Leticia Castillo

Cysteine metabolism and whole blood glutathione synthesis in septic pediatric patients.

ObjectiveTo investigate whole body in vivo cysteine kinetics and its relationship to whole blood glutathione (GSH) synthesis rates in septic, critically ill pediatric patients and controls. DesignProspective cohort study. SettingMultidisciplinary intensive care unit and pediatric inpatient units at a children’s hospital. PatientsTen septic pediatric patients and ten controls (children admitted to the hospital for elective surgery). InterventionsSeptic patients (age, 31 months to 17 yrs) and controls (age, 24 months to 21 yrs) received a 6-hr primed, constant, intravenous tracer infusion of l-[1-13C]cysteine. Blood samples were obtained to determine isotopic enrichment of plasma cysteine and whole blood [1-13C]cysteinyl-glutathione by gas-chromatography mass spectrometric techniques. The plasma flux and oxidation rate of cysteine and the fractional and absolute synthesis rates of GSH were determined. Septic patients received variable protein and energy intake, as per routine clinical management, and controls were studied in the early postabsorptive state. Measurements and Main Results Plasma cysteine fluxes were increased in the septic patients when compared with the controls (68.2 ± 17.5 [sd] vs. 48.7 ± 8.8 &mgr;mol·kg−1·hr−1;p < .01), and the fraction of plasma cysteine flux associated with oxidative disposal was similar among the groups. The absolute rates of GSH synthesis in whole blood were decreased (p < .01) in the septic patients (368 ± 156 vs. 909 ± 272 &mgr;mol·L−1·day−1). The concentration of whole blood GSH also was decreased in the septic group (665.4 ± 194 vs. 1059 ± 334 &mgr;M;p < .01) ConclusionsWhole blood glutathione synthesis rates are decreased, by about 60%, in critically ill septic children receiving limited nutritional support. Plasma cysteine fluxes and concentration of cysteine were increased in the septic patients, suggesting a hypermetabolic state with increased protein breakdown. The mechanisms whereby GSH synthesis rates are decreased in these patients are probably multifactorial, presumably involving an inflammatory response in the presence of limited nutritional support. The role of nutritional modulation and the use of cysteine prodrugs in maintaining GSH concentration and synthesis remain to be established.

Arginine, citrulline, and nitric oxide metabolism in end-stage renal disease patients

The kidneys are thought to be a major site of net de novo arginine synthesis, but the quantitative status of arginine metabolism and its substrate precursor relationship to nitric oxide (NO) synthesis in end stage renal disease (ESRD) patients have not been characterized. We have investigated kinetic aspects of whole body arginine metabolism in six patients with ESRD. They received two pre- and two post-hemodialysis intravenous tracer infusion studies with L-[guanidino-(15)N(2)]arginine and L-[(13)C]leucine during the first study, and L-[5-(13)C]arginine and L-[5-(13)C-ureido,5,5, (2)H(2)]citrulline during the second study. Arginine homeostasis in ESRD patients was found to be associated with a lower rate of arginine oxidation, and despite the decrease in renal function, the rate of de novo arginine synthesis appeared to be preserved. Plasma citrulline concentrations and flux were also elevated in these subjects compared with healthy adults. The rate of whole body NO synthesis was increased in the ESRD patients, but apparently not different pre- and post-hemodialysis therapy. The anatomic site(s) responsible for the maintenance of net de novo arginine synthesis and for the elevated NO synthesis and its pathophysiological importance in ESRD remain to be established.

Arginine and nitric oxide metabolism in critically ill septic pediatric patients.

ObjectiveTo investigate whole body, in vivo arginine metabolism and nitric oxide synthesis rates in septic, critically ill pediatric patients. DesignProspective study. SettingPediatric intensive care unit at a general hospital. PatientsTen consecutive septic patients age 6–16 yrs. InterventionsSeptic patients received an 8-hr primed, constant intravenous tracer infusion of l-[guanidino-15N2]arginine, l-[1-13C]leucine, and [13C]urea. A 24-hr urine collection was obtained for determination of [15N]nitrate enrichment (15NO3−) and urinary nitrogen. The next day they received an infusion of l-[5-13C]arginine and l-[5-13C-ureido, 5,5,2H2]citrulline. Blood samples were obtained for determination of plasma isotopic enrichment of the tracers given and of derived [15N]citrulline (nitric oxide synthesis), l-[13C-guanidino 5,5, 2H2]arginine (M+3 arg) (arginine synthesis), and [15N2]urea (urea formation). Data are compared with historic controls from studies in healthy young adults. Measurements and Main ResultsPlasma arginine fluxes were 67 ± 21 and 72 ± 17 &mgr;mol·kg−1·hr−1, respectively, for the [15N2 guanidino] and the [13C] arginine labels, which were not different from reported adult values. The rates of arginine oxidation were 22.9 ± 10.8 &mgr;mol·kg−1·hr−1 and were higher than de novo arginine synthesis rates of 9.6 ± 4.2 &mgr;mol·kg−1·hr−1 (p < .01); therefore, these patients were in a negative arginine balance. The rates of nitric oxide synthesis as estimated by the [15N]citrulline method were 1.58 ± 0.69 &mgr;mol·kg−1·hr−1 for septic patients and higher (p < .05) than values of 0.96 ± 0.1 &mgr;mol·kg−1·hr−1 in healthy adults. Septic patients were in a negative protein (leucine) balance of about −1.00 ± 0.40 g·kg−1·day−1. ConclusionsHomeostasis of plasma arginine in septic patients was impaired compared with reported adult values. The rates of arginine oxidation were increased whereas de novo net arginine synthesis was unchanged, leading to a negative arginine balance. The rates of nitric oxide synthesis and the fraction of plasma arginine used for nitric oxide and urea formation were increased. These findings suggest that under condition of sepsis, arginine becomes essential in critically ill children.

Whole Body Arginine Metabolism and Nitric Oxide Synthesis in Newborns with Persistent Pulmonary Hypertension

ABSTRACT: Despite the potential relevance of the L-arginine-nitric oxide (NO) pathway in the pathophysiology of pulmonary hypertension, no in vivo studies of the kinetics of arginine and NO have been conducted previously in this population. The terminal guanidino N-atom of L-arginine is the precursor for NO, which is oxidized to the stable inorganic nitrogen oxides, nitrite (NO2-) and nitrate (NO3-). Thus, synthesized NO is detected in serum or urine as NO2- and NO3-. The purpose of this investigation was to compare studies of whole body arginine metabolism twice in nine patients with persistent pulmonary hypertension of the newborn (PPHN), using a primed constant i.v. infusion of L-[guanidino-15N2,5,52H2]arginine and L-[5,5,52H3]leucine, first during acute pulmonary vasoconstriction and again during convalescence, and thereby to characterize quantitative aspects of whole body arginine kinetics and NO production, as estimated from the rate of transfer of the 15N-guanidino-label of arginine to urinary nitrate (15NO3-). Arginine flux rates were 84.1 ± 8.6 μmol-kg.-1h-1 (mean ± SEM) during acute pulmonary hypertension and increased to 125 ± 13.2 (p < 0.05) during convalescence, whereas leucine fluxes were unchanged (168.5 ± 15 versus 178.8 ± 10.2 μmol.kg.-1h-1), and comparable to those reported in healthy newborns. During convalescence total urinary nitrate excreted increased by 66% (p < 0.05), urinary 15NO3- increased from 0.29 ± 0.07 to 0.74 ± 0.15 μmol.d-1 (p < 0.05), and the rate of plasma arginine conversion to NO increased from 10.3 ± 2.2 to 45.6 ± μmol.d-1 (p < 0.05). This study indicates a decreased plasma arginine utilization for whole body NO synthesis during the acute vasoconstrictive state of PPHN and suggests that arginine availability may become an important factor in NO formation.

Plasma arginine and leucine kinetics and urea production rates in burn patients

We measured plasma arginine and leucine kinetics and rates of urea production (appearance) in 12 severely burned patients (mean body surface burn area, 48%) during a basal state (low-dose intravenous glucose) and while receiving routine, total parenteral nutrition ([TPN] fed state) including an L-amino acid mixture, supplying a generous level of nitrogen (mean, 0.36 g N.kg-1.d-1). The two nutritional states were studied in random order using a primed 4-hour constant intravenous tracer infusion protocol. Stable-nuclide-labeled tracers were L-[guanidino-13C]arginine, L-[1-13C]leucine, [18O]urea, and NaH13CO3 (prime only), with blood and expired air samples drawn at intervals to determine isotopic abundance of arginine, citrulline, ornithine, alpha-ketoisocaproate ([KIC] for leucine), and urea in plasma and 13CO2 in breath. Results are compared with data obtained in these laboratories in healthy adults. Leucine kinetics (flux and disappearance into protein synthesis) indicated the expected higher turnover in burn patients than in healthy controls. Mean leucine oxidation rates are also higher and compared well with values predicted from urea production rates, provided that urea nitrogen recycling via intestinal hydrolysis is taken into account. The plasma urea flux was also higher than for normal subjects. Arginine fluxes as measured in the systemic whole body, via the plasma pool, were correspondingly higher in burned patients than in healthy controls and were in good agreement with values predicted from leucine-KIC kinetics. However, systemic whole-body arginine flux measured via the plasma pool was only 20% of the arginine flux estimated from the urea flux plus the rate of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma arginine kinetics in adult man: Response to an arginine-free diet

To explore the response of whole-body arginine metabolism to a change in arginine intake, plasma arginine kinetics were investigated in eight healthy adult men who received an L-amino acid diet supplying an Arg-rich or Arg-free intake for 6 days before undergoing a tracer study on day 7. The tracer protocol lasted for 8 hours. For the first 3 hours subjects remained in the postabsorptive (fasted) state, and during the following 5 hours they consumed small meals at 30-minute intervals. Primed continuous intravenous infusions of L-[guanidino-13C]arginine, L-[5,5,5-2H3]leucine, and [15N2]urea were administered to estimate plasma amino acid fluxes and the rate of urea production. For the fasted and fed states, plasma arginine fluxes (mumol.kg-1.h-1, mean +/- SD) were 69 +/- 8 and 87 +/- 12 (P < .01), respectively, for the Arg-rich diet and 63 +/- 14 and 51 +/- 7 (P < .01, from Arg-rich) for the Arg-free diet. Compared with the Arg-rich results, fed-state plasma arginine and ornithine concentrations were decreased (P < .01) and citrulline concentration was increased (P < .01) during the Arg-free diet period. Leucine fluxes and rates of urea production did not differ between the diet groups. The lower fed-state arginine flux in subjects receiving the Arg-free compared with the Arg-rich diet appears to be entirely due to the decreased rate of entry of arginine from the intestine in the former group.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamine: precursor or nitrogen donor for citrulline synthesis?

Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton vs. nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, l-[2-(15)N]- and l-[5-(15)N]glutamine and (15)N-ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled the three nitrogen groups of citrulline almost equally. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the delta-nitrogen. D(5)-glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was utilized poorly, accounting for only 0.2-0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25-fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-(13)C(n) tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for approximately 40% of the circulating citrulline. Proline contribution was minor (3.4%), and glutamine was negligible (0.4%). However, the glutamine tracer resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.

Phenylalanine and Tyrosine Kinetics in Critically Ill Children with Sepsis

ABSTRACT: To better understand the impact of severe illness on the amino acid economy and nutritional needs of pediatric patients, we studied plasma phenylalanine and tyrosine kinetics in eleven critically ill patients (six full-term newborns and five young infants). Within 48 h of the diagnosis of sepsis they were given primed constant i.v. infusions of L-[1-13C]phenylalanine and L-[3,3,2H2]tyrosine for 4 h. Routine nutritional support continued during this period by parentcral administration of dextrose, lipid emulsion, and an amino acid mixture low in tyrosine. Phenylalanine and tyrosine fluxes and rate of phenylalanine hydroxylation did not differ significantly between the two age groups, and so the data were combined for evaluation. For the entire group, values (μmol-kg−1-h−1; mean ± SD) for phenylalanine and tyrosine fluxes and rate of phenylalanine hydroxylation were 132 ± 24, 66 ± 16, and 29 ± 12, respectively. Plasma phenylalanine to tyrosine concentration ratio was 1.67 ± 0.6. From a comparison of the rate of phenylalanine hydroxylation with measured phenylalanine intakes, it was concluded that their routine, clinical nutritional support was inadequate to achieve body phenylalanine balance. In comparison with published data, the relative rate of phenylalanine hydroxylation appears to be high. We speculate that tyrosine is a conditionally indispensable amino acid under these conditions; it would be desirable to establish the intake levels and ratio of phenylalanine to tyrosine that effectively support aromatic amino acid balance in these critically ill patients.

Current recommended parenteral protein intakes do not support protein synthesis in critically ill septic, insulin-resistant adolescents with tight glucose control

Objective:To investigate the effects of insulin infusion and increased parenteral amino acid intakes on whole body protein balance, glucose kinetics, and lipolysis in critically ill, insulin-resistant, septic adolescents. Design:A single-center, randomized, crossover study. Setting:A medicosurgical intensive care unit in a tertiary university hospital. Patients:Nine critically ill, septic adolescents (age 15.0 ± 1.2 yrs, body mass index 20 ± 4 kg m−2) receiving total parenteral nutrition. Interventions:Patients received total parenteral nutrition with standard (1.5 g·kg−1·day−1) and high (3.0 g·kg−1·day−1) amino acid intakes in a 2-day crossover setting, randomized to the order in which they received it. On both study days, we conducted a primed, constant, 7-hr stable isotope tracer infusion with [1-13C]leucine, [6,6-2H2]glucose, and [1,1,2,3,3-2H5]glycerol, in combination with a hyperinsulinemic euglycemic clamp during the last 3 hrs. Measurements and Main Results:Insulin decreased protein synthesis at standard amino acid and high amino acid intakes (p < .01), while protein breakdown decreased with insulin at standard amino acid intake (p < .05) but not with the high amino acid intake. High amino acid intake improved protein balance (p < .05), but insulin did not have an additive effect. There was significant insulin resistance with an M value of ∼3 (mg·kg−1·min−1)/(mU·mL−1) which was 30% of reported normal values. At high amino acid intake, endogenous glucose production was not suppressed by insulin and lipolysis rates increased. Conclusion:The current recommended parenteral amino acid intakes are insufficient to maintain protein balance in insulin-resistant patients during tight glucose control. During sepsis, insulin decreases protein synthesis and breakdown, and while high amino acid intake improves protein balance, its beneficial effects may be offset by enhanced endogenous glucose production and lipolysis, raising concerns that insulin resistance may have been exacerbated and that gluconeogenesis may have been favored by high amino acid intakes. Dose-response studies on the effect of the level of amino acid intakes (protein) on energy metabolism are needed.

Phenylalanine conversion to tyrosine: Comparative determination with l-[ring-2H5]phenylalanine and l-[1-13C]phenylalanine as tracers in man

The in vivo rate of conversion of phenylalanine to tyrosine (PheOH) can be estimated using combinations of stable isotope-labeled phenylalanine and tyrosine. We have compared in four healthy adult men the rates of phenylalanine conversion to tyrosine based on the following pairs of primed, continuous tracer infusions administered simultaneously: (1) L-[ring-2H5]phenylalanine and 2H2-tyrosine with a 2H4-tyrosine prime, and (2) L-[1-13C]phenylalanine and 2H2-tyrosine with a 1-13C-tyrosine prime. Phenylalanine oxidation was determined from measurement of 13CO2 excretion in expired air. Tracers were given for 8 hours, with subjects being in the postabsorptive state during the first 3 hours and in the fed state during the remaining 5 hours. Mean (+/- SD) rates (mumol.kg-1.h-1) of phenylalanine conversion to tyrosine for fasted and fed states, respectively, were 5.1 +/- 2.9 and 6.8 +/- 3.4 with 2H5-phenylalanine and significantly higher (P < .05) at 11.1 +/- 5.6 and 12.7 +/- 7.7 with 13C-phenylalanine as tracer. Phenylalanine oxidation was 9.9 +/- 2.0 and 13.5 +/- 2.6, respectively, for fasted and fed states, and these mean values did not differ (P > .1) from the rate of phenylalanine conversion to tyrosine determined using 13C-phenylalanine. These results indicate the need for caution in interpreting kinetic aspects of phenylalanine metabolism when based on isotopic data from multideuterated phenylalanine.(ABSTRACT TRUNCATED AT 250 WORDS)