Adults with long-term COVID can improve health and strength with l-arginine and vitamin C supplementation


Altered l-arginine metabolism has been described in patients with COVID-19 and has been associated with immune and vascular dysfunction.

28-day oral supplementation with l-arginine plus vitamin C restore circulating l-arginine levels and improve walking performance, muscle strength, endothelial function, and fatigue in adults with long COVID.

A new Italian randomized clinical trial shows that L-Arginine plus Vitamin C supplementation helps in long COVID.

The study findings were published in the peer reviewed International Journal of Molecular Sciences.

In the present investigation, we showed that l-arginine metabolism was altered in a group of adults with long COVID eight months after the diagnosis of COVID-19 compared with age- and sex-matched adults without history of SARS-CoV-2 infection.

l-arginine metabolism was still disrupted after 28-day supplementation with l-arginine plus vitamin C. However, in those who received the active intervention, both serum l-arginine concentrations and l-arginine/ADMA, a marker of NO biosynthetic capacity, significantly shifted towards healthy reference values compared with participants who were allocated to placebo.

Accumulating evidence indicates that arginine metabolism is altered in COVID-19 patients [17,19,31,32,33]. In particular, low circulating l-arginine levels and upregulated arginase activity have been associated with reduced NO bioavailability and immune and vascular dysfunction in acute COVID-19 [15].

Low arginine-to-ornithine ratio and low GABR, as well as two-fold increase in circulating levels of ADMA, were found in severely ill COVID-19 patients [34]. This suggests that SARS-CoV-2 infection may induce endothelial dysfunction and a pro-thrombotic vascular phenotype acting both on NOS substrate availability and enzyme activity.

Our findings show that similar perturbations in l-arginine metabolism may be found in adults with long COVID several months after the acute episode. Notably, low GABR was associated with the development of coronary artery disease and increased risk of major adverse cardiovascular events over a 3-year follow-up in a cohort of 1010 patients undergoing elective cardiac catheterization [35].

In addition, reduced GABR and l-arginine-to-ornithine ratio were associated with markers of endothelial dysfunction and increased risk of cardiovascular mortality in patients referred for coronary angiography [36]. Increased circulating levels of ADMA were found in conditions characterized by impaired NO synthesis and endothelial dysfunction, such as hypertension, diabetes, atherosclerosis, and cerebrovascular diseases [37,38,39,40,41,42,43].

Elevated ADMA levels increase the risk of recurrent cardiovascular events or death in patients with a history of acute coronary disease [44,45], unstable angina [46], or diabetes [47]. Low l-arginine/ADMA is an independent risk factor for atherosclerosis [48] and microangiopathy-related cerebral damage [49], and has shown to be a better predictor of all-cause mortality than ADMA alone [48,50].

In this scenario, the reduced indices of l-arginine/NO bioavailability found in adults with long COVID-19 in the present investigation suggest a role for altered l-arginine metabolism in increasing the risk of endotheliopathy and long-term cardiovascular events [51,52,53,54].

l-arginine plus vitamin C supplementation increased circulating levels of l-arginine and shifted l-arginine/ADMA values towards healthy reference. Owing to its arginine-like structure, ADMA may directly compete with l-arginine both for its transport into the cell via the cationic amino acid transporter and NOS binding [55,56].

It follows that NO bioavailability may be influenced by the balance between l-arginine and ADMA [1]. Low l-arginine/ADMA results in a net inhibition of NO production [57]. Oral l-arginine supplementation may re-equilibrate l-arginine/ADMA, increase NO synthesis, and improve endothelial function [1,58].

Our previous findings corroborate this hypothesis, since in adults with long COVID supplemented with l-arginine plus vitamin C, the increase in circulating l-arginine concentrations and l-arginine/ADMA was associated with a significant improvement in flow-mediated dilation (FMD), a measure of NO-dependent endothelial reactivity, compared with placebo [16].

These results are in line with those from a meta-analysis of randomized clinical trials showing that short-term oral l-arginine supplementation improved endothelial function in individuals with reduced FMD [59]. In this context, l-arginine supplementation may be particularly suited for people with ascertained endothelial dysfunction and low l-arginine/ADMA, such as those with long COVID, since l-arginine supplementation in individuals with high FMD and low ADMA levels (or normal l-arginine/ADMA ratio) failed to improve either NO bioavailability or endothelial function [59,60,61].

Some limitations should be considered in the interpretation of the study results. Due to the small number of participants and the single-center nature of the study, our results should be considered preliminary. Further investigation with larger populations, conducted in multiple centers, and using different study methodologies (e.g., longer intervention, crossover design) is warranted to confirm our findings.

The levels of physical activity as well as dietary habits of study participants may have influenced the concentration of l-arginine metabolites and the effects of interventions. However, participants were requested to refrain from exercising, limit the ingestion of foods rich in arginine, and taking substances with vasoactive properties for at least 12 h before study visits.

Due to the heterogeneity of data on vaccination status (e.g., timing, types of vaccine, number of doses, refusal to disclose vaccination status), this information was not accounted for in the analyses. The panel of metabolites assessed in the present investigation provided relevant information on differences in l-arginine metabolism between adults with long COVID and healthy controls and allowed for evaluating the effectiveness of the tested intervention.

However, we cannot exclude that a more comprehensive evaluation of NO metabolism (e.g., measurement of circulating levels of nitrite, nitrate, and NO derivatives), as well as the assessment of inflammatory, vascular, or neurological markers may provide further insights into the mechanisms by which l-arginine plus vitamin C supplementation affects the outcomes of interest.

Vitamin C levels were not quantified; thus, the relationship between circulating vitamin C concentrations and study outcomes could not be explored. Because l-arginine levels were not measured days after the end of the intervention, it was not possible to appreciate the duration of the beneficial effects of l-arginine plus vitamin C supplementation on the parameters of interest.

Finally, it cannot be ruled out that the co-administration of other nutraceuticals may convey additional beneficial effects on l-arginine metabolism and long COVID symptoms [22,62,63].

For instance, vitamin D may have positive effects on both NO synthesis and endothelial function [64]. Vitamin D deficiency is frequent in COVID-19 survivors and is associated with poor physical performance [65].

The combined use of l-arginine, coenzyme Q10, and vitamin D was found to reduce oxidative stress and stimulate NO synthesis in cardiac and endothelial cells to a greater extent than any of those compounds alone [66].

The combination has therefore been proposed as a cardiovascular protective remedy [66].

Further studies are needed to assess whether supplementation with different combinations of nutrients may be proposed as a remedy to restore l-arginine metabolism and limit post-acute COVID-19 sequelae.

Overview of Arginine Metabolism

Arginine is a cationic, semi-essential amino acid that plays an important role in regulating immune and vascular cell function [12,13]. Levels of free arginine within the body are derived from the diet, endogenous synthesis, and turnover of proteins. In healthy adults, endogenous synthesis is sufficient so that arginine is not an essential amino acid.

However, in cases of infection where catabolic stress occurs, arginine becomes conditionally essential as endogenous synthesis is inadequate to meet increases in metabolic demand. Arginine is involved in the synthesis of proteins and the removal of ammonia by the urea cycle in the liver, and serves as a precursor for several molecules, including NO, citrulline, proline, glutamate, polyamines, creatinine, agmatine, and homoarginine (Figure 1).

Figure 1
Outline of arginine metabolism via four distinct enzymatic pathways. Arginine catabolism in immune and vascular cells is largely driven by the enzymes nitric oxide synthase (NOS) and arginase (ARG). ADC, arginine decarboxylate; AGAT, arginine-glycine amidinotransferase; ASL, argininosuccinate lyase; ASS, argininosuccinate synthetase; GAMT, guanidinoacetate N-methyltransferase; ODC, ornithine decarboxylase; OAT ornithine aminotransferase; P5CR, pyrroline-5-carboxylate reductase; P5CD, pyrroline-5-carboxylate dehydrogenase.

Arginine is metabolized to NO and citrulline by NOS [12,13,14,15]. Aside from functioning as a substrate for the enzyme, arginine aids in the intracellular assembly of the functional dimeric form of NOS and contributes to the proper coupling between the reductive and oxidative domains of the enzyme.

Accordingly, the absence of arginine results in the uncoupling of the enzyme and the generation of superoxide rather than NO. The NOS product citrulline is subsequently recycled back to arginine by the serial action of argininosuccinate synthetase (ASS) and lyase. ASS is the rate-limiting enzyme in this salvage pathway, and it tightly controls NOS-derived NO synthesis [20].

There are three distinct isoforms of NOS: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). nNOS and eNOS are constitutively expressed as calcium-dependent enzymes that transiently release NO in responsive to specific physiologic stimuli.

In contrast, iNOS is a calcium-insensitive protein that is induced by proinflammatory cytokines and microbial-associated products. Once formed, iNOS generates large amounts of NO over a prolonged period. While nNOS-derived NO is implicated in synaptic plasticity and serves as a neurotransmitter for both the central and peripheral nervous system, NO generated by the high-output iNOS enzyme plays a critical role in host defense, exerting cytotoxic effects on bacteria, parasites, viruses, and tumor cells [21].

In addition, iNOS-derived NO contributes to the pathophysiology of inflammatory disease and is the predominant mediator of hypotension in septic shock. Alternatively, eNOS functions to maintain vascular health. The basal release of NO by ECs promotes blood flow by inhibiting arterial tone.

In addition, the luminal release of NO elicits a potent antithrombotic effect by inhibiting blood coagulation and platelet activation, adhesion, and aggregation, while the abluminal liberation of the gas limits the intimal thickening of blood vessels by blocking vascular SMC proliferation, migration, and extracellular matrix deposition.

EC-derived NO also prevents inflammation by retarding the synthesis of inflammatory cytokines and chemokines; the expression of surface adhesion receptors; and the recruitment, infiltration, and activation of leukocytes within the vasculature. In contrast, the loss of NO production causes endothelial dysfunction that is symbolized by impaired endothelium-dependent vasodilation, EC activation and apoptosis, endothelial barrier disruption, arterial stiffness, vessel wall thickening, and a prothrombotic and inflammatory state.

Arginine is also hydrolyzed to urea and ornithine by the manganese metalloenzyme ARG. There are two isoforms of ARG, ARG1 and ARG2, which are encoded by different genes mapped on separate chromosomes, but they share approximately 60% amino acid sequence homology.

Although they possess a similar mechanism of arginine metabolism, these isozymes differ in their tissue distribution, subcellular localization, and molecular regulation [22]. ARG1 is a cytosolic enzyme that is highly expressed in the liver where it catalyzes the final step of the urea cycle. ARG1 is also found outside the liver in various tissues, including myeloid cells.

Alternatively, ARG2 is a mitochondrial enzyme that is commonly expressed in extrahepatic tissues, most prominently in the kidney. While ARG1 plays a fundamental role in inflammation-associated immunosuppression, both ARG isoforms have been linked to vascular disease by triggering EC dysfunction [10,11,12,13,14,15].

ARG-derived urea is readily excreted by the kidneys while ornithine is further metabolized by ornithine decarboxylase (ODC) to putrescine and the downstream polyamines, spermine, and spermidine [23]. Ornithine is also catabolized by ornithine aminotransferase (OAT) to pyrroline-5-carboxylate, which is, in turn, converted to proline by pyrroline-5-carboxylate reductase or to glutamate by pyrroline-5-carboxylate dehydrogenase.

While polyamines play an essential role in cell growth, proline is used for the synthesis of many structural proteins, especially collagen, which is involved in fibrosis [12,13,24,25,26]. Arginine is also metabolized by arginine:glycine amidinotransferase to produce homoarginine or guanidinoacetate, and the latter is converted to creatine by N-methyltransferase. Finally, arginine may be catabolized by arginine decarboxylase to agmatine, which is converted to putrescine by agmatinase. However, the presence and functional significance of arginine decarboxylase in immune and vascular cells remains to be established.

There is substantial crosstalk between the two main arginine metabolizing enzymes: NOS and ARG. By restricting the availability of arginine, ARG promotes the uncoupling of NOS, thereby diminishing NO synthesis and elevating superoxide generation [27]. In addition, arginine depletion by ARG limits the translation of iNOS by activating the general control nonderepressible 2 (GCN2) kinase, while ARG-derived spermine inhibits the expression of iNOS, leading to further reductions of NO production [28,29].

Finally, the NOS-derived intermediate product N-ω-hydroxy-L-arginine directly inhibits ARG activity, whereas iNOS-formed NO selectively stimulates ARG1 activity by nitrosylating cysteine residues of the protein [30,31]. Thus, these two arginine-metabolizing enzymes show reciprocal and regulatory interactions that impact their activity.

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