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Strategies for Cardiovascular Disease: Closing the Statin Gap in Endothelial Dysfunction (LMR)

by Lara Pizzorno, MDiv, MA, LMT

This is a Longevity Medicine Review™ article.
For less technical content see the related articles at the bottom of this page.

Abstract

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

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

Introduction

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

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

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

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

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

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

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

Endothelial Function, L-arginine and the Nitric Oxide Pathway

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

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

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

Impairment of the Endothelial L-arginine/NO Pathway. A) Oxidized LDL upregulates arginase production, using up L-arginine stores. Thus, less L-arginine is available for NOS. B) When the ratio of L-arginine to ADMA is decreased, eNOS uncouples, promoting increased production of O2-, ROS & RNS, instead of NO. C) L-arginine stimulates insulin release, which is blocked by ADMA and is now thought to be largely due to ADMA inhibiting the neuronal isoform of NOS. Thus an elevated ratio of ADMA:L-arginine promotes both CVD and insulin resistance. (Flowchart by John Morgenthaler.)

L-arginine Supplementation Significantly Improves Endothelial Function …

In individuals with essential hypertension

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

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

In individuals with compromised flow-mediated dilation

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

In individuals with chronic heart failure

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

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

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

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

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

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

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

L-arginine may benefit men with erectile dysfunction

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

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

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

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

Impairment of the Endothelial L-arginine/NO Pathway. A) Oxidized LDL upregulates arginase production, using up L-arginine stores. Thus, less L-arginine is available for NOS. B) When the ratio of L-arginine to ADMA is decreased, eNOS uncouples, promoting increased production of O2-, ROS & RNS, instead of NO. C) L-arginine stimulates insulin release, which is blocked by ADMA and is now thought to be largely due to ADMA inhibiting the neuronal isoform of NOS. Thus an elevated ratio of ADMA:L-arginine promotes both CVD and insulin resistance. (Flowchart by John Morgenthaler.)

Primary Mechanisms through which L-arginine Improves Endothelial Function27

Antagonizes ADMA

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

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

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

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

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

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

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

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

Increases insulin secretion

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

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

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

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

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

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

Conclusion

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

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

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

Tetrahydrobiopterin (BH4) — key to L-arginine’s efficacy in established atherosclerosis1 6 2

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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);

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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

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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

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Methylating Factors6

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

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

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

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

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

Co-factors Essential for L-arginine Metabolism to NO. A) L-citrulline is converted into L-arginine by the enzymes arginosuccinate synthetase [ASS] and arginosuccinate lyase [ASL]. B) Homocysteine inhibits DDAH, so ADMA is not converted to citrulline, and AMDA levels build up. C) Methylation factors, used up in the conversion of L-arginine to creatine, are needed to metabolize homocysteine, which will otherwise inhibit DDAH, increasing ADMA. D) In conditions of BH4 deficiency eNOS leads to increased O2-. E) NO converts into Nitrite, Nitrate, and Peroxynitrites when ROS are present. (Flowchart by John Morgenthaler.)

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L-arginine Dosage Considerations

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

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

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

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

Potential side-effects of L-arginine supplementation

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

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

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

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

Citrulline – a Preferential Source of L-arginine?

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

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

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

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

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

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

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

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

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

Adding L-Arginine Closes the Statin Gap

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

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

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

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

Assessing Endothelial Function

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

Conclusion

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

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

References

  1. Statin Use in the Civilian Noninstitutionalized Medicare Population in 2002. Statistical Brief #96. U.S. Department of Health & Human Services Agency for Healthcare Research and Quality, accessed Oct. 4, 2009.

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