Association of l-Arginine Supplementation with Markers of Endothelial Function in Patients with Cardiovascular or Metabolic Disorders: A Systematic Review and Meta-Analysis

Abstract

l-Arginine supplementation is a potential therapy for treating cardiovascular and metabolic diseases. However, the use of distinct l-arginine sources, intervened populations, and treatment regimens may have yielded confusion about their efficacy. This research constitutes a systematic review and meta-analysis summarizing the effects of l-arginine supplementation compared to placebo in individuals with cardiovascular disease (CVD), obesity, or diabetes. Eligibility criteria included randomized clinical trials and interventions based on oral supplementation of l-arginine with a minimum duration of three days; comparison groups consisted of individuals with the same disease condition receiving an oral placebo substance. The primary outcome was flow-mediated dilation, and secondary outcomes were nitrite/nitrate (NOx) rate and asymmetric dimethylarginine (ADMA). Statistical heterogeneity among studies included in the meta-analyses was assessed using the inconsistency index (I2). Fifty-four full-text articles from 3761 retrieved references were assessed for eligibility. After exclusions, 13 studies were included for data extraction. There was no difference in blood flow after post-ischemic hyperemia between the supplementation of l-arginine and placebo groups before and after the intervention period (standardized mean difference (SMD) = 0.30; 95% confidence intervals (CIs) = -0.85 to 1.46; I2 = 96%). Sensitivity analysis showed decreased heterogeneity when the studies that most favor arginine and placebo were removed, and positive results in favor of arginine supplementation were found (SMD = 0.59; 95% CIs = 0.10 to 1.08; I2 = 75%). No difference was found in meta-analytical estimates of NOx and ADMA responses between arginine or placebo treatments. Overall, the results indicated that oral l-arginine supplementation was not associated with improvements on selected variables in these patients (PROSPERO Registration: CRD42017077289).

Keywords: asymmetric dimethylarginine; cardiovascular disease; flow-mediated dilation; nitric oxide; obesity; type 2 diabetes.

Figure 1

Mechanisms for lowering nitric oxide (NO) availability in cardiovascular and metabolic disorders (A). Increased angiotensin II (AngII) levels, asymmetric dimethylarginine (ADMA), and low plasma l-arginine concentration are all conditions likely to reduce NO· production. Inflamed adipose tissue (due to its expansion—obesity) can lead to (i) ↑ release of inflammatory cytokines; (ii) ↑ release of AngII; (iii) ↑ protein catabolism (due to the pro-inflammatory state); and (iv) ↑ activation of macrophages. Angiotensin II, acting through its receptor (AT1R), increases generation of superoxide (O²⁻), primarily through activation of reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase. O²⁻ reacts with NO∙ to form peroxynitrite (ONOO∙), a very reactive and destructive molecule, leading to decreased availability of NO∙. Moreover, superoxide is a known inhibitor of dimethylarginine dimethylhydrolase (DDAH), a key regulatory enzyme, which controls the metabolism of ADMA. ADMA is an endogenous methylated amino acid that inhibits the constitutive endothelial and neuronal isoforms of nitric oxide synthase (NOS). ADMA is released by protein hydrolysis; thus, increased catabolism induced by several inflammatory cytokines can elevate the ADMA levels. As DDAH uses ADMA as a substrate and regulates plasma levels of ADMA, it may influence the bioavailability of NO∙ and possibly contribute to changes in blood pressure. Homocysteine (an indirect substrate for the synthesis of ADMA) is also an inhibitor of the DDAH enzyme (via oxidation of a sulfhydryl group). Activation of macrophages (by pro-inflammatory cytokines) may lead to arginase secretion, an enzyme that metabolizes l-arginine to urea and l-ornithine. Chronically elevated arginase plasma levels can reduce plasma concentration and availability of l-arginine for NO∙ synthesis. l-Ornithine may also compete for the same transporter used by l-arginine (cationic amino acid transporter (CAT-1)) at the cell membrane level. In addition, low availability of l-arginine to NOS enzymes can increase superoxide synthesis via a mechanism known as endothelial NOS (eNOS) uncoupling (in the absence of sufficient l-arginine, the enzyme donates electrons to oxygen forming superoxide; however, considering the l-arginine concentrations in plasma/cells and the Michaelis constant (K_m) of eNOS, the contribution of this pathway to superoxide production is still under debate). How l-arginine supplementation can aid the condition (B). l-Arginine supplementation can restore the levels of l-arginine and NO∙ by (i) preventing eNOS uncoupling (thus reducing superoxide formation); (ii) providing enough l-arginine for NO synthesis according to the physiological requirements; (iii) increasing guanosine triphosphate (GTP) cyclohydrolase I activity )an enzyme that is activated by l-arginine, and is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4)—an essential co-factor for NOS activity). Both l-arginine and NO∙ have important metabolic functions, increasing protein synthesis pathways (by activating mammalian target of rapamycin (mTOR)) and through activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), lead to mitochondrial biogenesis. Metabolization of l-arginine by arginase may lead to polyamine synthesis, essential for cell growth and angiogenesis. Altogether, increased blood vessels and vasodilation (induced by normalization of NO∙ availability) will lead to increased nutrient delivery and oxygen consumption. For details on mechanisms, please read References [1,10,19]. This model describes several possibilities of mechanisms in a representative cell. Mechanisms may vary between cells due to the presence or absence of enzymes, receptors, and transporters.

Figure 1

Mechanisms for lowering nitric oxide (NO) availability in cardiovascular and metabolic disorders (A). Increased angiotensin II (AngII) levels, asymmetric dimethylarginine (ADMA), and low plasma l-arginine concentration are all conditions likely to reduce NO· production. Inflamed adipose tissue (due to its expansion—obesity) can lead to (i) ↑ release of inflammatory cytokines; (ii) ↑ release of AngII; (iii) ↑ protein catabolism (due to the pro-inflammatory state); and (iv) ↑ activation of macrophages. Angiotensin II, acting through its receptor (AT1R), increases generation of superoxide (O²⁻), primarily through activation of reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase. O²⁻ reacts with NO∙ to form peroxynitrite (ONOO∙), a very reactive and destructive molecule, leading to decreased availability of NO∙. Moreover, superoxide is a known inhibitor of dimethylarginine dimethylhydrolase (DDAH), a key regulatory enzyme, which controls the metabolism of ADMA. ADMA is an endogenous methylated amino acid that inhibits the constitutive endothelial and neuronal isoforms of nitric oxide synthase (NOS). ADMA is released by protein hydrolysis; thus, increased catabolism induced by several inflammatory cytokines can elevate the ADMA levels. As DDAH uses ADMA as a substrate and regulates plasma levels of ADMA, it may influence the bioavailability of NO∙ and possibly contribute to changes in blood pressure. Homocysteine (an indirect substrate for the synthesis of ADMA) is also an inhibitor of the DDAH enzyme (via oxidation of a sulfhydryl group). Activation of macrophages (by pro-inflammatory cytokines) may lead to arginase secretion, an enzyme that metabolizes l-arginine to urea and l-ornithine. Chronically elevated arginase plasma levels can reduce plasma concentration and availability of l-arginine for NO∙ synthesis. l-Ornithine may also compete for the same transporter used by l-arginine (cationic amino acid transporter (CAT-1)) at the cell membrane level. In addition, low availability of l-arginine to NOS enzymes can increase superoxide synthesis via a mechanism known as endothelial NOS (eNOS) uncoupling (in the absence of sufficient l-arginine, the enzyme donates electrons to oxygen forming superoxide; however, considering the l-arginine concentrations in plasma/cells and the Michaelis constant (K_m) of eNOS, the contribution of this pathway to superoxide production is still under debate). How l-arginine supplementation can aid the condition (B). l-Arginine supplementation can restore the levels of l-arginine and NO∙ by (i) preventing eNOS uncoupling (thus reducing superoxide formation); (ii) providing enough l-arginine for NO synthesis according to the physiological requirements; (iii) increasing guanosine triphosphate (GTP) cyclohydrolase I activity )an enzyme that is activated by l-arginine, and is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4)—an essential co-factor for NOS activity). Both l-arginine and NO∙ have important metabolic functions, increasing protein synthesis pathways (by activating mammalian target of rapamycin (mTOR)) and through activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), lead to mitochondrial biogenesis. Metabolization of l-arginine by arginase may lead to polyamine synthesis, essential for cell growth and angiogenesis. Altogether, increased blood vessels and vasodilation (induced by normalization of NO∙ availability) will lead to increased nutrient delivery and oxygen consumption. For details on mechanisms, please read References [1,10,19]. This model describes several possibilities of mechanisms in a representative cell. Mechanisms may vary between cells due to the presence or absence of enzymes, receptors, and transporters.

Figure 2

Flow diagram of search and selection of studies. ^*Nox and ADMA: Results of Schneider et al (2015) included two separated studies with two different types of patients: Coronary Artery Disease (CAD) and Peripheral Artery Disease (PAOD).

Figure 3

(A) Blood flow responses of individual studies: l-arginine vs. placebo treatment. (B) Sensitivity analysis of blood flow responses of individual studies: l-arginine vs. placebo treatment (removing the studies that most favor arginine and placebo). SD = standard deviation; CI = confidence interval.

Figure 4

(A) NOx and (B) ADMA responses of individual studies: l-arginine vs. placebo treatment.