l-5-oxoproline (l-5-OP) is an intermediate in glutathione synthesis, possibly limited by cysteine availability. Urinary 5-OP excretion has been proposed as a measure of glycine availability. We investigated whether 5 days of dietary sulfur amino acid (SAA-free) or glycine (Gly-free) restriction affects plasma kinetics of 5-OP and urinary excretion of l- andd-5-OP in 6 healthy men. On day 6, l-5-[1-13C]oxoproline and [3,3-2H2]cysteine were infused intravenously for 8 h (3 h fast/5 h fed). In a control study (adequate amino acid mixture), plasma oxoproline fluxes were 37.8 ± 13.8 (SD) and 38.4 ± 14.8 μmol ⋅ kg− 1 ⋅ h− 1; oxidation accounted for 85% of flux. Cysteine flux was 47.9 ± 8.5 and 43.2 ± 8.5 μmol ⋅ kg− 1 ⋅ h− 1for fast and fed phases, respectively. Urinary excretion ofl- and d-5-OP was 70 ± 34 and 31.1 ± 13.3 μmol/mmol creatinine, respectively, during days 3–5, and 46.4 ± 13.9 and 22.4 ± 8.3 μmol/mmol over the 8-h tracer study. The 5-OP flux for the Gly-free diet was higher (P = 0.018) and tended to be higher for the SAA-free diet (P = 0.057) when compared with the control diet. Oxidation rates were higher on the Gly-free (P = 0.005) and SAA-free (P = 0.03) diets. Cysteine fluxes were lower on the the Gly-free (P= 0.01) and the SAA-free diets (P = 0.001) compared with the control diet. Rates of l-5-OP excretion were unchanged by withdrawal of SAA or Gly for 5 days but increased on day 6(P = 0.005 and P = 0.019, respectively). Thus acute changes in the dietary availability of SAA and Gly alter oxoproline kinetics and urinary 5-OP excretion.
- oxoproline kinetics
- glutathione metabolism
- dietary intake
the tripeptide glutathione(γ-glutamyl-l-cysteinyl-glycine; GSH) is synthesized in virtually all cells from its constituent amino acidsl-glutamic acid, l-cysteine, and glycine by γ-glutamylcysteine synthetase (GCS) and GSH synthetase (1). Intracellular cysteine is thought to be the limiting substrate in the synthesis of GSH (25). It has been shown that GCS, which has been cloned (11), is under transcriptional and posttranslational regulation by, among other factors, insulin, stress hormones, and a high glucose concentration (25). GSH degradation is catalyzed by γ-glutamyl transpeptidase to cysteinylglycine and γ-glutamyl amino acid (5). The latter is converted to l-5-oxoproline (l-5-OP) via γ-glutamylcyclotransferase and then to glutamic acid by 5-oxoprolinase (5-OPase). Because the glutamic acid moiety can be re-used for de novo synthesis of GSH, the synthesis and degradation of GSH are linked by 5-OPase (5). The GSH level in normal cells does not necessarily depend on the activity of 5-OPase because it has no feedback inhibition on GSH synthesis, and glutamic acid from many other sources is usually sufficiently available. It seems, however, that under conditions of oxidative stress, the role of 5-OPase reaction and, subsequently, the salvage of l-5-OP are more important for maintenance of GSH level (5). After traumatic stress, such as burns, and after major surgery, in liver cirrhosis, inflammatory and infectious diseases, and also in protein malnutrition, tissue levels of GSH are depleted (12-14, 21, 26-28, 44). It has been demonstrated that the intra- and extracellular GSH deficiency in human immunodeficiency virus (HIV) infection is due to a reduced synthesis rate (20).
Jackson and co-workers have used l-5-OP excretion as a marker of glycine status (17). They have also reported increased urinary l-5-OP excretion in children gaining weight rapidly during recovery from severe malnutrition, where the increase inl-5-OP excretion was interpreted to indicate an insufficient glycine supply (36). Furthermore, studies in rodents indicate that cysteine availability is rate limiting in GSH synthesis (45), although the relevance of this to the physiology of human GSH metabolism remains to be evaluated.
From the foregoing, it would be desirable to explore the dynamic and quantitative nature of whole body l-5-OP metabolism and its relationship to altered glycine and cysteine intakes in healthy adults, including plasma l-5-OP kinetics and l-5- andd-5-OP excretion, because both enantiomers are excreted in measurable amounts (46). Previously we have shown that the rate of endogenous glycine synthesis is reduced when a diet low in nitrogen and without dispensable amino acids is given to healthy adults (49) for a relatively brief period. Although to our knowledge the effects of a specific glycine-devoid diet on glycine synthesis and availability have not been examined, we considered it desirable to examine first whether a diet specifically devoid of either glycine or cysteine and its precursor methionine, but otherwise adequate, affected the plasma kinetics and urinary excretion of l-5-OP.
We hypothesized that a deprivation of dietary glycine or cysteine would lead to a higher oxidation and flux rate of l-5-OP for the following reasons. 1) A relative glycine deficiency would result in an accumulation of γ-glutamylcysteine, which is converted to l-5-OP and then to glutamate by 5-OPase. This would lead to an increase of both the l-5-OP flux and oxidation rates, as well as a higher oxoproline urinary excretion. 2) A deficient dietary supply of methionine and/or cysteine could lead to an increased salvage of cysteine via GCS generated by the hydrolysis of cysteinylglycine after the degradation of GSH. The GSH synthesis rate is presumably reduced under these conditions, leading to a reduced reutilization of l-5-OP with increased l-5-OP flux and oxidation rate, as well as a higher oxoproline urinary excretion.
MATERIALS AND METHODS
Subjects and experimental design.
Six male students of the Massachusetts Institute of Technology (MIT) aged 21.5 ± 1.3 (SD) yr agreed to participate in the study that was performed at the Clinical Research Center of MIT (MIT-CRC). Their mean height was 1.77 ± 0.07 m; their body weight was monitored daily during the studies and did not change (mean body wt during periods 1, 2, and 3: 75.2 ± 7.3, 75.5 ± 7.1, and 75.7 ± 7.2 kg). They were healthy according to medical history, physical examination, vital signs, blood parameters, and urinalysis. They were nonsmokers and reported no or minimal alcohol consumption. The subjects were allowed to engage in their usual physical activity but were requested not to participate in competitive sports or strenuous exercise. The purpose of the study and potential risks involved were explained to each of the subjects before written consent was obtained. The study protocol was approved by the MIT Committee on the Use of Humans as Experimental Subjects and the MIT-CRC Advisory Committee. The subjects received financial compensation for their participation.
The protocol consisted of three separate dietary periods of 5 days (adaptation period) with an 8-h intravenous (3-h fast/5-h fed) tracer study being conducted on the 6th day. The reason for measuring OP kinetics during the fasted and fed states was to investigate whether the supply of the specific diet caused a different modulation of the oxoproline kinetics during the absorptive compared with the postabsorptive state. Subjects were studied during three dietary periods (control diet, sulfur amino acid-free diet, glycine-free diet; see Diets and tracer protocol); the order of the different periods was randomly assigned. A washout period of 3 wk was included between each of the experimental periods, during which subjects consumed their usual free-choice diets.
Diets and tracer protocol.
The isoenergetic, isonitrogenous experimental diets supplied ∼43 kcal ⋅ kg− 1 ⋅ day− 1and 160 mg N ⋅ kg− 1 ⋅ day− 1and were based on crystalline l-amino acids obtained from Ajinomoto USA (Teaneck, NJ). The composition of the amino acid mixture in the control diet was patterned approximately as in hen's egg (Table1). The other two amino acid mixtures used here were either sulfur amino acid (SAA) free or glycine (Gly) free (Table 1). To provide 160 mg N ⋅ kg− 1 ⋅ day− 1, 1.18 g of amino acid mixture ⋅ kg body wt− 1 ⋅ day− 1was consumed. The diet consisted of protein-free cookies, a carbohydrate (wheat starch and beet sugar) drink, and the flavored amino acid mixture, which was eaten as a mash mixed with water and sugar. The composition of the diet was described in detail recently (32). No other food or beverages were allowed, except tap water, decaffeinated coffee, and tea. Diets were consumed as three equal meals per day, with at least two being eaten under the supervision of the MIT-CRC dietary staff.
At 0700 on day 6, the subjects were admitted to the MIT-CRC after an overnight fast. Two small catheters were placed into veins of the nondominant arm by use of aseptic sterile procedures. One catheter was introduced into an antecubital vein for tracer infusion, and the other was placed into a superficial dorsal vein of the hand for blood collection. The hand was placed into a custom-made warming box (air temperature 65°C) 10 min before each blood drawing (“arterialized” blood). After collection of baseline blood and breath samples for baseline analyses, and after the subjects emptied their bladders, an 8-h infusion protocol was commenced at ∼0800, involving a continuous intravenous tracer infusion of l-5-[1-13C]oxoproline [l-2-pyrrolidone-5-[5-13C]carboxylic acid, 2 μmol ⋅ kg− 1 ⋅ h− 1; 99 atom percent (AP); MassTrace Woburn, MA] andl-[3,3-2H2]cysteine [1.5 μmol ⋅ kg− 1 ⋅ h− 1; 98 AP; Cambridge Isotope Laboratories (Woburn, MA)]. Approximately 1 min before the start of the tracer infusion (infusion rate was 10 ml/h), intravenous priming doses of NaH13CO3 (1.2 μmol/kg),l-5-[1-13C]oxoproline (2 μmol/kg), andl-[3,3-2H2]cysteine (1.5 μmol/kg) were administered. The tracers, previously confirmed to be sterile and pyrogen-free by an independent laboratory, were dissolved in sterile saline by use of sterile procedures.
During the first 3 h of the infusion study, no food was given, and during the remaining 5 h a total of 10 equal small meals of the experimental diet were consumed (one meal/30 min). The total intake corresponded to two-thirds of the daily nitrogen intake and 54% of the previous daily energy intake.
Blood samples (6 ml per time point) were collected half-hourly between 120 and 180 min (fasting) and 390 and 480 min (fed) into heparinized tubes. Measurements of the rate of CO2 production were made for 20 min during each hour by indirect calorimetry by use of a ventilated hood system (Deltatrac, Sensormedics, Anaheim, CA). Expired air samples for determination of 13C isotopic enrichment were collected at timed 30-min intervals, as described previously (7).
Urine collection and creatinine measurement.
Complete urinary output was collected, with HCl as preservative, starting in the morning of day 3 (0800). During days 3–5, timed 24-h collections were made, and during the day of the tracer study (day 6) urine was pooled separately during the 3-h fasting period and the 5-h fed period. Urine volumes were measured and aliquots were kept frozen until analysis. Urinary creatinine was measured on the basis of the Jaffe reaction (alkaline picrate and creatinine) as previously described (43).
Mass spectrometry analysis of oxoproline, cysteine, and13CO2.
Measurements of 5-OP plasma concentration and plasma enrichment were made as recently described (33). Briefly, after addition of internal standards (dl-5-[2,4,4-2H3]oxoproline,dl-[2,4,4-2H3]glutamic acid), plasma OP was eluted with water from a cation exchange column (AG 50W-X8 resin, Bio-Rad, Hercules, CA). Anotherl-5-[15N,U-13C5] oxoproline internal standard was added, and after evaporation, thetert-butyldimethylsilyl (t-BDMS) derivative was formed. Aliquots of 1 μl were injected into a gas chromatograph/electron impact-mass spectrometer (GC/EI-MS, HP 5890 series II, HP 5988 MSD, Hewlett-Packard, Palo Alto, CA) equipped with an HP-1 column (Hewlett-Packard). Under EI ionization, ion fragments for 5-OP derivatives from mass-to charge ratio (m/z) 300.1 to 306.1 were monitored. Plasma 13C 5-OP enrichment and plasma 5-OP concentration were calculated from response ratios against molar ratios (m+1/m+6 or m+0/m+6) after subtraction of the natural background enrichment. Enrichments were expressed as mole percent excess (MPE).
l- and d-5-OP concentration in urine was analyzed by isotope dilution. Two internal standards were added to 200 μl of acidified urine.l-[15N,U-13C5]glutamic acid (10 μl; 0.5 mg/ml) was used to control for a possible leakage of glutamic acid into the isolated fraction containing 5-OP, anddl-5-[2H3]oxoproline (10 μl; 0.1 mg/ml) served to quantify the d-5- andl-5-OP concentrations. The mixture was subjected to a cation exchange column (AG 50W-X2, Bio-Rad). 5-OP was eluted separately with 2 × 1 ml distilled water. Subsequently, the column was rinsed with 1 ml of 3 M ammonium hydroxide solution and then 0.5 ml of deionized water to elute glutamic acid and other amino acids. After evaporation of the samples under a stream of nitrogen, 100 μl of 3 N HCl were added to each tube (water and NH4OH eluates), and the mixtures were heated at 100°C for 2 h (18). This procedure converts oxoproline to glutamic acid, and it was confirmed that no racemization occurs during this step. The samples were dried again, and after addition of 0.5 ml of isopropanol-acetyl chloride (10:1, vol/vol), the sample was propylated at 80°C for 30 min. After evaporation, 100 μl of pentafluoroacetic anhydrate were added, and the mixture was derivatized at room temperature for 1 h to yieldN-pentafluoracetyl-isopropyl esters. The derivatives were diluted with acetonitrile after being dried in a gentle stream of nitrogen and then transferred to autosampler vials. All chemicals used were of analytical grade and purchased from Aldrich Chemical (Milwaukee, WI).
An aliquot of 1 μl was injected into a GC/EI-MS (HP 5890 series II, HP 5988 MSD) equipped with a Chirasil-Val column (25 m, 0.25 mm ID, 0.16 μm film thickness; Alltech, Deerfield, IL). 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 a carrier gas (head pressure 7 psi). Under these conditionsd-5-OP (measured after conversion to d-glutamic acid) was eluted after 12.5 min; the retention time ofl-5-OP (l-glutamic acid) was 12.9 min. Target ions were monitored between m/z 276 and 282. After correction for the leakage of glutamic acid into the oxoproline (water) fraction (<3%), urinary d- and l-5-OP concentrations (μM) were calculated from the area response m+0/m+3 ratios. Urinary d- and l-5-OP excretions were expressed as micromoles per millimoles urinary creatinine and also as daily urinary output (μmol ⋅ kg− 1 ⋅ h− 1).
Plasma cysteine enrichment was measured after conversion to thet-BDMS derivative of cysteine, as previously described (39). Ethanethiol was also used in the derivatization mixture to convert cystine to cysteine and to serve as an antioxidant. 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). Cysteine and [3,3-2H2]cysteine were monitored atm/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.
Breath CO2 was isolated cryogenically, and the13C/12C isotope ratio was determined by isotope ratio MS and mathematically converted to 13C AP excess (APE) over baseline (Delta E, Finnigan MAT, Bremen, Germany).
Plasma oxoproline and cysteine fluxes or rates of appearance (l-5-OP Ra and Cys Ra) were calculated as follows where i is the tracer infusion rate (μmol ⋅ kg− 1 ⋅ h− 1), Ei is the enrichment of the administered isotope (5-[13C]oxoproline or [2H2]cysteine), and Epis the enrichment of the tracer in plasma at isotopic steady state during fasting (120–180 min) and feeding (390–480 min). Absence of a statistically significant slope of plasma enrichment during the above time frames was confirmed by linear regression.
Oxoproline oxidation (l-5-OP Ox) was computed from separate 30-min intervals during the last hour of fasting (120–180 min) and the final 90 min during feeding (390–480 min) to reduce any possible effect of the 5-OP and bicarbonate prime where plasma 13C 5-OP enrichment is the average of the enrichments determining the specific half-hour intervals where # = APE × 1,000, and ## = recovery of13CO2 (as reported in Ref. 16; for fed state, 79%; for fasted state, 81%).
The nonoxidative oxoproline disposal (NOOPD), presumably via glutathione and protein synthesis (via glutamic acid), was computed according to the following equation The fraction of flux that was oxidized (FROX) during the experiment at isotopic steady state was calculated as follows
Data are presented as means ± SD. Oxoproline and cysteine kinetics and oxoproline excretion were analyzed with mixed-models ANOVA (proc mixed; SAS version 6.12). The models considered two within-subject factors (diet and metabolic state, or diet and day) and their interaction. A significant interaction was followed up with contrasts for the relevant pairwise differences. If the interaction was not significant, main effects were examined, and pairwise differences between diets were evaluated as appropriate. A P value <0.05 is considered to be statistically significant.
The steady-state isotopic enrichment values achieved after infusion of the labeled oxoproline and cysteine tracers are summarized in Table2. From these isotopic data, the oxoproline and cysteine kinetics were determined.
Oxoproline and cysteine kinetics and oxoproline excretion with the control diet.
In Table 3, the findings for oxoproline and cysteine kinetics are presented for the period when subjects consumed the adequate l-amino acid diet: the oxoproline flux did not differ significantly between the fed and fasted states and approximated 38 μmol ⋅ kg− 1 ⋅ h− 1. Similarly, oxoproline oxidation was not significantly different between the two metabolic states. The rate was ∼32 μmol ⋅ kg− 1 ⋅ h− 1and accounted for ∼84% of the oxoproline flux. Finally, the cysteine flux was 47.9 μmol ⋅ kg− 1 ⋅ h− 1in the fasted state and decreased significantly with ingestion of small meals (P = 0.023).
Plasma l-5-OP concentration was 61 μM before the infusion of labeled l-5-OP. The plasma values for the fast and fed periods of the infusion were similar and not significantly different from the preinfusion level (Table 3).
The rates of urinary excretion of l- and d-5-OP during the control diet period are summarized in Table4. The daily output of l-5-OP amounted to a mean value of 0.59 μmol ⋅ kg− 1 ⋅ h− 1or 70 μmol/mmol creatinine during the 3rd-5th days of the control diet period. During the 8-h tracer infusion period on day 6,the rate was 46.4 μmol/mmol creatinine, which tended to be lower (P = 0.062) than on day 5. l-5-OP excretion accounted for ∼1.5% of the plasma oxoproline flux and 15% of the NOOPD (Tables 3 and 4). During days 3–5 the rate of excretion of d-5-OP (Table 4) was 0.26–0.27 μmol ⋅ kg− 1 ⋅ h− 1, amounting to ∼30% of total oxoproline excretion. It was also lower during the 8-h infusion period on day 6, when expressed per unit creatinine excretion, than on the previous day (P = 0.032).
Oxoproline and cysteine kinetics and oxoproline excretion with the SAA-free and Gly-free diets.
Results for oxoproline and cysteine kinetics during the periods when subjects consumed the SAA-free and Gly-free diets are summarized in Table 3. The oxoproline fluxes were significantly affected by diet, independent of the metabolic state. The control diet flux was significantly lower than the Gly-free diet flux (P = 0.018) and tended to be lower than the SAA-free diet flux (P = 0.057). Oxidation rates were significantly higher for the Gly-free (P = 0.005) and SAA-free (P = 0.03) diets compared with the rate for the control diet. However, the FROX did not differ among the three diet groups. The NOOPD was also not significantly different among the three diet groups, but there was a tendency (P = 0.066) for this to be higher during the fed than during the fast period, independent of the diet.
Plasma oxoproline concentration was not significantly affected by feeding and was similar among all of the diet groups (Table 3).
Cysteine fluxes were lower for the Gly-free (P = 0.01) and SAA-free (P = 0.001) diets compared with the control diet, independent of metabolic state. Also, fluxes were significantly lower (P = 0.023) during the fed state (Table 3) on all diets.
The rates of excretion of l- and d-5-OP during the initial 5 days were apparently unaffected by the absence of the specific dietary amino acids. When expressed per unit of creatinine excretion, although l-5-OP excretion tended to be lower (P = 0.062) on day 6 than on day 5 for the control diet, it was significantly higher on day 6 than onday 5 for the Gly-free (P = 0.019) and SAA-free (P = 0.005) diets. Furthermore, on day 6, l-OP excretion per millimole creatinine was significantly higher for both the Gly-free (P = 0.005) and SAA-free (P = 0.005) diets compared with the control diet.
The excretion of d-5-OP expressed per unit body weight per hour was higher on day 6 than on day 5, independent of diet (P = 0.001). In relation to creatinine output,d-5-OP output on day 6 was lower than for day 5 for the control diet (P = 0.032), but there were no significant differences between days 5 and 6 for the Gly-free and SAA-free diets. Furthermore, on day 6 there were no significant differences in d-5-OP excretion among the three diets except for a tendency for the output on the control diet to be lower than for the Gly-free diet (P = 0.066) and the SAA-free diet (P = 0.102).
For our subjects, urinary creatinine excretion ranged between 11.5 and 21.6 mmol/24 h, and the subjects studied also could be grouped into “high” l-5-OP and “low” l-5-OP excretors. Three of the six subjects had a urinary l-5-OP excretion that was about twice that (control 80.8; SAA-free 85.5; Gly-free 98.9 μmol l-5-OP/mmol creatinine) for the other three subjects (control 41.4; SAA-free 39.2; Gly-free 41.1 μmoll-5-OP/mmol creatinine); the difference was statistically significant (P < 0.02; unpaired t-test). This applied also to d-5-OP excretion, with the output for “high” excretors (control 43.6; SAA-free 33.8; Gly-free 33.9 μmold-5-OP/mmol creatinine) being about two times the amount excreted by the “low” excretors. Again, the difference was significant (P < 0.01).
In this investigation, which is to our knowledge the first study to examine the effect of different amino acid patterns on oxoproline kinetics in healthy subjects, we have explored the effects on the oxoproline metabolism of two diets based on l-amino acid mixtures that were devoid of either sulfur amino acids (methionine and cysteine) or glycine. The period of dietary intervention was a relatively short total 6-day period, because we wanted to examine whether oxoproline kinetics would be acutely affected by these dietary conditions. More prolonged dietary studies in healthy volunteers at this early stage in our investigations did not appear warranted until we had accumulated initial data under the former experimental conditions. Furthermore, Meakins et al. (31) showed that significant changes in l-5-OP excretion occurred within a 5-day period of supplementation of a low-protein diet with urea or urea plus glycine. These investigators also observed a significant linear relationship between the urinary excretion of l-5-OP and urinary sulfate excretion after their subjects had consumed the experimental diet for 5 days. In this context, we observed previously a rapid and profound reduction of sulfate excretion when healthy subjects were given a sulfur-free amino acid diet (22). Also, Jackson et al. (18) reported a marked increase in urinary oxoproline excretion betweendays 4 and 5 from giving a low-protein diet (25 g/day) to healthy adult men.
Data on the kinetics of oxoproline metabolism in human subjects are extremely limited. Usingl-[14C]pyroglutamate (oxoproline), Eldjarn et al. (6) estimated a daily endogenous production of 85 g of oxoproline in a patient with a markedly elevated plasma level (∼400 μmol/l) and oxoprolinuria (35 g daily). Thus production was more than twice the amount excreted in the urine. In our control subjects, the endogenous oxoproline production rate was ∼38 μmol ⋅ kg− 1 ⋅ h− 1, or ∼45 times that excreted in urine. Clearly, the relevance of the study by Eldjarn et al. to the present conditions and for an interpretation of our data is questionable. Recently, we have presented initial data on l-5-OP 13C and 15N plasma fluxes and l-5-OP oxidation for healthy subjects receiving an adequate diet (33). The plasma l-5-OP flux and oxidation rate in the present study were similar to those in that recent study.
The specific amino acid-devoid diets resulted in increases in the plasma oxoproline flux and oxidation rate. We predicted that this would have occurred if the plasma flux of oxoproline reflected an increased salvage of the cysteine and glycine generated by dipeptidase action on cysteinylglycine, the degradation product of GSH (5). However, to explore this hypothesis in more detail, studies will need to be undertaken on the whole body kinetics and interorgan aspects of the metabolism of the intermediates of the γ-glutamyl cycle. This presently poses a difficult technical and modeling challenge.
Nevertheless, we (33) have made some comparisons with GSH pharmacokinetic data and excretion data. Lauterburg and co-workers [Burgunder and Lauterburg (4) and Heibling et al. (14)] estimated the plasma GSH flux in postabsorptive healthy control subjects to be ∼24 μmol ⋅ kg− 1 ⋅ h− 1. In cirrhotics and HIV patients, GSH flux was significantly lower (∼15 μmol ⋅ kg− 1 ⋅ h− 1), reflecting a probably decreased rate of hepatic and systemic production of GSH (4, 14, 20). Similarly, in another study (3), the endogenous plasma appearance rate of GSH in healthy controls was ∼19.4 μmol ⋅ kg− 1 h− 1. For comparison, we find in the present study that ∼38 μmol ⋅ kg− 1 ⋅ h− 1of oxoproline enters the extracellular space in healthy volunteers receiving the control diet. Therefore, it appears that the oxoproline rates of appearance or disappearance are approximately double those for GSH kinetics. They are also about one-half of the plasma glutamate flux (2). On the other hand, the nonoxidative oxoproline disposal rate was ∼6–7 μmol ⋅ kg− 1 ⋅ h− 1, or about one-third to one-quarter of the rate of appearance of GSH.
The functional and causal relationships between thesel-5-OP fluxes and oxidation rates and GSH metabolism will require integrated GSH and l-5-OP tracer studies. These comparisons also raise the question of the transport ofl-5-OP, and studies with renal brush-border vesicles have shown that oxoproline transport is sodium dependent (10). It has also been reported that oxoproline serves as an intracellular signal for amino acid transport in the mammary gland and at the blood-brain barrier (47, 23). γ-Glutamyl amino acids are formed at the outer surface of luminal membranes of endothelial cells by γ-glutamyl transpeptidase, which are then transported into the cell where the amino acid and oxoproline are released, and the l-5-OP can then be reused for GSH synthesis. Compared with the13CO2 enrichments that we usually find with a comparable infusion rate of leucine (∼4–5 APE × 1,000), we observed here relatively high 13CO2 breath enrichments after 13C oxoproline infusion (Table 2). This leads us to conclude that the transport of oxoproline from the plasma compartment into a cellular site, where oxidation occurs, proceeds without apparent restraint.
The cysteine flux with the control diet was similar to that in our previous studies (9, 15). In general, both of the specific amino acid-devoid diets resulted in lower cysteine fasted and fed fluxes compared with the control diet. This might be due to the reduced turnover of GSH, because we earlier concluded that GSH breakdown accounts for ∼50% of tracer-derived cysteine flux (9). In mature rats, plasma GSH serves as a principal source for maintaining plasma cysteine (35).
The rate of l-5-OP excretion varied markedly among our subjects. The mean excretion of l-5-OP for subjects consuming the control diet was determined here to be generally higher than that given in a number of published studies, although the reported rates of excretion both between and within subjects studied in different laboratories appear to be highly variable. Pitt and Hauser (38), for example, used for reference range purposes anl-5-OP excretion of <70 μmol/mmol creatinine. However, for healthy adults consuming adequate, usual diets, a basal excretion of 210 ± 60 μmol/mmol creatinine after an overnight fast has been reported (17), and values for controls in a 1990 study by Forrester et al. (8) approximated 2 μmol ⋅ kg− 1 ⋅ h− 1, both values being considerably higher than for the present control diet.
In a later study by Jackson et al. (18), healthy omnivorous adults consuming a diet providing ∼1 g protein ⋅ kg− 1 ⋅ day− 1for 5 days appeared to have excreted only 10% of this value (males 15.5, and females 16.7 μmol/mmol creatinine). For a mainly vegetarian, low-protein diet (67 mg N ⋅ kg− 1 ⋅ day− 1) given to young women for a period of 5 days, a mean l-5-OP excretion was 385 μmol/day or ∼30 μmol/mmol creatinine (31). During late pregnancy, Jamaican women apparently excreted 131 μmol oxoproline/mmol creatinine, or a rate apparently 3–10 times greater than in nonpregnant women (19). The same authors also investigated l-5-OP excretion in infancy. In children recovering from malnutrition, l-5-OP excretion ranged between 50 and 300 μmol/mmol creatinine, with a mean value of 172 μmol/mmol creatinine (36). The l-5-OP excretion value in urine samples of apparently healthy Jamaican babies at the age of 6 wk was compared with that for English babies; mean excretions of 525 (African-Caribbean descent born in Jamaica), 398 (African, Indian, or mixed descent born in Jamaica), and 194 (Caucasian parentage born in England) μmol l-5-OP/mmol creatinine were reported (37,24). In contrast, for a group of infants born in England of Indian parentage, excretion of 5-l-OP (155 μmol/mmol creatinine) was not different from infants of Caucasian parentage (24). The authors concluded that the observed difference was of environmental rather than ethnic origin. Thus the basis for the considerable differences among the various studies is not clear. Different methodologies have been used to determine l-5-OP excretion, and these could be important. However, despite changes made between studies by Jackson et al. in the analysis of urinary oxoproline, they have reported that no significant differences were observed between the different methods (18).
In patients with genetic defects in the γ-glutamyl cycle, such as glutathione synthetase deficiency and 5-OPase deficiency, but also urea cycle defects, such as ornithine transcarbamylase deficiency, and organic acidurias (tyrosinemia, propionic acidemia, methylmalonic aciduria), a transient oxoprolinuria is reported (30). Furthermore, a transient oxoprolinuria in an asymptomatic patient with normal activities of glutathione synthetase and 5-OPase was also observed. Of relevance is that three of our subjects excreted l-5-OP at a rate that was twice (∼85 μmol/mmol creatinine) that for the remaining subjects, and this difference was maintained during all three diet periods.
Patients with gastroenteritis were found to excrete high levels ofl-5-OP (∼3,700 μmol/mmol creatinine) when on a diet containing Nutramigen, a lactose-free, casein hydrolysate, compared with controls given a diet based on usual foods (∼90 μmol/mmol creatinine) (34). The authors suspected that this was due to 5-OP formation during casein hydrolysis and thus claimed that oxoprolinuria was of dietary origin. However, l-5-OP levels in the diet were not actually measured.
Acetaminophen administration depletes the kidney GSH and cysteine contents in a dose- and time-dependent manner in growing and mature mice. Recovery to near control values was seen by 24 h after administration (42). During high anion gap metabolic acidosis and intake of therapeutic doses of acetaminophen, transientl-5-oxoprolinuria (600–23,600 μmol/mmol creatinine) was observed in patients. The level returned to a reference range (<70 μmol/mmol creatinine) after metabolic acidosis disappeared (38). This indicates that acetaminophen intake and/or metabolic acidosis may lead to depletion of tissue GSH, which affectsl-5-OP excretion. Our clinical records indicate that two of the “high” excretors were given acetaminophen for headaches onday 3 of the dietary control period but felt well the next day and did not continue any medication during the remainder of the study. No increase of urinary l-5-OP excretion was seen in these subjects between day 3 and day 5 of the control dietary period.
Under the present experimental conditions, the removal of methionine and cysteine or of glycine from the diet did not result in an increased excretion of l-5-OP during the initial 5 days, although onday 6 there was a significant increase in l-5-OP excretion (Table 4) with these two amino acid-free diets but not for the control diet. In addition, l-5-OP excretion was higher on day 6 for the SAA-free and Gly-free diets compared with the control diet. Together with the differences in the plasma fluxes and oxidation rates of l-5-OP, it appears that the availability of dietary sulfur amino acids and of glycine alters the activity of the γ-glutamyl cycle and the rate of oxoproline excretion. Based on these observations and those of Jackson et al. (18), it is reasonable to speculate that a longer period of specific amino acid restriction may have resulted in a more profound change in l-oxoproline kinetics and excretion. Furthermore, we showed earlier (49) that the level of supply of nonspecific nitrogen was critical for glycine synthesis, when the dietary nitrogen was provided via indispensable amino acids alone and at a submaintenance nitrogen intake. Again it seems likely, from these results, that a diet also low in nonspecific nitrogen might well have had a greater impact on l-5-OP kinetics and excretion. It is also worth noting that dietary glutamic acid is almost completely removed and catabolized during its first pass in the splanchnic region (29, 40), and enteral glutamate is used preferentially for GSH synthesis in the gut and the liver (41). Thus the importance of dietary glutamate, as well as of glycine and the sulfur amino acids, in determining the activity of the γ-glutamyl cycle and of its intermediates, including the kinetics and fate ofl-5-OP, would be an interesting research extension of the present study.
Finally, we found that d-5-OP excretion accounted for ∼30% of total 5-OP excretion for all of the dietary periods. Its output was not affected by the composition of the diets. It is thought that the origin of the d-form is from diet as well as from the metabolic activity of the intestinal flora (21, 46). In the present case, however, the intake of d-glutamic acid can be taken to be very low, because the l-glutamic acid included in thel-amino acid diet was pharmaceutical grade, and we could not detect with the procedure described above anyd-glutamic acid in the product used to formulate the diets. Therefore, the significant output of d-5-OP by our subjects appears to be due to microbial metabolism within the gastrointestinal tract (46).
In conclusion, a relatively short period of dietary sulfur amino acid or glycine restriction in healthy adults affects l-5-OP kinetics and excretion. These changes imply that GSH homeostasis may be similarly affected by these dietary modifications, and studies are now required to interrelate the observed changes with estimations of GSH status and kinetics. This is important 1) to an understanding of the way by which nutritional factors affect and/or may be used to modulate GSH homeostasis in stressed patients (13, 14, 26, 27) and2) for developing appropriate metaprobes for assessing the activity of key biochemical reactions involved in GSH metabolism (48).
We thank the dietary and nursing staff of the MIT Clinical Research Center for excellent technical assistance, and we acknowledge gratefully the commitment made by the subjects who participated in the study.
This study was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-15856 and P-30-DK-40561, Shriners Hospitals for Children (Grants 8370 and 8470), and a grant of the Deutsche Forschungsgemeinschaft (Me 1420/1–1), Bonn, Germany.
Present address and address for reprint requests and other correspondence: C. C. Metges, Deutsches Institut für Ernährungsforschung (German Institute of Human Nutrition), Arthur-Scheunert-Allee 114–116, D-14558 Bergholz-Rehbrücke, Germany (E-mail:).
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