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Synthesis of erythrocyte glutathione in healthy adults consuming the safe amount of dietary protein^1,2,3

¹ From the Institute of Human Nutrition, University of Southampton, Southampton General Hospital, Southampton, United Kingdom (AAJ, NRG, and YL), and the Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston (NRG and FJ).

² Supported by a grant from the Biotechnology and Biological Scientific Research Council and by the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement no. 58-6250-1-003.

³ Reprints not available. Address correspondence to AA Jackson, Institute of Human Nutrition, University of Southampton, Southampton General Hospital (MP 113), Tremona Road, Southampton SO16 6YD, United Kingdom. E-mail: aaj{at}soton.ac.uk.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: The finding that plasma glutathione turnover decreases as dietary protein intake decreases suggests that the safe amount of dietary protein, although sufficient for maintenance of nitrogen balance, may be insufficient for maintenance of cellular glutathione.

Objective: Our objective was to determine the effect of the safe protein intake on the erythrocyte glutathione synthesis rate and its relation with urinary 5-L-oxoproline excretion.

Design: Erythrocyte glutathione synthesis and urinary 5-L-oxoproline excretion were measured in young adults (6 men and 6 women) by using an infusion of [¹³C₂]glycine on 3 occasions: initially during the subjects' habitual protein intake (1.13 g · kg^–1 · d^–1) and on days 3 and 10 of consumption of a diet providing the safe protein intake (0.75 g · kg^–1 · d^–1).

Results: Compared with baseline values, the fractional synthesis rate of erythrocyte glutathione was significantly lower (P < 0.05) on days 3 and 10 of the diet with the safe protein intake. Urinary 5-L-oxoproline excretion increased significantly (P < 0.05) above baseline by the third day of the diet with the safe protein intake and remained elevated. Erythrocyte glutathione concentrations and absolute synthesis rates decreased by day 3 but recovered to baseline values by day 10. Erythrocyte concentrations of cysteine, methionine, and serine remained unchanged, whereas erythrocyte concentrations of glycine, glutamic acid, and glutamine increased significantly by day 10.

Conclusion: During adaptation to the safe amount of dietary protein, there are changes in the concentration and kinetics of erythrocyte glutathione that suggest a reduced antioxidant capacity and possible increased susceptibility to oxidant stress.

Key Words: Glutathione • glycine • safe amount of dietary protein • amino acids • nitrogen balance • 5-L-oxoproline

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The dietary requirement and the safe amount of dietary protein for adults are based on the amount of protein that must be consumed to maintain body weight and nitrogen balance in healthy persons. The safe intake is currently set at 0.75 g · kg^–1 · d^–1 (1). The habitual intake of persons in the developed world is normally higher than this (2), and adaptive processes are brought into play when a healthy adult consumes a diet that provides the safe amount of protein (3). In young adults, a reduction in protein intake from the habitual intake (1.13 g · kg^–1 · d^–1) to the safe intake (0.75 g · kg^–1 · d^–1) elicits implementation of metabolic processes that lead to nitrogen conservation (4-7). The ability to achieve nitrogen equilibrium and maintain body weight confirms the adequacy of this protein intake. Nevertheless, it has been argued that achieving nitrogen balance is not a sufficient criterion of adequacy and that the ability to respond to the everyday stresses of life is also necessary.

It has been suggested that the safe amount of protein might not adequately provide the indispensable amino acids that are required for an effective defense response (8). In such an event, amino acids are required for the synthesis of acute phase and immunologic proteins. Furthermore, with the possible accumulation of toxins, a greater potential for oxidative stress also exists. The extent to which a person is able to withstand the potentially damaging effects of oxidative stress is determined by the balance between the rate at which the oxidant species are generated and the capacity of metabolic processes to produce antioxidants. An important cellular antioxidant is the tripeptide glutathione (-L-glutamylcysteinylglycine), and the glutathione redox cycle is a fundamental component of this cellular antioxidant defense system.

Glutathione is formed in a stepwise process from its constituent amino acids, glutamate, cysteine, and glycine (Figure 1) (9). Cellular free cysteine is maintained at low concentrations by incorporation into glutathione, and cysteine is generally considered to be the rate-limiting substrate for the formation of glutathione (10). However, glycine has been postulated as the limiting amino acid for glutathione synthesis under some circumstances (11). Because of the importance of a constant amino acid supply for glutathione production, feeding diets that are marginal in protein could conceivably lead to a problem in maintaining the cellular glutathione content. We hypothesized that when persons are faced with a reduced intake of protein, a component of the adaptive response may be to decrease the synthesis of cellular glutathione. In the present study, we measured the synthesis rate of erythrocyte glutathione by using stable-isotope methods. In a previous study, we showed that, when subjects first began consuming a lower-protein diet, all of them went into negative nitrogen balance but that nitrogen equilibrium was reestablished by the end of the study period (7). In the present study, we report the glutathione kinetics for the same healthy young adults during consumption of their habitual amount of dietary protein and during a short and a long period in which a diet that provided the safe amount of protein was consumed.

View larger version (10K): [in this window] [in a new window]   FIGURE 1. Relations between the amino acids that are used for the formation of glutathione (GSH), the generation of 5-L-oxoproline, and the factors that might increase the demand for glutathione. GSSG, oxidized GSH.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Table 1

Table 2

After a 12-h overnight fast, volunteers arrived at the metabolic unit of Southampton General Hospital. Intravenous catheters were inserted into superficial veins of both arms, one for continuous infusion of the tracer solution and the other for repeated blood sampling. After collection of baseline blood (10 mL), a priming dose of [¹³C₂]glycine (20 µmol/kg) was given and was followed immediately by continuous infusion of [¹³C₂]glycine (15 µmol · kg^–1 · h^–1) for 7 h. Blood samples (5 mL) were taken hourly from hours 3–5 and then every 0.5 h until the end.

Sample analyses
Blood for amino acid analysis was drawn in prechilled tubes (containing Na₂EDTA and a cocktail of sodium azide, merthiolate, and soybean trypsin inhibitor) and immediately centrifuged at 4 °C, and the plasma was removed and stored at –70 °C for later analysis. The hematocrit of each blood sample was determined by centrifugation at 13 460 x g for 5 min at 25 °C on a Micro Hematocrit Centrifuge (Damon/IEC Division, Needham Heights, MA).

Erythrocyte glutathione
A 1-mL aliquot of each blood sample was mixed immediately in a cryotube with 0.5 mL chilled, isotonic monobromobimane (MBB) buffer solution (pH 7.5) containing the following (in mmol/L): 5 MBB, 17.5 Na₂EDTA, 50 potassium phosphate, 50 serine, 50 boric acid. The whole blood–MBB mixture was centrifuged at 1000 x g for 10 min at 4 °C, and then the supernatant fluid was incubated in the dark for 20 min for development of the plasma MBB. Another 1.5 mL MBB buffer was added to the packed erythrocytes, which were immediately lysed by rapid freeze and thaw with the use of liquid nitrogen, and the lysed erythrocyte–MBB buffer mixture was shaken and left in the dark at room temperature for 20 min for development of the erythrocyte glutathione–MBB derivative. Proteins were precipitated by using 0.5 mL 2 mol perchloric acid/L, and the supernatant fluids were stored at –70 °C until further analysis.

Isolation of erythrocyte glutathione and measurement of its concentration were performed by using a Hewlett-Packard 1100 HPLC system (Hewlett-Packard, Avondale, PA) equipped with a reverse-phase ODS Hypersil column (5 µm, 4.6 x 200 mm; Agilent Technologies, Inc, Wilmington, DE) and fluorescence detector (model HP 1046A; Hewlett-Packard). Elution of the glutathione was accomplished by a 3–13.5% acetonitrile linear gradient in 1% acetic acid (pH 4.25) at a flow rate of 1.1 mL/min. The glutathione elution was collected by using a fraction collector, dried, and hydrolyzed for 4 h in 4 mol HCl/L at 110 °C.

Erythrocyte free amino acids
A 1-mL aliquot of blood was centrifuged at 1000 x g for 10 min at 4 °C, and the plasma was then removed and stored at –70 °C for later analysis. The remaining red blood cells were then washed 3 times with 3 mL sodium chloride solution (9 g/L) and centrifuged at 1000 x g for 10 min at 4 °C. The washed cells were then lysed by freeze-thaw action with the use of liquid nitrogen. Finally, the cellular proteins were precipitated by using 1 mL ice-cold 10% (1 mol/L) perchloric acid solution. After centrifugation at 1000 x g for 10 min at 4 °C, the supernatant fluid was used for erythrocyte free amino acid analysis.

Before derivatization for gas chromatographic–mass spectrometric analysis, erythrocyte free glycine was further isolated by cation-exchange (Dowex 200x; Bio-Rad Laboratories, Inc, Hercules, CA) chromatography. Samples of glycine derived from erythrocyte glutathione and plasma and erythrocyte free glycine samples were converted to the n-propyl ester, heptafluorobutyramide derivative. The ratio of tracer to tracee for glycine in various samples was determined by negative chemical ionization gas chromatographic–mass spectrometric analysis with selective monitoring of ions at mass-to-charge ratios of 293–295.

Erythrocyte amino acid concentrations were determined by HPLC analysis with a Waters Picotag system (Millipore Corp, Milford, MA). To assess the level of oxidative stress, the derivatives of reactive oxygen metabolites were determined in serum by using the method in which hydroperoxides are converted into radicals that oxidize N,N-diethyl-para-phenylendiamine, which is detected spectrophotometrically (12). The activities of erythrocyte glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) and plasma -glutamyl transpeptidase (EC 2.3.2.2) were measured spectrophotometrically (13).

Individual urine samples were collected in containers and stored at 4 °C during the day. Twenty-four–hour specimens were pooled and thoroughly mixed, and the total volume was recorded. A 20-mL sample of each 24-h collection was stored at –70 °C until analysis. Daily excretion of 5-L-oxoproline in urine was measured as described previously (14). In this procedure, 5-L-oxoproline was isolated from urine by using ion-exchange chromatography. The eluted 5-L-oxoproline was hydrolyzed to L-glutamic acid, which was assayed with L-glutamate dehydrogenase (EC 1.4.1.4). This assay does not measure the alternative enantiomer, 5-D-oxoproline, which is derived from the diet or microbial metabolism.

Calculations and statistics
Fractional synthesis rate of erythrocyte glutathione
The fractional synthesis rate (FSR) of erythrocyte glutathione (FSR_GSH) was derived by using a precursor-product relation (15).

(1)

where (E_t7 –E_t5) is the increase in the isotopic enrichment of glycine in erythrocyte glutathione between the fifth and seventh hours of infusion, E_RBC is the plateau enrichment of erythrocyte free glycine, and (t7 – t5) is the time interval between the fifth and seventh hours (ie, 2 h). The absolute synthesis rate (ASR) of erythrocyte glutathione (ASR_GSH) was calculated from the product of erythrocyte glutathione concentration and the FSR_GSH.

Statistical analysis
The data were analyzed by using analysis of variance with repeated measures or split-plot analysis of variance where appropriate with the use of SPSS version 11.5 (SPSS Inc, Chicago). When significant differences were identified, individual differences were assessed by using post hoc testing with Bonferroni correction for multiple comparisons. Significance was set at P < 0.05. The changes with time were not always linear, which suggested different patterns of interaction as the period of consumption of the safe amount of protein increased. The differences in the patterns of response were explored for each study day by using multivariate analysis of variance to explore the influence of other variables on the basis of a presumed a priori model of metabolic interrelations (Figure 1).

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
We previously reported that, when the subjects first began consuming a lower-protein diet, all of them went into negative nitrogen balance but that nitrogen equilibrium was reestablished by the end of the study period (7). The age and physical characteristics of the subjects are shown in Table 1. The men and women did not differ significantly in age ( ± SD: 25.6 ± 1.0 y) or body mass index. The men were significantly taller than the women and had significantly higher lean body mass and lean body mass index and significantly lower fat mass than did the women.

The metabolic variables at baseline and on days 3 and 10 of consumption of the diet that provided the safe amount of protein are shown in Table 3. The basal metabolic rate (BMR) of the men was significantly higher (30%) than that of the women. This difference could be attributed in part to the higher lean body mass of the men, and when an adjustment was made for lean body mass, there was a significant change in BMR with time. The BMR adjusted for lean body mass increased with time of consumption of the low-protein diet in the men but decreased progressively in the women. Hematocrit values were significantly higher in the men than in the women and decreased significantly over the study period. There were no significant differences in oxidative stress as measured by the derivatives of reactive oxygen metabolites or in the activities of the erythrocyte enzymes glutathione peroxidase, glutathione reductase, and -glutamyl transpeptidase over the study period. There were also no significant differences in plasma glycine flux or in erythrocyte concentrations of serine, methionine, and cysteine over the study period; however, on day 10 of the study, the concentrations of glycine, glutamic acid, and glutamine were significantly higher (12%) than those at baseline or on day 3.

Figure 2

View larger version (12K): [in this window] [in a new window]   FIGURE 2. Mean (± SEM) erythrocyte glutathione concentrations and mean (± SEM) fractional synthesis rates and absolute synthesis rates of erythrocyte glutathione (FSRGSH and ASRGSH, respectively) in 12 healthy adults (6 men and 6 women) during consumption of their habitual amount of dietary protein at baseline and on days 3 and 10 of consumption of a diet that provided the safe amount of protein. For glutathione concentration, sex was entered as a fixed variable, and height and weight were entered as covariates. *Significantly different from baseline, P < 0.05 (repeated-measures ANOVA followed by post hoc analysis with Bonferroni correction for multiple comparisons).

Figure 3

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Mean (± SEM) urinary 5-L-oxoproline excretion per day in 12 healthy adults (6 men and 6 women) during consumption of their habitual amount of dietary protein at baseline and during consumption of a diet that provided the safe amount of protein for 10 d. *Significantly different from baseline, P < 0.05 (repeated-measures ANOVA followed by post hoc analysis with Bonferroni correction for multiple comparisons).

Univariate analysis showed no significant interactions for glutathione concentration for any of the study days. For ASR_GSH, there was a significant univariate relation with erthyrocyte glutamic acid on day 10. There were univariate relations with 5-L-oxoproline on each study day: with body mass index, BMR, and ASR_GSH at baseline; with BMR, ASR_GSH, FSR_GSH, and erythrocyte glycine and serine on day 3; and with erythrocyte cysteine on day 10. The results for the multivariate analysis for ASR_GSH and urinary 5-L-oxoproline are shown in Table 4. For ASR_GSH, erythrocyte methionine, glycine, and cysteine and sex contributed at baseline; erythrocyte methionine and glycine contributed on day 3; and body mass index and sex contributed on day 10. In a multivariate model for 5-L-oxoproline, the most significant interactions were with ASR_GSH and oxidative stress, as measured by plasma derivatives of reactive oxygen metabolites, at baseline; with ASR_GSH, erythrocyte glycine, and BMR on day 3; and with ASR_GSH, erythrocyte cysteine, and age on day 10.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

The steady state level of any metabolite represents the balance between its rate of formation and its rate of degradation or utilization. The initial decrease in glutathione concentration on day 3 can be accounted for by a decrease in its rate of formation, at a time when nitrogen balance was negative. By the time nitrogen balance was reestablished on day 10, the steady state concentration of glutathione had recovered, although FSR_GSH remained decreased. The factors that might account for variability in glutathione synthesis and concentration differed between baseline and day 10, especially erythrocyte concentrations of methionine and glycine. These 2 amino acids are linked stoichiometrically within the methionine-homocysteine cycle through both the remethylation and the transulfuration pathways. In the remethylation pathway, conversion of homocysteine to methionine is associated with serine-glycine interconversion. In the transulfuration pathway, the degradation of methionine utilizes serine to form cysteine, which potentially consumes 2 mol glycine for every 1 mol cysteine generated. At baseline, the relation between ASR_GSH and concentrations of methionine, glycine, and cysteine suggests that the availability of methionine may have limited glutathione formation. By contrast, on day 3, the changed direction of the association of glycine and methionine with ASR_GSH suggests that the availability of glycine was limiting at this time. There is a substantial demand for the endogenous formation of glycine (16); formation decreases with diets that are low in protein, and the adaptive response takes some time to develop (14).

It has been proposed that when glycine availability for glutathione synthesis is limiting, there is a modest increase in urinary 5-L-oxoproline, which can be used as an index of glycine status (Figure 1) (11). The more recent finding of elevated 5-oxoprolinuria in subjects receiving a diet limited in sulfur-containing amino acids but containing normal amounts of glycine has raised the possibility that 5-oxoprolinuria may not be specific in this regard (17). However, glycine can be used for the endogenous formation of cysteine (2:1 on a molar basis), and therefore a diet limited in cysteine will generate a demand for available glycine (18). In the present study, ASR_GSH was related to 5-oxoprolinuria on each study day. At baseline, the interaction with reactive oxygen metabolites suggests that the demand for antioxidant protection interacted with the capacity for glutathione formation in determining 5-oxoprolinuria. By day 3, body composition and erythrocyte glycine concentration became significant. The relation with glycine was indirect, which supports the idea that limited availability of glycine is important in the generation of 5-oxoprolinuria. On day 10, erythrocyte cysteine concentrations were strongly and positively related to 5-oxoprolinuria, which supports the proposal that 5-L-oxoproline concentrations increase when cysteine cannot be effectively utilized for glutathione formation. Together, the data provide evidence that, at each stage of the process of adaptation to low-protein diets, 5-L-oxoproline in urine relates to different aspects of glutathione formation and to the relative availability of glycine and sulfur-containing amino acids.

There were limitations to the design of the present study. Measurements of glutathione kinetics in erythrocytes may not reflect other cells in the body, although in piglets, changes in erythrocyte glutathione kinetics induced by protein deficiency and inflammatory stress reflected changes in the gut mucosa (15). We studied men and women and identified seemingly important differences in the responses of BMR, hematocrit, and possibly glutathione, for which the basis is unclear and which require further detailed exploration. The results of the present study indicate that urinary 5-L-oxoproline carries promise as a marker of metabolic state, which might be measured in larger groups of subjects. To follow the time course of the response to the lower-protein diet, we chose an early time, at which the acute changes might best be characterized, and a later time, by which nitrogen balance would have been reestablished.

Metabolic adaptations to a lower protein intake are not without cost. When healthy adults consume a diet providing either a marginal amount of protein for 5 d (19) or the safe amount of protein (7), changes in the synthesis of hepatic secretory proteins occur. The present results show that even when nitrogen balance has been achieved, there may be functional changes, such as in glutathione kinetics and metabolism. These changes in glutathione imply a reduced capability to withstand stress and potentially greater susceptibility to environmental challenge (20). There is a need to determine the extent to which these metabolic changes interact in clinically stressful situations in which glutathione may be important, eg, in HIV infection (21), severe malnutrition (22), sepsis (23), and heart disease and cancer (24). In the definition of protein requirements, much emphasis has been placed on the need for indispensable amino acids; however, attention needs to be given to the amount of dietary protein that allows adequate endogenous formation of amino acids that are dietarily dispensable (6). The 3 amino acids required for glutathione formation fall into this category.

In summary, in the present study, we observed that adaptation to the safe amount of dietary protein (1) is associated with changes in glutathione kinetics that may have a functional cost. In part, the extent of these changes is related to the availability of the component amino acids. The urinary excretion of 5-L-oxoproline can be used to mark these processes, which suggests that the ability to maintain adequate endogenous formation of glycine to meet the needs for glutathione formation may be an important aspect of adaptation. Although it is possible for persons to achieve nitrogen balance while consuming a diet that provides the safe amount of protein (1), that this achievement is without metabolic consequence or cost cannot be presumed.

	ACKNOWLEDGMENTS

 
We are grateful to L Ware and C Persaud for their assistance with these studies and to Margaret Frazer and Melanie Del Rosario for their invaluable technical help with the specimen analyses.

All authors were engaged in the conception of the study, the clinical conduct, the analysis of specimens, and the preparation of the manuscript. The authors are not aware of any conflicts of interest in this work.

	REFERENCES

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