QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Courtney-Martin, G. Articles by Pencharz, P. B. Search for Related Content PubMed PubMed Citation Articles by Courtney-Martin, G. Articles by Pencharz, P. B. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-METHIONINE CYSTEINE Medline Plus Health Information Diets

---

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Methionine-Adequate Cysteine-Free Diet Does Not Limit Erythrocyte Glutathione Synthesis in Young Healthy Adult Men^1,2

³ Research Institute, Hospital for Sick Children, M5G 1X8 Toronto, Canada; ⁴ Department of Nutritional Sciences, and ⁵ Department of Paediatrics, University of Toronto, M5G 1X8 Toronto, Canada; ⁶ School of Dietetics and Human Nutrition, McGill University, H3A 2T5 Montreal, Canada; and ⁷ Department of Agriculture, Food and Nutritional Science, University of Alberta, T6G 2P5 Edmonton, Canada

^* To whom correspondence should be addressed. E-mail: paul.pencharz{at}sickkids.ca.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Most methods of determining amino acid (AA) requirements are based on endpoints that determine adequacy for protein synthesis. However, the sulfur AA (SAA) cysteine is believed to be the rate-limiting substrate for synthesis of the most abundant intracellular antioxidant, glutathione (GSH). Our objectives were to determine whether supplementation of cysteine in a diet containing adequate SAA for protein synthesis, as methionine, increased GSH synthesis by measuring the fractional and absolute synthesis rates, and if concentration of GSH changed in response to feeding 5 graded intakes of cysteine (0, 10, 20, 30, and 40 mg·kg^–1·d^–1) in a random order with a fixed methionine intake of 14 mg·kg^–1·d^–1 and a protein intake of 1 g·kg^–1·d^–1. Each subject received a multivitamin and choline supplement during the study. Four healthy adult men each underwent 5 isotope infusion studies of 7-h duration after a 2-d adaptation to the level of cysteine intake being studied on the isotope infusion day. The isotope used was [U-¹³C₂ –¹⁵N]glycine. Analyses included erythrocyte GSH synthesis rates and concentration and urinary sulfate excretion. The GSH synthesis rates and concentration, measured at a methionine intake of 14 mg·kg^–1·d^–1, did not change with increasing intakes of cysteine. Urinary sulfate excretion showed a significant positive relationship with cysteine intake (r = 0.92; P < 0.01). In conclusion, this study provides preliminary evidence that consumption of SAA adequate to meet the requirement for protein synthesis does not limit GSH synthesis in healthy adult men receiving an otherwise adequate diet.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

GSH status has been shown to be very sensitive to changes in cysteine intake (4,5) and cysteine is considered the rate-limiting substrate for GSH synthesis (6–8). Cysteine deficiency, as measured by changes is GSH synthesis and concentration, has been observed in healthy adults consuming a SAA-free diet (6), as well as healthy adults consuming an energy-adequate, low-protein diet.

In addition, cysteine deficiency has been observed in stress and disease states, including children with edematous malnutrition (9), adults with HIV (10), and sickle cell disease (11). The cysteine deficiency, as reflected by decreased GSH synthesis and concentrations in those studies, was mainly attributed to an increase in the cysteine requirement due to its increased utilization for GSH synthesis. The short-term supplementation of N-acetyl-cysteine in subjects in those studies (8,10) led to significant improvement in GSH synthesis and concentration.

There is some evidence from animal and human data that GSH synthesis is not adequate at SAA intakes adequate for protein synthesis and maintenance of nitrogen balance (4,7,8,12). These data have been interpreted to mean that protein synthesis has a higher priority for cysteine than does GSH synthesis (13). The findings by Kurpad et al. (14), that undernourished Indian men have the same requirement for total SAA (as provided by methionine alone without cysteine) as their well-nourished Indian counterparts and well-nourished North American men, therefore begs the question as to whether the SAA requirements as determined using currently available methods, which reflect the needs for protein synthesis, underestimates the SAA intake necessary to maintain a normal GSH status.

The goal of this study was to determine whether GSH synthesis would increase following supplementation of the level of total SAA (TSAA) intake that supported maximum protein synthesis. This was determined by measuring erythrocyte GSH fractional and absolute synthesis rates (ASR) as well as erythrocyte GSH concentration in healthy adult men fed a diet providing 1 g/kg protein in the presence of the mean population requirement for TSAA (as methionine only) of 14 mg·kg^–1·d^–1 and a varying additional cysteine intake.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Subjects

Ethical approval for the study was obtained from the Research Ethics Board at the Hospital for Sick Children. Written informed consent was obtained from each subject after the protocol was explained to them fully. Four young healthy adult men participated in this study. To participate in the study, each subject had to be in good health as determined by medical history and blood test, which included normal cell count and no evidence of anemia as determined by white and red cell count, hemoglobin, hematocrit, mean cell volume, and mean cell hemoglobin concentration within the normal range for age. Exclusion criteria were: presence of diseases known to affect GSH concentration (e.g. HIV and diabetes), anemia, medications known to affect protein and amino acid metabolism (e.g. steroids), substantial weight loss during the past month, consumption of weight-reducing diets, inability to tolerate the experimental diet, unwillingness to have blood drawn from a venous access during the study, substantial caffeine consumption [equivalent to >2 cups (500 mL) of coffee per day], or significant alcohol consumption [>1 drink per day, e.g. 1 beer (355 mL, 4.2 g alcohol), one-half glass of wine (125 mL, 5 g alcohol)]. Before the commencement of the study, height, weight, and body composition were measured for each individual (Table 1).

The 4 subjects each underwent 5 stable isotope infusion studies of 7-h duration, in a repeated-measures design, to measure GSH synthesis in response to 5 different intakes of dietary cysteine assigned in random order. The decision to use 4 subjects and 5 levels of cysteine intake per subject was carefully made. A repeated-measures design, in which each subject acts as their own control, is a powerful design to reduce the impact of subject variability, and this approach is also more sensitive to treatment differences than the alternative designs. In addition, we had predicted, based upon observations reported in the literature, that a curvilinear response was probable. We decided to use 5 cysteine intakes to ensure that we could adequately detect and prove a curvilinear response, if it occurred. Alternatively, with 5 increasing intakes of cysteine per subject, if there was a linear response in any of our measured variables, this would be clearly established.

Each individual study was carried out over a period of 3 d and individuals completed all their studies within a 3-mo period. The first 2 d were the adaptation days during which subjects were adapted to the level of cysteine administered for that study. Day 3 was the isotope infusion study day. A 2-d adaptation period was chosen because Lyons et al. (6) showed that the fractional GSH synthesis rate in blood was 65%, suggesting that the half-life of GSH in RBC was 18 h. In addition, data from Lee et al. (15) showed that the activity of the rate-limiting enzyme for GSH synthesis reached a new steady state in liver within 16 h of changing from a low- to a high-protein diet and that liver GSH and cysteine concentrations reached a new steady state within 12 h. In addition, Jackson et al. (7) found a significant decrease in GSH fractional synthesis rate (FSR) on d 3 after switching healthy participants from their habitual protein intake to the WHO-recommended intake of 0.75 g/d. In the same study, after a 9-d adaptation on the WHO 0.75-g/kg protein intake, the FSR was the same as that measured on d 3 of the diet. The suggestion is that the AA pool had already undergone an adequate adaptation after 2 d. Based on these data, we judged a 48-h adaptation period to be adequate to achieve equilibration of GSH in erythrocytes after each individual change in cysteine intake.

The diet was provided as an experimental formula and protein-free cookies developed for AA kinetic studies (16). Briefly, a liquid formula (protein-free powder, Product 80056, Mead Johnson) flavored with orange and fruit crystals (Tang and Kool-Aid, respectively; Kraft Foods) and protein-free cookies supplied the main source of energy in the diet (Table 2). The nitrogen content of the diet was provided as a crystalline AA mixture (1.0 g·kg^–1·d^–1) based on the AA pattern of egg protein (17). The reason for using egg protein composition is that most studies on AA requirement have used this pattern. Certainly, all studies from which the total SAA requirement was based for this current study used egg protein. For consistency and to allow comparisons to be drawn, the same AA pattern was chosen (17). The adaptation diet for the 2 d before the tracer infusion study provided energy content for resting energy expenditure (REE) x 1.7, whereas the energy content of the diet on the isotope infusion day, provided as REE x 1.5. REE, was measured by open circuit indirect calorimetry (Deltatrac; SensorMedics, Yorba Linda). The macronutrient content of the experimental diet, expressed as a percentage of dietary energy, was 53% carbohydrate, 37% fat, and 10% protein. The diet was weighed (Sartorius Balance model BP110 S; Sartorius) and prepared in the research kitchen at the Hospital for Sick Children.

During the adaptation days, the diet was provided as 4 equal meals per day to be consumed at the same time each day. Subjects were allowed water in their desired quantity, but caffeinated beverage, alcohol, or any other drinks except that provided by the diet were not allowed. On the isotope infusion day, the diet was provided as 10 isoenergetic, isonitrogenous meals, each representing one-twelfth of the subjects' total daily requirement. Subjects had free access to water on the study day.

Methionine was provided at an intake of 14 mg·kg^–1·d^–1, which is a mean of the 2 mean published estimates (using carbon oxidation techniques) for TSAA requirement in adult humans (19,21). We chose this level of methionine because it represented the mean estimated total SAA requirement for protein synthesis as estimated by nitrogen balance (22), indicator AA oxidation technique (21), and 24-h indicator oxidation-balance technique (19). The levels of cysteine studied were 0, 10, 20, 30, and 40 mg·kg^–1·d^–1. The 0 mg·kg^–1·d^–1 allowed for the estimation of GSH kinetics at the mean TSAA requirement of 14 mg·kg^–1·d^–1. The cysteine intake of 10 mg·kg^–1·d^–1 combined with the methionine intake of 14 mg·kg^–1·d^–1 provided the total SAA intake of 24 mg·kg^–1·d^–1, the estimated recommended daily allowance for total SAA requirement (19,21). The cysteine intakes of 20, 30, and 40 mg·kg^–1·d^–1 provided cysteine in an amount typically consumed in the western diet by individuals consuming a mixed protein diet providing >1.0 g.kg^–1.d^–1 protein (7,10). These intakes also represented 30, 50, and 65%, respectively, of the amount given in a supplementation study of children with malnutrition (8). The 40 mg·kg^–1·d^–1 of cysteine was chosen to represent the highest possible level beyond which increases in GSH synthesis would be unlikely in healthy subjects.

Glycine was provided at an intake of 69.5 mg·kg^–1·d^–1, which is twice that found in the high-quality egg protein at a protein intake of 1 g/kg. This was judged to be adequate to prevent it from being deficient for GSH synthesis. The amount of [¹⁵N – ¹³C₂]glycine provided on the study day was subtracted from the dietary provision to maintain the intake at 69.5 mg·kg^–1·d^–1. The glycine content of the diet was increased to ensure glycine was not limiting for GSH synthesis. The glycine content of egg protein is 3.8%, whereas the glycine content of human tissue protein is 7.2%. We chose to increase the glycine content to make it more comparable to the composition in human tissue (composition of the AA mixture presented in Table 3).

[U-¹³C₂ –¹⁵N]glycine (98% ¹³C₂, 98% ¹⁵N) was purchased from Cambridge Isotope Laboratories. Stock solutions were prepared in 0.9% sodium chloride (10 g/L) by the Research Pharmacy at the Hospital for Sick Children and were confirmed to be sterile and pyrogen free. We chose to use an M+3 glycine tracer as the GSH precursor to determine its incorporation into the GSH molecular ion by liquid chromatography tandem MS. In preliminary studies with M+2 glycine, we could not accurately detect the enrichment in the whole GSH molecule above background because of the high baseline M+2 GSH enrichment.

Each tracer infusion study was conducted on d 3, after completion of the 2-d adaptation on the liquid AA-based diet; subjects arrived at the Clinical Research Centre at the Hospital for Sick Children after a 12-h overnight fast. Ten hourly, isoenergetic, isonitrogenous meals were consumed beginning 3 h before the start of the i.v. isotope infusion. The cysteine content of each meal was dependent on the test level being studied. Because the amount of cysteine in the diet was manipulated, L-alanine was adjusted to keep the nitrogen content of the diet constant.

After consumption of the first 2 meals, i.v. catheters were inserted into superficial veins of both arms, 1 for continuous infusion of the tracer solution and the other for repeated blood sampling. Baseline blood (2.0 mL) was collected after the 3rd hourly meal. At the beginning of the 4th meal, a priming dose of [U-¹³C₂ –¹⁵N]glycine (40 µmol/kg) was given over a 15-min period, followed immediately by a continuous infusion of [U-¹³C₂ –¹⁵N]glycine (15 µmol·kg^–1·h^–1) for 7 h. Blood samples (1.5 mL) were taken hourly from the 3rd hour of the infusion until the 5th hour and then every 0.5 h until the end. To ensure arterialized blood, the hand was heated inside a thermostatic chamber maintained at 60°C for 15 min before the blood was sampled (23).

Urine samples were collected after each void for the 10 h of the study day. Samples were pooled and 2- x 2-mL aliquots were stored at –20°C for urinary sulfate analysis to relate changes in cysteine intake to sulfate excretion.

Sample analysis

Blood (0.5 mL) for hematocrit was collect into tubes containing Na₂EDTA and immediately sent to the clinical biochemistry laboratory at the Hospital for Sick Children for analysis. Briefly, the red cells were analyzed using an Abbott CELL-DYN Sapphire Hematology analyzer. Hematocrit was then calculated using the formula HCT (L/L) = (RBC x MCV)/1000, where HCT is hematocrit and MCV is mean corpuscular volume.

Erythrocyte GSH concentration and enrichment

All chemicals were purchased from Sigma-Aldrich Canada. A 0.5-mL aliquot of each blood sample collected in Na₂EDTA was centrifuged for 2 min at 13,000 x g (Beckman Microfuge-TM 11, Beckman Coulter Canada) within 5 min of collection. Each tube was weighed before and after blood collection to determine the amount of blood, because GSH concentration was normalized to hematocrit. After centrifugation, the plasma was immediately removed. Two hundred microliters of 100 mmol/L N-ethylmaleimide and 20 µL of 5 mmol/L -glutamyl-leucine (internal standard) were added to the separated RBC. The sample was then caped, mixed with a vortex mixer, and left for 10 min at room temperature. Cells were then lysed with 50 µL 0.4 mol/L ZnSO₄ and the protein precipitated with 1 mL ice-cold methanol. The sample was then vortexed, centrifuged at 13,000 x g for 2 min, the supernatant removed and stored at –80°C until analysis.

GSH concentration and enrichment were analyzed using a triple quadrupole mass spectrometer API 4000 (Applied Biosystems/MDS SCIEX) operated in positive ionization mode with the Turbo Ion Spray ionization probe source (operated at 5.8 kV). This was coupled to an Agilent 1100 HPLC system. All aspects of system operation and data acquisition were controlled using The Analyst NT v 1.4.1 software. GSH concentration was measured using an external standard curve and the ratio of the analyte (GSH) to the internal standard (-glutamyl-leucine). The parent-to-daughter transitions measured for GSH and the internal standard were 433.4 to 304.3 and 261.4 to 132.0, respectively. GSH enrichment was determined in the whole GSH molecule (as the tripeptide). There was no fractionation and hydrolysis step prior to measurement of enrichment. GSH enrichment was calculated as a ratio of the (enriched) M+3 to (unenriched) M peaks of the tripeptide molecule of GSH by measuring the transition of parent to daughter ions of 436.4 to 307.0 (M+3) and 433.4 to 304.3 (M+0) and was expressed as mole percent excess calculated from peak area ratios at the isotopic steady state of erythrocyte glycine enrichment in the last 2 h of the isotope infusion. Interassay precision for GSH concentration was between 2.3 and 4.8%, whereas interassay precision for GSH enrichment was 3.7 ± 7.3%. The accuracy of the instrument for GSH concentration was measured by spiking samples with a known amount of GSH and comparing to the unspiked sample. Concentrations were determined using a standard curve. The accuracy of the GSH concentration was between 90 and 108% of that expected. Accuracy for GSH enrichment was measured using enrichment curves, which were linear within the ranges of expected sample enrichment. The results of the enrichment curve was y = 0.94x + 0.097; r² = 0.99.

Erythrocyte-free glycine enrichment

Each sample was collected and centrifuged as above. Plasma was quickly removed and the cells washed twice with 300 µL ice-cold saline on each occasion. Samples were vortexed between each wash. Cells were then lysed and deproteinated as above, vortexed, centrifuged for 2 min at 13,000 x g and the supernatant stored at –80°C until analysis.

Fifty microliters of each sample was then dried under nitrogen at 35°C. One hundred microliters of butanol·HCl (Sigma-Aldrich Canada) was then added and the sample vortexed, topped with nitrogen, and heated for 20 min at 55°C. The sample was again dried under nitrogen, reconstituted in 0.1% formic acid (Sigma-Aldrich Canada). Samples were then analyzed using a triple quadrupole mass analyzer as described above. Glycine enrichment was calculated as the ratio of the (enriched) M+3 to (unenriched) M peaks of glycine after derivitization with butanol·HCL. The masses of the parent to daughter transitions of butylated glycine monitored were 135.2 to 79.0 (M+3) and 132.2 to 76.0 (M).

The intra-assay precision of the triple quadrupole mass analyzer for measurements of erythrocyte-free glycine enrichment was between 3 and 5%. Accuracy for erythrocyte-free glycine enrichment was measured using enrichment curves. Enrichment curves were linear within the ranges of expected sample enrichment. The result of the enrichment curve was y = 0.732x + 0.042, r² = 0.99 for glycine enrichment.

Urinary sulfate

Urinary sulfate was measured using the method of Swaroop (24). Briefly, a standard curve was made using known concentrations of Ba²⁺ with sodium rhodizonate to form a red-colored complex that was measured at 520 nm against water. A known amount of sulfate was then added to form a BaSO₄ precipitate, which resulted in a diminished color and absorbance. The standard curve was obtained by plotting the concentration of sulfate on the x-axis and differences in absorbance between blank (water) and the corresponding standard on the y-axis. One mL of each urine sample was then diluted to 200 mL with distilled water from which 0.05 mL was removed and 2.0 mL of ethanol added and vortexed. To each tube, BaCl₂ and sodium rhodizonate were added and the tubes vortexed. The tubes were then allowed to stand for 10 min in the dark, after which the red color produced was measured at 520 nm against water. The difference of absorbance between blank and sample was read on the graph and corrected for the dilution.

Calculations

    Fractional synthesis rate of erythrocyte GSH. The FSR of erythrocyte GSH (FSR_GSH) was calculated using the precursor-product method of Jahoor et al. (25):

where (E_t7 – E_t5) was the increase in the isotopic enrichment of erythrocyte GSH between the 5th and 7th h of infusion as a result of the incorporation of the labeled glycine; E_RBC was the intracellular glycine enrichment at isotopic steady state; and (t7–t5) was the time interval between the 5th and 7th h, when the incorporation of glycine into GSH was measured:

where GSH _mass = the product of the cell volume (or cell number or cell protein) and the concentration of GSH in the cell. Hematocrit was calculated using the formula HCT (L/L) = (RBC x MCV)/1000.

Statistical analysis

The data were analyzed by repeated-measures ANOVA with the PROC MIXED procedure to assess the effects of cysteine intake on GSH FSR, ASR, and concentration. Other independent variables tested were cysteine intake, subject and order of cysteine intake, as well as the interaction between cysteine intake and order.

Repeated-measures ANOVA with the PROC MIXED and PROC general linear model procedures was also used to assess the effect of cysteine intake on urinary sulfate excretion.

When significant differences were identified, individual differences were assessed by post hoc analysis with Bonferroni correction for multiple comparisons. Differences were considered significant at P 0.05. Data were analyzed using SAS version 9.1 for Windows (SAS Institute).

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
The age and physical characteristics of the 4 healthy, male subjects who participated in the study are presented in Table 1. Isotopic steady state was achieved in the erythrocyte-free intracellular glycine pool by 5 h after the start of the isotope infusion. This was determined by the absence of a significant slope between data points from 5 to 7 h using ANOVA (Fig. 1). Therefore, FSR was calculated based on the linear incorporation of glycine into GSH during the last 2 h of the infusion.

View larger version (6K): [in this window] [in a new window]   FIGURE 1  The net tracer:tracee molar ratio (mol% above baseline) of erythrocyte-free glycine (A) and the net tracer-to-tracee enrichment of erythrocyte glutathione (B) during a 7-h continuous infusion of [U-13C2 –15N]glycine in young, healthy, adult men. Values are means ± SEM, n = 20 (5 observations in 4 men).

View larger version (15K): [in this window] [in a new window]   FIGURE 2  Effect of 5 levels of cysteine intake on GSH concentration, FSR of GSH, and the ASR of glutathionine in 4 healthy young men with set methionine intake of 14 mg·kg–1·d–1, n = 20. Values are means ± SEM, n = 20 (5 observations in 4 men).

View larger version (25K): [in this window] [in a new window]   FIGURE 3  Effect of varying cysteine intakes on urinary sulfate excretion (normalized to creatinine) of 4 healthy young men. Using repeated-measures ANOVA, cysteine intake affected sulfate excretion (P < 0.01; r2 = 0.92). Values are means ± SEM, n = 20 (5 observations in 4 men). Means without a common letter differ, P < 0.05.

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

The inability to measure GSH kinetics in liver or muscle due to practical and ethical considerations is a potential limitation of in vivo studies with healthy subjects. However, erythrocyte GSH kinetics has been shown to respond to dietary changes in disease (10,11), malnutrition (8), and even to small decreases in protein intake (7), demonstrating that erythrocyte GSH is a sensitive pool from which to detect changes in GSH metabolism. At a protein intake of 0.75 g·kg^–1·d^–1, set by WHO as the safe intake (26), Jackson et al. (7) showed decreased erythrocyte GHS synthesis when compared with a habitual protein intake of 1.13 g·kg^–1·d^–1. In fact, this higher protein requirement was recently confirmed by our group, showing that a safe intake of protein is closer to 1 g·kg^–1·d^–1 (27). In addition, cysteine supplementation at only 15 mg·kg^–1·d^–1 produced significantly increased GSH synthesis in symptom-free HIV individuals (10), suggesting that at the cysteine intakes used in the current study, significant changes in GSH metabolism should have been observed had they occurred.

Although we did not observe changes in GSH metabolism in response to feeding graded intakes of cysteine to healthy adults receiving the previously derived mean methionine (TSAA) requirement (Fig. 2), we observed a significant linear increase in urinary sulfate production in response to graded cysteine intakes (Fig. 3). The precise mechanisms governing all aspects of SAA metabolism are not yet completely understood. However, the increases in urinary sulfate excretion in the current study can be partly explained by work conducted by Stipanuk et al (13). Because excess cysteine is considered toxic, the liver regulates cysteine concentration within a small range and maintains a plasma concentration within a 2.5-fold range (13). Cysteine concentration has been found to be the key regulator of it own metabolism (4,5,15,28–30). When protein and/or SAA intake is low, -glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis is upregulated, resulting in a greater partitioning of SAA toward GSH synthesis. On the other hand, when protein and/or SAA intake is increased, cysteine dioxygenase the enzyme that catalyzes cysteine to sulfate and taurine, is upregulated, resulting in greater partitioning of cysteine toward sulfate production. Thus, increasing urinary sulfate observed in the present study appears to be due to increased partitioning of dietary cysteine toward catabolism in response to graded intakes of cysteine.

However, a closer look at the pattern of the isotope results reveals a similar rate of erythrocyte GSH synthesis at cysteine intakes of 0, 20, 30, and 40 mg·kg^–1·d^–1, with an almost 45% increase from 0 at a cysteine intake of 10 mg·kg^–1·d^–1. This increase in GSH synthesis, although not significant (P = 0.49), may be of biological importance, especially because other investigators have shown significant results at lower changes in synthesis rates (6,7). The observed similar GSH synthesis rates at cysteine intakes of 20 mg·kg^–1·d^–1 and above to that observed at a 0 cysteine intake suggest a return to baseline at the higher cysteine intakes. This is supported by previous data that show that increasing the level of protein (soy and casein), as well as the SAA methionine and cysteine, in the diets of rats or the addition of SAA to the culture medium of primary rat hepatocyte results in increased cysteine dioxygenase and decreased -glutamylcysteine activity (5,28,29). In healthy humans, a deficient protein and or SAA intake has been shown to significantly decrease GSH synthesis (6,7). Although no comparison studies of graded protein or SAA intake have been published, supplemental cysteine has been found to restore GSH synthesis to that of controls in diseased individuals (8,10).

A potential limitation of our study is the number of subjects studied. There is a possibility that the small sample size could have given rise to a type II error, especially with regard to the seeming biologically important increase in GSH synthesis at a cysteine intake of 10 mg/kg. Other investigators have found significant results with a considerably lower change in GSH synthesis rate (6,7) (15–27% compared with 45% in the current study). Because the change in GSH synthesis at the other intakes of cysteine was clearly not different, we think the results from the current study provided preliminary data to suggest that at the recommended daily allowance for SAA (24 mg·kg^–1·d^–1), GSH synthesis may not be limiting. To prove a real biologically important effect at a cysteine intake of 10 mg·kg^–1·d^–1, a subsequent study is needed that uses cysteine intakes in the range of 5–15 mg·kg^–1·d^–1 in addition to a methionine intake of 14 mg·kg^–1·d^–1.

This is the first study, to our knowledge, to report on GSH kinetics in response to varying cysteine intakes in healthy adult men. These data provide a point of focus for the design of future experiments on adequate SAA requirement for the maintenance of whole-body antioxidant status. These data also provide preliminary evidence that the currently derived TSAA requirement for healthy adults, when provided in the presence of an adequate protein intake (27), does not limit GSH synthesis in that population. Nevertheless, the need for TSAA may be higher in disease states where there is increased oxidative stress. Studies in rats have suggested an increase in cysteine requirement in septic rats compared with controls as evidenced by an increase in methionine transsulfuration and an increase in both methionine and cysteine flux compared with controls (31). A later study showing increased GSH synthesis in septic compared with control rats was used as a possible explanation for the increased cysteine requirement in sepsis (32). In humans with HIV and malnutrition, decreased GSH metabolism was ameliorated by cysteine supplementation, which served to increase GSH synthesis and concentration to that of control subjects. However, our current results show that typical cysteine intake in not rate limiting for GSH synthesis in healthy adult men, but rather that GSH synthesis is maximized at protein and TSAA intakes equivalent to those required for adequate protein synthesis in healthy adult subjects.

	ACKNOWLEDGMENTS

 
We thank Maria Maione and Roberta Gardiner, of the Clinical Investigation Unit at The Hospital for Sick Children, for setting up the isotope infusion and drawing the blood on the study day.

	FOOTNOTES

 
¹ Supported by grant number FRN 12928 from the Canadian Institutes for Health Research. Mead Johnson Nutritionals (Canada) donated the protein-free powder for the adaptation and experimental diets. First author (G.C.M.) was supported by scholarships from the Clinician Scientist Training Program, Hospital for Sick Children and The Strategic Training Institute in Health Research, Canadian Institute of Health Research.

² Author disclosures: G. Courtney-Martin, M. Rafii, L. J. Wykes, R. O. Ball, and P. B. Pencharz, no conflicts of interest.

⁸ Abbreviations used: AA, amino acid; ASR, absolute synthesis rate; FSR, fractional synthesis rate; FSR_GSH, FSR of erythrocyte GSH; GSH, glutathione; REE, resting energy expenditure; SAA, sulphur amino acid; TSAA, total sulphur amino acid.

Manuscript received 23 May 2008. Initial review completed 18 June 2008. Revision accepted 13 August 2008.

	LITERATURE CITED

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 

1. Stipanuk MH. Metabolism of sulfur-containing amino acids. Annu Rev Nutr. 1986;6:179–209.[Medline]

2. Griffith OW. Mammalian sulfur amino acid metabolism: an overview. Methods Enzymol. 1987;143:366–76.[Medline]

3. Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983;52:711–60.[Medline]

4. Stipanuk MH, Coloso RM, Garcia RA, Banks MF. Cysteine concentration regulates cysteine metabolism to glutathione, sulfate and taurine in rat hepatocytes. J Nutr. 1992;122:420–7.[Abstract/Free Full Text]

5. Kwon YH, Stipanuk MH. Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes. Am J Physiol Endocrinol Metab. 2001;280:E804–15.[Abstract/Free Full Text]

6. Lyons J, Rauh-Pfeiffer A, Yu YM, Lu XM, Zurakowski D, Tompkins RG, Ajami AM, Young VR, Castillo L. Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet. Proc Natl Acad Sci USA. 2000;97:5071–6.[Abstract/Free Full Text]

7. Jackson AA, Gibson NR, Lu Y, Jahoor F. Synthesis of erythrocyte glutathione in healthy adults consuming the safe amount of dietary protein. Am J Clin Nutr. 2004;80:101–7.[Abstract/Free Full Text]

8. Badaloo A, Reid M, Forrester T, Heird WC, Jahoor F. Cysteine supplementation improves the erythrocyte glutathione synthesis rate in children with severe edematous malnutrition. Am J Clin Nutr. 2002;76:646–52.[Abstract/Free Full Text]

9. Reid M, Badaloo A, Forrester T, Morlese JF, Frazer M, Heird WC, Jahoor F. In vivo rates of erythrocyte glutathione synthesis in children with severe protein-energy malnutrition. Am J Physiol Endocrinol Metab. 2000;278:E405–12.[Abstract/Free Full Text]

10. Jahoor F, Jackson A, Gazzard B, Philips G, Sharpstone D, Frazer ME, Heird W. Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate. Am J Physiol. 1999;276:E205–11.[Medline]

11. Reid M, Badaloo A, Forrester T, Jahoor F. In vivo rates of erythrocyte glutathione synthesis in adults with sickle cell disease. Am J Physiol Endocrinol Metab. 2006;291:E73–9.[Abstract/Free Full Text]

12. Hunter EA, Grimble RF. Cysteine and methionine supplementation modulate the effect of tumor necrosis factor alpha on protein synthesis, glutathione and zinc concentration of liver and lung in rats fed a low protein diet. J Nutr. 1994;124:2319–28.[Abstract/Free Full Text]

13. Stipanuk MH, Dominy JE Jr, Lee JI, Coloso RM. Mammalian cysteine metabolism: new insights into regulation of cysteine metabolism. J Nutr. 2006;136:S1652–9.[Abstract/Free Full Text]

14. Kurpad AV, Regan MM, Varalakshmi S, Gnanou J, Young VR. Daily requirement for total sulfur amino acids of chronically undernourished Indian men. Am J Clin Nutr. 2004;80:95–100.[Abstract/Free Full Text]

15. Lee JI, Londono M, Hirschberger LL, Stipanuk MH. Regulation of cysteine dioxygenase and gamma-glutamylcysteine synthetase is associated with hepatic cysteine level. J Nutr Biochem. 2004;15:112–22.[Medline]

16. Zello GA, Ball RO. The design and validation of a diet for studies of amino acid metabolism in adult humans. Nutr Res. 1990;10:1353–65.

17. Geigy C. Scientific tables. 7th ed. Basle (Switzerland): Ciba-Geigy Limited; 1970.

18. Food and Nutrition Board IoM. Dietary reference intakes: thiamin, riboflavin, niacin, vitamin B-6, vitamin B-12, pantothenic acid, biotin, and choline. Washington, DC: National Academy Press; 1998.

19. Kurpad AV, Regan MM, Varalakshmi S, Vasudevan J, Gnanou J, Raj T, Young VR. Daily methionine requirements of healthy Indian men, measured by a 24-h indicator amino acid oxidation and balance technique. Am J Clin Nutr. 2003;77:1198–205.[Abstract/Free Full Text]

20. Storch KJ, Wagner DA, Burke JF, Young VR. Quantitative study in vivo of methionine cycle in humans using [methyl-2H3]- and [1–13C]methionine. Am J Physiol. 1988;255:E322–31.[Medline]

21. Di Buono M, Wykes LJ, Ball RO, Pencharz PB. Total sulfur amino acid requirement in young men as determined by indicator amino acid oxidation with L-[1–13C]phenylalanine. Am J Clin Nutr. 2001;74:756–60.[Abstract/Free Full Text]

22. Rose WC, Coon MJ, Lockhart HB, Lambert GF. The amino acid requirements of man. XI. The threonine and methionine requirements. J Biol Chem. 1955;215:101–10.[Free Full Text]

23. Zello GA, Smith JM, Pencharz PB, Ball RO. Development of a heating device for sampling arterialized venous blood from a hand vein. Ann Clin Biochem. 1990;27:366–72.[Medline]

24. Swaroop A. A micromethod for the determination of urinary inorganic sulfates. Clin Chim Acta. 1973;46:333–6.[Medline]

25. Jahoor F, Wykes LJ, Reeds PJ, Henry JF, del Rosario MP, Frazer ME. Protein-deficient pigs cannot maintain reduced glutathione homeostasis when subjected to the stress of inflammation. J Nutr. 1995;125:1462–72.[Abstract/Free Full Text]

26. FAO/WHO/UNU. Energy and protein requirements. Report of a joint FAO/WHO/UNU expert consultation. World Health Organ Tech Rep Ser. 1985;724:1–206.[Medline]

27. Humayun MA, Elango R, Ball RO, Pencharz PB. Reevaluation of the protein requirement in young men with the indicator amino acid oxidation technique. Am J Clin Nutr. 2007;86:995–1002.[Abstract/Free Full Text]

28. Bella DL, Hahn C, Stipanuk MH. Effects of nonsulfur and sulfur amino acids on the regulation of hepatic enzymes of cysteine metabolism. Am J Physiol. 1999;277:E144–53.[Medline]

29. Bella DL, Hirschberger LL, Hosokawa Y, Stipanuk MH. Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo. Am J Physiol. 1999;276:E326–35.[Medline]

30. Stipanuk MH, Londono M, Lee JI, Hu M, Yu AF. Enzymes and metabolites of cysteine metabolism in nonhepatic tissues of rats show little response to changes in dietary protein or sulfur amino acid levels. J Nutr. 2002;132:3369–78.[Abstract/Free Full Text]

31. Malmezat T, Breuille D, Pouyet C, Buffiere C, Denis P, Mirand PP, Obled C. Methionine transsulfuration is increased during sepsis in rats. Am J Physiol Endocrinol Metab. 2000;279:E1391–7.[Abstract/Free Full Text]

32. Malmezat T, Breuille D, Capitan P, Mirand PP, Obled C. Glutathione turnover is increased during the acute phase of sepsis in rats. J Nutr. 2000;130:1239–46.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y. Lamers, B. O'Rourke, L. R Gilbert, C. Keeling, D. E Matthews, P. W Stacpoole, and J. F Gregory III Vitamin B-6 restriction tends to reduce the red blood cell glutathione synthesis rate without affecting red blood cell or plasma glutathione concentrations in healthy men and women Am. J. Clinical Nutrition, August 1, 2009; 90(2): 336 - 343. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Courtney-Martin, G. Articles by Pencharz, P. B. Search for Related Content PubMed PubMed Citation Articles by Courtney-Martin, G. Articles by Pencharz, P. B. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-METHIONINE CYSTEINE Medline Plus Health Information Diets