Plasma L-5-oxoproline kinetics and whole blood glutathione synthesis rates in severely burned adult humans

Yong-Ming Yu, Colleen M. Ryan, Zhe-Wei Fei, Xiao-Ming Lu, Leticia Castillo, John T. Schultz, Ronald G. Tompkins, and Vernon R. Young

Shriners Burns Hospital and Trauma Services, Massachusetts General Hospital, Boston 02114; and Laboratory of Human Nutrition and Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Compromised glutathione homeostasis is associated with increased morbidity in various disease states. We evaluated the kinetics of L-5-oxoproline, an intermediate in the gamma -glutamyl cycle of glutathione production, in fourteen severely burned adults by use of a primed, constant intravenous infusion of L-5-[1-13C]oxoproline. In nine of these patients, whole blood glutathione synthesis and plasma kinetics of glycine and leucine were also measured with [15N]glycine and L-[2H3]leucine tracers. Patients were studied under a "basal" condition that provided a low dose of glucose and total parenteral nutrition. For comparison with control subjects, whole blood glutathione synthesis was estimated in six healthy adults. Burn patients in a basal condition showed significantly higher rates of plasma oxoproline clearance and urinary D- and L-oxoproline excretion compared with fasting healthy control subjects. Whole blood glutathione concentration and absolute synthesis rate in the basal state were lower than for control subjects. Total parenteral feeding without cysteine but with generous methionine did not affect oxoproline kinetics or whole blood glutathione synthesis. The estimated rate of glycine de novo synthesis was also lower in burn patients, suggesting a possible change in glycine availability for glutathione synthesis. The roles of precursor amino acid availability, as well as alterations in metabolic capacity, in modulating whole blood glutathione production in burns now require investigation.

flux; glycine; de novo synthesis; clearance; urinary excretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DIMINISHED LEVELS of the tripeptide glutathione (gamma -glutamyl-cysteinyl-glycine; GSH) in cells and organs are associated with compromised cell function, increased tissue damage, and greater morbidity in various disease conditions (14-16, 44). Martensson et al. (32) showed that burn injury in rats markedly reduces mitochondrial GSH levels in liver and in several peripheral tissues, including brain, heart, and skeletal muscle. These investigators proposed that mitochondrial GSH levels may be increased by giving cysteine precursors, because cysteine appears to be the limiting amino acid for GSH synthesis in most tissues, in the rodent at least (26) and also in humans (28). A depletion of skeletal muscle glutathione levels occurs after surgical trauma (27). There are reports that trauma and infection affect plasma levels and the metabolism of glycine, serine, and taurine (3, 24), which may have implications for cysteine utilization for GSH synthesis (29, 30). Also, Vina et al. (45) reported that there is an impairment of cysteine synthesis from methionine in rats exposed to surgical stress. As might be anticipated, therefore, Grimble and colleagues (11, 12, 38) have shown that dietary cysteine is of prime importance in facilitating an increase in liver glutathione, zinc, and protein content in rats in response to a challenge with tumor necrosis factor (TNF). Because Kupffer cells are a major target of TNF action in stimulating superoxide anion production, they may further deplete liver GSH and contribute to organ failure (4). Finally, severely burned patients show signs of altered sulfur amino acid metabolism, characterized by an increased activity of the methionine-homocysteine cycle (51) and enhanced urinary excretion of mercaptolactate (25, 33). All of these observations point to altered rates of sulfur amino acid metabolism in severe stress, with implications for modifying the rates of GSH synthesis in different tissues and organs.

GSH synthesis occurs intracellularly from its constituent amino acids via the reactions of the gamma -glutamyl cycle (2). An intermediate in this cycle is L-5-oxoproline, and genetic deficiencies in glutathione synthetase result in the accumulation of plasma oxoproline and in an oxoprolinuria (34). An increased urinary output of this metabolite also has been taken to reflect an inadequate cellular availability of glycine for GSH synthesis (19), and we have shown in healthy adults that a diet devoid of either methionine plus cystine or of glycine increases oxoproline excretion (36). Possibly, therefore, 5-oxoproline kinetics may serve as a marker of the status of GSH synthesis and/or availability of glycine/cysteine for GSH synthesis in the severely burned trauma patients. Our recent studies (28, 36) in healthy adults support this thesis. Therefore, we hypothesized that the stress of severe burn injury in humans would result in a diminished rate of whole blood GSH synthesis and that this would be reflected by an increase in plasma oxoproline flux and oxidation rate relative to values in healthy subjects. Before proceeding to study the effects of substrate or nutrient precursor administration, with or without other pharmacological interventions (46, 47) in critically ill patients, on blood and tissue GSH levels in metabolic stress, it was important to carry out this initial series of tracer studies. Our objective was to characterize and quantify oxoproline kinetics and the rate of whole blood GSH synthesis in severely burned patients. We compare our findings with those obtained in healthy adult volunteers by use of comparable tracer approaches and analytic techniques.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

L-5-[1-13 C]oxoproline [99% atom percent excess (APE)] and [1,2-13C2]glycine (99% APE) were purchased from MassTrace (Woburn, MA). [15N]glycine (99% APE), NaH13CO3 (98% APE), and L-[5,5,5-2H3]leucine (>98% 2H) were obtained from Cambridge Isotope Laboratories (Andover, MA). The isotopically labeled tracers were made into stock solutions at the pharmacy of the Massachusetts General Hospital (MGH). Before use, they were confirmed to be sterile and pyrogen free; then they were prepared for each tracer study, conducted either at MGH Burns and Trauma Unit or at the Massachusetts Institute of Technology (MIT) Clinical Research Center (CRC). The total parenteral nutrition (TPN) solutions for patient feeding were prepared in the Nutritional Support Unit of MGH. A Clinisol amino acid injection (Baxter Healthcare, Clintec Nutrition Division, Deerfield, IL) at 15% was used as the amino acid source, and the composition of the formula is shown in Table 1. Diets for the healthy volunteers are described in Healthy Control Subjects.

                              
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Table 1.   Amino acid composition of parenteral amino acid mixture

Burn Patients

A total of 14 burn patients were studied. Among these patients, the plasma kinetics of oxoproline were assessed in all the subjects, whereas GSH kinetics were determined in 9 of the 14 patients as a subgroup. The general characteristics of these patients are shown in Table 2. The mean age (±SE) was 46.6 (±5.3) yr for the whole group and 48.2 (±6.4) yr for the subgroup. On admission, the total percentage of body surface burned area (TBSA) for these patients was estimated to be 43 ± 6 and 42 ± 8% for the entire group and subgroup, respectively. Smoke inhalation injury, as diagnosed by admission bronchoscopy, was present in all of the patients studied. Three patients died subsequently from their injuries. All patients were treated with standard burn resuscitation and critical care measures, including serial excision and grafting procedures beginning early in their hospital course (mean of 2 days, ranging from 1 to 4 days after admission) (43). The experimental protocol was approved by the Subcommittee for Human Studies, Committee of Research, MGH, and the Partners Health Care System. Written consent was obtained, either from the patients or from one or more of the family members after being informed of the purpose, design, and possible hazards of the experiment.

                              
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Table 2.   General condition of patients studied

The patients were studied, on average, on days 14 and 16 postburn when their general clinical condition was stabilized but still in a severe catabolic state. For the total group of 14 burn patients, energy expenditure measured via indirect calorimetry and based on O2 consumption was equivalent to 35.6 ± 2.2 (mean ± SE) kcal · kg-1 · day-1 for the basal state and 38.5 ± 2.4 kcal · kg-1 · day-1 for the TPN phase. The corresponding values for the subgroup of 9 patients were 33.3 ± 1.6 and 35.7 ± 2.0 kcal · kg-1 · day-1, respectively. The general clinical condition of these patients was comparable to that of burned patients studied by us earlier (23, 51-54, 56). For each patient, 24-h urine collections were started before the day of tracer infusion and were continued for 2-4 consecutive days to evaluate the rates of total urinary creatinine, nitrogen, and D- and L-oxoproline excretion. The reported data are averages of these 24-h collections for each patient.

Healthy Control Subjects

To compare our findings in burn patients with those in healthy control subjects, two approaches have been taken here.

Whole body GSH (WBGSH) synthesis rate in healthy control volunteers had been estimated previously by us at the CRC of MIT by use of L-[1-13C]cysteine as tracer (28). However, for comparison with burn patients, we further studied six healthy adults by use of [1,2,-13C2]glycine as a tracer. This group included five men and one woman [age 26 ± 6 (SD) yr; body wt 69.7 ± 11 kg]. All were in good health, as determined by history and physical examination, analysis of cell blood count, biochemical profile, and urine analysis. Their daily energy intake was calculated to maintain body weight on the basis of a dietary history and an estimate of the subjects' usual level of physical activity. The subjects were encouraged to maintain their customary levels of physical activity but not to participate in competitive sports. The purpose of the study and the potential risks involved were fully explained to all subjects before they signed the written consent form. The study protocol was approved by the MIT Committee on the Use of Humans as Experimental Subjects and the Executive and Policy Committee of the MIT CRC. All subjects received financial compensation for their participation in the experiments, and all remained healthy throughout.

The subjects were given for 10 days before the tracer study a fully adequate diet, with the protein component in the form of a complete L-amino acid mixture. Details of the diet composition have been described in our previous publication (28).

For comparisons of oxoproline kinetics between burn patients and healthy adults, we refer to our recent reports on tracer studies of oxoproline kinetics in healthy subjects who received a fully adequate diet containing a complete L-amino acid mixture for 6 days (36).

Comparisons have been made between burn patients in the "basal" state and healthy adults under an overnight fasting state.

Tracer Studies in Burn Patients

Tracer studies were performed when the patients were in a relatively stable condition, as assessed by blood pressure, heart rate and cardiac function, respiration rate, body temperature, and liver and kidney functions.

As in our previous investigations, each patient was studied twice, once during a basal or "fast" phase and a second time while they were in a "fed" (TPN) phase. The two phases were randomized in order (Table 2) and usually conducted within 2 or 3 days of each other, with the maximum interval being 5 days. During the TPN condition, patients received nutritional support that had begun >= 2 days before the tracer study. The average intakes were 0.35 ± 0.02 g N · kg-1 · day-1, with nonprotein calories equivalent to 30.8 ± 3.5 kcal · kg-1 · day-1 being supplied by glucose. The basal condition was created by terminating the TPN ~10 h before the tracer studies were begun. To prevent hypoglycemia during the basal condition, the patients received an infusion of 5% dextrose supplying 6.2 ± 1.4 mg glucose · kg-1 · h-1. This rate of glucose administration was not expected to have any major impact on amino acid kinetics, as previously discussed (56). After the tracer infusion studies were completed, TPN feedings were either resumed or were replaced by enteral feeding in accordance with orders written by the attending clinicians.

Measurement of oxoproline kinetics. Oxoproline kinetics were determined using a primed, constant intravenous infusion of L-5-[1-13C]oxoproline tracer. The infusion studies were generally started between 0600 and 0700. For the first five patients, in whom WBGSH synthesis was not measured, the oxoproline tracer studies lasted for 240 min. Before the beginning of the tracer infusion, blood and expired air samples were taken for the determination of the baseline 13C enrichments. Additional blood samples (~3 ml) were taken at 180, 200, 220, and 240 min after commencement of the tracer infusion. Four sets of expired air samples were also taken concomitant with times of blood sampling for determination of the isotopic steady-state level of 13CO2 enrichment in the expired air. Timed expired air samples also were collected for determination of total O2 consumption and CO2 production, as described previously (52, 53). The targeted, but known, infusion rate of oxoproline was 3.6 µmol · kg-1 · h-1. Before the infusion was started, priming doses of L-5-[1-13C]oxoproline (3.6 µmol/kg) and 13C-labeled sodium bicarbonate (NaH[13C]O3, 1.3 µmol/kg) were also given. This tracer protocol is depicted in Fig. 1A. For the remaining nine patients, L-[1-13C]oxoproline was infused together with [15N]glycine and L-[5,5,5,-2H3]leucine tracers for a total of 360 min. Plateau level blood samples were taken at 180, 240, 270, 300, 330, and 360 min. Four sets of air samples were collected at the last four time points; otherwise, procedures were as for the initial five patients (Fig. 1B). Pilot studies have shown that L-[1-13C]oxoproline infusion at this rate does not result in detectable GSH enrichment.


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Fig. 1.   A: tracer study protocol for oxoproline kinetics in patients 1-5. B: tracer study protocol for oxoproline and glutathione (GSH) kinetics in patients 6-14.

Measurement of whole blood GSH synthesis rate and plasma kinetics of glycine and leucine. The rate of GSH synthesis was measured in 9 of the 14 patients by use of a primed, constant intravenous infusion of [15N]glycine tracer for 360 min. Blood samples were taken before commencement of the tracer infusion, for baseline enrichment, and then at 120, 180, 240, 270, 300, 330, and 360 min after the infusion was started, to measure the plateau level enrichment of [15N]glycine and the appearance of the [15N]glycine label ([15N]glycyl-GSH) in WBGSH. The targeted, but known, infusion rates of labeled glycine were ~21 µmol · kg-1 · h-1 (prime, 21 µmol/kg) for the basal state and 28.5 µmol · kg-1 · h-1 during TPN (prime, 28 µmol/kg).

Whole body protein kinetics were also measured by use of a simultaneous primed, constant infusion of L-[5,5,5 2H3]leucine tracer, with a targeted infusion rate and priming dose of 4.2 µmol · kg-1 · h-1 and 4.2 µmol/kg, respectively. Plasma enrichment of the labeled alpha -ketoisocaproate ([2H3]KIC) was determined from the blood samples taken at the time points mentioned. The enrichment of [2H3]KIC is used as a surrogate of intracellular leucine enrichment (31). Measurement of leucine kinetics serves two purposes: 1) to assess how the turnover rate of body proteins in the present group of patients is compared with earlier groups of burned patients studied at different times in our hospital, and 2) to calculate the glycine de novo synthesis rate.

Tracer Studies in Healthy Control Subjects

Measurement of oxoproline kinetics. The study procedures have been described in detail (35, 36). They involved a constant intravenous infusion of L-5-[1-13C]oxoproline for a total of 8 h; during the first 3 h of the tracer period, the subjects were studied in the postabsorptive state, and they received adequate, small, and equal meals at 30-min intervals during the subsequent 5 h. Oxoproline kinetics did not differ between these two studies (35, 36), and so we used the data in the fasting condition from one of these two experiments for comparison with burn patients (36).

Measurement of whole blood GSH synthesis and plasma kinetics. In these studies, a [1,2-13C2]glycine tracer was used. The isotope infusion period lasted for 6 h and was carried out when subjects were in the postabsorptive state. Details of the general procedures followed immediately before and during the infusion protocol have been presented (7, 8, 28). On day 10 of the adequate diet period, the subjects entered the infusion room at the MIT CRC at ~0700 and later received a priming dose of 20 µmol/kg [1,2-13C2]glycine, followed by a constant infusion of the same tracer (20 µmol · kg-1 · h-1) for the following 6 h. Blood samples were drawn at hourly intervals for determination of the isotopic enrichment in whole blood and in plasma, as well as that of [13C]glycyl-GSH in whole blood.

Analytic Methods

Details have previously been given for sample collection, preparation, and analysis of blood and urine samples for [13C]oxoproline (35) and [2H3]KIC (7). The procedure for analysis of whole blood GSH concentration and isotopic enrichment has been described in detail (28). The changes for the present studies were that tracers [15N]glycine and L-[1,2-13C2]glycine were used to label whole blood GSH. Briefly, 50 µl of whole blood samples were immediately placed into prechilled tubes containing 1 ml of ice-cold dithiothreitol (DTT, 20 mM in 1 M acetic acid) and frozen at -80°C until analysis. Upon analysis, the red blood cells were lysed by repeated freezing (-80°C) and thawing (room temperature) three times. After standing at room temperature for 4 h, they were centrifuged at 10,000 g for 5 min. The supernatant was transferred to an ion exchange column (1.5 ml of the AG 50W-X8 cation exchange resin, Bio-Rad, Hercules, CA). After being washed with Milli-Q water (2.5 ml × 3), the GSH was eluted from the column using NH4OH (3 M, 1.5 ml × 2) and collected into derivatization tubes (10 ml). The samples were dried under gentle nitrogen flow at 65°C. Each sample was then reacted with 2 ml of the same DTT solution at room temperature for 2 h to reduce any dimerized GSH under the alkaline condition due to the treatment by NH4OH in the prior step. The samples were then dried again in gentle nitrogen flow at 65°C. Then, each sample was reacted with 1 ml of methanol-acetyl chloride solution (10:1, vol/vol, prepared on ice) at room temperature for 1 h. After the excess reagent was removed by nitrogen flow at 65°C and cooled down to room temperature, the samples were further reacted with trifluoroacetic anhydride (0.5 ml) at room temperature for 1.5 h. The excess reagent was further removed by nitrogen flow at 65°C and then reconstituted with 50 µl of acetonitrile before injection into a gas chromatograph-mass spectrometer (GC-MS). The GC-MS analysis of GSH was carried out on an HP 5890 Series II gas chromatograph coupled to an HP 5988 mass spectrometer by use of a fixed silica capillary column of cross-linked polydimethylsiloxane (HP-2, 30 M × 25 mm ID, 0.25 µm film thickness). The samples were injected into a splitless injection port under the following operating conditions: column temperature programmed at 10°C/min from 160 to 290°C, followed by 5°C/min from 290 to 320°C; the injector and detector temperatures were 250°C. Helium and methane were separately used as carrier gas and reactant gas. The derivative, tentatively identified as a complex bicycloglutaramide (28), was measured under a negative chemical ionization condition by selective ion monitoring (SIM) at a nominal mass-to-charge ratio (m/z) of 477.1 to 480.1, corresponding to the most abundant and preponderant near-parent ion. The concentrations of whole blood glutathione were determined using synthetic [1,2-13C2-glycyl]GSH as an internal standard. In healthy controls with use of the [1,2-13C2]glycine tracer, whole blood GSH concentration was measured using the same internal standard on the three "baseline" samples taken before the beginning of tracer infusion. The GC-MS method is sensitive enough to determine tracer enrichments of [15N]GSH with a quantitation limit in the range of 0.3-0.5 mole % excess (MPE). For the [1,2-13C2]glycine and [15N]glycine tracers, plasma glycine was eluted from the ion exchange column with the same procedures as followed for GSH. Dried samples were reacted with n-propanol-HCl and heptafluorobutyric acid anhydride to yield N-heptafluorobutyryl-n-propanol ester. GC-MS analysis was performed on an HP 6890 series gas chromatograph coupled to an HP 5973 mass spectrometer with a fused silica capillary column of cross-linked polydimethylsiloxane (HP-1, 30 m × 0.25 mm ID, 0.25 µm film thickness). Glycine derivative was quantified under negative chemical ionization, with methane as reagent gas, by use of SIM at m/z 293 (M-HF, unlabeled glycine), 294 ([1-13C]glycine), and 295 ([1,2-13C]glycine). The concentrations of urinary urea nitrogen and creatinine were determined using routine methods in the Clinical Chemistry Laboratory of MGH by use of enzymatic methods.

The enrichment of 13CO2 in the expired air of the burned patients was measured using an isotope ratio mass spectrometer (7), and total CO2 production was determined by indirect calorimetry (7, 51).

Calculations

We have described, previously, calculations used to estimate plasma oxoproline flux and oxoproline oxidation (35, 36), plasma leucine flux (6, 7), and whole blood glutathione synthesis (28) on the basis here of the glycine enrichment in plasma in both burn patients and healthy controls. Plasma oxoproline clearance (l · kg-1 · h-1) was the product of the plasma oxoproline flux and concentration. For GSH we estimated the fractional synthesis rate (FSR) as well as the absolute synthesis rate (ASR), as previously described (28). We also corrected the ASR (ASRcorrected) for differences in the blood hematocrit between the patient and control groups. Glycine flux in the burn patients and healthy control subjects also was determined according to a standard stochastic equation (55). Because the plasma flux of a nutritionally dispensable amino acid is due to intake, its release via protein breakdown, and also its appearance via de novo synthesis, the rate of de novo glycine synthesis could be estimated (55). Thus we assumed that amino acids are released from proteins via proteolysis (B) or taken up for protein synthesis at rates proportional to their concentration (k) in body proteins (652 µmol glycine and 621 µmol leucine/g protein) (50). For a nutritionally indispensable amino acid, such as leucine
B<SUB>Leu</SUB><IT>=</IT>k<SUB>Leu</SUB><IT>×</IT>B<SUB>wb</SUB> (1)
where Bwb is the rate of whole body protein breakdown, expressed as g protein · kg-1 · h-1.

The rate of de novo glycine synthesis (Qdn), therefore, is calculated as follows
Q<SUB>Gly</SUB><IT>=</IT>B<SUB>Gly</SUB><IT>+</IT>I<SUB>Gly</SUB><IT>+</IT>Q<SUB>dn</SUB>, or Q<SUB>dn</SUB><IT>=Q</IT><SUB>Gly</SUB><IT>−</IT>B<SUB>Gly</SUB><IT>−</IT>I<SUB>Gly</SUB> (<IT>2</IT>)
where QGly is glycine flux, IGly is glycine supplied via intravenous feeding, and
B<SUB>Gly</SUB><IT>=</IT>glycine released by protein breakdown<IT>=</IT>B<SUB>Leu</SUB><IT>×</IT><FR><NU>652</NU><DE>621</DE></FR>
Finally, the FSR of whole blood GSH (FSRgsh; %/day) is calculated from the rate of increment in the enrichment of the glycine label in whole blood GSH (K, MPE/min) and the steady-state plasma enrichment (Ep) of the glycine tracer, an approach that has been described by Jahoor et al. (21). The calculations were made on the basis of plasma glycine enrichments in both burn patients and control subjects. Thus
FSR<SUB>gsh</SUB><IT>=</IT><FR><NU>K<IT>×</IT>1,440</NU><DE>E<SUB>p</SUB></DE></FR><IT>×</IT>100 (3)
The ASR of GSH in whole blood (ASRwb,gsh; µmol · l-1 · day-1) is calculated from the blood GSH concentration (Cgsh; µmol/l) and the FSR. Hence
ASR<SUB>wb gsh</SUB><IT>=</IT>C<SUB>gsh</SUB><IT>×</IT>FSR<SUB>gsh</SUB> (4)
The ASR corrected (ASRcorrected,gsh; µmol · l-1 · day-1) is calculated by
ASR<SUB>corrected,gsh</SUB><IT>=</IT><FR><NU>ASR<SUB>wb gsh</SUB></NU><DE>Hct</DE></FR> (5)
where Hct is hematocrit during the time of study, expressed as a fraction.

Evaluation of Data

Statistical evaluation of the data was performed using PROSTAT software (Poly Software International, Sandy, UT). All data were examined for normalcy of distribution before further comparison tests were carried out. Paired t-tests were used to compare the metabolic measurements between the basal and the TPN states. Kinetic parameters obtained in our current and earlier investigations with healthy adults were used here to help further evaluate the status of oxoproline and GSH metabolism in the burn patients; comparisons were made by unpaired t-test. For those data that did not follow a normal distribution, the nonparametric Wilcoxon sign-rank test was used for comparison between the burn and healthy subjects.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stable Isotope Abundance

The isotopic abundance of L-5-[1-13C]oxoproline and alpha -[2H3]ketoisocaproate (a surrogate of intracellular L-[5,5,5-2H3]leucine enrichment) in plasma of the burn patients (Fig. 2), [15N]glycine in burn patients, and [1,2-13C2]glycine in the healthy control subjects (Fig. 3) reached relatively steady levels within 120 min after the beginning of the tracer infusion. Linear regression analysis of the slope of each tracer enrichment was not statistically different from zero (P > 0.10). The enrichment of whole blood [15N]glycine-labeled GSH ([15N]glycine-GSH) in burn patients and [1,2-13C2]glycine-labeled GSH ([1,2-13C2]glycine-GSH) showed a steady rate of increase from 120 to 360 min after the beginning of the glycine tracers (Fig. 4).


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Fig. 2.   Plasma enrichments of [1-13C]oxoproline (OP, A) and [1-13C]ketoisocaproate (KIC, B) in burn patients during basal and total parenteral nutrition (TPN) conditions. Values are means ± SE.



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Fig. 3.   Plasma enrichment of L-[15N]glycine in burn patients in basal state (A) and TPN state (B) and in healthy control subjects (C). Values are means ± SE.



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Fig. 4.   Whole blood GSH enrichment measured during the tracer infusion studies in burn patients in the basal state (A) and the TPN state (B) and in healthy control subjects (C). Values are means ± SE.

Oxoproline Kinetics and Excretion

The results for the various parameters of oxoproline metabolism in the burn patients are summarized in Table 3. They are compared here with those obtained in healthy subjects, reported recently (36), by use of a comparable tracer approach and the same analytic techniques. Thus, by use of a primed constant infusion of L-5-[1-13C]oxoproline, the plasma oxoproline flux and oxidation rate, when expressed per unit of body weight in burn patients, were not apparently different from those for healthy control subjects. There was a tendency for the nonoxidative disposal rate to be higher in burn patients, but because of the high intersubject variation, it was not significantly different from that in healthy controls (P = 0.15). However, in burn patients, the plasma concentration of oxoproline in the basal state was significantly lower than the level for healthy subjects [P < 0.05, unpaired t-test (36)]. TPN did not significantly alter either plasma oxoproline concentration or kinetics. This parallels our observations (36) made in healthy subjects when they were given frequent small meals. Nevertheless, the calculated plasma clearance rate of oxoproline was significantly higher (P < 0.001) in burn patients than in healthy control subjects.

                              
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Table 3.   Oxoproline kinetics measured in burn patients and control subjects

The urinary excretion rates of D- and L-oxoproline were measured in 12 of the 14 burn patients (Table 4). The data are compared here with those previously obtained from healthy control subjects (36). The output of urinary L- and D-oxoproline increased to a level of approximately threefold in burn patients compared with healthy volunteers. The rate of urinary creatinine excretion was 13.9 ± 1.3 mmol/day, which is within the range of daily excretion rates, ~12 and 22 mmol/day, observed in our healthy controls (36). Thus the D- and L-oxoproline-to-creatinine ratios in burn patients were about three times higher than those for healthy subjects, indicating a substantially increased urinary loss of oxoproline after burn injury.

                              
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Table 4.   Urinary excretion of L- and D-oxoproline in burn patients and healthy control subjects

Leucine Kinetics

The kinetics of leucine (Table 5) were measured in the subgroup of nine of these patients. The total metabolic flux of leucine was (means ± SE; µmol · kg-1 · h-1) 184.1 ± 19.0 for the basal state and 231.8 ± 19.4 during the TPN state, when the leucine intake was 43.0 ± 3.6 µmol · kg-1 · h-1. Thus the rate of plasma leucine release from whole body protein breakdown was similar between the basal and TPN states, which further indicates a similar rate of whole body protein breakdown (7.1 ± 0.7 vs. 7.3 ± 0.8 g · kg-1 · day-1, respectively) under these two states. These values in burn patients are much higher than those in healthy subjects (9, 31, 50) but are close to those previously observed in burn patients (52, 53, 56). Finally, the lack of a difference in the rate of appearance of leucine via protein breakdown between the basal state and TPN feeding in burn patients is in agreement with our earlier observations (52-54, 56), as well as those of others (5, 13, 20) who have studied amino acid kinetics under conditions of parenteral feeding.

                              
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Table 5.   Leucine kinetics measured in burn patients

Glycine Kinetics

The plasma glycine kinetics measured in burn patients and in healthy controls are presented in Table 6. An estimate of de novo glycine synthesis can be made from these data, as described in MATERIALS AND METHODS. We have assumed a plasma leucine flux of 110 µmol · kg-1 · h-1 for estimating glycine synthesis in the controls. Hence, as summarized in Table 6, compared with the healthy postabsorptive controls, burn patients show a reduced rate of glycine de novo synthesis (P < 0.01).

                              
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Table 6.   Glycine and glutathione kinetics in burn patients and control subjects

Whole Blood GSH Kinetics

The whole blood GSH concentration in burn patients was not different between the basal and TPN states (Table 6). However, these values are significantly lower than those in healthy subjects (P < 0.05 by the Wilcoxon rank-sum test). The estimates for whole blood GSH synthesis rates in the nine burn patients are also summarized in Table 6. The FSR of whole blood GSH (means ± SE) in the basal state was 54 ± 9%/day. TPN did not change this value (53 ± 6%/day). Although there was a strong tendency, these values were not statistically different from those measured in the healthy fasted subjects (P = 0.055, by unpaired t-test). The whole blood ASR of GSH in burn patients was 345 ± 52.0 and 366 ± 52 µmol · l-1 · day-1, respectively, in the basal and TPN states. These values are significantly lower than for postabsorptive, healthy subjects (P < 0.01, by unpaired t-test). Because the Hct of burn patients was lower than that for normal subjects during the basal and TPN phases, we have made an estimated ASRcorrected for GSH by accounting for differences in the packed cell volume. These rates are significantly lower (P < 0.05) than the rate measured in healthy subjects. Hence, burn injury significantly reduces the GSH ASR in whole blood.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These are the first investigations, to our knowledge, to explore plasma oxoproline kinetics and determine blood GSH synthesis rates after severe trauma in human subjects. We have also, for purposes of comparison, generated some additional new data from a control group of healthy adults. Our studies reveal a reduced whole blood GSH synthesis rate and an increased plasma oxoproline clearance in the burn patients, compared with the present and our previous studies in healthy controls (28, 35, 36). These data, therefore, inform a further understanding of the diminished GSH concentrations in blood and perhaps other tissues under various stress and/or disease conditions. In addition, there is evidence that erythrocytes can provide antioxidant protection to other tissues in vivo (40). Hence, these findings of whole blood GSH kinetics in burn patients offer a possible basis for the diminished antioxidant capacity in severely stressed patients. Finally, our study also revealed a reduction in glycine de novo synthesis rate in burn patients, which together with an increased rate of urinary oxoproline excretion and diminished whole blood GSH synthesis, suggests a possible causal relationship between glycine kinetics and the altered GSH homeostasis in these severely injured patients.

Before proceeding further, it should also be recognized that these burn patients received their standard medical care, including nutritional support. The latter does not routinely include an exogenous source of preformed cysteine. However, the supply of methionine (~98 mg · kg-1 · day-1) given to our patients amounts to ~6-7 times the minimum requirement of the total sulfur amino acids (methionine + cysteine) for healthy subjects. At this level of intake, the supply of methionine would be expected to be in considerable excess of the methionine requirement for burn patients, and so it would serve also as an adequate source of cysteine for meeting the functional needs for this amino acid. However, it is uncertain whether the route of amino acid intake might be of importance in this context, because the burn patients were nourished via the parenteral route, whereas our healthy control subjects received their nutrients by mouth. This needs to be borne in mind, but it was first, for ethical reasons, necessary for us to study patients according to current clinical practice before we could justify a possibly significant change in the routine medical treatment and nutritional support.

There are a limited number of tracer-derived estimates of the rate of whole blood GSH synthesis in human subjects (21, 28, 39). Using [2H2]glycine, Jahoor et al. (21) found that the smaller erythrocyte GSH pool in symptom-free HIV-infected adult males was associated with a significantly lower ASR of GSH compared with controls (1.15 ± 0.14 vs. 1.71 ± 0.15 mmol · l-1 · day-1), although there was not a significant difference between the FSR values of erythrocyte GSH in these groups. N-acetylcysteine supplementation for 1 wk tended to increase the FSR (P = 0.07) and the ASR of WBGSH. In comparison, we find in burn patients that FSR tended to be lower (P = 0.055) when compared with the FSR for healthy controls. However, as in the findings of Jahoor et al. (21), the ASR of GSH was significantly depressed in burn patients. The ASR of WBGSH for our volunteers, when corrected for Hct, is very similar to the estimate of Jahoor et al. (21) for healthy subjects. Our present observations suggest, therefore, that the lower whole blood GSH concentration in burn patients is due to a lower ASR of this important critical intracellular thiol.

Because a severe stress created by major burn injury results in an increased oxidative load, it might be expected that it leads to an increased rate of consumption of GSH. To assess this hypothesis will require a direct estimate of the rate of disappearance of whole blood GSH. Nevertheless, our finding of an impaired rate of WBGSH synthesis in burn patients is consistent with a number of other observations. Thus, using a pharmacokinetic approach, Heebling et al. (15) concluded that GSH concentrations in patients with HIV infection are low because of a decreased systemic synthesis of GSH. Also, Luo et al. (27) found that the depletion of the GSH pool in skeletal muscle after surgical trauma is associated with a decreased activity of GSH synthetase, the rate-limiting enzyme in GSH synthesis. This strongly indicates a decreased GSH synthetic capacity in this tissue, although the precise status of GSH synthesis could not be determined from the data of Luo et al.

In the present study, we sampled the whole blood GSH pool because it can be accessed noninvasively under our clinical conditions. An important issue is whether changes in whole blood GSH concentration and kinetics in our burn patients reflect parallel changes in important organ systems such as heart, kidney, liver, lung, and intestinal tissues. In protein-deficient pigs, a turpentine-induced inflammatory stress (22) caused a significant increase in the erythrocyte GSH FSR but with no significant change in the ASR. Although both pre- and postinflammation ASRs of GSH synthesis in liver and gut mucosa were not available in that published study (22), the postinflammation rates of GSH synthesis in these organs were either not different or lower, respectively, compared with values for the well-nourished control subjects. Therefore, the changes in blood GSH kinetics may not necessarily be in proportion to those occurring in various organs or tissues. During the acute phase of sepsis in rats, Malmezat et al. (29) found a decreased ASR of GSH in blood (-47%), whereas there were increases in liver (+465%), spleen (+388%), and lung (+100%). However, the response of GSH metabolism differed in the later stages of the infection, according to these authors, although specific results were not presented. On the other hand, in preliminary studies by use of a burn rabbit model (18), we (Y-M Yu, ZW Fei, X-M Lu, A Rhodes, A Lu, C-L Chen, RG Tompkins, and VR Young; unpublished results) observed at 3 days postburn that the ASR of GSH was reduced by ~30% in whole blood and by ~80 and 70% in liver and lungs, respectively, with no apparent change in the kidney. Hence, alterations of GSH kinetics in different organ tissues may occur in a time-dependent fashion, and the time course may vary among individual organ tissues. The quantitative relationships among these organs and their relation to whole blood GSH metabolism in human subjects will require the development of novel tracer probes and application of new modeling paradigms (48-50). For now, supported by our findings in the burn rabbit model, we think that it is valuable to generate initial kinetic data on in vivo aspects of whole blood GSH metabolism, to the extent that they can be studied and serve as a basis for exploring some of the nutritional, metabolic, and functional corollaries of GSH homeostasis in the severely burned patients.

A GSH synthetase deficiency results in an increase in gamma -glutamylcysteine synthetase activity through a feedback mechanism, leading to an accumulation and excretion of 5-oxoproline (34). Hence, it is also of interest to learn how severe burn injury simultaneously affects the kinetics of oxoproline metabolism and, indeed, whether oxoproline kinetics might serve as a marker of changes in GSH status in burn patients. Using a primed, constant tracer infusion approach, we find here that the plasma flux and oxidation rates only tended to be higher than those for healthy controls. However, the nonoxidative disposal rate of oxoproline was higher and the plasma clearance rate almost twice as high in burn patients compared with those rates for healthy controls. This change could mean that a depressed rate of GSH synthesis leads to an increased rate of oxoproline production through the gamma -glutamylcyclotransferase reaction, followed by an increased rate of oxoproline urinary excretion. This possibility is supported by our findings of an accelerated rate of urinary D- and L-oxoproline excretion in these burn patients (Table 4). It should be emphasized that these initial observations on plasma oxoproline kinetics are based on a stochastic model, and it seems highly worthwhile now to explore the impact of burn injury on oxoproline kinetics by use of a bolus injection and compartmental modeling for this purpose (49, 50). We speculate that plasma or peripheral kinetics of oxoproline, which we assessed using the present tracer paradigm, may not fully reflect changes in oxoproline metabolism at more central sites.

Finally, because we used labeled glycine to estimate whole blood GSH synthesis rates, it is worth commenting on our findings for plasma glycine flux and the estimated de novo synthesis rates. In this study, plasma glycine fluxes did not differ significantly between burn patients and control subjects. Because the rate of whole body protein breakdown is higher in burn patients, the rate of de novo glycine synthesis is lower after burn injury than in healthy controls (Table 6). Whether this difference in glycine metabolism is causally related to the lower rate of GSH synthesis cannot be determined from these data, although they could be taken to imply a reduced metabolic availability of cellular glycine for supporting the activity of the gamma -glutamyl cycle. The increased urinary excretion of oxoproline supports this notion (19). We should note, however, that the present estimate of the glycine flux in healthy controls is higher than that which we reported a number of years ago (41, 51) and also compared with that of Jahoor et al. (21) and others (17, 57). It follows, then, that our estimate of the rate of de novo glycine synthesis in healthy control subjects is also higher than we determined in two earlier studies (41, 55), although it is comparable to that in another report (42). The basis for these differences is unclear, but we believe that we have ruled out the presence of analytic problems with the present samples. However, our conclusion that the de novo synthesis rate of glycine after burn injury is depressed should be viewed with due regard for differences among the various studies concerned with estimates of de novo glycine synthesis rates. We intend now to extend our studies of glycine metabolism to determine the nature of this variation in glycine kinetics among populations of healthy adults.

In summary, plasma oxoproline kinetics, urinary oxoproline excretion, and whole blood GSH synthesis rates are all altered after severe burn injury. It is concluded that the synthesis of whole blood GSH is depressed under these conditions. Whether the availability of glycine is limiting and/or that of one or more of the other precursors, cysteine and glutamate, including glutamine (1, 46), whose peripheral production is either insufficient (37) or diminished (10) in burn patients, is also limiting for maintenance of GSH homeostasis now deserves further investigation.


    ACKNOWLEDGEMENTS

We acknowledge the excellent work of Mary Ann Malloy and Laura Collier in conducting the tracer studies, and the assistance of the MGH Pharmacy in preparing the isotope tracer solutions. We thank Andrew Rhodes, Amy Lu, Sue Wong-Lee, and Mike Kenneway for their technical assistance in the analyses of samples. We also appreciate the assistance of the nursing and dietetic staff at the Burn Unit of MGH and the CRC at MIT.


    FOOTNOTES

These studies were financially supported by grants from the Shriners Hospitals for Children and the National Institutes of Health (DK-15856; GM-02700; P-30-DK-40561; RR-88).

Address for reprint requests and other correspondence: V. R. Young, Shriners Burns Hospital, 51 Blossom St., Boston, MA 02114 (E-mail: vryoung{at}mit.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00206.2001

Received 14 May 2001; accepted in final form 25 September 2001.


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Am J Physiol Endocrinol Metab 282(2):E247-E258
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