This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.ABSTRACTNitric oxide (NO) has many physiologic roles, including vasodilatation, immune function, neurotransmission, and inflammation. It is produced by the enzyme nitric oxide synthase (NOS), which converts arginine to citrulline and nitric oxide. However, the roles of arginine and NO in sepsis are not clearly understood, because there is limited in vivo data for septic humans. NO synthesis rates in septic children were significantly higher than in historic adult controls. However, as compared to healthy controls, hypotensive patients with sepsis had higher plasma NO metabolite concentration (NOx), a slower fractional synthesis rate of NOx, and a similar absolute synthesis rate of NOx. These different findings suggest that nitric oxide synthesis may be different in sepsis without hypotension compared to septic shock, and these differences may be related to these patients' underlying metabolic rate. Asymmetric dimethylarginine (ADMA) also plays a role in the arginine-nitric oxide pathway. ADMA inhibits nitric oxide synthase and acts as a competitive inhibitor of arginine for transport across the y+ transporter. ADMA has been shown to be a risk factor for intensive care unit (ICU) mortality, duration of ICU stay, duration of inotropic and vasopressor treatment, mean APACHE II score, and duration of ventilatory support. We will use stable isotope tracer and biochemical methods to study and compare three groups of subjects: normal controls, septic patients without hypotension, and patients with septic shock.
Specific aim #1 is to determine the differences in leucine kinetics in these patients. The hypothesis tested is that septic patients without hypotension have faster rates of whole body protein kinetics when compared to septic patients with hypotension.
Specific aim #2 is to determine the differences in arginine kinetics and the rate of arginine conversion to NO in these patients. The hypothesis tested is that septic patients with hypotension will have a slower rate of production of arginine and therefore a decreased rate of its conversion to NO as compared to septic patients without hypotension.
Specific aim #3 is to determine the plasma and urinary concentrations of ADMA in these patients. The hypothesis tested is that NO synthesis rate will be negatively related to plasma ADMA concentrations.HYPOTHESIS Hypothesis #1: Septic patients without hypotension have faster rates of whole body protein kinetics. Increased protein breakdown results in a faster rate of production of arginine, hence its increased rate of conversion to NOHypothesis #2: Septic patients with hypotension have slower rates of whole body protein kinetics. Slower protein breakdown results in a slower rate of production of arginine, hence its decreased rate of conversion to NOHypothesis #3: Plasma concentrations of ADMA are elevated in septic patients because of the faster rate of protein breakdown plus decreased renal excretion and hepatic metabolism. As a consequence, NO synthesis rate will be negatively related to plasma ADMA concentrations SPECIFIC AIMSStable isotope tracer and biochemical methods will be used to study 3 groups of subjects: normal controls, septic patients without hypotension, and patients with septic shock. The following measurements will be made:
Specific Aim #1 : Endogenous leucine flux, an index of whole body protein breakdown; leucine oxidation, an index of net protein catabolism; and non-oxidative leucine disposal, an index of protein synthesisSpecific Aim #2: Arginine flux, plasma concentration, and its rate of conversion to NO in the whole bodySpecific Aim #3: Plasma concentration and urinary excretion of ADMABACKGROUND AND SIGNIFICANCE Sepsis is defined as a systemic response to infection. Both arginine and nitric oxide appear to play a role in sepsis. However, the exact metabolism of arginine during sepsis remains unknown. Lower arginine levels have been found in patients who die of sepsis as compared to survivors of sepsis. Arginine is converted to NO and citrulline by nitric oxide synthase (NOS).Despite the in vitro evidence that NOS is induced by sepsis, there is limited in vivo data for septic humans. Several studies showed elevated levels of nitrite and nitrate, products of NO metabolism, in septic patients. In a study of 10 septic pediatric patients, Argaman et al demonstrated that NO synthesis rates in septic children were significantly higher than in historic adult controls. In addition, they found that plasma arginine flux in these septic children were similar to those observed in healthy adults, but that due to a higher rate of arginine oxidation as compared to synthesis, the septic patients were in a negative arginine balance. However, in another study of six adult patients in septic shock as compared to healthy controls, the septic patients had higher plasma NO metabolite concentration (NOx), a slower fractional synthesis rate of NOx, slower arginine flux, and lower plasma arginine concentration. In addition, significant positive and negative correlations between plasma NOx and creatinine concentrations and with GFR suggested that the higher plasma NOx levels were due to impaired excretion, rather than increased synthesis. Other studies have shown correlation between NOx and plasma creatinine.These different findings suggest that nitric oxide synthesis may be different in sepsis without hypotension compared to septic shock. Two different responses are seen in sepsis: a low flow and a high flow response. The low flow state is characterized by low cardiac output and low metabolic rate, while the high flow state is characterized by high cardiac output and high metabolic rate. The low flow state occurs later, as a result of failure to resolve infection or tissue damage. When compared to normal volunteers, septic patients have a significantly increased rate of protein turnover. An increased rate of protein catabolism is the result of a marked increase in whole body protein breakdown with a more modest increase in whole body synthesis. This increased whole-body protein catabolism persists despite nutritional support. The increased availability of arginine from increased protein breakdown during early sepsis may increase the rate of nitric oxide synthesis. However, the presence of hypotension will lead to decreased rate of metabolic processes, including protein breakdown and hence, arginine production. Depletion of arginine availability over time will therefore lead to decreased NO synthesis.Asymmetric dimethylarginine (ADMA) also plays a role in the arginine-nitric oxide pathway. Dimethylarginines are synthesized by the methylation of arginine residues in proteins and subsequent release when these proteins are hydrolyzed. ADMA inhibits all isoforms of nitric oxide synthase. It also acts as a competitive inhibitor of arginine for transport across the y+ transporter. The major mechanism for elimination of ADMA from the body is degradation by the enzyme arginine dimethylaminohydrolase (DDAH).ADMA appears to play an important role in critically ill patients. ADMA has been shown to be a risk factor for ICU mortality10 as well as duration of ICU stay, duration of inotropic and vasopressor treatment, mean APACHE II score, and duration of ventilatory support.11 In addition, hepatic failure as well as lactic acid and bilirubin, biochemical markers of hepatic function, were independent determinants of plasma ADMA concentration. Nijveldt et al hypothesized that because of the association between ADMA levels and outcome, ADMA plays a causative role in multisystem organ failure by interfering with nitric oxide (NO) production and the physiologic properties of NO. They also hypothesize that ADMA accumulates by increased proteolysis as well as decreased elimination during renal and hepatic failure.
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