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Article

Plasma L-5-Oxoproline Carbon and Nitrogen Kinetics in Healthy Young Adults

Cornelia C. Metges^*,1, Yong-Ming Yu, Wei Cai, Xiao-Ming Lu, Sue Wong, Alfred M. Ajami and Vernon R. Young^*,,23

^* Laboratory of Human Nutrition, School of Science and Clinical Research Center, Massachusetts Institute of Technology, Cambridge, MA 02139; Boston Burns Hospital, Boston MA 02114; MassTrace Inc., Woburn, MA 01801

³Correspondence to: Prof. Vernon R. Young, Laboratory for Human Nutrition, Bldg. E17-434, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, Phone: 617-253-5801; Fax: 617-253-9658; email: vryoung{at}mit.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
L-5-oxoproline (OP), an intermediate of the -glutamyl cycle of glutathione synthesis and degradation, may serve as a probe for the state of glutathione kinetics. We explored the whole-body carbon and nitrogen kinetics of OP in five male healthy subjects (75.2 kg; 181 cm; 26 y) after a 5-d adaptation to an adequate L-amino acid-based diet (160 mg N · kg^-1 · d^-1; 188 kJ · kg^-1 · d^-1), using a crossover design. On day 6 of the diet period, we carried out an 8-h tracer protocol (3 h fast; 5 h fed; 2/3 of daily nitrogen intake) with intravenous infusion of L-[1-¹³C]oxoproline and L-[3,3-²H]cysteine or, in randomized order, on the second occasion, L-[¹⁵N]oxoproline and L-[3,3-²H]cysteine. Plasma OP was isolated by cation exchange and after addition of internal standards (DL-[²H₃]-5-oxoproline; L-[¹⁵N, U-¹³C₅]-5-oxoproline; DL-[²H₃]-glutamic acid) derivatized to form TBDMS esters and measured by gas chromatography/mass spectrometry. Plasma OP concentration did not differ between fed and fasted state (fast: 59.4 ± 8.3; fed 59.2 ± 8.9 nmol/mL). ¹³C- and ¹⁵N OP flux during the fasted and fed state were 19 ± 3.6, 21.2 ± 3.2, and 22.6 ± 3.9, 25.8 ± 4.3 µmol · kg^-1 · 30 min^-1, respectively. OP oxidation was 15.6 ± 3.6 and 17.9 ± 3.5 µmol · kg^-1 · 30 min^-1, in fasting and feeding, respectively, (P < 0.05). More than 80% of the plasma flux was oxidized. These findings are compared with the published literature on GSH turnover in plasma of human subjects and underscore the need to define more completely the dynamic aspects of glutathione metabolism and of the intermediates of the -glutamyl cycle.

KEY WORDS: • oxoproline kinetics • glutathione • stable isotopes • oxidation • flux • humans

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
L-5-oxoproline (OP)⁴ is an intermediate in the -glutamyl cycle of glutathione synthesis (Meister and Anderson 1983 ). The tripeptide glutathione (-glutamyl-L-cysteinyl-glycine; GSH) is the most abundant intracellular thiol and plays a fundamental role in the regulation of numerous enzymatic reactions and acts synergistically with vitamins E and C to protect cells from oxidant injury (Dröge et al. 1994 , Winkler et al. 1994 ). Various inborn errors of glutathione metabolism result in elevated blood levels of OP and high rates of urinary OP excretion (Erasmus et al. 1993 , Meister and Larsson 1995 , Ristoff and Larsson 1998 ). Depletion of GSH renders cells more susceptible to the toxic effects of drugs and even to the products of normal cellular processes (e.g., hydrogen peroxide, superoxide). In critically ill patients and after trauma, reduced and total glutathione in skeletal muscle is depleted by about 50% (Hammarqvist et al. 1997 , Luo et al. 1996 , Luo et al. 1998 ). The rate of GSH synthesis is reduced when protein-deficient pigs are subjected to experimental inflammation (Jahoor et al. 1995 ). Golden has proposed that a reduced availability of glutathione is responsible for the classical clinical features of kwashiorkor (Golden et al., Jackson and co-workers used OP excretion as a marker of glycine status (Jackson et al. 1987 ) and reported increased urinary OP excretion in children gaining weight rapidly during recovery from severe malnutrition, where an increase in OP excretion was taken to indicate insufficient glycine supply (Persaud et al. 1996 ). Hence, it would be desirable to understand the dynamic and quantitative nature of whole body OP metabolism and its relationship to glycine metabolism and glutathione status under varying conditions known to affect OP excretion and/or GSH status. We were interested in studying OP kinetics, particularly because (i) the role played by 5-oxoprolinase (OPase) in the modulation of GSH has not been extensively studied (Chen et al. 1998 ) in contrast to the transcriptional and/or post-transcriptional regulation of the rate-limiting enzyme, -glutamyl cysteine synthetase (Lu 1998 ) and (ii) OPase links the pathways of GSH synthesis and degradation (Chen et al. 1998 ). Thus OP kinetics might allow an assessment of the flow of precursor for GSH synthesis and, potentially, status of GSH balance.

Because we were unable to locate any published studies on whole body OP kinetics in healthy adults, the objectives were to perform an initial study to explore the dynamic status of OP in young men who were adapted to a defined diet for 5 d prior to a tracer study. Here we report our initial findings, as a basis for the design of follow-up studies concerned with dietary interventions and OP kinetics. From studies in rodents, cysteine availability is thought to be rate-limiting in GSH synthesis (Taniguchi et al. 1989 ). Hence, cysteine flux was measured simultaneously to characterize its relation to OP kinetics under basal physiologic conditions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects.

The five male subjects who participated in this study were students at the Massachusetts Institute of Technology (MIT) or from the community of the Boston-Cambridge area. Their mean body weight was 75.2 ± 7.9 kg, height 181 ± 0.7 cm and age 26 ± 7.5 y. All were in good health as determined by medical history, physical examination, analysis for blood cell count, routine blood biochemical profile and urinalysis. Those who smoked cigarettes, consumed five or more alcoholic drinks per week or drank more than six cups of caffeinated beverages per day were excluded from participation in these studies.

An estimation of the daily energy intake required to maintain body weight, based on an estimation of basal metabolic rate and diet history, was 188 kJ · kg^-1 · d^-1 (44 Kcal · kg^-1 · d^-1). This level was given to each subject. They were asked to maintain their usual level of physical activity while avoiding excessive or competitive exercise. The purpose of the study and the risks involved were explained to each subject. The subjects signed a consent form and they were paid for their participation. The experimental protocol was approved by the MIT Committee on the Use of Humans as Experimental Subjects and the Advisory Committee of the MIT Clinical Research Center (CRC).

Diet and experimental design.

Each subject was given, during each of two experimental diet periods, separated by 3 wk, a weight-maintaining diet, based on an L-amino acid mixture for 5 d prior to the tracer study. The indispensable amino acid profile of the mixture was close to that for hen's egg protein (Paul and Southgate 1988 ). The L-amino acids were supplied by Ajinomoto USA Inc. (Teaneck, NJ). Protein-free, wheat-starch cookies and flavored drinks were given as the major source of energy, exactly as previously described in detail (Raguso et al. 1997 ). Nonprotein energy was provided in the form of lipid (40%) and carbohydrate (60%). Beet sugar and wheat starch were the main sources of dietary carbohydrate, to maintain a low ¹³C content in the diet and a steady background level of breath ¹³CO₂ enrichment over the 8-h period. Breath ¹³CO₂ enrichments that were obtained during the tracer studies were corrected to account for the small changes in the background ¹³CO₂ output that would be expected to occur without the L-[¹³C]5-oxoproline (¹³C OP) tracer, as described previously (Metges, C.C. et al. unpublished data). The L-amino acid mixture was given to the subjects to supply a daily nitrogen intake of 160 mg · kg^-1 · d^-1. Vitamins and minerals were supplied as a daily supplement to meet or exceed dietary allowances or the safe and adequate intakes (NRC 1989 ). Dietary fiber was given as 20 g microcrystalline cellulose daily, together with a choline supplement of 500 mg. During each day of the dietary period, the total daily intake was given as three isoenergetic, isonitrogenous meals at 0800, 1200 and 1800 h. Each morning, body weight and vital signs were monitored. At least two of the three daily meals were taken at the MIT Clinical Research Center (CRC) under the supervision of the dietetic staff.

Tracer protocol.

On day 6 of the experiment and following a 12-h overnight fast, a tracer study was carried out. On the first occasion an intravenous infusion of L-[1-¹³C]5-oxoproline and L-[3,3-²H₂]cysteine was administered. For the second occasion the same subject repeated the study according to the above design but received L-[¹⁵N]5-oxoproline (¹⁵N OP) and L-[3,3-²H₂]cysteine as tracers. The order of the two OP tracers studies was randomized. The major features of the 8 h (3 h fast; 5 h fed) stable isotope tracer infusion protocol used in this experiment have been described (Raguso et al. 1997 ). Subjects were admitted to the infusion room in the CRC at ~0630 h. Intravenous lines were inserted, and baseline samples of blood and breath were collected. The intravenous priming doses of the OP and cysteine tracers and of ¹³C labeled bicarbonate were administered; continuous infusions of the tracer infusions began immediately following the priming doses (for doses see below).

After the initial 3 h, subjects were given for 5 h a total of 10 equal small meals with the total intake corresponding to 2/3 of the daily "protein" and 54% of the usual daily energy supply. The latter accounted for the lower daily energy expenditure during this tracer day.

Infusates of the tracers were prepared from sterile powders of high chemical purity (99%), high optical purity and high isotopic enrichment. The L-[1-¹³C]5-oxoproline and the L-[¹⁵N]5-oxoproline (99 atom percentage; AP) were obtained from MassTrace Inc. (Woburn, MA), and the cysteine tracer was L-[3,3-²H₂]cysteine (98 AP; Cambridge Isotope Laboratories, Andover, MA). The bicarbonate pool was primed (1.2 µmol · kg^-1) with a sterile solution of sodium ¹³C bicarbonate (99 AP; Tracer Technologies, Inc., Somerville, MA) containing 25 g of sodium bicarbonate per liter. The labeled OP priming dose was equivalent to the amount of the tracer infused per hour. Target, but known, constant infusion rates for OP were 2 (n = 2) or 4 (n = 3) µmol · kg^-1 · h^-1 L-[1-¹³C]5-oxoproline and 4 µmol · kg^-1 · h^-1 L-[¹⁵N]5-oxoproline and 1.5 µmol · kg^-1 · h^-1 L-[3,3-²H₂]cysteine, respectively.

Between sampling of blood, the intravenous lines were kept open with a slow drip of sterile physiologic saline. Arterialized venous blood samples for determination of plasma OP and enrichment and concentration, and cysteine enrichment, respectively, were collected using a hand-warming device (65°C air temperature) at 15 min intervals between 120–180 min into the isotope infusion period and again during 360–480 min of the tracer infusion time period. Measurements of the rate of CO₂ production were made for 20 min during each hour by indirect calorimetry using a ventilated hood system (Deltatrac, Sensormedics, Anaheim, CA) (El-Khoury et al, 1994 ). Expired air samples for determination of ¹³C isotopic enrichment in CO₂, were collected at timed 30-min intervals.

Mass spectrometry.

Blood samples were collected into EDTA test tubes on ice containing 100 µL L-[3,3,4,4-²H₄]glutamine (Cambridge Isotope Laboratories) to correct for possible OP tracer dilution by glutamine/glutamate, and plasma was isolated immediately by refrigerated centrifugation at 1,500 x g for 10 min and stored at -20°C until analysis. Aliquots of plasma (200 µL) were placed in 13 x 100 mm tubes and the internal standards DL-[2,4,4-²H₃]-glutamic acid (Cambridge Isotope Laboratories) and DL-[2,4,4-²H₃]5-oxoproline were added followed by vigorous mixing. DL-[2,4,4-²H₃]5-oxoproline was generated from DL-[2,4,4-²H₃]glutamic acid by autoclaving for 2 h at alkaline pH (250°C). The plasma was then applied to columns containing Bio-Rad AG 50 W-X8 cation resin (1 mL; Bio-Rad, Melville, NY), OP was eluted with Milli-Q water (2 x 1 mL) and DL-[¹⁵N, U-¹³C₅]-5-oxoproline (MassTrace Inc.) was added to the water fraction as another internal standard. All tubes were evaporated at 65°C under a stream of nitrogen, N-methyl-N(tert-butyl dimethylsilyl) trifluoroacetamide (MTBDTFA) (Pierce Chemical Co., Rockford, IL) (50 µL)/acetonitrile (50 µL) was added to each tube and derivatization was performed at 65°C for 1 h. Aliquots of 1 µL OP samples were analyzed using gas chromatography/electron impact-mass spectrometry (GC/EI-MS).

The derivatized sample was injected into an HP gas chromatograph equipped with a mass selective detector (model 5890/5970; Hewlett-Packard, Palo Alto, CA) and a fused silica capillary column of cross-linked polydimethylsiloxane (HP-1, 30 m x 0.25 mm, 0.25 µm film thickness Hewlett-Packard). The injector and initial oven temperatures were set at 250 and 70°C, respectively. After splitless injection (0.95 min purge on time), the oven was programmed to 290°C at 11°C/min, followed by 310°C at 20°C/min, and held at 310°C for 1 min. Helium was used as carrier gas. Under electron impact ionization, the major ion fragment for OP derivatives were monitored from m/z 300.1 to 306.1 (¹³C OP or ¹⁵N OP measurements) and m/z 272.1 to 277.1 (¹⁵N OP measurement). Details of mass spectrometric characteristics of L-[¹³C]- and L-[¹⁵N]5-oxoproline and handling of potential interferences by glutamic acid and glutamine will be described elsewhere (Lu, X.-M., Fischman, A.J., Tompkins, R.G., Young, V.R.; unpublished data, 1999). Quantitative analysis of the concentration of tracee OP and tracers (L-[¹³C]- and L-[¹⁵N]5-oxoproline) was achieved by using calibration curves. The mass recoveries from the cation exchange column were corrected based on the m+3/m+6 ratio for individual samples. The response of plasma m+0 OP was kept within the linear range of the calibration curve (0.74 to 74 · 10^-12 mol). The plasma tracer and tracee levels were calculated from response ratios against molar ratios from any one of the two internal standards after subtracting the natural background enrichment. Enrichments were expressed as mole percentage excess (MPE).

We described previously, in detail, analysis of plasma free-labeled cysteine (Raguso et al. 1997 ). Briefly, MTBSTFA was used to form the tert-butyl dimethylsilyl derivative of cysteine. Ethanethiol was also used in the derivatization mixture to convert cystine to cysteine and to serve as an antioxidant.

It might be noted here that the cysteine bound to protein and dipeptides would not be recovered in this assay because the ethanethiol was added after the free amino acids had been extracted from the plasma. The cysteine isotope enrichments reflect, therefore, the combined free cysteine and cystine in plasma (i.e., total free plasma cysteine). Isotopic enrichments were measured by GC/EI–MS (HP 5890 Series II and HP 5988A; Hewlett-Packard). Cysteine and [3,3-²H₂]cysteine were monitored at m/z 406 and 408, respectively. The isotopic enrichment of the experimental samples was determined by a calibration curve of [3,3-²H₂]cysteine standards with known molar ratios from 0 to 0.1 after baseline subtraction. Slopes of calibration curves were between 1.03 and 1.08.

Breath ¹³CO₂ was isolated cryogenically and the ¹³C/¹²C ratio was measured using isotope ratio MS and enrichment was expressed as atom percentage excess (APE) over pre-infusion baseline (Delta E, Finnigan MAT, Bremen, Germany).

All chemicals used were of analytical grade and purchased from Alltech (Deerfield, IL) if not stated otherwise.

Evaluation of data.

Mean fasted and fed state ¹³C and/or ¹⁵N OP and ²H₂ cysteine plasma enrichments were calculated between 120 and 180 min and 390 and 480 min, respectively, for consecutive half-hourly intervals from measurements at 15-min intervals. ¹³CO₂ production and fluxes (Q) of OP and cysteine, and rate of OP oxidation during the fasted and fed states were computed all as previously described (Raguso et al. 1997 ). ¹³CO₂ production rate was corrected for the retention of ¹³CO₂ in the body during the experimental time frame, according to our previous short-term bicarbonate-infusion studies (Hoerr et al. 1989 ). The fraction of flux oxidized was computed as OP oxidation divided by OP flux and multiplied times 100. Nonoxidative OP disposal (NOOPD) was calculated as the difference between the ¹³C-OP flux and ¹³C-OP oxidation. This parameter would include the incorporation of the glutamate formed, via OPase, into the multiple pathways of glutamate metabolism (Young and Ajami 1999 ), including GSH synthesis, and the excretion of OP via the urine.

Data are reported as means ± SD. After checking for absence of crossover effect (period x treatment interaction) using ANOVA (SPSS for Windows Ver. 6.1) two-sided paired t-tests were used to compare the above variables between the two OP tracers and between fasting and feeding. A P value <0.05 was considered to be statistically significant. Means which were not statistically different (P 0.05) are labeled "NS."

	RESULTS

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¹³CO₂ production and plasma ¹³C OP and ¹⁵N OP as well as ²H₂ cysteine enrichments are shown in Figures 1 and2,respectively. There was no significant difference in ¹³CO₂ production between the fasting and feeding states, at both rates of ¹³C OP infusion (2 or 4 µmol · kg^-1 · h^-1) (Fig. 1) . At the infusion rate of 2 µmol · kg^-1 · h^-1 plasma ¹³C OP enrichment in fed state was significantly lower (P < 0.05) than during fasting (Fig. 1B) . This difference was not apparent with the infusion rate of 4 µmol · kg^-1 · h^{-1 13}C OP, although only two subjects received this higher rate. Fast and fed state plasma ¹⁵N OP enrichments were not significantly different (Fig. 2) .

View larger version (21K): [in this window] [in a new window]   Figure 1. Mean 13CO2 production during fasted and fed state in µmol · kg-1 · 30 min-1 (panel A) with intravenous infusion of 2 and 4 µmol L-[1-13C]oxoproline · kg-1 · h-1 and plasma [13C]oxoproline enrichment in MPE (mole percent excess) with both infusion rates (panel B). Timepoints of small meal ingestion are indicated by (x). Values are means ± SD, n = 2 and 3, respectively. *P < 0.05 vs. infusion rate of 2 µmol L-[1-13C]oxoproline · kg-1 · h-1; + P < 0.05 vs. fed state at isotopic steady state.

View larger version (14K): [in this window] [in a new window]   Figure 2. Mean plasma [15N]oxoproline and [2H2]cysteine enrichment during fasted and fed states. Timepoints of small meal ingestion are indicated by (x). Values are means ± SD, n = 5.

Tables 1

The plasma concentration of OP was not different between the fast and fed states (Table 2) ; it approximated 60 µmol/L during both tracer studies.

The cysteine flux, as well as the plasma total OP concentration, did not differ between fasted and fed state under the experimental conditions (Table 2) . Data for cysteine flux, measured in conjunction with ¹³C OP infusion were essentially identical to those given in Table 2 and so are not repeated here.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
L-5-oxoproline is an intermediate in the -glutamyl cycle of glutathione synthesis. The latter occurs predominantly in the liver (Griffith and Meister, 1979 ), but also in the gut mucosa, and it is catalyzed by the enzymes -glutamyl-cysteine synthetase and glutathione synthetase (Meister and Anderson 1983 ). Arterio-venous difference studies in the rat showed that more than 50% of the cysteine taken up by the liver is used for GSH synthesis (Garcia and Stipanuk 1992 ) and in the fed piglet mucosal GSH is synthesized from enterally supplied glutamate (Reeds et al. 1997 ). GSH is removed from the plasma mainly by the kidney and other organs that have -glutamyl-transpeptidase activity (Griffith and Meister 1979 ). Glutathione homeostasis is of great importance for the pro- and antioxidative balance of the body. As shown in a number of studies, during severe stress such as in burns, surgical trauma, strenuous exercise, and in inflammation and HIV infection there is depletion of GSH tissue and plasma levels (e.g., Helbling et al. 1996 , Jahoor et al. 1995 , Luo et al. 1996 , Luo et al. 1998 , Martensson et al. 1992 , Sen et al. 1994 ). GSH deficiency, in experimental animals, produces tissue damage (Martensson et al. 1990 , Martensson and Meister 1991 ), and the morbidity in burn trauma might be a consequence of inappropriate GSH levels. Finally, oxoprolinuria occurs in generalized GSH synthetase deficiency and in OPase deficiency (Mayatepek 1999 , Ristoff and Larsson 1998 ).

For these various reasons, together with the fact that OP plays a role in the transport of amino acids at the blood–brain barrier (Lee et al. 1996 ), it appeared worthwhile to characterize OP kinetics in healthy well-nourished adults before proceeding to an examination of dietary (amino acid) and other factors that might affect GSH metabolism, and in consequence, OP kinetics.

Hence, we undertook this initial study of the dynamic status of whole-body OP kinetics in young adults, with the purpose of eventually examining whether OP kinetics offer a means of probing the dynamic status of glutathione metabolism. We wanted to learn, at first, (a) whether there were differences in the carbon and nitrogen metabolism of this glutathione intermediate during the fed and fasted state and (b) about some of the characteristics of plasma OP kinetics and how they relate to the published information on rates of glutathione synthesis and turnover in human subjects.

The plasma ¹³C OP flux approximated 20 µmol · kg^-1 · 30 min^-1 with no observable effect of feeding on this rate. This was also found with ¹⁵N OP tracer, but the flux in this case was somewhat higher (about 20%), in the fast condition, as compared with that found with the ¹³C tracer; for the fed state there was a trend (0.05>P < 0.1) toward a difference between the two tracers. Because the OPase reaction, involving conversion of OP to glutamate, is not reversible, the reason for this small and/or apparent difference between the ¹³C OP and ¹⁵N OP plasma fluxes is not entirely clear. While the glutamate that is formed from OP can be used for GSH synthesis and subsequently reused for this purpose, via a turn of the -glutamyl cycle (Chen et al. 1998 ), this fact would not really offer a plausible explanation unless this glutamic acid was channeled through a small metabolic compartment where some transamination occurred while also remaining in tight proximity with the interrelated reactions of the -glutamyl cycle. Another possibility is that the difference reflects the multiple organ sites at which differing levels of OPase activity (Chen et al. 1998 ) and rates of GSH synthesis (Smith et al. 1996 ) occur and again in relation to the dynamic status and compartmentation of glutamate metabolism (Young and Ajami 1999 ).

We view these comparative flux data as suggesting to us that the ¹³C OP tracer might be the one of choice, at least at this stage, particularly since it also provides information about the relative disposal of OP via oxidative and nonoxidative routes. Therefore, some further comments on this aspect of whole body (plasma) OP kinetics might be made.

From the rate of appearance of ¹³CO₂ in expired air and the plasma enrichment of the tracer, we estimated that OP was terminally oxidized at a mean rate of 15.6 and 17.9 µmol · kg^-1 · 30 min^-1 for the fast and fed states. This represented about 81–84% of the plasma flux, with the approximate rate for the nonoxidative disposal of OP being 3.3 µmol · kg^-1 · 30 min^-1. Labeling studies in rats (Ramakrishna et al. 1970 ) and mice (Van der Werf and Meister 1975 ) similarly reveal an extensive oxidation of OP. It is evident from these human, as well as animal, data that OP turnover in the circulating blood plasma is largely associated with the oxidative disposal of OP. With reference to our longer term objectives, these rates cannot yet be integrated with tracer kinetics of GSH synthesis and turnover since these studies have yet to be done in human subjects. Nevertheless, some comparisons with pharmacokinetic data and excretion data can be made.

Thus, using a pharmacokinetic approach, Lauterburg and co-workers (Burgunder and Lauterburg 1987 , Helbling et al. 1996 ) estimated the plasma GSH flux in postabsorptive healthy control subjects to be about 24 µmol · kg^-1 · h^-1 (SD approximately 25%). In cirrhotics and HIV patients, GSH flux was significantly lower (about 15 µmol · kg^-1 · h^-1), reflecting a probably decreased rate of hepatic and systemic production of GSH (Burgunder and Lauterburg 1987 , Helbling et al. 1996 , Jahoor et al. 1999 ). Similarly, using a two-step constant infusion protocol, Bianchi et al. (1997) estimated the endogenous plasma appearance rate of GSH in healthy controls to be about 19.4 µmol · kg^-1 · h^-1, with a significant reduction, to about 7.5 µmol · kg^-1 · h^-1 in patients with liver cirrhosis. For comparison, we determined, for the fasted state, that about 38 µmol · kg^-1 · h^-1 of OP enters the extracellular space in healthy volunteers. Nitrogen OP flux was found to be slightly higher than the carbon flux as discussed above. Therefore, it appears that the basal OP rate of appearance, or disappearance for our conditions, which were taken to be steady state, are approximately double those for GSH kinetics. They are also about one-half of the plasma glutamate flux (Battezzati et al. 1995 ). On the other hand, the nonoxidative OP disposal rate was about 6.6 µmol · kg^-1 · h^-1 or about one-third to one-quarter of the rate of appearance of GSH. Of course, a determination of the functional and causal relationships between these OP fluxes and oxidation rates will require integrated GSH and OP tracer studies, but it is of interest to find that the OP appearance rate in the circulation exceeds that for GSH.

OP is removed, in part, from the plasma compartment via the kidney and excretion in urine. Hence, it is also of possible interest that the nonoxidative disposal rate of 6.6 µmol · kg^-1 · h^-1 is much higher than the reported rates of daily excretion rate of OP. For omnivorous males, Jackson et al. (1996) report a daily output of 217 µmol/day (about 0.13 µmol · kg^-1 · h^-1), and in vegetarian males the value was 404 µmol/day (about 0.24 µmol · kg^-1 · h^-1). In nonpregnant women, urinary OP output amounts to about 3–3.4 µmol · kg^-1 · d^-1 (0.13 µmol · kg^-1 · h^-1), and this increases to about two to three times this level by late pregnancy (Jackson et al. 1997 ). In women given a low-protein diet, it approximates 0.26 µmol · kg^-1 · h^-1 (Meakins et al. 1998 ). Therefore, on the bases of these urinary excretion values the disposal of OP via this route only accounts for ~2–4% of the nonoxidative disappearance of OP from the circulation. It is apparent then that the renal losses of OP account for a relatively small component of the daily turnover of whole body OP which might suggest a significant salvage of OP for GSH synthesis via OPase reaction or non-GSH utilization of glutamate. Nevertheless, the urinary output changes with the physiological (Jackson et al. 1997 , Jackson et al. 1997a ) and metabolic condition/nutritional state (Persaud et al. 1996 ) of the individual, and it will be interesting to determine the metabolic association between such variable urinary losses and rates of synthesis and turnover of OP. We assume, from studies on the tissue distribution of OPase that there is significant oxidation of OP in the liver, kidney and intestine (Chen et al. 1998 , Weber et al. 1999 ); how these tissues are differentially affected by different metabolic/nutritional conditions and whether changes in kidney OP metabolism might more closely reflect or be responsible for altered rates urinary losses are areas for subsequent investigation. This is particularly important both with respect to a test of the hypothesis that urinary OP output is a marker of tissue glycine insufficiency (Jackson 1991 ) and a further understanding of the role played by OP formation and its metabolism via 5-oxyprolinase in the maintenance of GSH homeostasis. It has been stated that under normal conditions cells do not necessarily depend on 5-oxoprolinase for maintenance of GSH content because glutamate is free available from many sources for purposes of GSH synthesis (Chen et al. 1998 ). However, OPase capacity might well be important under conditions of oxidative stress and in tumor-bearing animals, for example (Chen et al. 1998 ).

The plasma cysteine flux in our subjects was ~44 µmol · kg^-1 · h^-1, which agrees with our earlier estimates (Fukagawa et al. 1998 , Hiramatsu et al. 1994 , Raguso and Young 1999 ). However, in this study we did not observe the decline in the fed state flux which we had seen in our previous studies. Nevertheless, with reference to our interest in the activity of the -glutamyl cycle, we showed that an infusion of glutathione increases the plasma cysteine flux (Fukagawa et al. 1996 ). On the basis of these findings, we concluded that a significant fraction of the plasma cysteine flux, about 50%, is generated via the turnover of GSH. Indeed, one of the most widely considered functions of extracellular GSH is as a source of cysteine in overall sulfur amino acid balance (Smith et al. 1996 ). It might be important, therefore, that the OP flux approximated that of cysteine and both were about twice that of the GSH flux, as derived from literature reports. Chen, Carystinos and Batist (1998) view the -glutamyl cycle as consisting of two pathways, one catalyzing the de novo synthesis of GSH and the other a degradation pathway, linked by OPase and conversion of OP to glutamate. Further these investigations point out that cysteine and glycine generated via the degradation of GSH can be reused in the synthesis of GSH. Hence, there is also a salvage pathway of GSH synthesis. Thus, it might be that the plasma OP flux reflects these interrelated components of the -glutamyl cycle, and this possibility could be explored through an appropriate multitracer protocol including specifically labeled OP and GSH, if the latter were to become available at reasonable cost. We have prepared labeled GSH in the laboratory (Lu et al. 1997 ) with this purpose in mind but have not yet succeeded in producing amounts of purified material necessary for a series of human metabolic experiments.

Finally, the plasma OP concentration as determined by our GC/MS method was about 60 µmol/L and it did not differ between the fed and fasted states. Reported plasma or blood OP concentrations in the literature are highly variable, probably related, in part, to the use of various analytical methods; the range appears to be from about 14 to 216 µmol/L in control subjects (Oberholzer et al. 1975 , Palekar et al. 1975 , Pitt and Hauser 1998 , Wolfersberger and Tabachnik 1973 ). On the other hand, human plasma GSH levels are about 2–7 µmol/L (Aebi et al. 1991 , Bianchi et al. 1997 , Helbling et al. 1996 , Yoshida et al. 1995 ). Hence, the fractional appearance/clearance rates of GSH, from these and the foregoing plasma GSH appearance/disappearance rates, appear to be about five to ten times for OP. Again, the eventual insight that this comparison might bring a better understanding of the physiology of human GSH metabolism and the extent to which OP kinetics can be used as a proxy for GSH kinetics and metabolism will require studies in which OP kinetics are measured under conditions of increased GSH requirement, such as in burn trauma. These measurements would then be related to simultaneous, or parallel, measurements of GSH kinetics and its dipeptides, as well as plasma and tissue levels of these compounds. Hum et al. (1991 , Hum et al. 1992 ) showed in rats that changes in plasma GSH turnover reflect changes in hepatic GSH content and they have proposed that measurement of plasma GSH kinetics might find an eventual clinical application in the noninvasive determination of liver GSH status in humans. For these various reasons, we have commenced upon a series of investigations along these general lines in both healthy controls and in burn trauma patients.

	ACKNOWLEDGMENTS

 
The authors wish to thank the nursing and dietetic staff of the MIT Clinical Research Center for their considerable help in making this study possible. We also appreciate the commitment made by the volunteers.

	FOOTNOTES

 
¹ Present address: Deutsches Institut für Ernährungsforschung (The German Institute of Human Nutrition), Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany, e-mail: metges{at}www.dife.de

² This work was funded by NIH grants DK 15856, RR 88 and P-30-DK-40561; Shriners Hospitals for Children (Nos. 8370, 8470) and the Deutsche Forschungsgemeinschaft, Bonn, Germany (Me 1420/1-1).

⁴ Abbreviations used: ¹³C OP, L-[¹³C]-5-oxoproline; ¹⁵N OP, L-[¹⁵N]-5-oxoproline; AP, atom percentage; GC/EI–MS, gas chromatography/electron impact mass spectrometry; GSH, glutathione; MPE, moles percent excess; MTBSTFA, N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide; NOOPD, nonoxidative oxoproline disposal; OP, L-5-oxoproline; OPase, 5-oxoprolinase; TBDMS, t-butyldimethylsilyl.

Manuscript received May 3, 1999. Initial review completed June 25, 1999. Revision accepted August 3, 1999.

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