The main finding of our study is that the ratio of arginine to its endogenous metabolic inhibitors, ADMA and SDMA, is associated with severe sepsis, severity of illness, and independently predicts both hospital mortality and risk of death over the 6 months after diagnosis. Our results indicate that this estimate of systemic arginine availability could be a prognostically useful and pathophysiologically significant biomarker in severe sepsis, with potential therapeutic implications. Similar measurements and ratios have been calculated to estimate systemic arginine availability in a variety of cardiopulmonary disorders (5
), but to our knowledge such estimates have not been reported in septic patients.
The relationship between lower Arg/DMA and higher mortality suggests that impaired capacity for NO synthesis is a clinically relevant and detrimental feature of sepsis pathophysiology. This concept is supported by the direct correlation between Arg/DMA and urine nitrate shown in our post-hoc analysis and previous experimental studies. For example, Mittermayer and colleagues showed that arginine and the arginine/ADMA ratio decline within 4 hours of intravenous endotoxin in humans (14
). Human endothelial cells exposed to lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) in vitro develop increased arginase activity and decreased endothelial NOS activity, suggesting diversion of arginine away from NO production (27
). Similar experiments showed that TNF-α decreases endothelial dimethyl-aminohydrolase (DDAH) activity, the enzyme responsible for ADMA breakdown, resulting in higher ADMA concentrations (28
). The hemodynamic consequences of these biochemical perturbations has been shown in rats treated with arginase (diverting arginine away from NO synthesis) and ADMA (blocking NO synthesis), causing decreased cardiac output, increased systemic vascular resistance, and decreased blood flow to liver, kidneys, and spleen (15
). Additional detrimental effects of low systemic arginine availability may include immune deficiency, impaired wound healing, and oxidative stress (29
We find that arginine levels are lower in sepsis patients than in healthy subjects, consistent with several previous studies (7
). Previous kinetic studies show that arginine decrements in sepsis are related to both impaired production (8
) and accelerated clearance (10
). Importantly, enhanced arginine clearance appears to result from enhanced protein synthesis in sepsis (9
), not augmented NO production (8
). Like our study, none of these papers report a significant relationship between arginine concentration and severity of illness or outcome.
ADMA levels have been associated with severity of illness in septic patients (13
) and poor outcome in unselected critically ill patients (11
). Prior measurements of ADMA and SDMA in critical illness have used either an enzyme-linked immunoassay (ELISA) or HPLC. To our knowledge, our study is the first to measure methylarginines using HPLC-MS in sepsis. This method has significant advantages in separating and specifically quantifying ADMA and SDMA (32
Unlike ADMA, SDMA does not inhibit NOS (2
). Until recently SDMA was thought to have little role in NO physiology and was largely ignored except as a novel measure of renal function (33
). However, recent studies show that SDMA inhibits NO synthesis and increases reactive oxygen species production in a dose-dependent fashion in endothelial cells (3
). The inhibitory effect of SDMA on NO production occurs through competitive inhibition of cellular arginine uptake via the cationic amino acid transporter system (4
). SDMA also independently predicts coronary artery disease severity, cardiovascular events, and dialysis-associated hypotension (35
). These data show that SDMA is metabolically active and independently associated with cardiovascular disease. Importantly, the association we observed between the Arg/DMA ratio and mortality persisted even after controlling for serum creatinine concentration.
We found that the Arg/(Cit+Orn) ratio was similar in sepsis patients vs. controls. While this estimate of systemic arginine availability was lower in non-survivors compared to survivors, it was not an independent predictor of outcomes in multivariable analysis. This ratio of reactant (arginine) to enzymatic products (citrulline and ornithine) has been used previously to estimate systemic arginine availability because a lower ratio should reflect lower reactant availability. However, citrulline is metabolized back to arginine under physiological conditions (9
). This inter-conversion between product and reactant probably undermines the ability of this ratio to estimate systemic arginine availability.
Another limitation of the Arg/(Cit+Orn) ratio is that it will not reflect diversion of arginine metabolism from NOS to arginase, since products of both enzymes are in the denominator. Notably, ornithine was the only amino acid significantly different in non-survivors vs. survivors at Measurement 2 (higher in non-survivors). The Arg/Orn ratio has previously been validated as an estimate of arginase activity (6
). Although there was no difference in this ratio at the first measurement, the rising ornithine concentration was greater over time in the non-survivors, resulting in a lower Arg/Orn ratio in non-survivors vs. survivors at Measurement 2. These results suggest a link between arginase activity and worsening sepsis pathophysiology in non-survivors.
The therapeutic implications of our findings are worthy of consideration. Arginine supplementation has been studied in critically ill patients with mixed and controversial results (29
), some studies suggesting harm particularly in patients with severe sepsis (39
). Although these clinical trials are limited by co-administration of several immunonutrients, insufficient power, and methodological weaknesses (37
), they are supported by experimental sepsis studies showing that arginine therapy is harmful (41
). Published nutritional guidelines recommend against arginine supplementation in critically ill septic patients (42
Our results confirm low arginine levels in severe sepsis but indicate that outcomes are predicted only by the Arg/DMA ratio. Moreover, although we found that arginine levels rise over time during the course of sepsis, the Arg/DMA ratio remains low in non-survivors, while it improves in survivors. These findings suggest the potential for novel therapeutic interventions that could specifically increase systemic arginine availability in severe sepsis, without administering exogenous arginine. For example, oxidative stress decreases DDAH activity, slows ADMA clearance, and increases ADMA production (43
). These effects are prevented by antioxidant therapy. Anti-oxidant therapy has been studied in sepsis and a compilation of the evidence suggests benefit (45
). The Arg/DMA ratio could be used to specifically identify patients for enrollment in clinical trials of anti-oxidant therapy. Sepsis is a heterogeneous syndrome, and biomarker-based identification of patients eligible for tailored therapies offers promise (1
). In addition, the declining Arg/Orn ratio in non-survivors could warrant consideration of arginase inhibition as a novel therapeutic strategy that could be offered later in the course of sepsis (46
Our study shares the limitation of all observational research in that it determines associations, not causality. Therefore, the possible detrimental effects of low Arg/DMA discussed herein are speculative and must be further evaluated.
It is important to emphasize that the Arg/DMA ratio only estimates systemic arginine availability. The Arg/DMA ratio does not include monomethylarginine (MMA), another PRMT-catalyzed arginine metabolite that inhibits NOS (47
) and was included in the denominator of the ratio reported by Lara, et al (5
). However, MMA concentrations are approximately one-tenth those of either ADMA or SDMA, so its exclusion has only a small effect on the magnitude of the ratio. The Arg/DMA ratio also does not provide precise insights about the metabolic fate of arginine in severe sepsis. The latter requires radio-isotope studies that were beyond the scope of this study. However, such studies have been performed previously in smaller cohorts and support the concept that arginine bioavailability and nitric oxide production are reduced in sepsis (8
). Likewise, we also could not determine whether DMA concentrations were correlated with the extent of protein breakdown because we did not measure nitrogen balance or other indices of protein catabolism.
It is also important to note that urine nitrate concentrations only roughly estimate systemic NO production (48
). We were unable to determine which of the nitric oxide synthase isoforms were most active in our septic patients, and whether they may have been affected differently by altered concentrations of arginine and its metabolites. In addition, the urine nitrate measurements were performed post-hoc, so these findings serve only to generate hypotheses and require further study.
Finally, because we chose a relatively healthy control group, we cannot rule out the possibility that our findings are applicable to non-septic critically ill subjects. This healthy control group was necessary to establish expected ranges of arginine metabolites in subjects with age and gender distributions similar to our severe sepsis patients. The possibility that the Arg/DMA ratio is a useful predictor of illness severity and outcomes in non-septic critically ill patients warrants additional study.