Arginine Metabolism: Enzymology, Nutrition, and Clinical Significance

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1 Arginine Metabolism: Enzymology, Nutrition, and Clinical Significance Elimination of Asymmetric Dimethylarginine by the Kidney and the Liver: A Link to the Development of Multiple Organ Failure? 1,2 Robert J. Nijveldt,* Michiel P. C. Siroen,* Tom Teerlink, and Paul A. M. van Leeuwen* 3 Departments of *Surgery and Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands ABSTRACT Asymmetric dimethylarginine (ADMA) is a recently recognized endogenous inhibitor of nitric oxide production. Its role in cardiovascular disease is emerging, and ADMA appears to be an important causal factor in dysfunction of the vascular system. Several studies show that ADMA accumulates during renal failure, and ADMA has been identified as causing the cardiovascular complications accompanying renal failure. In addition to the kidney, we recently suggested an important role for the liver as an ADMA-eliminating organ. In a population of critically ill patients, hepatic failure was the most prominent determinant of ADMA concentration, and, notably, high ADMA concentration proved to be a strong and independent risk factor for intensive care unit mortality in these patients. We here summarize the role of both the kidney and the liver in the regulation of ADMA levels. In addition, the potential central role of ADMA as a causative factor in the development of multiple organ failure is discussed. J. Nutr. 134: 2848S 2852S, KEY WORDS: asymmetric dimethylarginine dimethylarginine dimethylaminohydrolase nitric oxide liver kidney multiple organ failure The arginine nitric oxide (NO) pathway plays a crucial role in several pathophysiological aspects of critical illness, such as infection, inflammation, organ injury, and transplant rejection. NO is synthesized from the amino acid arginine by the action of NO synthases (NOSs), 4 a family of enzymes with endothelial, neuronal, and inducible isoforms. Endothelium-derived NO causes vasodilation, prevents cellular adhesion to the vascular wall, inhibits platelet aggregation, and limits the development of neointimal hyperplasia by inducing apoptosis of vascular smooth muscle cells. Furthermore, NO reduces superoxide radical generation, inhibits oxidation of LDLs, and is involved in host defense by acting as a cytotoxic agent. Considering these important physiological functions, it is conceivable that reduced NO availability may result in impaired function of the cardiovascular and immune system. Interestingly, in 1992 it was discovered that asymmetric dimethylarginine (ADMA) plays a regulatory role in the arginine-no pathway, by 1 Prepared for the conference Symposium on Arginine held April 5 6, 2004 in Bermuda. The conference was sponsored in part by an educational grant from Ajinomoto USA, Inc. Conference proceedings are published as a supplement to The Journal of Nutrition. Guest Editors for the supplement were Sidney M. Morris, Jr., Joseph Loscalzo, Dennis Bier, and Wiley W. Souba. 2 R.J.N. is the recipient of a fellowship from the Council for Medical Research of the Netherlands Organisation for Scientific Research. 3 To whom correspondence should be addressed. pam.vleeuwen@vumc.nl. 4 Abbreviations used: ADMA, asymmetric dimethylarginine; DDAH, dimethylarginine dimethylaminohydrolase; MMA, monomethylarginine; MOF, multiple organ failure; NO, nitric oxide; NOS, nitric oxide synthase; SDMA, symmetric dimethylarginine. inhibiting all isoforms of the enzyme NO synthase (1). Since this observation, there has been growing interest in ADMA, especially with respect to cardiovascular disease. In asymptomatic humans with hypercholesterolemia, elevated ADMA levels were found, and ADMA levels were associated with impaired endothelium-dependent vasodilation and reduced nitrate excretion (2). ADMA levels are also increased in elderly patients with peripheral arterial disease and generalized atherosclerosis (3). Miyazaki et al. (4) measured plasma ADMA levels in 116 human subjects who had no sign of coronary or peripheral artery disease. They found that ADMA levels were positively correlated with age, mean arterial pressure, and glucose tolerance. Most intriguingly, ADMA levels proved to be correlated with carotid artery intima-media thickness in stepwise regression analysis. Although the cross-sectional nature of this study limited the ability to assess temporal relations, it suggests that ADMA may be a marker of atherosclerosis. Moreover, in a prospective nested case-control study in middle-aged nonsmoking men from eastern Finland, high concentrations of ADMA were associated with an increased risk of acute coronary events (5). Accumulation of ADMA has therefore been linked to endothelial dysfunction, and, as recently reviewed by Böger (6), the role of ADMA as a novel cardiovascular risk factor is emerging. In this article, we briefly focus on the origin and fate of ADMA, on the roles of the kidney and the liver as potential determinants of plasma ADMA concentration, and on the causative role of ADMA in the development of multiple organ failure (MOF) /04 $ American Society for Nutritional Sciences. 2848S

2 THE ROLE OF ADMA IN THE DEVELOPMENT OF MULTIPLE ORGAN FAILURE 2849S Origin and fate of ADMA Methylarginines are synthesized by posttranslational modification, involving the addition of methyl groups to arginine residues in proteins by enzymes called protein arginine methyltransferases (Fig. 1). These methylated proteins are predominantly found in the nucleus and play a role in RNA processing and transcriptional control (7). Methylarginines are released when these proteins are hydrolyzed, thereby being an obligatory product of protein turnover. In humans, 3 distinct methylated arginines are found in the circulation: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) (Fig. 2). Like ADMA, MMA also directly inhibits NOS, whereas SDMA has no direct effect on this enzyme. In addition, all 3 analogs interfere with NO synthesis by competing with arginine for cellular transport across cationic amino acid transporters of system y (8). Because physiological concentrations of ADMA are 10-fold higher than those of MMA, ADMA can be regarded as the predominant endogenous inhibitor of NO biosynthesis (9). Both ADMA and SDMA are eliminated from the body by urinary excretion. However, the most important metabolic pathway for ADMA is degradation by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which has been isolated from rat kidney and is colocalized with the various NOS enzymes (10 13). This enzyme converts ADMA into dimethylamine and citrulline and is mainly present in the liver, in the kidney, in the pancreas, and in endothelial cells (14,15). Overexpression of human DDAH in transgenic mice causes a decrease in plasma ADMA levels with a concomitant increase in tissue NOS activity, providing strong evidence for an important role of endogenous ADMA in regulating NO synthase activity (16). The role of the kidney In 1992, Vallance et al. (9) reported elevated levels of ADMA in patients with renal failure, and Kielstein et al. (17) FIGURE 1 The metabolism of ADMA. Methylarginines are synthesized via addition of methyl groups to arginine residues in proteins by protein arginine methyltransferases (PRMTs) and are released when these methylated proteins are hydrolyzed. Free ADMA is able to inhibit NOS. ADMA is eliminated from the body by urinary excretion, but the most important eliminatory pathway is degradation by DDAH. FIGURE 2 Biochemical structures of arginine analogs showed that ADMA was higher in dialysis patients with clinically manifest atherosclerosis than in those without atherosclerotic disease. This suggests that elevated levels of ADMA may contribute to the high cardiovascular morbidity in these patients. Indeed, Zoccali et al. (18) studied the relation between cardiovascular risk factors and plasma ADMA concentration in a cohort of 225 hemodialysis patients and found that plasma ADMA was a strong and independent risk factor of overall mortality and cardiovascular outcome. In another study in patients with end-stage renal disease, the same investigators reported that elevated ADMA plasma concentration was associated with left ventricular dysfunction and left ventricular hypertrophy, important risk factors for mortality in these patients (19). Interestingly, in patients with chronic renal failure, a sharp rise in concentration of SDMA, the stereoisomer of ADMA, has also been reported (20). Although SDMA has no inhibitory activity toward the enzyme NOS, Fleck et al. (20) pointed out the potential importance of SDMA, and concluded, in their study of a large population of renal failure patients, that not only ADMA levels but also SDMA levels were likely responsible for hypertension, possibly by competition for reabsorption between SDMA and arginine in the kidney. In the past, several groups demonstrated that the human kidney is capable of excreting both ADMA and SDMA (21,22). However, no conclusions on net renal extraction of dimethylarginines could be drawn from these data, because not only urinary excretion but also metabolic pathways within the kidney seem to determine renal dimethylarginine handling. We recently confirmed the role of the kidney as an ADMAeliminating organ in healthy humans (23). Plasma concentrations of dimethylarginines were measured in both arterial and renal venous blood in 20 fasting patients with normal renal function. Renal extraction of ADMA, as a measure of ADMA elimination, was calculated as the arteriovenous concentration difference divided by the arterial concentration times 100%. We found a notable net renal extraction of both dimethylarginines. Interestingly, net renal extraction of ADMA was greater than that of SDMA (Table 1). In addition, arterial SDMA concentration, but not ADMA concentration, correlated with arterial creatinine concentration (r 0.607, P 0.005). We concluded that the kidney contributed to the regulation of plasma levels of dimethylarginines. In addition, the greater renal extraction of ADMA strongly suggested the presence of an additional catabolic pathway for ADMA in the kidney. These data prompted us to study the role of the kidney in more detail in a rat model. Theoretically, reduced DDAH activity may be the cause of elevated ADMA concentrations (24). DDAH activity is in-

3 2850S SUPPLEMENT Model TABLE 1 Renal extraction of ADMA and SDMA1 ADMA Renal extraction2 SDMA Human study * Rat model Control LPS Values are means SEM; * different from ADMA, P 0.05; different from control, P Expressed as percentage of supply. fluenced by such factors as oxidative stress and inflammation (25,26). We intended to modulate DDAH activity in rats by endotoxin-induced inflammation. We found a net uptake of both ADMA and SDMA by the kidney (Table 1) (27). Furthermore, we found strong evidence for differential renal handling of the 2 dimethylarginines. The elimination of ADMA by the kidney could not be explained by urinary excretion, because the urinary concentration of unchanged ADMA was negligible. This finding points to a high metabolic turnover of ADMA in the kidney, which fully accounts for the observed net renal uptake of ADMA. Thus, in contrast to humans, in rats the kidney is not capable of excreting unchanged ADMA (23,28), which indicates a differential handling of dimethylarginines. Interestingly, both renal fractional extraction rate and net renal uptake were significantly lower in rats treated with endotoxin (Table 1). The role of the liver As mentioned, ADMA is not only dependent on renal function but is also subject to enzymatic degradation by DDAH. The recognition of DDAH as a potentially important regulator of plasma ADMA concentration widens the field of research and points out that other organs may also be involved in the metabolism of dimethylarginines. Carnegie et al. (28) indicated the potential role of the liver in the metabolism of dimethylarginines by reporting a decreased urinary excretion ratio of SDMA to ADMA in patients with chronic active hepatitis, owing to an increased output of ADMA. However, no precise data on the hepatic metabolism of dimethylarginines can be derived from this study, because only urinary concentrations were measured. Therefore, we designed an organ balance study in rats to assess arteriovenous concentration differences, together with blood flow measurement using radiolabeled microspheres. The combination of arteriovenous concentration differences and blood flow determination allows calculation of net organ fluxes and fractional extraction rates for the liver and the kidney. The main finding of that study was the high uptake of ADMA by the liver (Fig. 3) (29). The magnitude of hepatic uptake of ADMA was further clarified by estimating daily hepatic ADMA extraction. Accordingly, the liver extracted nmol ADMA/d, which is 700 times the amount of circulating plasma ADMA in rats. Notably, and in contrast to ADMA, the liver barely affected SDMA concentration (Fig. 3). Therefore, the probable explanation for the elimination solely of ADMA is intense catabolism by the enzyme DDAH. Hypothetically, reduced DDAH activity due to impaired liver function could lead to elevated % ADMA concentration. Interestingly, a very recent study by Tsikas et al. (30) reported increased concentrations of ADMA and the oxidative stress marker 15(S)-8-iso-PGF 2 in the plasma and urine of patients with end-stage liver disease. The role of the liver in the metabolism of ADMA was further substantiated in humans in a population of critically ill patients. Hepatic failure was independently correlated with ADMA concentration, as were lactic acid and bilirubin concentrations, both useful indicators of impaired liver function in critically ill patients (31). Moreover, ADMA level was the strongest predictor of outcome in these patients. Thus, this study showed that the role of ADMA is not only confined to chronic diseases such as cardiovascular disorders and renal failure, but may also be of relevance in the development of MOF during critical illness. ADMA and MOF We reported elevated ADMA levels in critically ill patients. Interestingly, plasma ADMA concentration ranked as the first and strongest predictor for outcome, with a 17-fold increased risk for death in the intensive care unit among patients who were in the highest quartile for ADMA (Table 2) (31). In this population of critically ill patients, the main determinant of plasma ADMA concentration proved to be the presence of hepatic failure. This finding suggests that proper liver function may be a prerequisite for the maintenance of normal ADMA levels. Multiple organ failure is the simultaneous dysfunction of several organs. MOF is the most intriguing problem in patients admitted to the intensive care unit. MOF is associated with a very high mortality rate in patients on intensive care units, ranging from 30 to 80% (32). Sepsis and severe trauma seem to be the main predisposing factors for the development of MOF. However, a unifying mechanism of pathogenesis for MOF is still lacking. As organ systems responsible for the development of MOF, the gastrointestinal tract and the liver have received considerable attention. Recent data demonstrate that the development of hepatic dysfunction reflects the severity of injury and is associated with a worse outcome after traumatic injury (33). NO produced by the constitutively expressed endothelial NOS (enos) is an important regulator of blood pressure; it FIGURE 3 Net liver flux of ADMA and SDMA. BW, body weight.

4 THE ROLE OF ADMA IN THE DEVELOPMENT OF MULTIPLE ORGAN FAILURE 2851S Risk factor TABLE 2 Risk factors for ICU death1,2 ICU death OR 95% CI P ADMA (highest quartile) SDMA (highest quartile) Hepatic failure Renal failure Neurological failure Respiratory failure Coagulation failure Cardiovascular failure Total SOFA score Age Gender Calculated by stepwise logistic regression analysis. 2 Abbreviations: OR, odds ratio; SOFA, sequential organ failure assessment. plays a crucial role in the preservation of organ perfusion and in the interaction of the vascular endothelium with blood platelets and leukocytes. Because of these properties, the role of the arginine NO pathway in sepsis and organ dysfunction and injury has been subject to extensive research. Overproduction of NO as part of the inflammatory or immune response to infection is implicated in the hypotension and hyporesponsiveness of septic shock, which raises the suggestion that NOS inhibitors may have therapeutic potential in septic shock. However, a phase III trial in septic patients of N G -monomethylarginine (NMMA), a pharmacological inhibitor of NOS, demonstrated increased mortality rates (34). In animal studies documenting adverse effects of NO inhibition, the inhibitors were nonselective, inhibiting both enos and inducible NOS. Thus, there is ample evidence that inhibition of the constitutively expressed isoform is unfavorable in sepsis and organ failure. The high ADMA levels in critically ill patients, in combination with the association between ADMA levels and outcome, prompted us to hypothesize a causative role for ADMA in the development of MOF by interfering with important physiological roles of NO production. In our hypothesis, ADMA accumulates in critically ill patients by the combination of increased proteolysis and decreased eliminatory pathways during renal and hepatic failure (Fig. 4) (35). Increased protein breakdown mediated by a diverse panel of cytokines is a characteristic response to injury, and allows the release of free methylated arginines from proteins. In addition, inflammation and oxidative stress may decrease DDAH activity; accordingly, ADMA elimination may be severely impaired. Plasma ADMA is strongly correlated with parameters of hepatic function and very high ADMA concentration in critically ill patients with hepatic failure (31). As discussed in the preceding section, ADMA accumulation in hepatic failure is most likely explained by the crucial role of the liver in the metabolism of ADMA. Accordingly, decreased hepatic DDAH activity causes elevated ADMA levels. Elevated ADMA concentrations may interfere with important physiological functions that are dependent on constitutive NO production. Recent work by Osanai et al. (36) demonstrated that the release of ADMA by vascular endothelial cells increases after shear stress 15 dyne/cm 2, which is comparable to mechanical forces on the arterial wall under physiological conditions, but is not affected by shear stress at 25 dyne/cm 2.In addition, DDAH activity is enhanced by shear stress at 25 dyne/cm 2, but is not affected by shear stress 15 dyne/cm 2. Because enos is also stimulated by shear stress, ADMA and NOS activity might antagonistically regulate production of NO in the systemic circulation. Furthermore, myocardial blood flow is regulated by local activity of the ADMA-degrading enzyme DDAH (37), and patients with vasospastic angina have higher coronary sinus ADMA concentrations than control patients (38). Interestingly, intravenous administration of ADMA in healthy humans reduces heart rate and cardiac output, but increases mean blood pressure and systemic vascular resistance at rest. During exercise, cardiac output only mildly increases in subjects given ADMA, compared with control subjects (39). These findings emphasize the potency of ADMA to influence physiological functions of the cardiovascular system. Hypothetical consequences of nonselective inhibition of NOS, ultimately leading to organ failure, include reduced perfusion of organs, reduced cardiac output, cardiac ischemia, capillary leakage, thrombocyte aggregation, reduced glomerular filtration rate, pulmonary hypertension, and increased adhesiveness of leukocytes. In our opinion, this hypothetical central role of ADMA as a causative factor in the pathophysiological alteration of organ failure syndromes demands further studies in the near future. Summary As recently reported, endogenously produced ADMA may play a causative role in cardiovascular disease. The mechanism by which ADMA accumulation detrimentally affects cardiovascular health seems to be nonselective inhibition of NO FIGURE 4 The hypothesized role of ADMA in multiple organ failure: Severe inflammation and organ injury may cause increased proteolysis, a decreased DDAH activity in the liver and other sites (e.g., kidney and endothelium), and decreased urinary excretion of ADMA. These processes lead to ADMA accumulation. In turn, increased ADMA concentration induces inhibition of NOS enzymes, thereby interfering with important physiological roles of NO. Consequently, NOS inhibition results in injury to important organs by such processes as reduced organ blood flow, endothelial damage, and adhesion and migration of leukocytes in tissues, and also in an incremental inflammatory response. Once again, inflammation and organ injury cause further accumulation of ADMA. The gray arrows highlight the hypothesized role of liver dysfunction in the development of the MOF cascade.

5 2852S SUPPLEMENT production, thereby increasing vascular tone and accelerating the process of atherosclerosis. Renal failure has received considerable attention in the literature as a cause of ADMA accumulation in plasma. Indeed, net extraction of ADMA by the kidney occurs in both animal models and humans, although the exact mechanism of renal ADMA handling, including the factors influencing this process, needs further study. More recently, we focused on the liver as a potentially important organ in the metabolism of ADMA, and demonstrated a high net hepatic uptake of ADMA in an animal model study. In addition, increased ADMA levels in patients with hepatic failure support the concept of the liver as an important player in the regulation of systemic ADMA concentration. This role of the liver is further substantiated by the strong relation between indicators of hepatic function and ADMA concentration in critically ill patients. 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