vitamin K is efficiently extracted and used to carboxylate the hepatic VKD proteins of the clotting cascade. This “triage effect” promotes subclinical vitamin K insufficiency since only after these Gla-proteins are fully carboxylated is any remaining vitamin K1 converted in the liver to MK-4, incorporated into low-density lipoproteins, and secreted from the liver into the bloodstream for delivery to extra-hepatic tissues*. In contrast, the longer-chain MKs (MK-7,MK-8, MK-9) derived from the diet are preferentially used to activate the extra-hepatic Gla-proteins needed to build bone, prevent arterial calcification, promote the production of myelin and other neuroprotective compounds, and help avert cancer.

*K1 not required for full carboxylation of VKD clotting proteins may also be sent back into the circulation in triglycerides and locally converted into MK-4 via the activity in certain tissues (brain, bone, adipose tissue) of a recently identified prenylation enzyme called UbiA prenyltransferase containing 1 (UBIAD1). ⁶³

Thus, elevated levels of undercarboxylated osteocalcin (ucOC) or MGP (ucMGP), which have been conclusively associated with osteoporosis, and cardiovascular disease and mortality, respectively, serve as early warning indicators of subclinical vitamin K2 deficiency. Since low K2 intake has also been shown to be associated with increased incidence of, and mortality due to, hepatocellular and prostate cancers, elevated levels of two other uncarboxylated Gla-proteins, ucTgfbi and ucPeriostin (Tgfbi, a VKD protein required to maintain genomic stability during mitosis, binds to periostin, another VKD protein, which is therefore also necessary for Tgfbi’s stabilizing effects on the mitotic spindle) may also serve as early warning signals of increased cancer risk due to vitamin K2 insufficiency. ⁶¹, ⁶², ⁶⁴

Micronutrient Requirements Increase with Age

In addition to triage, aging, in itself, constitutes an indication for increased vitamin requirements. In earlier research discussed in detail in Beyond the Mitochondrial Tune Up: Part I Ames demonstrated that the function of aging mitochondria can be rejuvenated by increasing the availability of nutrients that serve as the substrates or co-factors for key enzymes. This phenomenon is due to the fact that with age, increased oxidative damage to proteins causes increasing, albeit typically slight, deformations in enzymes’ structure, which results in decreased binding affinity for their co-enzyme (i.e., the nutrient co-factor for the enzyme), and thus a decrease in enzyme function.

Another way of stating this is that elders’ enzymes have an increased Michaelis constant/(Km), which is defined as the substrate or co-factor concentration required for half-maximal enzyme activity. The diminished kinetics of enzymes that occur with aging can be counteracted by supplying greater amounts of the co-factor and/or substrate, thus restoring the velocity of enzymes’ activity (Km) -- not only in the mitochondria, but, in the case of the VKD Gla-enzymes, in systemic cellular mitosis, bone, the brain and the vasculature.

VKD Proteins Highly Vulnerable to “Kinetics of Aging”

Furthermore, as pointed out by Vermeer and Theuwissen (2011), ⁶⁵ the VKD proteins are particularly vulnerable to “the kinetics of aging” because vitamin K metabolism is affected by an increased Km requirement for more than one enzyme. Vitamin K acts as both a co-factor for γ-glutamyl carboxylase, and as substrate for DT-diaphorase and vitamin K epoxide reductase (VKOR), the enzymes that recycle vitamin K epoxide into its active hydroquinone form. ³³

As noted in Vitamin D and Vitamin K Team Up to Lower CVD Risk, Part II: The Vitamin K Connection to Cardiovascular Health, vitamin K is so critical to survival that the body very efficiently conserves this micronutrient via a cyclic interconversion called the vitamin K cycle. In this recycling process, vitamin K’s quinone form is reduced by the FAD-containing enzyme DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase ) into vitamin K hydroquinone (KH2), which then serves as the cofactor for the 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 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.

The fact that three enzymes are involved in its metabolism renders vitamin K particularly susceptible to the “kinetics of aging.” This translates into an even greater increase in our requirements for vitamin K to maintain optimal function as we age.

K2, Particularly the Longer Chain Menaquinones, Crucial for Healthy Aging

K2, But Not K1, Carboxylates the VKD-Proteins that Prevent Chronic Age-Associated Disease

Several recently published papers confirm that K2, and specifically the longer-chain menaquinones (MK-7, MK-8, MK-9), but not K1, provide effective protection against cardiovascular disease and osteoporosis.

Gast and de Roos, et al. (2009), ⁶⁶ examined the relationship between dietary intake of K1 and K2, and K2 subtypes, and CHD incidence in the Prospect-EPIC cohort, which consisted of 16,057 women aged 49-70 years, free of CVD at baseline and followed an average of 8.1 years. Mean vitamin K1 intake was 211.7 +/- 100.3 mcg/d; vitamin K2 intake was 29.1 +/- 12.8 mcg/d. After adjustment for traditional risk and dietary factors, K1 intake was not found to be related to CHD, despite the fact K1 not used for clotting factors in the liver is converted to MK-4; but a significant inverse association was seen between dietary vitamin K2 intake and CHD risk, with a Hazard Ratio (HR) pf 0.91 per 10 mcg/d vitamin K2 intake. Furthermore, and perhaps most importantly, this association was principally due to intake of the longer-chain menaquinones, MK-7, MK-8 and MK-9, primarily supplied by cheese (54%) and milk products (22%) in the Netherlands where this research was conducted. Meat and eggs, which contain MK-4 and MK-7, but primarily MK-4, accounted for only ~15% of dietary intake of K2. Vegetables contributed 82% of vitamin K1 intake.

Other recent study results from the Netherlands show similar inverse associations for dietary intake of the longer-chain menaquinones with coronary calcification. ⁶⁷ This accumulating evidence helps explain why the inverse association between vitamin K intake and CHD was much less obvious in the two population studies conducted in the U.S., both of which evaluated only K1 and did not collect data on K2. ⁶⁸, ⁶⁹ In 2004, the Rotterdam study also reported a strong inverse association between vitamin K2 intake and CHD, but a much less significant association for K1. ¹⁵

The fact that the women in the Gast, de Roos et al. 2009 study were postmenopausal renders this subject population of particular relevance for understanding the protective impact of K2 on not only CHD, but osteoporosis since, during the menopausal transition, bone loss accelerates to a rate of 3-10%. A number of other recent studies (discussed in Vitamin D and Vitamin K Team Up to Lower CVD Risk, Part II: The Vitamin K Connection to Cardiovascular Health), also indicate that reduced bone mineral density is a risk factor for CVD.

One cogent example is the MORE study, which included 2,576 postmenopausal women (mean age, 66.5 years. Those with osteoporosis had a 3.9-fold increased risk for cardiovascular events compared to those with low bone mass (BMD); a total hip BMD T score < or = -2.5 versus a T score between -2.5 and -1 was associated with a 2.1-fold increase in risk; and presence of ≥1 vertebral fracture versus no vertebral fracture at baseline was associated with a 3.0-fold increase in risk. Risk of cardiovascular events increased incrementally with the number and increasing severity of baseline vertebral fractures (both p < 0.001). ⁷⁰

Most recently, Gerber et al. (July 2011) reported a striking association between myocardial infarction (MI) and osteoporotic fractures in a case-controlled study involving 6,642 residents of Olmsted County, Minnesota. Those who had suffered an MI were found to have a 32% increased risk of fracture. ⁷¹

As mentioned above, the dietary sources of vitamin K2 (cheese, milk products and meat) differ greatly from those of K1 (leafy greens and unhydrogenated oils) and thus subjects’ higher levels of these forms of vitamin K reflect different dietary patterns. Since a diet rich in K2 contains much more saturated fat (and if cheese is featured, salt, two well-accepted risk factors for cardiovascular disease), than a diet in which K1 predominates, this suggests that the risk reduction in CVD seen with the long-chain MKs, but not K1, is a result of their specific biological effects, i.e., K2 carboxylates extra-hepatic VKD proteins, while hepatic VKD proteins utilize virtually all available K1, with the result that far too little remains available to be sent back into the circulation for intestinal conversion to MK-4 sufficient to meet extra-hepatic Gla-protein needs.

The Body's Different Uses for K1 and K2 are Demonstrated by Their Different Distributions Over Plasma Lipoproteins

K1 is primarily transported with the triacylglycerol-rich lipoprotein fraction, which is quickly cleared by the liver, promoting its utilization of K1 as a cofactor for the VKD proteins required for blood coagulation and leaving little K1 for use in extra-hepatic tissues. ⁵⁸

K2 is found in both triacylglycerol-rich lipoprotein and low-density lipoprotein, the latter being its major transport system to extra-hepatic tissues. ⁵⁸ Animal experiments have confirmed not only that extra-hepatic tissues preferentially accumulate K2, but that K2 (added to the animals’ feed as MK-4 [in pharmacological amounts] in these experiments), but not K1, inhibits warfarin-induced coronary calcification. ²⁹

K1 and MK-4 Share Similar Structure, Thus Both Have a Shorter Half-Life than Long-Chain MKs

As noted above, the longer-chain MKs have a much longer half-life than either K1 or MK-4, which share a very similar molecular structure (both contain 4 isoprenoid residues, 3 of which are saturated in K1 but contain a double bond in MK-4) and therefore similar physiokinetics. Following intestinal absorption, all forms of vitamin K are taken up in the triglyceride fraction from which they are rapidly cleared by the liver, but only the longer-chain menaquinones are redistributed via low-density lipoproteins. This results in both K1 and MK-4 being cleared by the liver within a matter of hours. In contrast, the longer-chain MKs are significantly more lipophilic and are handled differently. Their incorporation into low-density lipoproteins results in their much slower hepatic clearance, several day half-lives, much more stable serum levels and accumulation to 7- to 8-fold higher levels during prolonged intake. ¹⁵, ⁵⁸, ⁹

In the human population studies discussed above, the K2 subtypes found to be responsible for lowering CHD risk were MK-7, MK-8 and MK-9. The stronger protective effect of the long-chain MKs is thought to be due to their much longer half-life than that of either K1 or MK-4, which renders the long-chain MKs available for carboxylation reactions for a significantly greater time period. ⁷², ⁵, ⁶⁵, ⁴⁵, ¹⁷

Another reason why the long-chain MKs (MK-7, MK-8, MK-9) play a more important physiological role than K1 (and its use for the endogenous production of MK-4) than one might conclude -- if looking solely at the relatively lesser amounts of the long-chain MKs present in the diet compared to K1-- is that vitamin K’s bioavailability depends not only on intake, but also absorption. K1 is tightly bound to chloroplast membranes in plant cells, from which it is absorbed with low efficiency. After ingestion of a standardized amount of spinach, circulating levels of K1 may be increased by at least 3-fold if the greens are accompanied by a little vegetable oil (unhydrogenated is preferred for the reasons discussed earlier); however, despite the fact that simultaneous intake of a small amount of fat improves K1 absorption significantly, absorption of MKs, which are derived mainly from fats and dairy products (including low-fat dairy products, which still contain some fat), is much better. ¹⁵

Vitamin K1’s Contributions to Healthy Aging

K1 Promotes Bone Quality not Quantity

It is becoming widely accepted that, in addition to BMD, healthy bone includes other qualitative properties, such as elasticity, structure and micro-architecture, which are now considered to be independent risk factors for bone fracture. Qualitative Ultrasound Assessment (QUS), in which these properties are evaluated, has therefore been suggested to provide better information than just BMD for estimating bone strength, and some consider QUS a promising alternative to DXA since it shows a significant correlation with DXA values in the lumbar spine. Several recent studies utilizing QUS suggest that K1 promotes better bone quality. ⁷³, ⁷⁴, ⁷⁵, ⁷⁶, ⁷⁷

Both the Nurses’ Health Study and the Framingham Heart Study found a significantly lower relative risk of hip fracture in subjects with higher intake of K1. K1 intake of >242 μg/d was reported in the Nurses' Health Study to lower RR of hip fracture by 30%, and in the Framingham Heart Study, the mean vitamin K intake, 254 μg/d, lowered RR by 65%. ⁷⁸, ⁷⁹, ⁸⁰

Results from the ECKO trial also provide evidence of K1’s beneficial effect on bone quality. This was a 2-year double-blind randomized, placebo-controlled trial involving 440 postmenopausal women with osteopenia. K1 (at a pharmacological dose of 5 mg/day) did not provide any protection against age-related decline in BMD or bone resorption markers—even if the women’s vitamin D levels were adequate; however, K1 was found to provide significant protection against clinical fractures: only 9 women given vitamin K1 vs. 20 given placebo experienced fractures (and fracture prevention is the reason we treat!), suggesting that K1’s effect on bone health is not due to its effects on BMD or bone turnover. ¹¹

Lead author, Cheung said she could not account for these highly beneficial outcomes. In an LMR review of this study, Vitamin K2, not KI, Helpful for Bone Density, we proposed that research indicating that vitamin K1 significantly lowers cytokine production and overall inflammation may provide one reason. It is well known that inflammation increases as estrogen levels decline with menopause, and that increased production of pro-inflammatory cytokines is associated with increased differentiation and activation of osteoclasts. Vitamin K lessens oxidative stress and down-regulates expression of pro-inflammatory cytokines. K2 also provides anti-inflammatory effects via carboxylation of Gas6 (growth arrest specific gene 6), which then speeds the phagocytosis of apoptotic cells (an essential process for normal tissue development and maintenance), and promotes cell survival in a wide range of cell types.

A more recent study, (Bullo et al., 2011), provides insight into how vitamin K1 supports elders’ bone health. This 2 year study followed a cohort of 200 people, average age 67 years, who had good nutritional status and consumed a traditional healthy Mediterranean diet rich in vitamin K. Analysis of bone health using QUS assessment revealed significant correlations between higher K1 intake and superior bone quality properties, lower losses of bone mineral density, and smaller increases in the porosity and elasticity attributed to aging. The authors note: “Since the participants in this analysis already ate a healthy diet rich in K1, even more beneficial effects may be seen in populations with a lower intake of vitamin K or poor nutrition. ⁷⁸

K1’s Anti-Inflammatory Actions Protective vs. Osteoarthritis

An analysis of data collected on 672 participants in the Framingham Offspring cohort found a strong inverse association between circulating K1 and the prevalence of OA, occurrence of osteophytes (bone spurs) and joint space narrowing, indicating an association between low plasma levels of K1 and increased prevalence of OA in the hand and knee. ⁸¹

Vitamin K Promotes Detoxification

Vitamin K upregulates production of CYP3A isoforms by binding to and activating the steroid and xenobiotic receptor (SXR), a nuclear receptor that acts as a sensor for the presence of many drugs (e.g., phenobarbital, taxol, rifampicin) and initiates the transcription of CYP450 enzymes and transporter molecules that clear these substances from the body. MK-4, which has an unsaturated side chain, activates nuclear receptor SXR (which then induces CYP450 enzymes),8 -10 fold, while K1, which has a saturated side chain, causes a lesser 2-5 fold activation. ⁸²

Vitamin K2’s binding to the SXR also induces bone-specific genes that favor the expression of osteoblastic markers – yet another way in which K2 (as MK-4 in this in vitro study) promotes bone formation. ⁸³

Conclusion

Supplementation with Physiological Doses of Vitamin K -- K1 plus MK-7 -- Rather than Pharmacological Doses of MK-4, May Be Preferable

K1 can be consumed in sufficient amounts to carboxylate hepatic VKD proteins by eating leafy greens, whose increased consumption should be encouraged because they are also low in calories, high in fiber, and rich in numerous other micronutrients essential for healthy aging. However, while natto-eating Japanese can easily ingest sufficient amounts of natto to provide >150 mcg of MK-7 on a daily basis (1.5 ounces of natto contains ~ 435 mcg of MK-7), ⁸⁴ the Western diet not only affords much lesser amounts of the MKs necessary for the carboxylation of the extra-hepatic VKD proteins, but its K2-rich foods are concentrated sources of calories and (with the exception of low-fat dairy products) saturated fat. Thus, the Western diet cannot provide adequate amounts of K2 to fully carboxylate extra-hepatic VKD proteins without increasing other risks for chronic degenerative disease. Ensuring protective levels of Vitamin K2 via a supplement along with low-fat dairy foods (for those not lactose-intolerant, nor reactive to dairy protein), is therefore advisable. The question then becomes, which supplemental form(s) of MK at what dosage would be best? We suggest somewhere in the range of 45 - 90 mcg K1 plus 100 - 200 mcg K2 as MK-7/day. The lower end of the dosage range is likely to be sufficient for apparently healthy individuals, particularly since MK-7 accumulates in tissues to provide a reserve, ⁹ while those with conditions related to vitamin K insufficiency may require the higher dose to help promote reversal of pathology.

In contrast to K1 and MK-4, which are cleared from the body within a matter of hours, the longer-chain MKs’ incorporation into LDL gives these forms of K2 several day half-lives during which they are transported from the liver to other tissues where they can be used to build and maintain a reserve that promotes optimal activation of the extra-hepatic VKD proteins. Expressed as AUC over 24 hours, the availability of MK-7 is 2.5-fold better than that of K1; expressed as AUC over 96 hours, it is 6-fold better. This greatly extended availability is what enables the long-chain MKs to optimize activation of extra-hepatic VKD proteins at a dose that could feasibly be supplied by the diet (150 mcg/day of MK-7) rather the pharmaceutical dose (15 mg t.i.d, i.e., 45 mg/day) required for MK-4. ⁵⁸, ⁹

In experiments monitoring the efficacy of K1 and MK-7 for osteocalcin carboxylation (using the ratio between circulating cOC and ucOC), both vitamins induced increased OC carboxylation within 3 days, but only with MK-7 did ratio between cOC and ucOC continue to increase during the entire study periods, of 40 and 84 days, respectively. ⁹, ¹³ And the difference in carboxylation efficacy was quite significant: in both trials, MK-7 increased carboxylation 5-fold more than K1.

Present recommendations for vitamin K intake range from 90 to 120 mcg/day. In a dose of 45 mg/day, which must be given as 15 mg 3 times per day due to MK-4’s 6-8 hour biological half-life, the vitamin is being used as a drug not a nutritional supplement. In addition to the problems with patient compliance sure to occur with this 15 mg/t.i.d. regimen, MK-4’s short half-life will necessarily result in fluctuating K2 serum levels. At this time no data are available on the efficacy of MK-4 at lower doses.

In many species including humans, K1 is a minor constituent of hepatic vitamin K content with the majority being long-chain menaquinones, MK-7 through to MK-13. ⁵ Typically the ratio is about 90% menaquinones:10% phylloquinone. It is this author’s belief that the human body does not make mistakes. The fact that the body so quickly clears K1 and MK-4, while allowing the longer-chain MKs to accumulate, raises the question of “Why?” Why are these different forms of vitamin K treated so differently? Might there be untoward repercussions from ingestion of pharmacological levels of K1 or MK-4 over many years? The fact that vitamin K, at levels potentially consumed in a healthful diet, activates PXR, which then induces production of CYP450s responsible for the elimination of xenobiotics, suggests that pharmacological amounts of these vitamins might be considered by the body as potentially harmful xenobiotics. ⁸² Why take this risk when much smaller doses of MK-7, which could conceivably be ingested via consumption of a healthful diet, have been shown to accumulate in the body and effectively carboxylate extra-hepatic VKD proteins?

One Caveat for Patients on Coumarins (Oral Anticoagulants)

Supplemental (and dietary) vitamin K can interfere with the anticoagulant action of coumarin derivatives, but lower doses appear to be safe. Experiments have shown that only at a dose of 315 mcg/d did K1 affect INR, decreasing it from 2.0 to 1.5. As one might expect, MK-7 turned out to be much more potent; a comparable decrease in INR was reached at an intake of 130 mcg/d. For this reason, Schurgers et al. recommend an upper safety limit of 50 mcg/d for supplemental long-chain menaquinones (i.e., MK-7) in patients on oral anticoagulant treatment. This dose is comparable to the menaquinone content of 75 to 100 g of cheese, and research evaluating the effect on INR of gradually increasing doses of MK-7 indicate 50 mcg/day would lead to a disturbance of the INR value of no more than 10%. The 96 hour half-life of MK-7, however, suggests that regular intake of MK-7 in combination with properly adapted coumarin doses should result in more stable INR values. ⁹ Thus carefully monitored patients on courmarin derivatives may be able to take a higher daily dose of MK-7, a preferable prescription given the greatly increased risk of vascular calcification with coumarin use.

As we reported more than a year ago in our February 2010 LMR review -- Managing Erectile Dysfunction -- When Viagra Doesn’t -- 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). Why? Because the PDE-5 inhibitors help restore erectile function by selectively blocking normal hydrolysis of cGMP, thus promoting cGMP accumulation and partially reversing deficiencies in the NO/cGMP pathway. The key word here is “partially” since insufficient production of NO precludes the formation of sufficient cGMP to enable benefit from a PDE-5 inhibitor.

NO is the physiologic signal essential to penile erection because NO is required for the activation of soluble guanylate cyclase to convert guanosine triphosphate to cyclic guanosine monophosphate (cGMP). cGMP, in turn, triggers penile smooth muscle relaxation. Thus, if PDE-5 inhibitors are to be of any help to men unresponsive to these drugs, the amount of available NO must be increased in their penile tissue. ¹

Recent studies show that natural agents, specifically, the NO-producing substrates, L-arginine and L-citrulline, can do so. L-arginine is produced in the body from L-citrulline, and the most recently published research (discussed below) suggests supplementation with L-citrulline, rather than L-arginine, is the better option.

Although supplemental L-arginine is readily absorbed and well tolerated in single doses of 3-6 grams,² ~50-70% of ingested L-arginine is rapidly converted in the body to ornithine, primarily by the enzyme arginase.³ Arginase 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 of not only overt cardiovascular disease, but also of subclinical systemic vascular disease.⁴,⁵ Thus, in the man with ED, arginase is highly likely to be upregulated, which translates to a greater than “normal” conversion of supplemental L-arginine to ornithine and urea.

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).

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, significantly lessens the likelihood of optimal improvement in NO production from oral supplements of L-arginine alone.

In contrast, L-citrulline escapes intestinal or liver metabolism, and enters the kidneys where it is rapidly converted into L-arginine. Oral L-citrulline supplementation (3 grams b.i.d.) was recently shown in a double-blind, randomized, placebo-controlled cross-over study to increase the plasma L-arginine concentration and cGMP, and to augment NO-dependent signaling much more effectively than L-arginine.⁶

Encouraged by this research, which confirmed the rationale for oral L-citrulline supplementation as a donor for the L-arginine/NO pathway in patients with ED, researchers in the Department of Urology at the University of Foggia, Foggia, Italy, conducted a single-blind trial, in which 24 men with mild ED (erection hardness score of 3), mean age 56.5 years, received a placebo for 1 month and L-citrulline, 1.5 g/d in 2 doses, for another month.⁷ The erection hardness score, number of intercourses per month, treatment satisfaction, and adverse events were recorded. All patients completed the study with no adverse events.

Improvement in the erection hardness score from 3 (mild ED--penile rigidity that still allowed some kind of vaginal penetration, but not satisfactory penetration or completion of successful intercourse) to 4 (normal erectile function) occurred in 12 (50%) of the men when taking L-citrulline, and in 2 of the 24 men when taking placebo. All patients reporting an erection hardness score improvement from 3 to 4 were highly satisfied. At the month 2-visit, the men were informed they had received a commercially available nutrient and given the option to continue it or to receive a prescription for a PDE-5 inhibitor. All 12 men who had benefitted chose L-citrulline.

L-citrulline corrects the cause of ED, PDE5-inhibitors do not

Evidence has shown that oral L-citrulline supplementation can reverse the endothelial dysfunction associated with sickle cell disease (2001)⁸ and pulmonary hypertension (2006)⁹, confirming that L-citrullline effectively activates the L-arginine/NO/cGMP/VEGF pathway.

In 2008, it was shown that increased NO production induces corpus cavernosum smooth muscle cell synthesis and the secretion of vascular endothelial growth factor (VEGF), which can restore impaired endothelial function. This provides a rationale for L-citrulline supplementation to not only manage, but reverse penile endothelial dysfunction by correcting the proximate cause of ED. ¹⁰

Clinical Pearl: L-citrulline may be of great benefit for your female postmenopausal patients as well. One reason estrogen is cardioprotective is that it inhibits arginase. Researchers have recently shown a close relationship between atherosclerosis and endothelial senescence--and that NO can prevent it—especially in a diabetic model. ¹⁶

Supplementation with the NO-boosting substances, L-citrulline and L-arginine, along with antioxidants, has been demonstrated to delay endothelial senescence despite high glucose levels or a high-cholesterol diet. This delay in endothelial senescence through NO and/or eNOS activation may have clinical utility in the prevention and treatment of atherosclerosis associated with menopause and diabetes, and with aging. ¹⁷,¹⁸

PDE5 inhibitors are not innocuous:

Conclusion

Given PDE5-inhibitors’ potential for adverse side-effects, a trial of supplementation with L-citrulline, which has been proven to be safe, effective, and psychologically well accepted by patients, and is significantly less expensive than the PDE5-inhibitor drugs, certainly deserves consideration.

Even more importantly for your patient’s long term health and longevity, PDE5-inhibitors do nothing to improve NO production, while using L-citrulline to increase L-arginine availability does.

by Lara Pizzorno, MDiv, MA, LMT with Barry Wheeler, ND

Salivary Hormone Testing

Advantages

In males, saliva testing may be helpful for monitoring cortisol circadian rhythm. For the four-point (6am, 12pm, 6pm, and 12am) cortisol assay required to evaluate cortisol circadian rhythm, salivary testing is the only practical option.

Disadvantages

While saliva testing can help determine the circadian rhythm of cortisol, it cannot provide information on tetrahydrocortisol (THE), tetrahydrocortisone (THF), and allo-tetrahydrocortsiol (5a-THF) levels, which is needed to determine adrenal reserve, ¹ ,² and thus, whether prescribing herbs, such as licorice, withania somiferum (ashwaganda), Panax ginseng or others, will be potentially beneficial or if replacement with hydrocortisone is indicated. Furthermore, it is difficult to estimate total production of cortisol since a night time spike, which is common in those with sleep disturbances, may be missed.

As discussed in detail in Part I of this review, salivary testing has repeatedly been shown to be highly unreliable for testosterone.

Like serum testing, saliva tests offer only a ‘snapshot’ look at hormones that ebb and flow throughout a 24-hour period, for which reason its diagnostic value for testosterone, estradiol, progesterone, dehydroepiandrosterone (DHEA) and aldosterone is compromised by rapid fluctuations in salivary concentrations of these steroids. Samples would need to be taken multiple times throughout the day and night to obtain reliable information.

More importantly, saliva tests cannot capture hormone metabolites, so cannot provide the clinician with information essential to safe hormone replacement therapy, e.g.:

In sum, in the male patient, salivary steroid testing may be useful for assessing circadian patterns for cortisol. For this indication, collection of unstimulated saliva into small plain tubes and storage at −20° C is recommended to avoid known pitfalls (See Part I). Saliva testing is not a reliable option for even a snapshot evaluation of androgen levels.

Blood (Serum) Hormone Tests

Advantages

Serum testing offers a reliable and is the preferred option for testing a number of hormones including:

Measurement of these compounds in serum is the best choice because most are proteins and thus do not show up in urine in significant quantities if the kidneys are functioning normally. Others, e.g., reverse T3, are extremely small, and thus more easily measured in serum than urine. Additionally, DHT, which is very difficult to measure in any medium, is most accurately measured in serum using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).⁵ , ⁶

Disadvantages

Serum hormone tests directly assess the amount of hormones in the circulation; however, significant limitations must be noted.

Most important is that measurement of sex hormones in serum is necessarily a ‘snapshot’ peek at hormones that fluctuate significantly during the day both pre-andropause and in men using any form of hormone replacement therapy (as indicated by Figure 1 below). The testes, in which Leydig cells produce 95% of the body’s testosterone, secrete hormones in pulses throughout the day. In men given testosterone injections, levels increase dramatically, peak within two to four days, then drop rapidly. Men supplemented with natural testosterone may be applying transdermal creams or gels once or twice daily or may be using a patch or sublingual tablet; regardless of the delivery form chosen, testosterone levels will fluctuate throughout the day.

Adding to the “snapshot” limitation is the fact that -- with the exception of testosterone for which both free and total values are typically reported -- most serum tests report only “total” hormone values, i.e., the sum of free, conjugated and bound forms of the hormones. Free estrone, free estradiol, and free progesterone are rarely measured in serum. In men, the ratio of free testosterone : free estrogens should be ≥4. If <4, this indicates excessive aromatization and possible insulin resistance.

Another distinction not taken into consideration in serum tests is that among free, conjugated and bound forms. Bound forms have been completely inactivated, but conjugated steroids (which have combined with simple molecules, i.e., glucuronic acid or sulfates in Phase II liver conjugation) can be activated by glucuronidases and sulfatases. Sulfatases are not only highly prevalent in breast tissue in men as well as women, but are also found in numerous other tissues including prostate bone, and brain. Quantitative data show that the sulfatase pathway, which transforms estrogen sulfates into bioactive unconjugated estradiol, is actually 100–500 times more active than the aromatase pathway, which converts androgens into estrogens. Thus, the distinction between conjugated and bound forms of estradiol is clinically important in men since the sulfatase pathway is a significant contributor to body load of bioactive estradiol. Conjugated estradiol is a factor in estrogen-related prostate cancer (via its potential for metabolism to 16α-OH estrone).¹²

In addition, conjugated estrogens can be re-activated in the intestines and returned to circulation via the action of beta-glucuronidase enzymes, which are produced by E. coli, Clostridum and Bacteroides ¹³ ; thus intestinal dysbiosis may increase a man’s circulating levels of active estrogens). For these reasons, evaluation of hormone replacement therapy in men should assess not only testosterone, but both free and conjugated estrogens. The 24-hour Urine Test does so; serum tests do not.

As a consequence of all of the above, serum testing does not provide the clinician with adequate information to safely prescribe or evaluate the effects of androgen replacement therapy. For example, an andropausal man whose serum test results indicate total testosterone at normal levels may still be experiencing loss of muscle and libido, prostate enlargement, and erectile dysfunction, along with increased risk of cardiovascular disease, osteoporosis and depression, if most of his testosterone is bound, which becomes increasingly likely in men >50, the age at which testosterone levels typically begin to decline, while levels of sex hormone binding globulin (SHBG) increase with age.¹⁴

Prescribing testosterone to this patient to alleviate his symptoms may result in increasing his levels of estradiol if his aromatase enzyme, which readily converts testosterone to estradiol, is overactive. Excessive aromatization, in addition to its “feminizing” effects, may increase prostate cancer risk since estradiol can be metabolized to estrone, which can then be converted to either protective 2-hydroxyestrone (2-OH) or Estriol (E3) OR into potentially pro-carcinogenic 16α-OH estrone. On the other hand, insufficient aromatization not only promotes bone loss in men since some 16α-OH is required for bone remodeling, but has recently been shown to promote prostate cancer by inhibiting the production of 3β-adiol (discussed next). Serum testing will not indicate which estrogen metabolites a man is producing.

When testing male hormones in serum, a morning blood draw is recommended since that is when they are typically at their highest levels. More typically, serum free testosterone will be low in an adropausal man experiencing the symptoms noted above. Nevertheless, to reduce risks, more information is needed than typical serum testing can provide.

Testosterone Metabolites Serum Testing

A recently developed serum test, the Testosterone Metabolites Profile, does provide significant insight into prostate cancer risk, however. This test captures levels of not only 5α-DHT, but its metabolites: 5α-Androstane-3α-17β-diol (aka 3α-adiol) and 5α-Androstane-3β-17β-diol (aka 3β-adiol).

Low levels of 5α-DHT are indicative of over-inhibition of 5α-reductase (5AR), the enzyme that converts testosterone to 5α-DHT, which is ~2-3 times as potent as testosterone due to 5α-DHT’s greater affinity for androgen receptors.¹⁵

Drugs designed to inhibit 5AR (e.g., finasteride [Propecia, Proscar], dutasteride [Avodart]) and botanical supplements (e.g., Serenoa repens [Saw palmetto]) are taken by hundreds of thousands of men around the world to combat benign prostatic hypertrophy and androgenic alopecia (male pattern baldness). It has also been assumed that 5α-DHT is a key contributor to prostate cancer initiation and progression, so 5AR inhibitors are commonly used to treat prostate cancer. However, recent studies show a correlation between low levels of DHT and decreased survival in prostate cancer patients, rendering this assumption highly questionable.¹⁶ ,¹⁷ , ¹⁸ , ¹⁹ According to one recent paper, “Low dihydrotestosterone in the prostate is probably sufficient to propagate the growth of aggressive prostate cancer.” ²⁰

The American Society of Clinical Oncology/American Urological Association 2008 Clinical Practice Guideline states: “Men who are taking 5-ARIs for benign conditions such as lower urinary tract symptoms (LUTS) may benefit from…understanding that the improvement of LUTS relief should be weighed with the potential risks of high-grade prostate cancer from 5-ARIs.” ²¹

Mechanisms through which 5α-reductase inhibitors promote prostate cancer

Over-inhibition of 5AR results in increased availability of testosterone, which may be shunted, via aromatase, to estradiol, causing “feminization,” e.g., gynecomastia, one of the side effects of 5AR inhibitor drugs; and decreased production of not only 5α-DHT, but its metabolites, 5α-Androstane-3α-17β-diol (aka 3α-adiol, a storage form of 5α-DHT), and 5α-Androstane-3β-17β-diol (aka 3β-adiol), an estrogen receptor beta (ERβ) ligand that promotes normal cellular differentiation, thus lessening risk of benign prostatic hypertrophy and prostate cancer.²² ,²³, ²⁴

ERβ is involved in several biological functions in the prostate, including regulation of cell proliferation, apoptosis and differentiation. 3β-adiol, as an alternative ligand to estradiol of ERβ (the concentration of 3β-adiol in prostate is 100-fold higher than that of estradiol), mediates the majority of these effects. ERβ expression has been revealed to be greatly decreased in malignant prostate tissue, reaching nearly undetectable levels with tumor progression in more than 75% of cases. Reintroduction of ERβ triggers apoptosis, and decreases proliferation and invasiveness of malignant cells. In addition, prostate hyperplasia has also been described in mice lacking ERβ, and 3β-adiol has been shown to more potently induce expression of ERβ in the rat prostate than estradiol or 5α-DHT.²³

3β-adiol has lately also been revealed to be a key means through which the neuroendocrine response to stress is modulated in males. 3β-adiol suppresses excessive hypothalamic-pituitary-adrenal (HPA) axis activation, which would otherwise promote depression and neurodegeneration.²⁵ , ²⁶

Balancing Men’s Hormones Summary:

Issues to Consider

Under activity of 5α-Reductase (5AR):

Since 5AR is the enzyme that converts testosterone to 5α-DHT, excessive 5AR activity promotes benign prostatic hypertrophy and male pattern baldness; however, 5AR underactivity will inhibit 5α-DHT production, and therefore production of its protective metabolite, 3β-adiol, and thus can promote prostate cancer.
Solution—check for 5AR inhibitors and reduce or eliminate. Increase 5AR expression with exercise. Increase 5AR activity with DHEA and free testosterone; both upregulate 5AR activity in the prostate. Check free testosterone levels via 24 Hour Urine test; SHBG increases after age 50, resulting in decreased levels of free testosterone, so serum levels (total testosterone) in “normal” range do not confirm adequacy.

Over activity of 5α-Reductase:

If levels of androsterone are high on the 24 Hour Urine test, this may indicate that 5AR activity is elevated, especially if androsterone is high in relation to etiocholanolone. Excessive DHEA supplementation (>50mg/day in males) is a possible cause.
Solution—if 5AR activity is excessive, it may be lessened by zinc, Gamma-linolenic (GLA), docosahexaenoic acid (DHA), green tea extract, saw palmetto, and progesterone; and will be strongly inhibited by finasteride and dutasteride.
The nutrients (GLA, DHA, vitamin D3, and zinc) are unlikely to cause over-inhibition of 5AR and are suggested to use to replace the more potent "patent drugs" like finasteride. Also since these nutrients are required by the body, it is preferable not to decrease their intake (unless excessive), but to add Myomin and/or address the insulin resistance with diet and exercise.
Always recheck within 3 months to ensure over-inhibition has not occurred since 5AR over-inhibition can promote aggressive prostate cancer.

Excessive aromatization:

Excessive testosterone supplementation (≥75 mg/day) can result in excessive aromatase activity and elevated total estrogens in men.
Excessive aromatase activity is almost always associated with insulin resistance. Check for insulin resistance.
Solution: In addition to diet and exercise, supplementation with vitamin D, chromium, (if patient is insulin-resistant & when combined with biotin), and berberine .
Solution: Myomin (2 to 3 capsules BID) and Chrysin (liposomal drops, 6 drops sublingually BID) will also lessen aromatase activity.
Use of 5AR inhibitors may also result in excessive aromatization. 5AR inhibitors include not only the drugs Finasteride (Proscar, Propecia) and Dutasteride (Avodart), but the botanical medicine, Serenoa repens (Saw palmetto); the nutrients, Gamma-linolenic acid (GLA) ,Eicosapentaenoic acid (EPA), Alpha-linoleic acid, and Oleic acid (olive oil); Progesterone , and heavy metals, e.g., cadmium and arsenic.
Solution: Check for use of Finasteride (Proscar, Propecia), Dutasteride (Avodart), Saw palmetto and reduce dosage or discontinue. Check for excessive intake of GLA, EPA, ALA, olive oil and lessen intake. Check for heavy metals and promote detoxification with NAC, DMSA, and infared sauna if indicated.
Also check cortisol levels; in excess, cortisol promotes insulin resistance and many features of the metabolic syndrome (e.g., glucose intolerance, hypertension, dyslipidemia).
Retest with 24-Hour Urine after 3 months to ensure sufficient 16α-OH estrone is present for bone health; run Serum Testosterone Metabolites to check 3β-adiol
One indication of excessive aromatization would be low Lutenizing Hormone (should be checked with serum test) – if accompanied by a decrease in the testosterone : estradiol ratio (as shown on a 24 Hour Urine) – as this would suggest suppression of testosterone by estradiol and thus excessive aromatization.

Dysbiosis:

Conjugated estrogens can be re-activated in the intestines and returned to circulation via the action of beta-glucuronidase enzymes, which are produced by E. coli, Clostridum and Bacteroides ; thus intestinal dysbiosis may increase a man’s circulating levels of active estrogens. This would be seen as a decrease in the testosterone : estradiol ratio (as shown on a 24 Hour Urine).
Solution – run Comprehensive Digestive Stool Analysis to identify pathogens. Use Institute for Functional Medicine “5 R” Program (available at http://www.functionalmedicine.org/ ):

Remove toxins: i.e., allergic foods, parasites, pathogenic bacteria or yeast overgrowth). This might involve using an allergy “elimination diet” or prescribing drugs or herbs to eradicate a particular bug. It might also involve making healthier dietary choices: a whole foods, Mediterranean style, lower carb diet; less processed, high glycemic index foods.
Replace: replace digestive enzymes, hydrochloric acid and bile acids whose secretion may have been compromised by diet, drugs, diseases, aging.
Reinoculate: with probiotic foods (e.g., yogurt with active cultures), supplements that contain bifidobacteria and lactobacillus species, and consumption of the high soluble fiber foods that contain fructooligosaccharides (FOS) and other prebiotics.
Repair: supplement with key nutrients utilized by GI tract cells for repair, e.g., zinc; vitamins A, C, and E; fish oil; glutamine.
Rebalance: lifestyle choices – sleep, exercise and stress -- can all affect the GI tract.

Imbalanced 2/16α ratio:

While conversion of some estrone to 16α is required for bone health, high levels of 16α, which promote prostate cancer, are caused by obesity, hypothyroidism, pesticides, alcohol and cimetidine.
Estrone’s conversion to (cancer preventive) 2-OH estrone is promoted by indole-3-carbinol (I3C), 3.3- diindoylmethane (DIM), flax, soy, omega-3 fatty acids and exercise.
Levels of 16a-OH E1 can also be lowered by increasing its conversion to estriol. This conversion is stimulated by iodine and iodide.
Supplementation with Calcium D-glucarate supports proper elimination of estrogen by inhibiting beta-glucuronidase, an enzyme produced by colonic microflora (e.g., E.coli) that hydrolyzes glucuronide conjugates, thus returning estrogen into circulation. (The major route of estrogen elimination in humans is through Phase II glucuronidation in the liver.)

Promoting 3β-adiol Production

Monitor Impact of All 5α-Reductase Inhibitors

Given the importance of 3β-adiol for prostate cancer prevention, stress reduction and prevention of age-related cognitive decline, men using any type of 5AR inhibitor -- natural compounds as well as patent medicines -- should be monitored for over-inhibition. If over-inhibition is found: (1) use a lower dose of, or a less potent, 5AR inhibitor (2) replace 3β-adiol by asking your compounding pharmacy to include it in your patient’s testosterone cream.²⁷

5α-Reductase Inhibitors

5α-Reductase Agonists

DHEA as well as free testosterone upregulates 5AR activity in the prostate³⁴, ³⁵; levels of both are assessed on a 24-Hour Urine Hormone Profile.
Exercise, even a single session on a treadmill, significantly increases 5AR expression—at least in male and female mice. ³⁶

Indirect Assessment of 5α-Reductase Activity

A 24-Hour Urine test provides two ratios that assess 5AR activity: Andosterone:Etiocholanone and Allo-tetrahydro-cortisol:Tetrahydro-cortisol. These are discussed in Part II of this review.
Very low ratios of the above hormones in urine indicate low 5AR activity and are suggestive of very low 5α-DHT production. Follow-up testing with a serum Testosterone Metabolites Profile can confirm since serum testing, which allows assessment of two other important ratios: the ratio of Testosterone:5α-DHT and that of 5α-DHT:3β-adiol, is a better indicator of elevated, low or normal levels of DHT.

The serum Testosterone Metabolites Profile also allows assessment of two other important ratios: the ratio of Testosterone:5α-DHT and that of 5α-DHT:3β-adiol:

Testosterone:5α-DHT: In men, (average age 64 +/- 9 years), a higher testosterone to 5α-DHT ratio has been associated with a 42% decreased risk of BPH.³⁷ A significant inverse association has also been shown between Testosterone:5α-DHT and lower urinary tract symptoms (LUTS). Additionally, men with higher concentrations of bioavailable testosterone had a 56% decreased risk of LUTS compared with those with hypogonadal concentrations.³⁸

As early as 1966, a study found an inverse relationship between tumor volume and the testosterone : DHT ratio in men screened for prostate cancer. Lower DHT values were seen in more advanced tumors. Testosterone levels were also lower in patients with cancer than in the control group. An inverse relationship was also found between tumor volume and 5α-DHT level; 5α-DHT levels tended to be lower among cases and in those with more advanced tumors, and the testosterone : 5α-DHT ratio was higher in patients with more advanced tumors.³⁹

More recently, a 2008 study found that plasma 5α-DHT levels were reduced when testosterone was given to elderly men with below normal levels of testosterone and elevated 5α-DHT levels at baseline. ⁴⁰

Several possible mechanisms could explain why: (1) Since 5α-DHT is ~3x as potent as testosterone, 5α-reductase activity may be increased to compensate for the low testosterone levels pre-treatment. When testosterone is given, the need to compensate via increased production of 5α-DHT is removed; the androgen receptors are now also being activated by testosterone, resulting in signaling for reduced synthesis of 5α-reductase and/or reduced 5α-reductase activity.⁴¹ In sum, the research suggests that testosterone replacement promotes normalization of 5α-reductase activity, 5α-DHT levels and its metabolism to 3β-adiol.

5α-DHT : 3β-adiol : While 5α-DHT has been seen only as a potential promoter of proliferation and growth in the prostate, its metabolite 3β-adiol is a differentiating agent that activates ERβ and may help prevent cancer. A growing body of evidence suggests that the ratio of 5α-DHT to 3β-adiol may much more important than their absolute values.²²,²³,²⁴,⁴² However, the ratio of 3β-adiol : (5a DHT + 3α-adiol) may be even more useful since 3α-adiol serves as a “reservoir” which re-cycles back to 5α-DHT.²³

Urine Hormone Testing

Advantages

As discussed in Part II of this review, urine hormone testing using both gas chromatography-mass spectrometry (GS-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well-established in the medical literature as a reliable method of assessing levels of active (free & conjugated) hormones and their metabolites, correlates well with patient symptoms, and reflects the beneficial impact (or lack of benefit and potentially oncogenic effect) of therapeutic interventions.⁴³, ⁴⁴⁴ Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA, also known as enzyme immunoassay [EIA]) analysis may not be accurate. The clinical takeaway: ask the lab you use what procedures they are using.⁴⁵, ⁴⁶, ⁴⁷

A 24-hour urine sample is non-invasive, easy for the patient, and provides a more accurate indication of hormonal output since it averages out the hour-to-hour fluctuations seen in both serum and salivary measurements.

In addition, a 24-Hour Urine test captures metabolites that are not measurable in saliva and cannot be reliably measured by a single, or even multiple, blood draws, e.g., 16α-OH estrone, an estrogen metabolite shown to impact bone loss in men, and a number of glucocorticoid and mineralocorticoid metabolites (discussed in Part II of this review) that provide greater insight into long-term adrenal health, short term stress response, the cortisol/cortisone balance, and other measures of adrenal health and function than assessment of cortisol alone.

Disadvantages

Circadian fluctuation of cortisol cannot be measured using 24-hour urine collection. To prevent sample contamination, patients applying transdermal cream to the scrotum or testicles should be instructed to apply to the perianal mucosal area on the day of collection.

The 24-Hour Urine Test: Analyte Interpretation and Evaluation for Men

Following is an explanatory listing of the steroids evaluated by the most comprehensive of the 24-hour urine tests, the Comprehensive PLUS Hormone Profile with hGH. Levels of all steroids reported indicate free plus conjugated forms.

For a schematic overview of the metabolism of these steroids, please see Part II of this review, Figure 1: “Metabolism of Select Steroids.”

24 Hour Urine Test analytes and adult reference ranges for men are summarized in Figure 2 below.

24 Hour Urine Test Analytes

Creatinine: a break-down product of creatine phosphate in muscle, which is produced by the body at a fairly constant rate and filtered out of the blood by the kidneys. Creatinine concentration provides a marker for a full – neither under nor over – urine collection.

The adult reference range is 0.5 – 2.0 gm/24hr. Since it is a muscle breakdown product, creatinine is typically higher in men than women (especially if the patient exercised on the day of collection).

Total urine volume: ranges from 1,200 – 3,000 mL; most likely to be ≥1,500 in men.

SEX HORMONES: ESTROGENS

Estrone (E1): The most potent estrogen, estrone is derived from testosterone via prior aromatization to estradiol and estradiol’s conversion by 17β-hydroxysteroid dehydrogenase (17β-HSD) to estrone. Because of its bone-building actions, small amounts of the estrone metabolite, 16α-OH estrone (which is produced by Phase I metabolism) are required by men. The adult male reference range is 3 – 11.4 μg/24 hr.

Estradiol (E2): Estradiol is produced from testosterone via aromatase. Estradiol is also readily converted to estrone via 17β-HSD, which is reversible. Small amounts are also produced in the adrenal cortex, adipose tissue, brain, arterial wall and, in males, in the testes. Unless converted to estrone, estradiol is metabolized into protective 2-OH estradiol (Phase I) and 2-methoxyestradiol ([2-CH3O estradiol], Phase II). The adult male reference range is 0.8 – 4.6 μg/24 hr.

Estriol (E3): Derived from estrone through the intermediate (bone-building but potentially oncogenic) 16α-OH estrone, estriol is the weakest estrogen with 20-30% less affinity for estrogen receptors. Also estriol stimulates ERβ and has anti-proliferative effects in estrogen sensitive prostatic tissues. The adult male reference range is 3.5 – 13.7 μg/24 hr.

Elevated total estrogens in men may be due to excessive testosterone supplementation (≥75 mg/day) or excessive aromatase activity, which is nearly always associated with insulin resistance.

If excessive aromatase activity is indicated, check for insulin resistance. The most accurate and definitive test for insulin resistance is the procedure published by Kraft.⁴⁸

In addition to diet and exercise, supplementation with vitamin D,⁴⁹, ⁵⁰; chromium⁵¹,⁵², (most likely to be effective in insulin-resistant vs. insulin-sensitive patients and when combined with biotin⁵³,⁵⁴); and especially the natural plant extract berberine are very likely to be helpful.⁵⁵, ⁵⁶, ⁵⁷, ⁵⁸, ⁵⁹, ⁶⁰, ⁶¹, ⁶²

Supplementation with Myomin (a combination of Chinese botanicals, 2 to 3 capsules BID⁶³ and Chrysin (liposomal drops, 6 drops sublingually BID⁶⁴,⁶⁵ will also lessen aromatase activity; however, retest with 24-Hour Urine after 3 months to ensure sufficient 16α-OH estrone is present for bone health; run Serum Testosterone Metabolites to check 3β-adiol levels for protection against prostate cancer.

2/16α ratio: The ratio of 2-hydroxyestrone (2-OH E1) to 16α-hydroxyestrone (16α-OH E1) is one indicator of a man’s risk of developing prostate cancer. 2-OH E1, a Phase I metabolite of estrone, has weak estrogenic activity and is associated with protection against prostate cancer. 16α-OH E1, also a Phase I estrone metabolite, is an estrogen agonist, stimulates cell mitosis and proliferation, and is associated with increased risk of prostate cancer. However, 16α-OH estrone is also involved in bone building, so very low levels indicate increased risk for osteopenia/osteoporosis in men as well as women.⁶⁶, ⁶⁷, ⁶⁸, ⁶⁹, ⁷⁰, ⁷¹, ⁷²

The ideal 2/16α Ratio range is 2 to 4. A 2/16α Ratio of <2 indicates increased risk for prostate and laryngeal cancer. A 2/16α Ratio of >4 indicates increased risk for osteopenia, osteoporosis.

Fortunately, the 2/16α ratio is highly modifiable with natural interventions. Estrone’s conversion to (pro-carcinogenic) 16α-OH E1 is promoted by obesity, hypothyroidism, pesticides, alcohol and cimetidine. Conversion to (protective) 2-OH E1 is promoted by indole-3-carbinol (I3C), 3.3- diindoylmethane (DIM), flax, soy, omega-3 fatty acids and exercise. (I3C and DIM are phytochemicals in Brassica vegetables that induce CYP1A1, a Phase I enzyme that stimulates formation of 2-OH E1 while downregulating formation of 16α-OH E1. DIM forms spontaneously from dimerization of I3C in the presence of stomach acid and is thought to be the primary active agent in I3C.)⁷³, ⁷⁴, ⁷⁵, ⁷⁶, ⁷⁷, ⁷⁸, ⁷⁹

Levels of 16a-OH E1 can also be lowered by increasing its conversion to estriol. This conversion is stimulated by iodine and iodide.⁸⁰

The major route of estrogen elimination in humans is through Phase II glucuronidation (conjugation with glucuronic acid) in the liver. Glucose and inositol are oxidized in the liver to glucuronic acid and then bound into uridine diphosphate glucuronate (UDG), which is used by the enzyme uridine diphosphate transglucuronylase to convert 2-OH estrone, 4-OH estrone, 16α-OH estrone, estradiol or estriol into water-soluble glucuronides, which can be excreted through the urine as well as bile.

Calcium-D-glucarate is the calcium salt of D-glucaric acid. Oral supplementation with calcium-D-glucarate has been shown to inhibit beta-glucuronidase, an enzyme produced by colonic microflora (e.g., E.coli) that is capable of hydrolyzing glucuronide conjugates, thus returning estrogen readied for excretion back into circulation. Elevated beta-glucuronidase activity is associated with an increased risk for various cancers, particularly hormone-dependent cancers such as prostate, and colon cancers.⁸¹, ⁸²

SEX HORMONES: PROGESTERONES

Progesterone: The major naturally occurring member of a class of structurally very similar hormones; in males, progesterone is produced by the brain.

Progesterone is an aromatase inhibitor,⁸³ so may be useful for treating benign prostatic hyperplasia (BPH). Follow-up testing with the Serum Testosterone Metabolites assay is recommended to ensure over-inhibition of 5AR is not occurring.

Pregnanediol, an inactive progesterone metabolite, is used to provide an indirect (but accurate) measure of progesterone levels in the body since progesterone’s structure prevents it from being eliminated in urine in significant quantities.⁸⁴, ⁸⁵
In men, the Reference Range for pregnanediol is70-1050 μg/24hr.

SEX HORMONES: ANDROGENS

DHEA: produced in the adrenals, DHEA is the most abundant steroid in the human body. Levels peak between 25 to 30 years of age and then decline. After conversion into androstenedione, DHEA may be used to produce testosterone and its metabolites (for which reason DHEA replacement may help boost testosterone levels) or estrone, and thus 16α-OH estrone⁸⁶.

For this reason, DHEA helps build new bone tissue and has been shown to significantly improve bone mineral density in older adults.⁸⁷, ⁸⁸, ⁸⁹ Low levels are associated with increased risk of fracture and osteoporosis.⁹⁰

DHEA and its metabolite etiocholanolone (also reported by the 24-Hour Urine Test, see below), inhibit glucose-6-phosphate dehydrogenase (G6PD), an enzyme that plays a key role in anerobic glycolysis, the major route of energy production for cancer cells.⁹¹, ⁹²

DHEA has significant immune-modulatory functions—both immune-stimulatory and anti-glucocorticoid effects. Evidence is accumulating that DHEA may be effective as a treatment for the immunological abnormalities that arise in subjects with low circulating levels of this hormone, including the impaired immune response of older individuals and immune dysregulation in patients with chronic autoimmune disease.⁹³

In men, as noted above, DHEA is a 5AR agonist, thus promoting conversion of testosterone to DHT, and has also been shown to exert anti-atherogenic effects; higher serum levels of DHEA-S correlate inversely with carotid artery intima-media thickness, plaque and blood flow volume.⁹⁴

Etiocholanolone: One of two DHEA metabolites reported on the 24-Hour Urine Test, etiocholanolone, as noted above, has cancer-preventive anti-proliferative effects via its inhibition of G6PD. Etiocholanolone is produced from androstenedione by the enzyme 5β-reductase followed by 3α-hydroxysteroid dehydrogenase (3α-HSD). Excessive DHEA supplementation (>50 mg/day in males) may be the cause of high etiocholanolone levels.

Androsterone: A DHEA metabolite derived from androstenedione via the activity of 5AR followed by 3α-hydroxysteroid dehydrogenase (3α-HSD), and therefore useful for monitoring 5AR. If androsterone is high in relation to etiocholanolone, 5AR activity may be elevated, resulting in increased conversion of testosterone to dihydrotestosterone (5α-DHT).

5α-DHT is ~3 times more powerful than testosterone and unlike testosterone, cannot be aromatized to estradiol. Thus, excess 5AR is associated with male pattern baldness. Excessive DHEA supplementation (>50 mg/day in males) is a possible cause of high androsterone levels. However, if androsterone is low in relation to etiocholanolone in men, then 5AR activity may be low as well, resulting in insufficient production of 3β-adiol (thus increasing prostate cancer risk) and leaving more testosterone to be aromatized to estradiol.

Testosterone: Primarily secreted by the testes in men, but also produced in the adrenals, liver, skin and brain, testosterone is important not only for its effects on the male libido⁹⁵,⁹⁶, but on lean body mass and muscle strength, cardiovascular health, cognitive function and bone mineral density.⁹⁷

The range is wide because of the many different body types included in its calculation, which is based on 30+ years of 24 Hour Urine tests on thousands of men. Some men may produce less testosterone than other men and may have more active 5AR to compensate. For anti-aging, we generally recommend that, with treatment, men be between 80 - 150μg/24 hr. While some clinicians prefer to see testosterone levels in the upper end of the reference range to help with anti-aging, even though the patient could likely be symptom free with less HRT, we believe the key question is: “Is the testosterone dose sufficient to render the patient symptom-free?” In addition, hormones should always be evaluated comprehensively because of they constitute an interactive web. We can often get more benefit with less because when they are all balanced, they “cooperate" with each other. In terms of testosterone, if the patient is in the upper end of the reference range, it is essential to also run the Serum Testosterone Metabolite profile to check DHT and its metabolites.

5α-Androstanediol: On the 24 Hour Urine test, the 5α-androstanediol analyte contains both the 3α-adiol (aka 5α-Androstane-3α-17β-diol) and 3β-adiol (aka 5α-Androstane-3β-17β-diol) subcomponents, and does not differentiate between these final downstream metabolites of 5α-DHT. The 3α-adiol and 3β-adiol metabolites are able to be further differentiated and are reported on the Serum Testosterone Metabolite profile. 5α-Androstanediol is a testosterone/5α-DHT metabolite produced via the activity of 5AR. High levels indicate testosterone is primarily being metabolized through 5α-DHT, especially if levels of 5β-androstanediol are low. As noted above, 5α-DHT is highly potent: ~3 times more powerful than testosterone. Elevated 5AR activity may be linked to increased risk for male pattern baldness and benign prostatic hypertrophy. Over-inhibition of 5AR, however, has been linked to increased risk of aggressive prostate cancer. (As explained above, a Serum Testosterone Metabolite test will give you more insight into testosterone’s metabolization and potential over-inhibition.)

5β-Androstanediol: a testosterone/5β-DHT metabolite produced via the activity of 5β-reductase. High levels indicate testosterone is being metabolized through 5β-DHT, especially if levels of 5α-androstanediol are low. 5β-DHT is a weak testosterone metabolite.

GLUCOCORTICOIDS

Pregnanetriol: A progesterone metabolite and indicator of sufficient substrate for the cortisol pathway. The Reference Range for pregnanetriol is quite wide (200 – 1,500 μg/24hr). Mid-range is desirable.

Cortisol: After DHEA, cortisol is the second most plentiful steroid in a healthy person. Cortisol increases gluconeogenesis, affects protein and fat metabolism, and thyroid metabolism. (Both excess and insufficient cortisol inhibit, while normal levels promote the conversion of T4 to T3.)⁹⁸, ⁹⁹ Cortisol has potent immunosuppressive and anti-inflammatory activity.

In excess, however, cortisol promotes insulin resistance and many features of the metabolic syndrome (e.g., glucose intolerance, hypertension, dyslipidemia). High levels of cortisol also decrease the ability of osteoblasts to synthesize new bone and interfere with absorption of Ca2+ from the gastrointestinal tract.

In 21st century life, cortisol is often elevated due to unremitting stress and sleep deprivation, and in “Catch 22” fashion, elevated cortisol promotes both. Elevated cortisol (or cortisone, see below) is associated with Cushing disease, unipolar depression, sleep deprivation, anxiety, panic disorder, PTSD in its early stages, exogenous cortisol supplementation, high-dose licorice root supplementation, intense physical exercise, and acute ingestion of alcohol.

Clinical signs of adrenal excess include insomnia, anxiety, insulin resistance, obesity (especially truncal adiposity), hyperglycemia, hypertension, easy bruising in the extremities due to loss of subcutaneous adipose and connective tissue, bone loss, muscle weakness and sarcopenia. If causes are not addressed, adrenal fatigue and cortisol insufficiency is the likely outcome.

The Reference Range for cortisol is 30 – 170μg/24hr, which is quite wide and does not represent optimal levels. Optimal range for Cortisol is ~90μg/24hr.

Cortisone is the inactive metabolite of cortisol and serves as a “cortisol reserve,” in the body. Cortisone is produced by the action of 11β-hydroxysteroid dehydrogenase (11β-HSD), an enzyme with two isoforms, the first of which, 11β-hydroxysteroid dehydrogenase I (11β-HSD I) catalyzes cortisone into cortisol, enabling rapid supply of the active hormone as needed. The second isoform, 11β-HSD II, inactivates cortisol to cortisone (an action that is reversible via the activity of 11β-HSD I).

Decreased cortisol or cortisone seen with adrenal insufficiency is associated with Chronic Fatigue Syndrome, fibromyalgia, rheumatoid arthritis, and late stage Panic Disorder. Clinical signs of adrenal insufficiency include fatigue, exercise intolerance, hypoglycemia, salt craving, insomnia, depression, irritability, positive Hippus test (greater light exposure should result in pupil contraction), and low blood pressure.

The Reference Range for cortisone is 31 - 209μg/24 hr, which is quite wide and does not represent optimal levels. Optimal range for Cortisone is ~120-130μg/24hr.

Cortisol : Cortisone Ratio: The ideal cortisol : cortisone ratio is 0.7 (i.e., cortisone should be ~30% higher than cortisol), as this indicates slightly more storage (cortisone) than active (cortisol) hormone. Sleep problems are common when this ratio gets to ≥1.

A ratio greater than 1.4 is considered possibly suspicious for the hypertensive syndrome “Apparent Mineralocorticoid Excess Type 2” (AME Type 2 is a much milder version of AME Type 1, a severe and lethal congenital deficiency of 11β HSD II).

Tetrahydrocortisone, Tetrahydrocortisol, and Allo-tetrahydocortisol: are metabolites of cortisone (tetrahydrocortisone), and cortisol (tetrahydrocortisol, and allo-tetrahydocortisol), and can be used to determine daily cortisol output.

When their 24 Hour Urine Test values are added together, these three metabolites account for approximately half of daily cortisol output. Taking the sum of the three, doubling it and moving the decimal 3 points to the left will give, in milligrams, about how much cortisol is being made each day. In men, the three metabolites should add up to between 5,000 and 10,000, which corresponds to a cortisol output of 10-20 mg/day.

Low levels are a very strong indication of weak adrenal function. (If allo-tetrahydrocorticotsterone (5α-THB), tetrahydrocorticosterone (THB) and 11-dehydrotetrahydrocorticosterone (THA) levels are also low, this is a very strong indication of long term adrenal insufficiency. These analytes are discussed below.)

11β-OH Androsterone and 11β-OH Etiocholanolone: terminal metabolites of cortisol. Their values will confirm if cortisol production is excessive or insufficient. Often, as patients become insufficient, cortisol levels may still appear within normal range, but downstream metabolites will be low.

MINERALOCORTICOIDS

Aldosterone: The major mineralocorticoid, aldosterone is part of the renin-angiotensin system and acts on the distal tubules and collecting ducts of the nephron (the functional unit of the kidney) to cause conservation of sodium, secretion of potassium, increased water retention, and increased blood pressure. Aldosterone levels are usually a reliable indication of whether a person is on a normal, low or high salt diet.

Aldosterone reverses certain types of hearing loss inn experimental animals¹⁰⁰, ¹⁰¹, ¹⁰², ¹⁰³, and levels of serum aldosterone have been inversely correlated with degree of hearing in humans.¹⁰⁴ Case studies at Tahoma Clinic, Renton, Washington, have found that a significant percentage of individuals with hearing loss and low levels of aldosterone recover some of their hearing when exogenous aldosterone is taken.¹⁰⁵

(For further discussion and possible mitigating circumstances, see “Activity of 11β-Hydroxysteriod dehydrogenase I and II” below.) Reference ranges are: Normal Diet = 6.0 -25.0 μg/24hr; Low Salt = 17.0 -44.0μg/24hr; High Salt = 0.0 – 6.0μg/24hr.

Allo-tetrahydrocorticostersone (5α-THB), Tetrahydrocorticosterone (THB) and 11-dehydrotetrahyrdocorticosterone (THA): metabolites of aldosterone that serve as sensitive markers for monitoring acute adrenal stress. These metabolites are the first to rise in the ACTH stimulation test; high levels suggest acute stress at the time of collection. Low levels are a good indication of chronic adrenal fatigue. References ranges: 5α-THB = 130 – 600μg/24hr; THB = 30 - 240μg/24hr; THA = 62 - 293μg/24hr.

The most comprehensive 24-Hour Urine Test also reports levels of human Growth Hormone, free T3 and T4 (the thyroid hormones).

Human Growth Hormone: Produced in the anterior pituitary and regulated from hypothalamus by growth hormone releasing hormone and growth hormone inhibiting hormone (aka somatostatin), human growth hormone (hGH, aka somatotrophin) enters the circulation and is delivered to the liver where it is converted to growth factors that initiate muscle, bone, and cartilage production; improve kidney function, skin elasticity, and cell repair and regeneration. One thing growth hormone does not increase is body fat; hGH decreases adipose tissue.

IGF1, used to evaluate hGH levels in serum tests, is not considered a reliable marker¹⁰⁶, ¹⁰⁶; urine hGH is a better indicator.¹⁰⁸, {ref109, ¹¹⁰, ¹¹¹, ¹¹²

Growth hormone levels are increased by deep sleep,¹¹³ arginine (more effective in younger men, but useful in elders as well)¹¹⁴, ¹¹⁵, glutamine (helpful for older people, 2 grams at bedtime),¹¹⁶ and ornithine alpha-ketoglutarate (may boost the use of glutamine in arginine metabolism, 0.25 mg/kg)¹¹⁷, resistance training, short intense bursts of exercise, and vigorous aerobic exercise¹¹⁸, ⁴¹¹⁹, (although the effect of exercise is more pronounced in younger subjects)¹²⁰, and adequate protein.¹²¹

A number of hormones increase hGH secretion including testosterone (the most potent secretagogue for hGH), estrogen, progesterone, thyroid, melatonin, and growth hormone releasing hormone (GHRH).¹²², ¹²³, ¹²⁴, ¹²⁵ hGH is decreased by a sedentary lifestyle, inadequate protein, poor sleep, and insufficient endogenous hormones.

Thyroid Hormones

Upon stimulation by thyroid-stimulating hormone (TSH), the thyroid produces two main hormones: thyroxine (T4), the major form of thyroid hormone in the blood, and triiodothyronine (T3), the active hormone (three to four times more potent than T4), which primarily regulates the metabolic machinery inside cells. (The ratio of T4 to T3 released into the blood is roughly 20 to 1.) Thyroid hormones’ effects include controlling the speed of protein synthesis, energy use, and sensitivity to other hormones.

Both thyroid hormones combine tyrosine with iodine (T4 with 4 iodine molecules, and T3 with 3 iodine molecules), thus iodine insufficiency prevents adequate thyroid hormone formation.

Even if TSH is effectively signaling the thyroid gland, and iodine is present in sufficient amounts for adequate production of T4 and T3 (blood test levels thus appearing normal), intra-cellular conversion to T3 may not occur for several reasons. T4 is converted to the T3 within cells by deiodinases (5'-iodinase), enzymes for which selenium is the required cofactor. Cortisol is also required for the conversion of T4 to T3; long-term stress, which depletes adrenal reserves of cortisone, will therefore cause inhibition of the T4 to T3 conversion. Inflammatory cytokines (notably interleukin-2) can promote formation of autoantibodies to the thyroid, again inhibiting the T4 to T3 conversion.¹²⁶ Thus, a patient can have hypothyroid symptoms despite normal serum levels of thyroid hormones. The 24-Hour Urine test gives a better indication of what is happening inside the cell.¹²⁷

Ideally, one wants to see higher levels of free T3 than free T4. The richest food source of selenium, Brazil nuts may help improve conversion of T4 to T3, and may also help lessen inflammation since selenium is a cofactor for reduction of glutathione peroxidases. One Brazil nut provides ~ 100 mcg of selenium; 200 mcg is the recommended dosage. Excessive selenium can interfere with enzyme systems related to sulfur metabolism and can be toxic in amounts greater than 900 mcg/day.¹²⁸, ¹²⁹, ¹³⁰, ¹³¹

Urinary Minerals

Urinary levels of sodium and potassium clearly reflect dietary intake. The ideal ratio of sodium: potassium is 1.5. Due to the typical Western diet, which contains a disproportionate amount of high-sodium processed foods and few servings of potassium-rich green leafy and other vegetables, the ratio of sodium: potassium is typically elevated.

Bringing this ratio into ideal range is well recognized to be of vital importance in the prevention of hypertension, myocardial infarction, stroke and kidney failure.¹³², ¹³³, ¹³⁴, ¹³⁵

When salt intake is high, even a modest reduction for a duration of 4 or more weeks has a significant and important beneficial effect on blood pressure in individuals with normal as well as elevated blood pressure. A modest and long-term reduction in population salt intake could significantly reduce strokes, heart attacks and heart failure.¹³⁶ Decrease sodium by avoiding processed foods; increase potassium by increasing green leafy vegetable intake.

On the other hand, a 24 Hour Urine sodium on the low end of the reference range is not uncommon in people with low adrenal function. Many people have heard that it is beneficial to reduce salt and are over-zealous about it, and thus are not getting the sodium they require for good adrenal function.

Enzyme Activity

5α-Reductase is the enzyme that converts testosterone to the more potent 5α-DHT. Upregulated 5α-DHT activity promotes BPH and male pattern baldness, and is associated with insulin resistance and obesity.

(1) Androsterone/Etiocholanolone Ratio and (2) Allo-tetrahydrocortisol/tetrahydrocortisol Ratio.

If excessive, 5AR can be down-regulated by zinc¹³⁷, ¹³⁸, GLA,¹³⁹ EPA,¹³⁹ vitamin D3,¹⁴⁰ saw palmetto¹⁴¹, progesterone¹⁴², ¹⁴³, green tea extract¹⁴⁴, finasteride, and dutasteride.

Over-inhibition of 5AR, however, can promote aggressive prostate cancer. In the Prostate Cancer Prevention Trial, a study involving more than 18,000 men, subjects taking dutasteride had a lower rate of prostate cancer, but a significantly higher rate of aggressive prostate cancers than the placebo group. This resulted in a higher absolute number of aggressive prostate cancers in the dutasteride group than in the placebo group – even though the placebo group had a higher rate of prostate cancer.¹⁴⁵ (See above “Mechanisms through which 5α-reductase inhibitors promote prostate cancer”) (Neal, please make this a link to this section above) Thus, as we have seen elsewhere, e.g., rofecoxib (Vioxx®), celecoxib (Celebrex®), balance in biological processes is more beneficial than absolute interruption.)

11β-Hydroxysteriod dehydrogenase I and II are the enzymes that convert cortisone to cortisol and vice versa.

11β-Hydroxysteriod dehydrogenase I converts cortisone to cortisol. 11β Hydroysteroid dehydrogenase II converts cortisol to cortisone. 11β HSD I is inhibited by estradiol and HGH. 11β HSD II is inhibited by licorice and cadmium; this latter inhibition explains some of the hypertensive effects of these two substances.

11β-HSD I is highly expressed in key metabolic tissues including the liver, adipose tissue, and the central nervous system, where it reduces cortisone into the active hormone, cortisol.

11β-hydroxysteroid dehydrogenase II (11β-HSD II) is found in salivary glands and aldosterone-selective tissues, e.g. kidneys, where it oxidizes cortisol to cortisone to prevent activation of the mineralocorticoid receptor. As noted in Part I on this review, the presence of 11β-HSD II in salivary glands can invalidate salivary cortisol measurements.¹⁴⁶

Activity of 11β-Hydroxysteriod dehydrogenase I and II is reflected by two ratios provided by the 24-Hour Urine Test:

This ratio of hormone reserve (cortisone) to active hormone (cortisol) shows enzyme activity in adipose tissue and kidneys, and provides significant insight into the patient’s adrenal health. The ideal ratio is 0.7. Licorice, which contains glycyrrhetinic acid, can inhibit 11 β-HSD II, causing increased conversion of (storage) cortisone to (active) cortisol.

(2) Tetrahydrocortisol + allo-tetrathydrocortisol/Tetrahydrocortisone Ratio

Low ratios for these enzymes are associated with obesity and insulin resistance, while elevated ratios are associated with low-renin hypertension, high dose licorice, and exogenous cortisol. Ideal ratio is 0.9.

In patients with essential hypertension, elevated ratios may also be a sign of primary aldosteronism (PA), for which recent reports suggest incidence may be as high as 10-15% in hypertensive patients. PA may be missed in these patients because it can exist for many years before hypokalemia is demonstrable. Such patients are often mis-placed on anti-hypertensive medications, which do not prevent progression of the hypertensive vascular complications induced by hyperaldosteronemia (i.e., heart attack, stroke, kidney failure). If 11β-HSD ratios are elevated, hyperproduction of aldosterone will be detectable by ACTH-stimulated venous sampling.¹⁴⁷

CONCLUSION

Serum and 24-Hour Urine Hormone Testing—Sine Qua Non for Optimal Men’s BHRT

Despite the brevity of this introductory overview of a very complex subject—how to safely and effectively monitor BHRT in your aging male patients—the necessity of assessing hormone metabolites has, hopefully, been demonstrated. By utilizing the latest serum Testosterone Metabolites tests in combination with the most comprehensive 24-Hour Urine tests, physicians can gain unprecedented insight into their male patients’ health, ensuring safe and effective BHRT.

by Lara Pizzorno, MDiv, MA, LMT, with Pushpa Larsen, ND

Part I: The Advantages and Disadvantages of Saliva, Serum and Urine Tests

Treating the sequelae of the age- and stress-related decline in adult hormones with bio-identical hormone replacement (BHRT) can restore more youthful hormone levels and significantly alleviate symptoms associated with “normal” aging, optimizing health, happiness and quality of life. Successful and safe BHRT, however, necessitates laboratory testing to assess the patient’s current hormonal status, monitor treatment, and ensure that hormones are being metabolized in ways that reduce risks for cancer, cardiovascular disease, osteoporosis, other age-related diseases and declines in cognitive and sexual function.

Hormones can be assayed using saliva, blood (serum), and urine. Each testing method has advantages and disadvantages. Which of the three hormone test methods, or which combination of tests, you will wish to utilize will depend upon what information you need in a given clinical situation.

Salivary Hormone Testing

For the four-point (6am, 12pm, 6pm, and 12am) cortisol assay required to evaluate cortisol circadian rhythm, salivary testing is the only practical option.

Saliva testing may also be useful, and is surely a more practical option than blood draws, to assess the cyclical estrogen and progesterone pattern throughout the month in a cycling or peri-menopausal woman. Saliva samples have been demonstrated to enable differentiation between the follicular and luteal phase for both estradiol and progesterone. Saliva collected daily throughout the menstrual cycle shows a specific pattern, with a mid-cycle rise and a peak in the early luteal phase. Mean salivary progesterone concentrations in the follicular phase range from 20 to 100 pmol/L, whereas peak concentrations during the peri-ovulatory period may attain 300 pmol/L. This significant difference is consistently reported and allows for an assessment of ovarian function.¹

The clinician can see if the patient is cycling, where hormone levels are off during the cycle, and can get an overview of the cycle’s pattern.

Saliva test readings may be unreliable. Evaluation of saliva samples obtained from the same subject at the same time and sent to two different labs has been shown to produce results with rates of variation from 35 to 75% for estradiol, 8 to 103% for progesterone, and 13 to 40% for testosterone.²

Salivary testing has repeatedly been shown to be highly unreliable for testosterone. Salivary testing of postmenopausal women receiving transdermal testosterone supplementation found no correlation with salivary testosterone levels for any of the serum testosterone subtypes (total testosterone, bioavailable testosterone [free and albumin-bound testosterone], and free testosterone.)³

It has been well documented that salivary testosterone measurements, more so than other commonly measured salivary analytes (i.e., cortisol), can be substantially influenced during the process of sample collection. Materials commonly used to absorb the saliva sample (cotton and polyester swabs) or stimulate saliva flow (powdered drink-mix crystals and/or chewing gum) can artificially inflate testosterone results as much as two- to three-fold.⁴

Salivary testosterone levels are also susceptible to distortion resulting from the leakage of blood (plasma) into saliva as a result of micro-injury to the oral mucosa. In one study confirming this, the micro-injury group (n=35) donated a baseline saliva sample, then manually brushed their teeth for 2 minutes using an ADA recommended medium bristle brush without toothpaste. Saliva samples were collected immediately after brushing and then every 15 minutes for one hour. A control group (n=10) provided saliva samples without brushing teeth. In the tooth-brushing group, the resulting micro-injury elevated the presence of blood and its components in saliva within minutes, and correspondingly, testosterone levels in saliva increased and remained elevated over baseline well after micro-injury, even in samples that did not appear visually contaminated with blood. The effect of micro-injury was specific for testosterone; neither salivary cortisol nor dehydroepiandrosterone (DHEA) levels differed from baseline after brushing.⁴

In addition to possible blood contamination, the presence of both corticosteroid-binding globulin and sex hormone binding globulin (SHBG) in uncontaminated saliva renders questionable whether salivary steroids can accurately reflect circulating free steroid levels ¹(particularly in the older patient since, as noted earlier, SHBG levels increase with age).

Another potential confounder is the presence of the enzyme that converts cortisol to cortisone, 11β-hydroxysteroid dehydrogenase II (11β-HSD II), in salivary glands. Significant conversion of cortisol to cortisone (the inactive / storage form) may occur in the salivary glands via the activity of 11β-HSD II. This enzymatic conversion is often underappreciated in salivary cortisol measurements with the result that the data reflect not cortisol, but falsely increased measurements of both glucocorticoids together.¹ Furthermore, ascertaining the ratio of cortisol: cortisone (the ratio of active hormone: hormone reserves) provides significant insight into the patient’s adrenal health. (The cortisol: cortisone ratio is easily determined using the 24-Hour Urine test, discussed below.)

Storage temperatures can also contribute to error in the values estimated for salivary testosterone. One group of researchers evaluating the reliability of salivary testosterone found a striking linear increase in testosterone levels across four weeks for samples stored at 4 °C. On average, after one week of storage at 4 °C, measured testosterone levels increased 20.59% (9.1 pg/ml); by four weeks, testosterone levels had increased 330.77% (150.5 pg/ml) over baseline!⁴

Artificially elevated results have been reported not only for testosterone, but DHEA, progesterone and estradiol using cotton absorbent materials. On the other hand, salivary cortisol values are reduced by more than 50% when saliva is not retrieved immediately from cotton buds.⁵⁶

In an effort to mitigate the numerous problems with saliva sample reliability, a recent review recommends collection of unstimulated saliva into small plain tubes and storage at −20° C as the best method to avoid known pitfalls. If long term storage is anticipated, storage at −80° C is advised.⁶

However, there are additional problems for which no recourse is currently available. As we age, we produce less saliva, fewer hormones, and more sex hormone binding globulin (SHBG). Since saliva tests reflect only the metabolic process of salivary glands and a limited passive diffusion of the hormones from the bloodstream, levels may be below detection limits in elderly patients.

On the other hand, saliva testing has repeatedly been found to indicate significantly higher than physiological levels of hormones assayed in samples from patients using transdermal creams to deliver bio-identical hormone replacement therapy. This gives a false impression of overdosing.⁶ A possible explanation may be that red blood cells passing through capillaries rapidly uptake steroid hormones, which are lipophilic, and quickly transport them to salivary glands and other tissues. This results in elevated hormone concentrations in saliva, while serum and urine levels remain low.⁷⁸⁹

Saliva tests cannot capture hormone metabolites, so they do not provide the clinician with information essential to safe hormone replacement therapy: e.g., what percentage of estrogens are being converted into carcinogenic rather than protective compounds; the extent of adrenal fatigue; the activity level of 5α-reductase (the enzyme that converts testosterone to the more potent [and potentially prostate-carcinogenic] DHT; DHT also promotes benign prostatic hypertrophy and male pattern baldness in men, and hirsutism and polycystic ovarian syndrome [PCOS] in women.) The 24-Hour Urine test reports all of these variables.

Like serum testing, saliva tests also offer only a ‘snapshot’ look at hormones that ebb and flow throughout a 24-hour period. Due to this fact, the conclusion drawn in a 2009 review entitled, “Salivary steroid assays - research or routine?” was: “The diagnostic value of salivary estradiol, progesterone, testosterone, dehydroepiandrosterone and aldosterone testing is compromised by rapid fluctuations in salivary concentrations of these steroids. Multiple samples are required to obtain reliable information, and at present the introduction of these assays into routine laboratory testing is not justified.”¹⁰

In sum, salivary steroid testing may be useful for assessing circadian patterns for cortisol and for evaluating estrogen and progesterone patterns (ovarian function) in cycling and peri-menopausal women. For these indications, collection of unstimulated saliva into small plain tubes and storage at −20° C is recommended to avoid known pitfalls. Saliva testing is not a reliable option for even a snapshot evaluation of androgen levels or for monitoring the absorption of sex hormones from transdermal creams, which are likely to show elevated salivary hormone levels, while plasma and urinary levels remain low.¹

Blood (Serum) Hormone Tests

Serum testing offers a reliable and is the preferred option for testing a number of hormones including:

Measurement of these compounds in serum is the best choice for two reasons. Most of these hormones are proteins and therefore do not show up in urine in significant quantities if the kidneys are functioning normally. Others, e.g., reverse T3, are extremely small, so are more easily measured in serum than urine.

Additionally, while most testosterone metabolites are easily measured in urine, dihydrotestosterone (DHT) is not. DHT is very difficult to measure in any medium, but most accurately measured in serum using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Blood (serum) hormone tests directly assess the amount of hormones in the circulation; however, significant limitations must be noted.

Most important is that measurement of sex hormones in serum is necessarily a ‘snapshot’ peek at hormones that fluctuate significantly during the day both premenopause/andropause and in individuals using any form of hormone replacement therapy. In premenopausal women, the ovaries secrete hormones in pulses. In men, the testes, in which Leydig cells produce 95% of the body’s testosterone, perform similarly. Postmenopausal women using BHRT (or HRT) typically take their replacement hormones once or twice daily, as do men using BHRT (fortunately for 21st century men, the formerly patented, carciogenic testosterone analogue, methyltestosterone—widely and enthusiastically prescribed for men in the 1940s and early 1950s, as was Premarin® for women from the 1980s until 2002—is hardly every prescribed at present).

Adding to the “snapshot” limitation is the fact that most serum tests report only “total” hormone values, i.e., the sum of free, conjugated and bound forms of the hormones—again, with the exception of testosterone for which both free and total values are typically reported. Free estrone, free estradiol, and free progesterone are rarely measured in serum. Serum hormone tests typically include measurements only of total estradiol (E2), although total estrone (E1, the most potent and potentially oncogenic estrogen) may be available. Since bound estradiol and estrone are inactive, serum tests do not provide feedback regarding levels of active hormone.

Another distinction not taken into consideration in serum tests is that among free, conjugated and bound forms. Unlike bound forms, which have been completely inactivated, conjugated steroids, which have combined with simple molecules—glucuronic acid or sulfates in Phase II liver conjugation—can be activated by glucuronidases and sulfatases. Sulfatases are not only highly prevalent in breast tissue, but are also found in numerous other tissues including the endometrium, ovaries, bone, brain, and prostate. Quantitative data show that the sulfatase pathway, which transforms estrogen sulfates into bioactive unconjugated estradiol, is actually 100–500 times more active than the aromatase pathway, which converts androgens into estrogens. Thus, the distinction between conjugated and bound forms of estradiol is clinically important since the sulfatase pathway is a significant contributor to body load of bioactive estradiol. Conjugated estradiol cannot be discounted as a factor in estrogen-related oncogenesis.¹¹

In addition, conjugated estrogens can be re-activated in the intestines and returned to circulation via the action of beta-glucuronidase enzymes, which are produced by E. coli, Clostridum and Bacteroides¹², thus intestinal dysbiosis may increase circulating levels of active estrogens). For these reasons, hormone replacement therapy assessment (whether BHRT or HRT) should incorporate both free and conjugated estrogens. The 24-hour Urine Test does so; serum tests do not.

Levels of unconjugated estriol (E3, considered protective and the weakest form of estrogen) are sometimes available in serum tests. While measurement of unconjugated estriol may be helpful in terms of evaluating a woman’s risk of cancer (an “Estrogen Quotient” [estriol divided by the sum of estrone and estradiol] of ≤ 1.0 suggests lower cancer risk), its usefulness is significantly hampered because 90% of estriol is conjugated.

As a consequence of all of the above, serum testing does not provide the clinician with adequate information to safely prescribe and evaluate the effects of hormone replacement therapy (BHRT or HRT). For example, a menopausal woman whose serum test results indicate total estradiol at normal levels may still be experiencing hot flashes and other common climacteric symptoms if most of her estrogen is bound, which it likely is, as levels of sex hormone binding globulin (SHBG) increase with age.¹³

In addition, prescribing estradiol to this patient to alleviate her symptoms may result in increasing her levels of estrone since estradiol readily converts to estrone. This may not be advisable since estrone may be metabolized to either protective 2-hydroxyestrone (2-OH), 2-methoxyestrone (2-CH3O) or Estriol (E3) OR into pro-carcinogenic 4-hydroxyestrone (4-OH estrone) and 16α-OH estrone. Serum testing will not indicate which pathways are capturing her estrogens, so will not provide the clinician with the insight necessary to safely alleviate this woman’s symptoms. Furthermore, BHRT typically involves using a compounded bi-est containing a combination of estriol and estradiol (often 80% and 20%, respectively). Yet serum testing does not usually measure estriol.

To be able to safely and effectively prescribe hormone replacement therapy (BHRT or HRT) will necessitate knowing not only how much estrone, estradiol and estriol are in circulation, but how much is active (free plus conjugated [potentially active]), and into what forms each estrogen is being metabolized: Is she predominantly producing protective or carcinogenic estrogen metabolites? This data is provided by the 24-Hour Urine Test (see below).

Urine Hormone Testing

Urine hormone testing is well-established in medical literature as a reliable method of assessing levels of active (free & conjugated) hormones and their metabolites, and has been shown in clinical settings to correlate well with patient symptoms and reflect the beneficial impact (or lack of benefit and potentially oncogenic effect) of therapeutic interventions.¹⁴¹⁵

Measuring steroid hormones via urine testing using both gas chromatography-mass spectrometry (GS-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been shown to produce highly accurate results, but radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA, also known as enzyme immunoassay [EIA]) analysis may not be accurate. Comparisons of RIA and ELISA (methods routinely used to measure estrogen metabolites in blood and urine due to efficiency and low cost) with GS-MS and LC-MS/MS have shown RIA and ELISA estrogen metabolite measures to be much less accurate, especially at the low estrogen metabolite levels characteristic of postmenopausal women. The clinical takeaway: ask the lab you use what procedures they are using.¹⁶¹⁷¹⁸

A 24-hour urine sample is practical (collection is non-invasive and easy for the patient) and provides a more accurate indication of hormonal output since it averages out the hour-to-hour fluctuations seen in both serum and salivary measurements.

In addition, a 24-Hour Urine test captures numerous metabolites that are not measurable in saliva and cannot be reliably measured by a single, or even multiple, blood draws. Estrogen metabolites that have been shown to impact estrogen-related cancers (more detail provided in Part II of this article) offer a prime example:

(1) The 2/16α ratio: a decrease in this ratio of the estrone metabolites, 2-hydroxyestrone and 16α-hydroxyestrone, is associated with an increased risk of breast and cervical cancer. Optimal ratio is 2.0 – 4.0.

(2) The “Estrogen Quotient” (EQ): also available only via the 24-hour urine test since it requires assessment of the levels of free estrone, free estradiol and free estriol. Another indicator of breast cancer risk, the EQ, is calculated by dividing the amount of estriol by the sum of that of estrone and estradiol. An EQ <1.0 is suggestive of increased breast cancer risk.

Measurement of urinary estrogens also provides insight into liver and gut function. Urinary levels of Phase I and Phase II estrogen metabolites serve as a functional indication of liver detoxification capability. Abnormal levels may indicate exposure to compounds that stress liver function, such as heavy metals, environmental chemicals, or pharmaceuticals including conjugated equine estrogens and progestins.

Urinary estrogen levels outside the reference range may also be suggestive of intestinal dysbiosis since beta-glucuronidase enzymes, which are produced by E. coli, Clostridum and Bacteroides in the intestines, can de-conjugate conjugated estrogens, enabling their return to the circulation.¹² For these reasons, interventions that improve liver and gut function may assist in gradual normalization of estrogen levels.

A number of glucocorticoid and mineralocorticoid metabolites measured in a 24-hour urine hormone profile, but not in a serum or saliva assay, provide greater insight into long-term adrenal health, short term stress response, the cortisol/cortisone balance, and other measures of adrenal health and function than assessment of cortisol alone. These include cortisone, tetrahydracortisone, allo-tetrahydrocortisol, tetrahydrocortisol, aldosterone, allo-tetrahydrocorticosterone, tetrahydocorticosterone and 11-dehydrotetrahydrocorticosterone, and are discussed in Part II of this review.

Circadian fluctuation of cortisol cannot be measured using 24-hour urine collection, nor can it easily show the monthly cyclical pattern in estrogen and progesterone production in a menstruating or peri-menopausal woman. You could, technically, do 24-hour urine collections for each of the days (as many as 11) tested in a saliva panel for this. But consider the logistics and expense! A single 24-hour urine test cannot provide the clinician with insight into where hormone levels are off during the cycle or whether the patient is ovulating; however, very low progesterone levels in a 24-hour urine sample collected during the mid-luteal phase (days 19-21) would suggest anovulation, and the timing and type of symptoms can give further clues to hormonal imbalance.

The physician should confirm that patients have a clear understanding of collection instructions since sample collection should occur during the mid-luteal phase (days 19-21) if the patient is premenopausal, or postmenopausal and on hormone replacement therapy; if postmenopausal and not on hormone replacement therapy, sample collection may be taken on any day.

To prevent sample contamination, patients applying transdermal cream to the vaginal labia should be instructed to apply to the perianal area or inner thigh on the day of collection.

Part II: The 24-Hour Urine Test: Analyte Interpretation and Evaluation for Women

This article discusses 24-Hour Urine Test results primarily in relation to female patients. Part III of this review will focus on BHRT serum and 24 Hour Urine test results in relation to male patients.

For a schematic overview of the metabolism of these steroids, please see Figure 1, “Metabolism of Select Steroids.”

24 Hour Urine Test Analytes

Creatinine: a break-down product of creatine phosphate in muscle, which is produced by the body at a fairly constant rate and filtered out of the blood by the kidneys. Creatinine concentration provides a marker for a full – neither under nor over – urine collection.

The adult reference range is 0.5 – 2.0 gm/24hr. Because it is a muscle breakdown product, creatinine will typically be higher in men than women (especially if the patient exercised on the day of collection).

Estrone (E1): The most potent estrogen, estrone is synthesized from androstenedione by aromatase in the ovaries and adipose tissue in premenopausal women, and in adipose tissue in postmenopausal women. Estrone, which

Micrograms of Vitamin K Present in Foods (mcg in 100 g or 100 ml)*
Food	K1	MK-4	MK-7,8,9
Meats	0.5-5	1-30	0.1-2
Fish	0.1-1	0.1-2
Green vegs	100-750
Natto	20-40		900-1200
Cheese	0.5-10	0.5-10	40-80
Other dairy	0.5 15	0.2-15	0-35
Eggs	0.5-2.5	10-25

24-Hour Urine Comprehensive Profile: Analytes & Reference Ranges for Men
*Analyte*	Adult Reference Range*
Creatinine	0.5 – 2.0 gm/24hr
Total urine volume	1,200 – 3,000 mL
*Steroid*	*Amount excreted in μg/24 hr*
Sex Hormones
Estrone (E1)	3.0 – 11.4
Estradiol (E2)	0.8 -4.6
Estriol (E3)	3.5 – 13.7
Total Estrogens	7.3 -29.7
2-OH Estrone (Phase I metabolite)	1.9 – 15.8
16α-OH Estrone (Phase I metabolite)	0.2 – 5.9
2/16α Ratio (Ideal = 2 - 4)	1.2 – 4.9
Pregnanediol (progesterone metabolite)	70 – 1,050
DHEA	100 – 2,000
Androsterone (DHEA metabolite)	2,000 – 5,000
Etiocholanolone (DHEA metabolite)	1,400 – 5,000
(Androsterone:etiocholanolone ratio, if low, suggestive of low 5AR activity, low 5α-DHT, low 3β-adiol)
Testosterone	20 - 200
5α-Androstanediol	22 - 131
5β-Androstanediol	40 - 401
Glucocorticoids
Pregnanetriol ( want ≥mid-range, indicator of substrate availability for cortisol pathway )	200 – 1,500
Cortisone (optimal range ~120-130μg/24hr)	31 - 209
Cortisol (optimal range ~90μg/24hr)	30 - 170
Cortisone: Cortisol Ratio (always interconverting)	Ideal = 0.7
Tetrahydrocortisone (THE, cortisone metabolite)	2,100 – 7,400
Allo-tetrahydrocortisol (5α-THF, cortisol metabolite)	700 – 3,800
Tetrahydrocortisol (THF, cortisol metabolite)	1,200 – 4,500
(THE + 5α-THF + THF) x2 = daily cortisol output	8,000 – 10,000
11β-OH Androsterone (terminal metabolite of cortisol; low value confirms insufficiency)	613 – 2,298
11β-OH Etiocholanolone (terminal metabolite of cortisol, low value confirms insufficiency)	153 - 950
Mineralocorticoids
Aldosterone(indicator of salt level in diet, low may indicate adrenal fatigue)	Normal Diet = 6.0 – 25.0 Low Salt: 17.0 – 44.0 High Salt: 0.0 – 6.0
Allo-tetrahydrocorticosterone (5α-THB, low = long term adrenal insufficiency, high = acute stress at collection)	130 – 1600
Tetrahydrocorticosterone (THB, low = long term adrenal insufficiency, high = acute stress at collection)	30 -240
11-dehydrotetrahydrocorticosterone (THA, low = long term adrenal insufficiency, high = acute stress at collection)	76 - 456
Human Growth Hormone	1,065 – 4,722
Thyroid Hormones
Free T3	470 – 1,750
Free T4	430 -3,200
T3 + T4 (if <1000 adrenal support may be indicated)	~1000
Urinary Sodium and Potassium
Sodium	40 - 220
Potassium	25 – 150
Sodium:Potassium Ratio (ideal 1.5, if low consider malabsorption)	1.2 – 4.8
Enzyme Activity
5α-Reductase enzyme (converts testosterone to more potent 5α-DHT) activity as shown by:
Androsterone/Etiocholanolone Ratio	Ideal = Mid-range 0.7
Allo-tetrahydrocortisol /Tetrahydrocortisol Ratio	Ideal = Mid-range 0.6
Elevated 5AR activity associated BPH, premature baldness, obesity, insulin resistance in men Low 5AR activity suggestive of reduced conversion of testosterone to DHT, undervirilization, low 3β-adiol production, increased prostate cancer risk

11β-Hydroxysteriod dehydrogenase I and II (isoforms of enzyme that converts cortisol to cortisone) activity shown by:
Cortisol: Cortisone Ratio	Ideal 0.7
Tetrahydrocortisol + allo-tetrahydrocortisol: Tetrahyrdocortisone Ratio	Ideal 0.9
Low ratios associated w/obesity & insulin resistance. Elevated ratios w/low-renin hypertension, high dose licorice, exogenous cortisol.
*Reference ranges vary by lab and can may be expressed in different units of measure

24-Hour Urine Comprehensive Profile: Analytes & Reference Ranges for Women
*Analyte*	Adult Reference Range*
Creatinine	0.5 – 2.0 gm/24hr
Total urine volume	1,200 – 3,000 mL
*Steroid*	*Amount excreted in μg/24 hr*
Sex Hormones	Luteal (Days 17-26)	Postmenopausal
Estrone (E1)	3.3 - 44.6	1.0 - 7.0
Estradiol (E2)	1.4 - 12.2	0 - 4
Estriol (E3)	6.1 - 32.4	0 - 30
Total Estrogens	10.8 - 89.2	0 - 41
Estrogen Quotient (E3 / E1 + E2)	>1.0
2-OH Estrone (Phase I metabolite)	3.8 - 38.1	0.2 - 5.4
16α-OH Estrone (Phase I metabolite)	2.1 – 7.9	0.15 – 3.5
2/16α Ratio (Ideal = 2 - 4)	1.8 – 5.5	0.6 – 5.0
4-OH Estrone (Phase I metabolite)	0.8 – 5.9	0.05 – 1.1
2-methoxyestrone (Phase II metabolite)	2.2 – 14.4	0.3 – 4.1
2-methoxyestradiol (Phase II metabolite)	0.2 – 2.2	0.03 – 0.54
Pregnanediol (progesterone metabolite)	1450 – 6140	200 – 1000
DHEA	100 - 2000
Androsterone (DHEA metabolite)	500 – 3200
Etiocholanolone (DHEA metabolite)	500 - 5000
Testosterone	5.0 – 35.0
5α-Androstanediol	3.0 – 35.0
5β-Androstanediol	13.0 – 180.0
Glucocorticoids
Pregnanetriol ( want ≥mid-range, indicator of substrate availability for cortisol pathway )	100 – 1500
Cortisone (optimal range ~120-130μg/24hr)	31 - 209
Cortisol (optimal range ~90μg/24hr)	30 - 170
Cortisone: Cortisol Ratio (always interconverting)	Ideal = 0.7
Tetrahydrocortisone (THE, cortisone metabolite)	1700 - 4200
Allo-tetrahydrocortisol (5α-THF, cortisol metabolite)	400 – 2100
Tetrahydrocortisol (THF, cortisol metabolite)	900 - 2600
(THE + 5α-THF + THF) x2 = daily cortisol output	5000 - 7000
11β-OH Androsterone (terminal metabolite of cortisol; low value confirms insufficiency)
11β-OH Etiocholanolone (terminal metabolite of cortisol, low value confirms insufficiency)
Mineralocorticoids
Aldosterone(indicator of salt level in diet, low may indicate adrenal fatigue)	Normal Diet = 6.0 – 25.0 Low Salt: 17.0 – 44.0 High Salt: 0.0 – 6.0
Allo-tetrahydrocorticosterone (5α-THB, low = long term adrenal insufficiency, high = acute stress at collection)	130 – 1600
Tetrahydrocorticosterone (THB, low = long term adrenal insufficiency, high = acute stress at collection)	30 -240
11-dehydrotetrahydrocorticosterone (THA, low = long term adrenal insufficiency, high = acute stress at collection)	62 - 293
Human Growth Hormone	1065 - 4722
Thyroid Hormones
Free T3	470 - 1750
Free T4	430 -3200
T3 + T4 (if <1000 adrenal support may be indicated)	~1000
Urinary Sodium and Potassium
Sodium	40 - 220
Potassium	25 – 150
Sodium:Potassium Ratio (ideal 1.5, if low consider malabsorption)	1.2 – 4.8
Enzyme Activity
5α-Reductase enzyme (converts testosterone to more potent 5α-DHT) activity as shown by:
Androsterone/Etiocholanolone Ratio	Ideal = Mid-range 0.7
Allo-tetrahydrocortisol /Tetrahydrocortisol Ratio	Ideal = Imd-range 0.6
Elevated 5α-reductase activity associated w/PCOS, hirsutism, obesity, insulin resistance in women

11β-Hydroxysteriod dehydrogenase I and II (isoforms of enzyme that converts cortisol to cortisone) activity shown by:
Cortisol: Cortisone Ratio	Ideal 0.7
Tetrahydrocortisol + allo-tetrahydrocortisol: Tetrahyrdocortisone Ratio	Ideal 0.9
Low ratios associated w/obesity & insulin resistance. Elevated ratios w/low-renin hypertension, high dose licorice, exogenous cortisol.
*Reference ranges vary by lab and can may be expressed in different units of measure

Adrenal Fatigue: The 21st Century Stress Syndrome

Vitamin K2: Optimal Levels Essential for the Prevention of Age-Associated Chronic Disease

Abstract

Introduction

Vitamin K Protects against Degenerative Diseases of Aging by Activating Vitamin K-Dependent Proteins

Vitamin K Needs in Apparently Healthy Humans Unmet for Extra-Hepatic Vitamin K-Dependent Proteins

Vitamin K: A Key Player in the Longevity Game

K2’s Regulation of the Body’s Use of Calcium Critical for Bone, Vascular and Kidney Health

Vitamin K Insufficiency – A Risk Factor for Kidney Disease?

K2 Supplementation Essential in Patients Supplementing with Calcium and Vitamin D

Vitamin K Insufficiency – A Risk Factor for Alzheimer’s Disease?

Vitamin K’s Neuroprotective Activities

Vitamin K2 Insufficiency – A Risk Factor for Type 2 Diabetes?

The Osteocalcin Connection to Insulin Sensitivity

K1’s Anti-Inflammatory Actions May Promote Insulin Sensitivity

MK-7 Likely More Effective in Carboxylating Osteocalcin than K1 or MK-4

Vitamin K2 Insufficiency – A Risk Factor for Cancer

Earliest Indicator of Vitamin K Insufficiency: Incomplete Carboxylation of Extra-Hepatic VKD-Proteins

Micronutrient Requirements Increase with Age

VKD Proteins Highly Vulnerable to “Kinetics of Aging”

K2, Particularly the Longer Chain Menaquinones, Crucial for Healthy Aging

K2, But Not K1, Carboxylates the VKD-Proteins that Prevent Chronic Age-Associated Disease

The Body's Different Uses for K1 and K2 are Demonstrated by Their Different Distributions Over Plasma Lipoproteins

K1 and MK-4 Share Similar Structure, Thus Both Have a Shorter Half-Life than Long-Chain MKs

Vitamin K1’s Contributions to Healthy Aging

K1 Promotes Bone Quality not Quantity

K1’s Anti-Inflammatory Actions Protective vs. Osteoarthritis

Vitamin K Promotes Detoxification

Conclusion

Supplementation with Physiological Doses of Vitamin K -- K1 plus MK-7 -- Rather than Pharmacological Doses of MK-4, May Be Preferable

One Caveat for Patients on Coumarins (Oral Anticoagulants)

L-Citrulline: Restoring Erectile Function (Viagra Doesn’t)

L-citrulline corrects the cause of ED, PDE5-inhibitors do not

PDE5 inhibitors are not innocuous:

Conclusion

Bio-Identical Hormone Replacement: Selecting the Right Hormone Test(s) for Your Male Patient

by Lara Pizzorno, MDiv, MA, LMT with Barry Wheeler, ND

Salivary Hormone Testing

Advantages

Disadvantages

Blood (Serum) Hormone Tests

Advantages

Disadvantages

Testosterone Metabolites Serum Testing

Mechanisms through which 5α-reductase inhibitors promote prostate cancer

Balancing Men’s Hormones Summary:

Issues to Consider

Promoting 3β-adiol Production

Monitor Impact of All 5α-Reductase Inhibitors

5α-Reductase Inhibitors

5α-Reductase Agonists

Indirect Assessment of 5α-Reductase Activity

Urine Hormone Testing

Advantages

Disadvantages

The 24-Hour Urine Test: Analyte Interpretation and Evaluation for Men

24 Hour Urine Test Analytes

SEX HORMONES: ESTROGENS

SEX HORMONES: PROGESTERONES

SEX HORMONES: ANDROGENS

GLUCOCORTICOIDS

MINERALOCORTICOIDS

Thyroid Hormones

Urinary Minerals

Enzyme Activity

CONCLUSION

Serum and 24-Hour Urine Hormone Testing—Sine Qua Non for Optimal Men’s BHRT

Ensure Effective Bio-Identical Hormone Replacement: Select the Right Hormone Test for Your Patient

by Lara Pizzorno, MDiv, MA, LMT, with Pushpa Larsen, ND

Part I: The Advantages and Disadvantages of Saliva, Serum and Urine Tests

Salivary Hormone Testing

Blood (Serum) Hormone Tests

Urine Hormone Testing

Part II: The 24-Hour Urine Test: Analyte Interpretation and Evaluation for Women

24 Hour Urine Test Analytes