Homoarginine in the cardiovascular system: Pathophysiology and recent developments

Upcoming experimental and epidemiological data have identified the endogenous non‐proteinogenic amino acid L‐homoarginine (L‐hArg) not only as a novel biomarker for cardiovascular disease but also as being directly involved in the pathogenesis of cardiac dysfunction. The association of low L‐hArg levels with adverse cardiovascular events and mortality has proposed the idea of nutritional supplementation to rescue pathways inversely associated with cardiovascular health. Subsequent clinical and experimental studies contributed significantly to our knowledge of potential effects on the cardiorenal axis, acting either as a biomarker or a cardiovascular active agent. In this review article, we provide a comprehensive summary of the L‐hArg metabolism, pathophysiological aspects, and current developments in the field of experimental and clinical evidence in favor of protective cardiovascular effects. Establishing a reliable biomarker to identify patients at high risk to die of cardiovascular disease represents one of the main goals for tackling this disease and providing individual therapeutic guidance.


| INTRODUCTION
Accounting for more than 6 million deaths every year, cardiovascular diseases remain the leading cause of death in the European Union. 1 Recent data estimate costs of more than €200 billion a year, representing a high socioeconomic burden for healthcare systems worldwide. 2 Current research focuses on identifying novel biomarkers and treatment targets to improve the survival of patients with cardiovascular disorders and establish new therapeutic approaches. Over the last decades, amino acids increasingly attracted scientific attention because of their proposed interactions with pathways in metabolic and cardiovascular pathophysiology. In this context, several previous studies have shown that low concentrations of L-homoarginine (L-hArg) are associated with myocardial dysfunction, an increased risk of major cardiovascular events, and stroke. [3][4][5] The underlying mechanisms remain to be further elucidated but accumulating evidence suggests that L-hArg may modulate the metabolism of nitric oxide (NO) due to its structural similarities with arginine by serving as an alternative substrate for the NO synthase. 6 Apart from its effects on the NO pathway, new light has been shed on the connection of L-hArg to energy and creatine metabolism.
Arginine-glycine amidinotransferase (AGAT) is not only the first and rate-limiting key enzyme in the synthesis of L-hArg from lysine but is also critically involved in the generation of the energy metabolites guanidinoacetate and creatine. 4 It has also been shown that dietary supplementation with L-hArg seems to directly influence the cause of heart failure in different scenarios, including a murine model of post-myocardial infarction heart failure, 7 aortic banding, 8 or renal insufficiency. 9 Establishing a reliable biomarker to identify patients at high risk to die of cardiovascular disease represents one of the main goals for tackling this disease and providing individual therapeutic guidance.
In this review article, we aim to summarize current findings and developments regarding molecular pathways and clinical applications of L-hArg in the cardiorenal system.

| ENDOGENOUS SYNTHESIS OF L-HOMOARGININE AND EXOGENOUS SOURCES
The synthesis of L-hArg is known to take place in different organs of humans and animals (e.g., small intestine, kidney, liver, or brain) using mainly arginine and lysine as substrates. 6 Previously, two large genomewide association studies (GWAS) have independently identified associations of L-hArg with single nucleotide polymorphisms (SNPs) for the gene encoding the enzyme AGAT, which is known to catalyze the generation of L-hArg from its key substrates arginine and lysine in animals and humans. 4,10 The reaction comprises the transfer of the amidino group from L-arginine to the amino group of L-lysine to finally generate L-hArg with ornithine being a co-product. 11 Consequently, lymphoblasts of a patient with AGAT deficiency were not able to synthesize L-hArg. 12 In an animal study, deficiency of AGAT in mice caused a substantial reduction in systemic L-hArg levels, indicating the important role of this enzyme in the regulation of L-hArg homeostasis. 4 Another metabolic route for L-hArg formation includes enzymes of the urea cycle. 13,14 In this pathway, homocitrulline is generated from lysine by ornithine transcarbamoylase (OTC), transformed to homoargininosuccinate by argininosuccinate synthase (ASS), and finally converted into L-hArg by argininosuccinate lyase (ASL). 15 However, especially the role of the second metabolic route involving OTC should be elucidated regarding its contribution to endogenous L-hArg production. Additionally, L-hArg may be directly generated through transamidination of lysine by glycine transamidinase. 16,17 Plants and especially seeds contain a magnitude of naturally occurring non-proteinogenic amino acids. 18 Moreover, L-hArg was identified as the major free nonproteinogenic amino acid in different sorts of grain legumes, such as Lathyrus species (grass pea seeds) that contain approximately 1-2% of L-hArg. 19 3 | PHYSIOLOGY OF L-HOMOARGININE L-hArg [n6-(aminoiminomethyl)-L-lysine] is an endogenous, non-proteinogenic amino acid derivative structurally related to L-arginine and present in human plasma at levels of 1 to 2 μmol/L. 20 In a study on rats with isoprenaline-induced takotsubo cardiomyopathy, intraperitoneally injected L-hArg at doses of 20, 220, or 440 mg/kg body weight had an elimination half-life of 20 to 40 min. 16,21 In another study on 20 healthy young volunteers, the half-life period of supplemented hArg could not be determined. 22 Regarding its special chemical characteristics, it is known to be solid, slightly water-soluble and has a melting point of 213-215 C at a molecular weight of 188.2 g/mol. 23 It is produced from its precursor lysine by the mitochondrial enzyme AGAT, which is expressed in the kidney or liver, as well as tissues with high energy requirements like striated muscle. 4,15,24,25 Apart from its role in L-hArg synthesis, AGAT participates in energy metabolism by creating creatine through the final methylation of guanidinoacetate by guanidinoacetate N-methyltransferase (GAMT). 20 Metabolic studies have shown that humans and mice with AGAT deficiency are not able to generate L-hArg, consequently developing muscular dystrophy with complete reversal upon creatinine supplementation. 4,12 However, creatine at presumably high concentrations may inhibit AGAT-mediated L-hArg formation as demonstrated in AGAT +/+ mice and previously published work using tissues from chicken and rats. 4,[26][27][28] Contrary, the administration of sodium nitrate (NaNO 3 ) has been shown to influence the homeostasis of L-hArg with remarkably higher plasma concentrations compared to placebo-treated healthy volunteers. 29 It seems to shift AGAT-mediated reactions toward L-hArg synthesis by decreasing N-methyltransferase-catalyzed synthesis of guanidinoacetic acid (GAA) and subsequently creatine.
In the case of dietary intake, L-hArg can be incorporated from the lumen of the small intestine via cationic amino acid transporters. 30 Subsequently, it is not only supposed to affect plasma concentrations and synthesis of endogenous L-hArg but also those from its metabolites arginine, asymmetric dimethylarginine (ADMA), creatine, GAA, and NO. 21 Animal studies confirmed dose-dependent increasements in L-hArg plasma concentrations after supplementation of dietary L-hArg or L-arginine. 8,31 Both genetic differences of AGAT and miscellaneous conditions were supposed to be responsible for variances in plasma L-hArg concentrations. While fasting was reported to go along with increased serum L-hArg concentrations in humans and rats, 32,33 smokers showed decreased L-hArg levels. 34 Possible molecular mechanisms by which L-hArg might act in cardiovascular disorders were proposed in several previous in vitro and in vivo studies. 4,13,16,22,[35][36][37][38] To date, three different pathways for the catabolism of L-hArg were reported. 13 Given its structural similarity to L-arginine with an additional methylene group (CH 2 ) in its main chain, L-hArg is supposed to act as an alternative substrate for NO synthase and to be directly involved in the generation of the potent vasodilator NO. 16,[39][40][41] Apart from being a precursor of NO, it may increase its availability by inhibiting arginase, which leads to increased plasma concentrations of L-arginine, the main substrate for NO synthase. 42 In this context, NO is synthesized from arginine in a two-stage reaction carried out by NO synthase. First, NO synthase hydroxylates L-arginine to Nω-hydroxy-L-arginine. In the second step, NO synthase oxidizes Nω-hydroxy-L-arginine to L-citrulline and NO. 43,44 Asymmetric dimethylarginine (ADMA), NG-monomethyl-L-arginine (L-NMMA), and symmetric dimethylarginine (SDMA) are methylated analogs of arginine and important modulators of the NO pathway. 45 For example, ADMA is a naturally occurring endogenous inhibitor of NO synthase. 46 The methyl groups are provided from the methyl donor Sadenosylmethionine involving the enzymes protein arginine methyltransferase type 1 and 2 (PRMT1, PRMT2). While PRMT1 catalyzes the generation of L-NMMA and ADMA, PRMT2 is involved in the formation of SDMA and L-NMMA. 46 Despite its similarity to L-hArg, several previous cardiovascular studies on the long-term effects of L-arginine supplementation failed to prevent cardiac remodeling and heart failure. [47][48][49][50][51][52] NO exerts well-investigated cardioprotective effects and takes part in the regulation and maintenance of cell viability, endothelial function, and vascular homeostasis. 53 However, the importance of L-hArg as a direct substrate for NO synthase has been questioned considering its lower catalytic efficiency and the approximately 20 times lower concentrations in biological fluids and tissue (plasma concentration of approximately 2 μmol/L) compared to L-arginine (plasma levels of 100-250 μmol/L). 13,40,54 On the other side, experimental data in mice revealed a prolonged NO activity with NO concentrations being elevated even 8 h after L-hArg supplementation in comparison to L-arginine treated animals going to baseline levels after 4 h. 55 Furthermore, binding affinity (K m ) as one essential kinetic parameter for the NO synthase-dependent oxidation is known to be higher for L-hArg than for L-arginine (e.g., for NO synthase-I 23 ± 5 vs. 2.7 ± 0.5 μmol/L). 40 Vmax values of 4380 ± 140 and 34 ± 3 μmol/min/mg protein for L-arginine and L-hArg, respectively, were reported for rat liver arginase. 56 Regarding its action as a weak competitive inhibitor of arginase, 3,57 the relevance of possible arginine elevations as causal mechanisms of L-hArg-induced cardiovascular safeguards seems uncertain. This was also underlined by a study that investigated the inhibition of the two arginase isoforms in mammals, arginase 1 and arginase 2, by L-hArg. 42 Whereas arginase 1 is predominantly expressed in blood and liver cells, arginase 2 plays an important role in extra-hepatic tissues, such as the kidneys. 58 The authors observed L-hArg-mediated arginase inhibition at concentrations that are significantly higher than those observed in plasma and found no association between plasma L-hArg concentrations and the L-arginine/ornithine ratio in healthy older adults. 42 They concluded that arginase inhibition is unlikely to represent a key mechanism in the putative cardioprotective effects of L-hArg. Available studies on the affection of arginase activity by L-hArg are contradictory since stimulatory as well as inhibitory effects have been reported. 42,56,57,[59][60][61] Another catabolic pathway of L-hArg is catalyzed by L-arginase hydrolyzing L-hArg to urea and lysine. 62 However, the ability of L-hArg to serve as an alternative substrate for arginase requires further research.
The last and quite recently identified catabolic route involves alanine-glyoxylate aminotransferase 2 (AGXT-2) which converts L-hArg to 6-guanidino-2-oxocaproic acid (GOCA). 13 In vivo murine studies revealed increasements in intracellular L-hArg concentrations in the scenario of AGXT-2 deficiency, proposing that this mitochondrial enzyme seems to be also required for the maintenance of systemic L-hArg levels by changing its expression. Interestingly, administration of labeled L-hArg in mice resulted in similarly high plasma levels of labeled GOCA as the concentrations of the other labeled L-hArg metabolites L-homocitrulline and Llysine. This observation under experimental conditions suggests that the proposed enzymatic clearance of L-hArg through AGXT-2 seems to be comparable to previously reported metabolic pathways of L-hArg that encompass arginase or NO synthase. Genome-wide association studies confirmed these findings by showing a correlation between SNPs located at AGXT-2 and L-hArg levels in humans. 4,10 Individuals from the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study being heterozygous or homozygous for the AGXT-2 rs37369 T-allele showed elevated L-hArg plasma concentrations by 8% and 12%, respectively. 10,13 Since L-hArg is not degraded by enzymes in animal tissues in the presence of physiological concentrations of other amino acids, the in vivo catabolism of the amino acid seems limited. 31 Several studies have reported on the importance of the kidneys in the regulation and maintenance of L-hArg concentrations. 11,24,[63][64][65] It is supposed that approximately 95% of orally administered L-hArg is excreted in the urine of pigs and rats, respectively. 31 Therefore, renal excretion of L-hArg might be a reliable indicator of dietary intake and intrinsic production.
In addition to the involvement of L-hArg in the cardiovascular systems, early investigations reported inhibitory effects on alkaline phosphatase isoenzymes in the liver, intestine, or bone. [66][67][68] However, previous studies have demonstrated inhibitory effects of L-hArg on the activity of alkaline phosphatases in the millimolar range, 69,70 such as in smooth muscle cells at supraphysiological concentrations of 10 mmol/L. 71 Therefore, physiological L-hArg concentrations might be insufficient to induce a significant suppression.
A simplified illustration of the metabolic pathways of L-hArg is provided in Figure 1.

| EPIDEMIOLOGICAL ASSOCIATIONS WITH L-HOMOARGININE DEFICIENCY
With the observation of flow-mediated vasodilatation upon increased plasma concentrations during the second and third trimenon of pregnancy (4.8 ± 1.7 and 5.3 ± 1.5 μmol/L, respectively), L-hArg has been supposed to be involved in the maintenance of endothelial function. 72 Furthermore, L-hArg was found to be inversely associated with the endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in a cohort of 3305 subjects referred for coronary angiography (LURIC study). 3 In 2010, Maerz et al. were the first to describe that L-hArg plasma levels are independently associated with cardiovascular and all-cause mortality in patients referred for coronary angiography and in patients undergoing haemodialysis. 3 Their prospective study investigated the correlation between L-hArg concentrations and cardiovascular outcomes in two large cohorts of 3305 subjects referred for coronary angiography (LURIC study) and 1255 diabetic patients undergoing haemodialysis (4D study). Especially in patients with impaired renal function, L-hArg levels were found to be reduced and inversely associated with markers of endothelial dysfunction.
Studies in the following years revealed an inverse correlation of L-hArg with cardiovascular events and mortality in miscellaneous clinical scenarios, such as heart failure, 73,74 acute chest pain, 38 stroke, 4 peripheral artery disease, 75 or critically ill patients in general. 76 However, data from patients with chronic kidney disease (CKD) are inconsistent. While several studies demonstrated the ability of L-hArg to predict cardiovascular mortality and progression to dialysis in patients with renal insufficiency, 63,77,78 another publication did not observe a significant relationship between L-hArg concentrations and mortality. 65 Previous studies have demonstrated an organ-specific pattern of AGAT expression with the highest levels in renal tissue from patients with end-stage heart failure. 25 Consistent with these findings, L-hArg levels were positively correlated F I G U R E 1 Illustration of the L-hArg metabolism. L-hArg is either ingested or intrinsically formed from lysine and arginine by the enzyme AGAT. Other metabolic routes include the formation of L-hArg through enzymes of the urea cycle or through the transamidination of lysine by glycine transamidinase. The amino acid derivative serves as a substrate for L-arginase but can also be catabolized by NO-synthase to NO, or by AGXT-2 to GOCA. Abbreviations: AGAT, arginine-glycine amidinotransferase; AGXT-2, alanine-glyoxylate aminotransferase 2; ASL, argininosuccinate lyase; ASS, argininosuccinate synthase; GAA, guanidinoacetate; GAMT, guanidinoacetate methyltransferase; GOCA, 6-guanidino-2-oxocaproic acid; L-hArg, L-homoarginine; NO, nitric oxide; OTC, ornithine transcarbamylase to left ventricular ejection fraction and pronounced in patients with decreased compared to those with normal kidney function. Due to the association of AGAT with energy metabolism, patients with diminished kidney function and consequently lower AGAT activity might be more vulnerable to myocardial energy depletion. 77 A study on the association of L-hArg with cardiovascular outcomes in 1255 diabetic haemodialysis patients identified L-hArg as a strong risk factor for sudden cardiac death and death due to heart failure. 64 In this context, patients in the lowest L-hArg quintile showed a more than twofold and threefold increased risk of sudden cardiac death or death due to heart failure, respectively, than patients in the highest quintile. Furthermore, low L-hArg concentrations were not only found to be lower at decreased kidney function but also to be associated with the progression of chronic kidney disease. 78 Considering the participation of AGAT in creatine formation, L-hArg is supposed to play a role in heart failure pathophysiology. 25,79 Cullen et al evaluated the expression and role of AGAT in 12 patients with nonischemic cardiomyopathy requiring the implantation of a left ventricular assist device (LVAD) due to deteriorating clinical status despite inotropic support and 10 patients with stable end-stage heart failure (ESHF). 25 The study showed that myocardial concentrations of AGAT mRNA were increased in response to heart failure in both patient populations (4.3-fold [p < 0.001] and 2.7-fold [p < 0.005] in LVAD and ESHF relative to donors, respectively), as well as in rat myocardia. Interestingly, after receiving a combination therapy consisting of mechanical support and standard pharmacological therapy, followed by the administration of clenbuterol to stimulate hypertrophy and improve cardiac function, AGAT activity returned to baseline levels. The findings indicate that the myocardium might compensate for the energy loss in the context of heart failure by the induction of locally elevated AGAT mRNA expression to normalize intracellular creatine levels. Another study investigating patients with takotsubo cardiomyopathy also revealed reduced plasma L-hArg concentrations in comparison to healthy subjects (1298 ± 112 vs. 2094 ± 321 nmol/L, respectively). 79 Whereas epidemiological evidence suggests a strong relationship between low L-hArg concentrations and adverse cardiovascular outcomes, a recently published Mendelian randomization analysis revealed no causal relationship between L-hArg and any of the studied cardiometabolic outcomes. 80 The study investigated three different SNPs located in GATM (glycine amidinotransferase), CPS1 (carbamoyl-phosphate synthase 1), and AGXT-2 (alanine-glyoxylate aminotransferase 2) that were all associated with serum L-hArg concentrations in a recent genome-wide association study (GWAS) of 5143 individuals. 10 However, the study had several limitations that are general to all Mendelian randomization analyses. One assumption that might influence studies with this principle is that endpoints for outcome prediction should be independent of confounders. However, all three genes exercise certain regulatory functions that are known to be independent of L-hArg. Another possible explanation is that other metabolites related to L-hArg might be responsible for the poor outcome associated with low L-hArg plasma concentrations. In this context, several other metabolites with a strong correlation to increased cardiovascular risk and poor outcomes have been reported. A summary is provided in Table 1. Finally, the study cohort consisted of patients aged 24-39 years who typically did not suffer from cardiovascular diseases or had associated risk factors. In light of the many study limitations, the role of L-hArg and its association with poor outcomes still needs to be further explored.

| EXPERIMENTAL EVIDENCE
Apart from several epidemiological investigations on L-hArg as a cardiovascular risk marker, the amino acid has been tested in different animal studies for potential direct effects on cardiac function and remodeling. Cardiac remodeling represents a complex adaptation process in response to miscellaneous pathological stimuli aiming at maintaining cardiac output. 88,89 It is accompanied by an increase in myocardial size and interstitial fibrosis on a microscopic level and a reduction of cardiac function, compliance, and elasticity on a macroscopic level. 90 At later stages, the cardiac remodeling process flows into replacement fibrosis and cell death which are considered major drivers of the transition to symptoms, heart failure, and adverse cardiovascular events. 91 The loss of cardiomyocytes is irreversible as evidenced by the persistence of focal replacement fibrosis seen on cardiac magnetic resonance through the detection of LGE (late gadolinium enhancement). 92 Since cellular hypertrophy and diffuse fibrosis are plastic processes with a certain reversibility-as seen in patients after valve replacement-L-hArg attracted a lot of interest as a potential new drug therapy acting as a protective factor in cardiovascular and renal diseases. 93 Mice with homozygous AGAT knockout (AGAT À/À ) and consequently L-hArg and creatine deficiency showed decreased stroke volumes and neurological deficits after supplementation with L-hArg in a model of experimental stroke, while the normalization of brain creatine had no neuroprotective effects. 4 Compared to wild-type mice with normal AGAT enzyme activity, AGAT À/À mice present a haemodynamic phenotype that is characterized by lower LV systolic pressure with impaired contractility and relaxation. 94 In contrast to creatine supplementation, which was not able to fully rescue this cardiac phenotype, administration of L-hArg via drinking water at a concentration of 14 mg/L for 10 days normalized all inotropic and lusitropic parameters. On the cardiomyocyte level, L-hArg deficiency per se resulted in impaired cardiac function with reduced myocyte shortening and relengthening velocities. 94 Using a murine model of post-myocardial infarction heart failure, another study investigated possible protective effects of L-hArg by supplementing adult female mice with 14 mg/L L-hArg for 4 weeks before myocardial infarction surgery and for another 6 weeks follow-up. 7 Dietary L-hArg supplementation has led to preserved contractile reserve and improved diastolic indices under stimulated conditions. However, remodeling of the left ventricle and the global function as assessed by magnetic resonance imaging differed not between controls and HA-treated animals.   81 Nitrogen source for NO synthesis NO is synthesized from arginine in a two-stage reaction carried out by NO synthases. First, NO synthase hydroxylates L-arginine to Nω-hydroxy-L-arginine. In a second step, NO synthase oxidizes Nω-hydroxy-L-arginine to L-citrulline and NO No improvement in cardiovascular outcomes after arginine supplementation. No correlation with the prevalence of CAD L-citrulline 82 Intermediate non-proteinogenic amino acid in the arginine pathway Arginine is metabolized by NO synthase to NO and L-citrulline High L-citrulline levels were associated with the development of CAD and an increased risk for major adverse cardiovascular events GOCA 83 Transamination product of L-hArg Transamination of L-hArg catalyzed by AGXT-2 leads to the formation of GOCA GOCA levels in the highest tertile (≥2.13 nmol/L) were associated with increased renal and cardiovascular risk. A further study on this topic utilized a different model of heart failure induced by aortic constriction to investigate L-hArg mediated attenuation of cardiac remodeling in rats. 8,37 The authors reported dosedependent improvements in ejection fraction and a decreased morphological and molecular response to pressure overload as evidenced by reduced hypertrophy and fibrosis. Co-administration of standard heart failure medication resulted in a more pronounced attenuation of adverse morphological changes without any side effects. The cardioprotective effects of L-hArg on cardiac remodeling hold true in rats undergoing 5/6 nephrectomy with induced cardiorenal syndrome. 9 Using this classic model of renal insufficiency, L-hArg treatment was found to increase LV contractility and attenuate cardiac remodeling processes.
Dellera et al investigated the effects of in vivo L-hArg supplementation on smooth muscle cell proliferation after arterial balloon angioplasty with balloon-induced injury of the left carotid artery in rats. 95 Intravenously administered L-hArg via the right jugular vein at a concentration of 30 mg/kg/day over 14 days was able to reduce neointimal hyperplasia as evidenced by a significant decrease of the intima/media ratio.
A recently published study on female ApoEdeficient mice supplemented with L-hArg (14 mg/L) in drinking water reported a molecular explanation for the proposed atheroprotective effects of L-hArg. 96 Suggesting a T-cell-related mechanism of action due to a substantial reduction of CD3+ T-cells in atherosclerotic lesions, the authors observed a profoundly modulated spatial organization of the T-cell actin cytoskeleton and increased filopodia formation. Further analysis revealed inhibition of T-cell proliferation and impaired migratory capacities of T-cells due to chemokine release by L-hArg.
Since the first report on the role of L-hArg as a noncompetitive inhibitor of tissue-nonspecific alkaline phosphatases (TNAP), 66 several studies have demonstrated an association of elevated TNAP expression with accelerated vascular calcification and mortality. [97][98][99][100][101][102][103] TNAP is found in miscellaneous tissues, including the liver, kidney, endothelium, and bone. 104 Experimental findings in a murine model, in which coronary atherosclerosis was induced by endothelium-specific transgenic overexpression of TNAP (eTNAP), atherogenic diet, and a mutation in the LDL receptor gene, showed that 4-week HA treatment preserved LV ejection fraction, protected from LV dilatation, and decreased myocardial fibrosis. 35 Beyond those effects, no changes in coronary artery calcification and atherosclerosis were registered when compared to placebo animals without lipid abnormalities. However, the exact pathways underlying TNAPmediated cardiovascular changes remain elusive and require further research. Using a similar experimental setting with diet-induced obese mice, glucose-lowering effects of L-hArg have been reported, suggesting a compensatory response under conditions of severely impaired glucose homeostasis and in patients with type 2 diabetes mellitus. 105 Considering the proposed interactions of L-hArg with NO metabolism, one could infer that L-hArg might also affect blood pressure. Literature about the association between L-hArg and blood pressure is still controversial at present. Whereas cohort studies with patients referred for coronary angiography (LURIC) or elderly participants (Hoorn) reported positive correlations of L-hArg with systolic and diastolic blood pressure, 5,[106][107][108] it has also been shown that salt-sensitive hypertensive rats experienced lowered blood pressure following intravenous L-hArg infusions together with an increased urinary excretion pointing toward an enhanced NO synthesis. 109 In addition, another experimental setting in rats undergoing aortic banding with subsequent 4-week L-hArg treatment reported lowered blood pressure from 108 ± 3 to 96 ± 3 mmHg following a dose of 800 mgÁkg À1 Áday À1 . 8 To summarize, the majority of aforementioned experimental studies corroborate clinical evidence of an association between L-hArg levels and cardiovascular health, fueling the hypothesis of direct protective effects rather than being only a biomarker for risk assessment. Furthermore, they underline the potential use of L-hArg as a therapeutic option for patients with cardiorenal disorders.

| APPLICATION OF L-HOMOARGININE IN CLINICAL TRIALS
A clinical study investigated the kinetic and dynamic properties of an orally applied dose of 125-mg L-hArg in 20 young healthy volunteers. 22 Once daily administered over 4 weeks, L-hArg plasma concentrations were increased sevenfold over baseline levels compared to a fourfold increase after a single dose. The study group of young individuals (mean age of 35 years) showed mean L-hArg concentrations of 2.87 ± 0.91 μmol/L at baseline, whereas a previous study from 786 healthy individuals (aged from 35 to 54 years) reported plasma concentrations of 1.88 μmol/L L-hArg [25th and 75th percentile 1.47 and 2.41 μmol/L]. 110 The higher L-hArg concentration at baseline might be attributed to the younger age and normal kidney function of participants but HA levels might also be affected by sex or AGAT SNPs. 111 L-hArg supplementation was well tolerated without vascular or neurological abnormalities or any other significant side effects.
These promising study findings provide a clear rationale to conduct prospective studies with more study participants and longer treatment periods to examine mechanistic pathways and the effects of L-hArg in patients with miscellaneous cardiovascular diseases and metabolic disorders. A randomized double-blind placebo-controlled study has been registered to examine the oral supplementation of L-hArg in patients with acute ischemic stroke (ClinicalTrials.gov identifier: NCT03692234).

| CONCLUSION
Results from clinical and experimental studies increasingly move the naturally occurring amino acid L-hArg into the spotlight of cardiovascular prevention. Despite associations between low L-hArg levels and increased cardiovascular and all-cause mortality in patients at cardiovascular risk, the exact pathophysiological mechanisms remain to be elucidated. However, the hypothesized direct cardioprotective effects of dietary L-hArg supplementation in different animal models are quite encouraging and promising to investigate its effects in larger randomized human trials. To date, clinical and experimental data have reported good tolerability of dietary intake without causing any significant side effects. Further research will result in a better characterization of mechanistic pathways and strengthen our understanding by which L-hArg exerts its direct protective effects in different patient populations including those with renal insufficiency.

ACKNOWLEDGEMENTS
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