Despite their efficacy in lowering cholesterol, statins remain ineffective for the primary or secondary prevention of myocardial infarction (MI) in two-thirds of patients, and cardiovascular disease (CVD) remains the #1 cause of death in the U.S. A key reason for statins’ failure to reduce MI incidence is that they ameliorate neither endothelial dysfunction, nor its corollary, hypertension. Additionally, statins do not impact insulin resistance, a major contributing factor to CVD as well as diabetes, which itself quadruples risk for CVD. Statins’ lack of efficacy against these risk factors constitutes a treatment gap that results in high risk of morbidity notwithstanding low cholesterol levels—a gap that has now been connected to elevated levels of a recently identified CVD risk factor, asymmetric dimethylarginine, whose deleterious effects can be overcome by treatment with L-arginine, which has been shown to significantly improve endothelial function both alone and when added to statin therapy, particularly if accompanied by necessary co-factors, including tetrahydrobiopterin and the methylating factors, B6, B12 and folate. Part I of this review summarizes the latest research on L-arginine’s beneficial effects on nitric oxide production, endothelial function and insulin resistance. Part II reviews the research on the co-factors without which L-arginine supplementation may not only lack efficacy, but could promote CVD; improvement in CVD outcomes from combined statin and L-arginine therapy; and why L-citrulline may offer an even better option.
BH4 is a necessary cofactor for endothelial nitric oxide synthase (eNOS) to produce NO. Furthermore, in conditions of deficient BH4, eNOS generates superoxide (O2-) instead of NO; therefore, it is particularly important to use BH4 in conjunction with L-arginine.
NO is synthesized by the family of oxidoreductase enzymes, the NO synthases, which utilize L-arginine as their principal substrate, oxidizing it to L-citrulline and NO, in a process involving the cofactors reduced nicotinamide adenine dinucleotide phosphate (NADPH) [which requires niacin], flavin adenine dinucleotide (FAD) [which requires riboflavin], and tetrahydrobiopterin (BH4).
Insufficiency of endothelial NO can occur as a result of decreased synthesis due to decreased expression of eNOS and/or insufficient substrate (L-arginine) and/or co-factors (tetrahydrobiopterin, niacin, riboflavin); or an increase in NO’s oxidative inactivation to nitrite, nitrate, and peroxynitrites due to the presence of increased ROS in the vasculature, including O2-, H2O2, derivative hydroxyl radical (OH-); lipid peroxides (e.g., malondialdehyde) and derivative peroxyl radicals; or both. Under normal circumstances, these highly reactive derivatives of oxidative metabolism are neutralized by antioxidant enzymes (e.g., superoxide dismutases and glutathione peroxidases) and antioxidants (e.g., -tocopherol and ascorbate neutralize ROS; γ-tocopherol neutralizes reactive nitrogen species [RNS]) (Vitamin A – Tolerance Extends Longevity); however, virtually all risk factors for CVD, as well as frank CVD, increase the load of ROS and RNS in the vasculature. eNOS itself will produce O2- by reducing oxygen when eNOS becomes "uncoupled" by limited availability of its cofactor tetrahydrobiopterin or of its substrate, L-arginine.
In established atherosclerosis, expression of the inducible form of NOS ( iNOS) is strongly upregulated. iNOS is present in activated macrophages, is found within atherosclerotic lesions, and is proatherogenic. iNOS produces greater amounts of NO compared to eNOS—a big problem in an atheromatous phenotype in which ROS are abundant because the large increase in NO production results in NO reacting with O2- and lipid peroxyl radicals to yield the RNS, peroxynitrite and lipid peroxynitrites.
These RNS are potent oxidants and inactivate NO because peroxynitrites oxidize tetrahydrobiopterin (BH4), which uncouples eNOS. The uncoupling of eNOS not only decreases NO production, but the uncoupled enzyme itself increases production of O2-. This promotes a vicious cycle in which levels of reactive species build, causing further eNOS uncoupling, insufficient NO production, and oxidative damage to proteins in the vessel wall—a recipe for CVD progression.3
Even if ROS are controlled by antioxidants, in the absence of sufficient BH4, the oxidation of L-arginine is no longer coupled to NADPH consumption, so NOS isoforms catalyze the formation of O2- at the oxygenase domain. In fact, BH4 levels appear to regulate the ratio of O2- and NO made by NOS enzymes. When BH4 levels are adequate, eNOS produces NO; when BH4 levels are limiting, eNOS becomes enzymatically uncoupled and generates O2-, contributing to vascular oxidative stress and endothelial dysfunction. Thus, insufficient BH4 can be a key cause of NO-related endothelial dysfunction.
BH4 levels can be preserved by supplementation with ascorbate and 5-methyltetrahydrofolate (5-MTHF), the circulating form of folate. Ascorbate does not fully protect BH4 from oxidation by perxoynitrite (ONOO-), but effectively recycles BH3 radical back to BH4.4 5-MTHF is a strong peroxynitrite scavenger, which has been shown to increase vascular BH4 and the BH4/total biopterin ratio.5
Since, as noted above, iNOS promotes CVD progression, researchers hypothesized that providing supplemental L-arginine to apoE null mice [animals bred to quickly develop atherosclerosis] also lacking iNOS would decrease plaque burden. It didn’t; in fact, it increased atheromatous burden to levels observed in the apoE null mice, completely offsetting the benefit of eliminating iNOS.7
The most likely explanation for this outcome is related to the fact that approximately 10-fold more L-arginine is metabolized to creatine than is used for NO synthesis,8 9 and creatine synthesis requires the methylation of guanidinoacetate by S-adenosyl-methionine in the liver, which yields S-adenosyl-homocysteine. S-adenosyl-homocysteine is then hydrolyzed to adenosine and homocysteine by S-adenosyl-homocysteine hydrolase.
Homocysteine can either be metabolized by methylation to methionine or undergo transsulfuration to cysteine, which can be utilized to form glutathione. However, converting homocysteine to methionine requires adequate methylation support, which may be in short supply because -- although the source of methyl groups is in a separate, although metabolically linked cellular process -- as much as 70% of accessible methyl groups may be used up for creatine synthesis.10
In addition, vascular cells are unable to transsulfurate homocysteine to cysteine (and thence to glutathione), so their only option for neutralizing homocysteine is remethylating it to methionine.7 Thus, if nutrients necessary for methylation (B6, B12, folate) are in short supply, local concentrations of homocysteine, a highly atherogenic amino acid, will increase in the vasculature. To cap it all off, homocysteine increases plasma concentrations of ADMA by inhibiting dimethylarginine dimethylaminohydrolase, the enzyme that metabolizes this NOS inhibitor to L-citrulline.11
The clinical takeaway here is to ensure adequacy of both tetrahydrobiopterin and methylating factors when supplementing L-arginine, not only to promote L-arginine’s efficacy in improving endothelial function, but because inadequacy of these factors may result in increased CVD risk.
Co-factors Essential for L-arginine Metabolism to NO
Flowchart by John Morgenthaler
A recent study evaluating the responses of healthy individuals to increasing doses of L-arginine suggests that supplementation with 3 grams bid is optimal. This level, which is about twice the amount of L-arginine present in a typical Western diet, was associated with no adverse side effects.12
Study participants were instructed to take L-arginine for 1-week periods at daily doses of 3, 9, 21, and 30 grams. Ten of the 12 subjects reported adverse gastrointestinal side effects at higher dosages, five subjects at 21 grams/day, and five subjects at 30 grams/day. It was hypothesized that a large bolus of L-arginine may disturb the acid–base balance of the stomach and GI tract, thus provoking gastrointestinal symptoms.
Mean L-arginine concentrations peaked at 9 grams/day (169 μmol/L), were found to be significantly higher than values at baseline (101 μmol/L) or 3 grams/day (110 μmol/L), and also slightly higher than L-arginine levels at 21 g/day (164 μmol/L).
Availability of L-arginine, relative to that of ADMA, increased significantly at both 9 grams/day and 21 grams/day. As ADMA is produced by proteolysis of proteins containing methylated arginine residues, its levels would not be expected to be altered by L-arginine supplementation and, in fact, were not. Indeed, lack of effect on ADMA levels is an aspect of the practical utility of L-arginine supplementation, which increases the substrate available to NOS without raising the level of inhibitors. Any increase in arginine accessibility when ADMA levels remain constant or decline slightly will favor NO production.
L-arginine induces water and electrolyte secretion that is mediated by NO, which acts as an absorbagogue at low levels and as a secretagogue at high levels. The action of many laxatives is NO mediated, and diarrhea following oral administration of arginine in single doses >9 grams has been reported.
The clinical data cover a wide span of arginine intakes from 3 grams/day to>100 grams/day. Single doses of 3-6 grams rarely provoke side effects. Single doses >9 grams are more likely to provoke gastrointestinal symptoms in healthy athletes than diabetic patients, which may relate to the effects of disease on gastrointestinal motility and pharmacokinetics. Most side effects have occurred at single doses of >9 grams in adults, often when part of a daily regime of >30 grams/day. Adverse effects seemed dependent on the dosage regime and disappeared if divided doses were ingested.13
L-arginine may promote replication of herpes simplex virus 1 (HSV-1) in already infected individuals. HSV-1 multifunctional regulatory protein ICP27 shuttles between the nucleus and cytoplasm in its role as a viral mRNA export factor. Arginine methylation has been shown to regulate protein ICP27 export and is thus required for efficient HSV-1 replication. Therefore, individuals infected with oral and/or genital herpes may need to reduce L-arginine dosage or avoid foods high in L-arginine when under high stress, fighting off a viral infection, or otherwise immune-challenged.14
On the other hand, arginine has recently been investigated as an antiherpetic agent against HSV-1. Arginine suppressed the growth of HSV-1 concentration-dependently and was particularly effective when added within 6 hours post-infection. When administered in the early stages of the infection, the latent period was significantly extended; the rate of viral replication decreased, and the final yield of viral progeny decreased to 1%. However, when arginine was added at 8 hours post infection after the completion of viral DNA replication, the amount of viral progeny produced in the subsequent 4 hours reached normally expected levels.15
Supplemental L-arginine is readily absorbed; however, ~50% of ingested L-arginine is rapidly converted in the body to ornithine, primarily by the enzyme arginase.16 As noted above, in the presence of oxidized LDL, arginase activity is increased, and arginase competes with NOS for L-arginine in its role as a substrate for NOS, which results in impaired production of NO and increased production of ROS by NOS.
In addition to supplemental L-arginine, arginase expression and activity is enhanced by diseases associated with endothelial dysfunction, including hypertension, heart failure, atherosclerosis, diabetic vascular disease and ischemia-reperfusion injury, thus further reducing the effectiveness of L-arginine therapy in those who need it most.17
Furthermore, L-arginine, in addition to its role as a substrate for NOS, is also a precursor for the synthesis of proteins, urea, creatine, vasopressin, and agmatine. Thus, L-arginine that escapes metabolism by arginase is targeted for processing by four other enzymes: NOS (to become NO); arginine:glycine amidinotransferase (to become creatine); arginine decarboxylase (to become agmatine); and arginyl-tRNA synthetase (to become arginyl-tRNA, a precursor to protein synthesis).17
Because of L-arginine’s fast turnover, sustained-release preparations are thought to be a better way to maintain blood levels over time, yet arginase’s substantial intestinal and hepatic metabolism of L-arginine to ornithine and urea, and L-arginine’s use for other functions in the body, impede the likelihood of optimal efficacy from oral supplements of L-arginine.18
L-citrulline may be a better option. L-citrulline is readily absorbed and efficiently converted to L-arginine. But its conversion does not take place in the intestine or liver, and therefore not only does not induce tissue arginase, but inhibits its activity. Upon entering the kidney, vascular endothelium and other tissues, L-citrulline is readily converted to L-arginine, raising plasma and tissue levels of L-arginine and enhancing NO production.14 Published data indicates that L-citrulline has relatively better absorption and systemic bioavailability than L-arginine.19
In a double-blind, randomized, placebo-controlled cross-over study, 20 healthy volunteers were given 6 different dosing regimens of placebo, citrulline, and arginine. After one week of oral supplementation, the plasma L-arginine/ADMA ratio was measured. L-citrulline dose-dependently increased plasma L-arginine concentration more effectively than L-arginine. The highest dose of citrulline (3 grams bid) improved the L-arginine/ADMA ratio from 186 +/- 8 (baseline) to 278 +/- 14, a 49% increase.20
In another recent study, L-citrulline supplementation was shown to attenuate brachial blood pressure and aortic hemodynamic responses to stress induced by the cold pressor test (CPT) in 17 young (average age 21.6 years) normotensive men. Subjects were randomly assigned to 4 weeks of oral L-citrulline (6 grams/day) or placebo in a crossover design. Hemodynamic responses to CPT were evaluated after each treatment. Compared to placebo, oral L-citrulline decreased brachial systolic blood pressure (-6 +/- 11 mm Hg), aortic systolic blood pressure (-4 +/- 10 mm Hg), and aortic pulse pressure (-3 +/- 6 mm Hg) during CPT but not at rest, suggesting improved induction of NO under stress.21
L-citrulline’s beneficial effects on NO were also demonstrated in a group of 17 male professional cyclists in whom oral L-citrulline administration prior to a cycling race increased plasma arginine availability for NO synthesis and immune cells’ (polymorphonuclear neutrophils [PMNs]) priming for oxidative burst without oxidative damage. Cyclists were randomly assigned receive 6 grams L-citrulline-malate or placebo, after which they participated in a race. Blood samples were taken in basal conditions, immediately after and 3 hours post-race. Citrulline supplementation significantly increased plasma concentration of both arginine and citrulline post-race. In controls, PMNs responded to exercise with a progressive decrease in immune-defensive ROS production, while PMNs in those supplemented with L-citrulline significantly increased ROS production after exercise compared to basal values and then diminished to values lower than basal at recovery.22 L-citrulline’s enhancement of immunity may benefit endurance athletes as it has long been noted that immune defenses drop post-event, increasing susceptibility to infection.23 24 25 26 27
Citrulline’s potential benefit to endothelial function is also evidenced by the fact that impairment in the activity or expression of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that metabolizes ADMA to L-citrulline and dimethylamine, results in elevated ADMA concentrations and reduced NO synthesis, and promotes the onset and progression of atherosclerosis in experimental models. AMDA elevation may be a marker for insufficient availability of citrulline due to DDAH activity and/or expression, which then contributes to the pathogenesis of endothelial dysfunction in various diseases.18 28
Despite the fact that statins increase eNOS expression (by stabilizing eNOS mRNA) and enhance eNOS activity (by decreasing caveolin, which otherwise forms an inhibitory complex with eNOS and impairs NO release), statins have failed to improve endothelial function in the majority of studies in which their effects on the endothelium have been evaluated.2 29 30
A key reason for statins’ lack of efficacy in this regard is ADMA, which inhibits eNOS by a mechanism unaffected by statins, but reversible by L-arginine.31 It was therefore hypothesized, and has now been demonstrated in several studies, that providing both a statin and L-arginine to individuals with high levels of AMDA can close the statin gap.
In a study involving 98 clinically asymptomatic elderly subjects, those in the highest and lowest quartiles of the ADMA distribution were given, in a randomized order, simvastatin (40 mg/day), L-arginine (3 grams/day), or a combination of both, each for 3 weeks. Endothelium-dependent vasodilation (EDD) was assessed by brachial artery ultrasound. While simvastatin alone had no effect on EDD in subjects with high ADMA (6.2 at baseline vs. 6.1 after simvastatin treatment), simvastatin plus L-arginine significantly improved EDD (9.8 at baseline vs. 5.3 after treatment). In subjects with low ADMA, L-arginine alone, simvastatin alone, or the two combined improved EDD. When given alone, only L-arginine, but not simvastatin, improved endothelial function in both groups.30
L-arginine has also been shown to enhance the triglyceride-lowering effect of simvastatin in a 2-arm, randomized, double-blind study of 33 hypertriglyceridemic patients that consisted of a 6-week run-in phase, 6 weeks of treatment with L-arginine (1.5 grams bid) or placebo, and a 6-week extension period in which simvastatin (20 mg q.d.) was added. The combination of L-arginine with simvastatin led to a significantly stronger reduction in triglycerides compared to placebo plus simvastatin (-140.5 +/- 149.2 mg/dL vs. -56.1 +/- 85.0 mg/dL). L-arginine also attenuated simvastatin-induced increases in aspartate transaminase (elevated levels of this enzyme indicate acute liver, cardiac and skeletal muscle, blood cell, kidney and/or brain tissue damage) and also in fibrinogen (the protein in plasma from which fibrin is generated in the process of clot formation), but had no triglyceride-lowering effects when given alone.32
Although FMD is the “gold standard” to evaluate endothelial function, this technique requires specialist imaging equipment and great attention to detail in order to obtain reproducible results. An alternative approach, suitable for assessing pre-clinical atherosclerosis and evaluating the effect of interventions on endothelial function, e.g., L-arginine supplementation, has recently become available. Vascular tone of the small arteries can now be easily and inexpensively measured in-office by checking the digital pulse wave reflection index, which has been shown to correlate with FMD and has high sensitivity and specificity in detecting arterial distensibility and stiffness, and abnormal endothelial function as defined by FMD.33 34 35 In recently published studies using pulse wave methodology, L-citrulline has been shown to lower aortic pulse pressure and improve hemodynamic responses to stress in normotensive men, and L-arginine to improve carotid-femoral pulse wave velocity, an index of aortic stiffness, in healthy smokers at rest and after acute smoking.36 37
Lifetime risk for CVD is currently 49% for men and 32% for women ≥age 40—statistics that are likely to increase exponentially given the fact that individuals with diabetes are two to four times more likely to develop CVD, and, as of 2006, 66.7% of the adult population in the United States was overweight or obese, 34.6% had MetS, and 5.9% had been diagnosed with type 2 diabetes.
Despite their efficacy in lowering cholesterol, statins are unable to improve endothelial function, a risk factor for CVD-related mortality on a par with hypercholesterolemia. Nor do statins impact insulin resistance, a key cause of endothelial dysfunction. Recent studies indicate that endothelial dysfunction, as reflected by FMD (or pulse wave reflection index), is a more powerful prognosticator of future cardiac events than carotid artery plaque burden, and patients with high FMD have low cardiovascular event rates irrespective of their degree of carotid atheroma. Statins’ lack of efficacy against endothelial dysfunction constitutes a gap in the treatment of CVD that results in high risk of morbidity notwithstanding low cholesterol levels—a gap that is largely due to elevated levels of ADMA and may be overcome by supplementation with L-arginine and/or L-citrulline.
Read Part I: L-Arginine Closes the Statin Gap by Overcoming ADMA’s Promotion of Endothelial Dysfunction and Insulin Resistance
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