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
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ABSTRACT |
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Compromised glutathione homeostasis is
associated with increased morbidity in various disease states. We
evaluated the kinetics of L-5-oxoproline, an intermediate
in the -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
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INTRODUCTION |
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DIMINISHED LEVELS of
the tripeptide glutathione (-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 -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.
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MATERIALS AND METHODS |
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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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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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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 · kg1 · 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 · kg1 · 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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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 · kg1 · h
1 (prime,
21 µmol/kg) for the basal state and 28.5 µmol · kg
1 · h
1 during
TPN (prime, 28 µmol/kg).
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 · kg1 · 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 atThe 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
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(1) |
The rate of de novo glycine synthesis (Qdn), therefore, is
calculated as follows
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(3) |
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(4) |
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(5) |
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 |
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Stable Isotope Abundance
The isotopic abundance of L-5-[1-13C]oxoproline and
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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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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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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
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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
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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 ![]() |
DISCUSSION |
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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 · kg1 · 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 · l1 · 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
-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
-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
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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