Although several studies have documented intra- and extracellular glutathione (GSH) deficiency in asymptomatic human immunodeficiency virus (HIV) infection, the mechanisms responsible for the altered GSH homeostasis remain unknown. To determine whether decreased synthesis contributes to this alteration of GSH homeostasis, a primed-constant infusion of [2H2]glycine was used to measure the fractional and absolute rates of synthesis of GSH in five healthy and five symptom-free HIV-infected subjects before and after supplementation for 1 wk withN-acetylcysteine. The erythrocyte GSH concentration of the HIV-infected group was lower (P < 0.01) than that of the control group (1.4 ± 0.16 vs. 2.4 ± 0.08 mmol/l). The smaller erythrocyte GSH pool of the HIV-infected group was associated with a significantly slower (P < 0.01) absolute synthesis rate of GSH (1.15 ± 0.14 vs. 1.71 ± 0.15 mmol ⋅ l−1 ⋅ day−1) compared with controls. Cysteine supplementation elicited significant increases in both the absolute rate of synthesis and the concentration of erythrocyte GSH. These results suggest that the GSH deficiency of HIV infection is due in part to a reduced synthesis rate secondary to a shortage in cysteine availability.
- glutathione synthesis
- human immunodeficiency virus infection
- stable isotope
glutathione (γ-glutamylcysteinylglycine, GSH), a tripeptide present in high concentrations in all mammalian cells, has many critical protective and metabolic functions. It detoxifies electrophilic metabolites of xenobiotics and protects cells from the toxic effects of free radicals and reactive oxygen compounds (1). It is also important in the immune response to infections, because it is necessary for lymphocyte proliferation, antibody-dependent and cell-mediated cytotoxicity, and protection of lymphocytes against superoxides produced to destroy invading pathogens (6, 23).
There have been numerous reports of GSH deficiency in human immunodeficiency virus (HIV) infection. The concentration of GSH is lower in plasma, lung epithelial lining fluid, and peripheral blood mononuclear cells (PBMC) of HIV-infected individuals (2, 4, 22). Moreover, in vitro studies have shown that low GSH levels impair T cell function (22) and also promote HIV expression (17), suggesting a link between GSH deficiency and progression of HIV disease. This was confirmed by a recent report of poor survival rate of HIV-infected individuals with lower GSH levels and improved survival when GSH was replenished and maintained (12).
Despite numerous reports of the compromised GSH status of HIV infection, the in vivo kinetic mechanism(s) responsible for glutathione deficiency has not been determined. Two obvious general mechanisms are suppressed synthesis and/or increased consumption relative to synthetic capacity. Inability to sustain the normal rate of synthesis of GSH can result from either a shortage in the supply of one of the precursor amino acids or a defect in its biosynthetic pathway. Alternatively, because GSH levels fall under conditions of increased oxidative stress, such as HIV infection, it can be proposed that a persistent oxidative load leads to an accelerated rate of consumption of GSH that is not matched by an equal increase in the rate of synthesis of the tripeptide.
To determine which of these mechanisms is responsible for the altered GSH homeostasis of HIV infection, it is necessary to measure glutathione kinetics in vivo in HIV-infected and uninfected individuals. This is now possible because we have developed a stable isotope tracer method to measure GSH synthesis rate in vivo (15), which was employed in the present study to measure GSH synthesis rates in the erythrocytes of HIV-infected and normal healthy subjects.
Five male subjects were recruited from HIV-infected patients attending the St. Stephen’s Clinic at Chelsea and Westminster Hospital (London, UK). HIV infection was confirmed by enzyme-linked immunosorbent assay. Based on their medical history, all of the HIV-infected subjects had previously met the Centers for Disease Control (CDC) criteria for acquired immunodeficiency syndrome (AIDS) (3). At the time of the study, however, none had any signs or symptoms of secondary infections. Five normal healthy subjects (3 males, 2 females) were recruited from the staff of the Clinical Nutrition and Metabolism Unit, University of Southampton (Southampton, UK). They were in good health on the basis of a complete medical history and physical examination. The physical characteristics of the subjects are shown in Table 1. All subjects were within the normal range of ideal body weight.
Diet and drug treatment.
As shown in Table 1, the two groups had similar habitual energy and protein intakes as assessed by the 3-day weighed record method and food composition tables. The intakes of methionine plus cysteine, serine, and selenium (nutrients that affect GSH synthesis) were calculated from food composition tables (5, 13). There was no difference between the methionine, cysteine, serine, and selenium intakes of the two groups (Table 1). At the time of the study, all HIV-infected subjects were being treated with antiviral drugs. Each subject was receiving one or more of the following drugs: loviride, cetirizine, atenolol, forceval, stanozolol, zidovudine, and itraconazole.
Hematology, CDC classification.
The hematocrit of the HIV-infected group, 41.6 ± 3%, was normal and not different from that of the controls, 40.5 ± 3%. The total white blood cell (WBC) count, lymphocyte profile, and CDC classification of the HIV-infected subjects are shown in Table2. Only one subject had a WBC count below the normal range. All of the HIV-infected subjects had previous clinical conditions attributed to HIV infection that met the CDC criteria for AIDS (3). As shown in Table 2, one of the HIV-infected subjects had a CD4+ T cell count >500 cell/μl (CD4+ T cell category 1), three had CD4+ T cell counts >200 cell/μl (CD4+ T cell category 2), and one had a CD4+ T cell count <200 cell/μl (CD4+ T cell category 3) (3).
The protocol was approved by the ethical committees of all participating institutions. Signed informed consents of all subjects were obtained after the nature of the study had been explained to them. The HIV-infected subjects were studied at the Chelsea and Westminster Hospital, and the healthy subjects were studied in the metabolic ward of the Clinical Nutrition and Metabolism Unit at the University of Southampton.
Isotope infusion protocol.
The rate of synthesis of erythrocyte GSH was measured from the rate of incorporation of [2H2]glycine into the tripeptide by use of erythrocyte-free glycine isotopic enrichment to represent the enrichment of the glycine precursor pool from which erythrocytes synthesize GSH.
After a 10-h overnight fast, the subjects’ weights and heights were measured, and venous catheters were inserted under local anesthetic into each forearm. One catheter was used for infusion of isotope and the other for blood sampling. The hand and forearm with the sampling catheter were wrapped in a heating pad to arterialize venous blood. A sterile solution of [2H2]glycine (Cambridge Isotope Laboratories, Woburn, MA) was prepared in 4.5 g/l of saline and infused continuously for 6 h at 15 μmol ⋅ kg−1 ⋅ h−1through the catheter in one forearm after a priming dose of 15 μmol/kg was injected. A 6-ml blood sample was drawn before the start of the infusion and at hourly intervals after 2 h of infusion.
The exact infusion protocol was repeated in the HIV-infected subjects after 1 wk of dietary supplementation with 20 mg ⋅ kg−1 ⋅ day−1of N-acetylcysteine (NAC). This amount of NAC supplied 15 mg ⋅ kg−1 ⋅ day−1of cysteine, which represented a 33% increase in the dietary intake of cysteine (see Table 1).
A 2-ml aliquot of each blood sample was placed immediately in an equal volume of isotonic ice-cold monobromobimane (MBB, obtained from Calbiochem, La Jolla, CA) buffer (pH 7.4) solution (in mM: 5 MBB, 17.5 Na2EDTA, 50 potassium phosphate, 50 serine, and 50 boric acid) for GSH derivatization and isolation. The whole blood-MBB buffer mixture was centrifuged immediately at 4°C, and the supernatant was removed. The packed red blood cells were lysed immediately with 1 ml of acetonitrile, 2 ml of MBB buffer were added, and the mixture was left in the dark at room temperature for 20 min for development of the erythrocyte GSH-MBB derivative. Proteins were then precipitated with ice-cold 1 mol/l perchloric acid, and the supernatant solution was stored at −70°C until analyzed. Hematocrit was determined on each blood sample with a Micro Hematocrit Centrifuge (Damon/IEC Division, Needham Heights, MA).
The remainder of the blood sample also was centrifuged immediately at 4°C, and the plasma was stored at −70°C for later analysis. The packed erythrocytes were washed several times with ice-cold 9 g/l saline, and proteins were precipitated with ice-cold 0.6 mol/l trichloroacetic acid. The protein-free supernatant containing the free amino acids was stored at −70°C for later determination of the isotopic enrichment of the erythrocyte-free glycine.
Plasma amino acid concentrations were determined by ion exchange HPLC. The concentration measurement of erythrocyte-free cysteine and GSH, plasma GSH, and the isolation of erythrocyte-free GSH was performed on a Hewlett-Packard 1090 HPLC equipped with a model HP 1046A fluorescence detector (Hewlett-Packard, Avondale, PA) as previously described (15). Reverse-phase separation of thiol compounds was performed on an ODS Hypersil column, 5 μm, 4.6 × 200 mm (Hewlett-Packard). Elution of the thiols was accomplished over 30 min by a linear gradient of 3% acetonitrile to 13.5% acetonitrile in 1% acetic acid in water, pH 4.25, at a flow rate of 1.1 ml/min. Standards included known concentrations of cysteine, GSH, andd-penicillamine (Sigma, St. Louis, MO) prepared and diluted in the same manner as the samples. Under these conditions, cysteinyl-MBB elutes at 11.98, and GSH-MBB elutes at 15.5 min, as shown in Fig. 1. The GSH-containing fractions were collected from 15 to 16 min on a fraction collector and processed for gas chromatography-mass spectrometric (GC-MS) analysis of GSH-bound glycine. The GSH-containing fractions were dried, and the peptide was hydrolyzed for 4 h in 6 mol/l HCl at 110°C.
Erythrocyte-free glycine was extracted from the protein-free supernatants and from 50 μl of plasma by cation exchange chromatography. Glycine from GSH, the erythrocytes, and plasma were converted to the n-propyl ester heptafluorobutyramide derivative, and the isotope ratio was measured by negative chemical ionization GC-MS on a model 5989B GC-MS system (Hewlett-Packard, Palo Alto, CA) equipped with a 30-m HP-5 column, monitoring ions at mass-to-charge ratios ofm/z 293–295.
Calculations and statistics.
The fractional synthesis rate (FSR) of erythrocyte-GSH was calculated according to the precursor-product equation, as described previously (15) where − is the increase in the isotope ratio of erythrocyte GSH-bound glycine over the period t 6 tot 4 h of the infusion, when the isotope ratio of erythrocyte-free glycine (IRrbc) had reached a steady state. The absolute synthesis rate (ASR) of erythrocyte GSH was calculated as the product of the erythrocyte GSH concentration and the FSR, using the equation ASR = erythrocyte GSH concentration × FSRGSH. The units of ASR are expressed as millimoles per liter of packed erythrocytes per day (mmol ⋅ l−1 ⋅ day−1).
The standard steady-state equation was used to calculate the flux of glycine in the circulation (15) where IEInf and IEplat are the isotope enrichments of the tracer amino acid in the infusate and in plasma at isotopic steady state, and D is the rate of infusion of the tracer in micromoles per kilogram body weight per hour (μmol ⋅ kg body wt−1 ⋅ h−1). The units of flux are micromoles per kilogram per hour (μmol ⋅ kg−1 ⋅ h−1).
Data are expressed as the means ± SE for each group. Differences between groups were detected by the nonpairedt-test. The pairedt-test was used to detect differences in the data obtained before and after supplementation with NAC in the HIV-infected group. A probability of 5% (P < 0.05) was assumed to represent statistical significance.
There was no difference between the habitual dietary intakes of energy and protein between the two groups (Table 1). Similarly, there was no difference in the intakes of methionine, cysteine, glycine, serine, and selenium, nutrients that are known to have an effect on GSH kinetics, between the two groups (Table 1).
There was no difference in plasma glycine, methionine, and cysteine concentrations between the two groups of subjects (Table3), although there was a trend toward lower cysteine and methionine concentrations in the HIV-infected group. One week of NAC supplementation did not change the concentration of any of these amino acids in the HIV-infected group. RBC-free cysteine concentration was, however, significantly lower (P < 0.05) in the HIV-infected group compared with the control group, and after 1 wk of NAC supplementation it increased significantly (P < 0.01) to a level that was not different from the value of the control group. Similarly, plasma free GSH concentration was significantly lower (P < 0.05) in the HIV-infected group compared with the control group, and after 1 wk of NAC supplementation it increased significantly (P < 0.01) to a level that was higher but not significantly different from the value of the control group.
The net tracer-to-tracee molar ratio (mol % above baseline) of free glycine from the deproteinized extract of RBC reached a steady state after 3 h of the isotope infusion in all three studies, and there was a linear increase in the amount of labeled glycine incorporated into GSH extracted from RBC during this period of time (Fig.2). Hence, the FSR of GSH was calculated from the rate of incorporation of labeled glycine into the peptide during the last 2 h of the isotope infusion.
As shown in Fig.3 A, the erythrocyte GSH concentration of the HIV-infected group, 1.4 ± 0.16 mmol/l, was significantly lower (P < 0.01) than that of the control group, 2.4 ± 0.08 mmol/l. Although there was no difference in the FSR of erythrocyte GSH between the two groups (Fig. 3 B) because of the smaller erythrocyte GSH pool of the HIV-infected group, their ASR of GSH was significantly slower (P < 0.01; Fig. 3 C). After 1 wk of NAC supplementation, the erythrocyte GSH concentration of the HIV-infected group increased significantly (P < 0.05) to 1.6 ± 0.1 mmol/l (Fig.3 A). Concurrently, there was a 25% increase in the FSR of GSH (P = 0.07), and the amount of synthesized GSH increased significantly (P < 0.05) from 1.15 ± 0.14 to 1.61 ± 0.3 mmol ⋅ l−1 ⋅ day−1, a rate of synthesis not different from that of the control group.
The glycine flux of the HIV-infected group, 181 ± 14 μmol ⋅ kg−1 ⋅ h−1, was almost identical to that of the control group, 179 ± 7.6 μmol ⋅ kg−1 ⋅ h−1, and it did not change significantly after NAC supplementation, 165 ± 10 μmol ⋅ kg−1 ⋅ h−1. The ratios of the isotopic enrichments of intracellular glycine to plasma glycine were 0.37 ± 0.02 for the control group and 0.30 ± 0.02 and 0.32 ± 0.04 for the first and second studies of the HIV-infected group.
Although the control and HIV-infected groups were not evenly matched for gender, our results suggest that this was not an important factor because GSH kinetics were not unduly influenced by the sex of the subject. That is, the individual data obtained for the two female controls were not different from those of the three male controls. For example, mean data for female vs. male for GSH concentration was 2.6 vs. 2.4 mmol/l, for FSR was 71.5 vs. 71%/day, and for ASR was 1,833 vs. 1,780 μmol ⋅ l−1 ⋅ day−1.
The fact that GSH status of HIV-infected individuals is compromised, and that this may play a role in the pathogenesis of the disease, is well documented. However, the mechanism(s) responsible for GSH deficiency has remained elusive because of the lack of a technique to measure GSH synthesis rate in vivo. We used such a method in this study to determine the rates of synthesis of GSH in the erythrocytes of HIV-infected and healthy noninfected individuals. The data reported here demonstrate that erythrocyte GSH ASR is considerably lower in HIV-infected than in normal individuals. They also demonstrate that supplementation of HIV-infected subjects with NAC restores the erythrocyte GSH ASR to that observed in controls.
Although it may be argued that findings in erythrocytes are not necessarily relevant to those in other types of cells, the 40% reduction in both plasma and erythrocyte GSH concentration observed in the present study is almost identical to the degree of depletion reported by others for plasma (11), CD8 and CD4 T cells (20, 22), and lung epithelial lining fluid (2), suggesting that HIV infection elicits a generalized alteration of GSH homeostasis. Hence, the changes in GSH kinetics observed in erythrocytes are likely to reflect the changes in other cell types. Furthermore, in a previous study in piglets, we showed that changes in erythrocyte GSH concentration and rate of synthesis induced by protein deficiency and by the stress of an inflammatory stimulus reflected changes in the gut mucosa (15). Similarly, in other conditions such as diabetes mellitus, it is well established that the lower GSH concentration of erythrocytes is accompanied by lower GSH concentrations in retinal tissue and the liver (9, 16, 18, 19, 21). Hence, most studies of GSH metabolism in diabetes mellitus have been performed on erythrocytes (e.g., Refs. 16,21).
The concentration of any metabolite represents the balance between its rate of synthesis and the rate at which it is consumed. Hence, to understand the mechanisms responsible for the maintenance and/or depletion of GSH, either its synthetic rate or its rate of consumption must be known. Because of the likelihood that HIV infection results in an increased oxidative load, it has been suggested that increased conversion of GSH to its oxidized form glutathione disulfide (GSSG), which can be exported from the cell, leads to increased consumption of GSH (24). The data reported here do not necessarily refute this possibility. However, they demonstrate that erythrocyte GSH synthesis is lower in HIV-infected subjects and that this can be increased to control values by NAC supplementation. The fact that NAC supplementation increases erythrocyte GSH concentration in parallel with the increase in GSH synthesis suggests that the rate of GSH consumption of symptom-free HIV-infected individuals differs minimally, if at all, from that of healthy individuals.
The in vitro finding that erythrocyte GSH peroxidase activity of symptom-free HIV-infected individuals is not different from that of controls (20) also suggests that HIV infection alone does not increase GSH oxidation appreciably. In another study of GSH metabolism in symptom-free HIV-infected individuals, it was also concluded that GSH consumption is not increased (11). On the basis of the observation that infused GSH was removed more slowly by symptom-free HIV-infected individuals, Helbling et al. (11) ruled out an increased consumption of GSH in symptom-free HIV infection. They concluded that GSH is low in HIV infection because of an overall decrease in its synthesis rate (11). Regardless of rates of GSH consumption, which are likely to increase with secondary infections, our finding that the lower erythrocyte GSH concentration of asymptomatic HIV-infected individuals is associated with lower rates of synthesis of the tripeptide demonstrates conclusively that impaired GSH synthesis contributes to the GSH deficiency associated with HIV infection.
On the basis of the observation that GSH concentrations decrease as protein intake decreases from an adequate to a deficient level (10,14), it is reasonable to assume that the reduction in erythrocyte GSH synthesis represents a response to a restricted intracellular supply of the component amino acids. Two of the component amino acids of GSH, i.e., glycine and cysteine, can become conditionally essential in certain pathological conditions (10). However, because plasma glycine flux was virtually identical in HIV-infected and control subjects, it seems unlikely that glycine supply was limiting in the HIV-infected subjects. In addition, the finding that there was no difference between the ratios of the isotopic enrichment of intracellular glycine to plasma glycine of the two groups suggests that there was no impairment in the transport of glycine into the erythrocytes of HIV-infected individuals. Rather, the lower intracellular RBC cysteine concentration of the HIV-infected group, together with the increased GSH concentration and synthesis rate in response to NAC supplementation, suggests that an inadequate intracellular supply of cysteine was the limiting factor. Furthermore, because NAC supplementation raised RBC GSH synthesis of the HIV-infected subjects to the same rate observed in controls, it is likely that the lower rate of GSH synthesis in HIV-infected individuals is due to a shortage of intracellular cysteine rather than impairment of the GSH biosynthetic pathways. Several studies have also reported lower intra- and extracellular cysteine concentrations in HIV infection, suggesting reduced cysteine supply for GSH synthesis (4, 6, 7, 8, 22). The surfeit level of habitual protein intake, 1.6 g ⋅ kg−1 ⋅ day−1, of the HIV-infected subjects suggests that any reduction in intracellular cysteine availability cannot be due to a compromised dietary intake of protein, because the methionine plus cysteine intakes of the HIV-infected subjects were not different from those of the controls (Table 1). Rather, this finding suggests that the lower RBC intracellular cysteine concentrations observed in patients with HIV infection result from a failure to synthesize this amino acid in sufficient quantities to satisfy ongoing needs, including the demand for glutathione synthesis.
In the present study, 1 wk of NAC supplementation caused a 93% increase in RBC intracellular cysteine concentration but had no effect on plasma cysteine concentration. We are not aware of any study of the long-term effect of NAC administration on plasma and RBC intracellular cysteine concentrations. In a study of the acute effect of a single dose of NAC on plasma and PBMC cysteine concentrations in HIV-infected subjects, de Quay et al. (4) reported transient increases in concentration that returned to baseline by 4 h. The failure of NAC supplementation to elicit a change in plasma cysteine concentration was therefore not surprising, because in the present study the subjects did not receive any NAC supplements during the course of the infusion protocol. The replenishment of intracellular cysteine concentrations in response to NAC in this study, the transient increase in plasma and intracellular cysteine concentrations in other studies (4, 7, 22), and the marked stimulation of GSH synthesis in response to NAC supplementation in this study support the proposal that NAC alleviates GSH by restoring intracellular cysteine supply for GSH synthesis.
Although the present study did not address the underlying mechanism responsible for the decrease in intracellular cysteine supply in the HIV-infected subjects, data from several studies in the literature (6,7, 8) suggest that the rate of synthesis of cysteine is impaired. The supply of any nonessential amino acid represents the balance between its dietary intake plus rate of synthesis and the rate at which the amino acid is utilized. In the present study, the HIV-infected subjects had a surfeit dietary intake of cysteine, suggesting that the lower RBC cysteine concentration was due to a reduction in the rate of synthesis. Cysteine can be synthesized de novo from methionine or from the reduction of cystine. Droge and co-workers (6, 8) have proposed that, in HIV infection, there is a decrease in intracellular cysteine synthesis from cystine because intracellular transport of cystine is competitively inhibited by higher extracellular glutamate, which uses the same membrane transport system as cystine. They based this proposal on the observation that HIV-infected patients have elevated plasma glutamate concentrations and the in vitro observation in cultures of macrophages, peripheral blood monocytes, and murine fibroblast cells that higher concentrations of glutamate in the medium inhibited the intracellular transport of cystine and the release of cysteine (6-8).
Because GSH is essential for lymphocyte proliferation and function, restricts HIV replication, and, above all, improves the survival of HIV-infected individuals (12, 24), the results of this study suggest that HIV-infected individuals should be treated with cysteine to restore and maintain GSH homeostasis. This may be particularly important for those with secondary infections. Although the data reported here provide no evidence for (or against) a rate of increased GSH consumption in the pathogenesis of GSH deficiency in symptom-free HIV-infected patients, secondary infections are likely to generate a further increase in oxidative load, which in turn will increase GSH consumption, leading to further depletion of GSH. Further studies of GSH metabolism are therefore necessary in this condition.
Finally, although the present data support our proposal that GSH depletion results from impaired synthesis secondary to a reduction in intracellular cysteine, we cannot rule out the possible involvement of other mechanisms. First, it is possible that impaired GSH reductase activity may be responsible for the lower intracellular levels of GSH in the HIV-infected subjects. If this mechanism is involved in the lowering of GSH, however, one would expect both intracellular and plasma GSSG concentrations to be higher in HIV-infected individuals. To the contrary, the studies by Buhl et al. (2) and Helbling et al. (11) have shown that both intracellular and plasma GSSG concentrations are lower in HIV-infected individuals compared with the values of controls. Second, because GSH is irreversibly consumed during the hepatic detoxification of toxins and drugs, it is possible that there is an increased consumption of GSH in HIV-infected patients because of the intensive drug therapies of these patients. Unfortunately, there are no data in the literature on the possible effects of these drugs on GSH utilization. The finding of Helbling et al. (11), however, that infused GSH was removed more slowly by symptom-free HIV-infected individuals, rules out an increased consumption of GSH in symptom-free HIV infection.
The authors thank Leslie Loddeke for editorial assistance and O’Brian Smith for statistical advice.
Address for reprint requests: F. Jahoor, Children’s Nutrition Research Center, Dept. of Pediatrics, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030–2600.
This research was supported with funds from the US Department of Agriculture/Agricultural Research Service, the Wessex Medical Trust, and the Charing Cross and Westminster Research Fund.
This is a publication of the US Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.
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