JAIDS Journal of Acquired Immune Deficiency Syndromes:
Plasma Ascorbate Deficiency Is Associated With Impaired Reduction of Sulfamethoxazole-Nitroso in HIV Infection
Trepanier, Lauren A. DVM, PhD*; Yoder, Andrea R.*; Bajad, Sunil PhD*; Beckwith, Michelle D. RN†; Bellehumeur, Jennifer L. RN†; Graziano, Frank M. MD, PhD†
From the *Department of Medical Sciences, School of Veterinary Medicine; and the †Medical Sciences Center, Medical School, University of Wisconsin–Madison, Madison, WI.
Received for publication September 22, 2003; accepted March 29, 2004.
Funded by a grant from the National Institutes of Health (GM61753).
The authors have no conflicts of interest, financial or otherwise, related to the data presented in this article.
Reprints: Lauren A. Trepanier, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, 2015 Linden Drive, Madison, WI 53706-1102 (e-mail: LATrepanier@svm.vetmed.wisc.edu).
Objective: The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions.
Methods: Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro.
Results: Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 ± 22.3 μM) than in healthy subjects (54.8 ± 22.3 μM; P = 0.0005) and patients taking 500–1000 mg of ascorbate daily (82.5 ± 26.3 μM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98 ± 0.32 mM) than in healthy subjects (1.45 ± 0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4 ± 3.9 μM) than in controls (10.3 ± 4.3 μM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4 ± 10.5%) than in healthy controls (4.0 ± 6.2%), even in the subset of patients taking ascorbate (8.4 ± 9.4%).
Conclusions: Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500–1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.
Sulfamethoxazole (SMX) is the drug of choice for the prevention and treatment of Pneumocystis jiroveci (formerly carinii) pneumonia in immunocompromised patients, particularly those with AIDS. SMX is more effective and less expensive than alternative drugs such as dapsone, pentamidine, and atovaquone. 1–5 The use of SMX, however, is limited by the development of delayed hypersensitivity reactions in some patients. These hypersensitivity reactions most commonly manifest as fever and morbilliform skin rash but may include neutropenia, thrombocytopenia, hepatopathy, pneumonitis, nephritis, or Stevens–Johnson syndrome. 6–9 The reported incidence of hypersensitivity reactions leading to sulfonamide discontinuation in HIV-infected patients is 25%–55%, 8,10–12 which is much higher than that reported for HIV-negative patients given sulfonamides (~3%). 13,14
The pathogenesis of sulfonamide hypersensitivity is not completely understood, but there is considerable evidence to suggest that its oxidative metabolite, SMX-nitroso (SMX-NO), is the proximate toxin (Fig. 1). SMX-NO, unlike the parent compound, can haptenize lymphocytes, 15–17 is cytotoxic in vitro, 18,19 and is immunogenic in vivo. 18,20 In addition, mono-nuclear leukocytes from HIV-negative patients with sulfonamide hypersensitivity show increased susceptibility to cytotoxicity in the presence of this metabolite or its immediate precursor, SMX-hydroxylamine (SMX-HA), compared with nonhypersensitive patients, and this susceptibility is also seen in related family members never exposed to sulfonamides. 21,22 This has lead to the hypothesis that HIV-negative patients with sulfonamide hypersensitivity have a genetic defect in the detoxification of SMX-NO or SMX-HA.
In HIV-positive patients, enhanced in vitro leukocyte cytotoxicity has also been associated with prior or subsequent development of sulfonamide hypersensitivity, 23,25 although 1 study in which HIV-positive patients were sampled >1 year after clinical toxicity did not show a difference between hypersensitive and tolerant patients. 26 Therefore, HIV-infected patients may also have a defect in the detoxification of sulfonamide metabolites, but this defect, if present, is likely to be acquired and may be labile.
The primary pathway(s) of the detoxification of SMX-NO have not been completely characterized; however, both glutathione (GSH) and cysteine have been shown to mediate the reduction of SMX-NO back to either the HA or the parent compound, depending on stoichiometry (Fig. 1). 15,27,28 Specifically, GSH forms a semimercaptal conjugate with SMX-NO, and this can decompose to SMX-HA (if GSH is in excess; eg, mM concentrations) or be converted to SMX via a sulfinamide intermediate (if GSH is limiting). 27 SMX-HA can then be further reduced enzymatically to the parent sulfonamide 29,30 or excreted unchanged in the urine. 31 Ascorbate has been shown to stabilize SMX-HA and prevent its auto-oxidation to SMX-NO, 30,32 but its direct role in nitroso reduction has not been explored.
In preliminary experiments in vitro, we observed that the nonenzymatic reduction of SMX-NO was mediated by ascorbate at a 1:1 stoichiometry. Because ascorbate deficiency has been reported in HIV infection, 33,34 the purpose of these studies was to determine the role of ascorbate deficiency in HIV-positive patients in the defective detoxification of SMX-NO. In addition, we determined the relationships among plasma ascorbate, ascorbate supplementation, intracellular GSH, extracellular cysteine, and dehydroascorbate (the oxidized form of ascorbate).
Monobromobimane was obtained from Molecular Probes (Eugene, OR). SMX-HA and SMX-NO standards were purchased from Dalton Chemicals (Toronto, CA). Phosphate-buffered saline (pH 7.4) and Hanks balanced salt solution were obtained from Gibco-BRL (Grand Island, NY), and lymphocyte separation media was obtained from Mediatech (Herndon, VA). Tris[2-carboxyethyl] phosphine was purchased from Pierce Biotechnology (Rockford, IL). High-performance liquid chromatography (HPLC) solvents and perchloric acid were obtained from Fisher Scientific (Hanover Park, IL). Ascorbate, dehydroascorbate, dithiothreitol, EDTA, N- ethylmorpholine, metaphosphoric acid, Tris HCl, SMX, 5-sulfosalicyclic acid, trichloroacetic acid, and all other reagents were obtained from Sigma Chemical Company (St. Louis, MO).
Healthy subjects were recruited from the university student and staff populations and were excluded if they were taking ascorbate supplements. Patients with HIV infection were recruited from the University of Wisconsin–Madison Hospital and Clinics Outpatient HIV Clinic. Patients were eligible for enrollment if they were 18 years of age or older, had stable enough conditions to be treated as an outpatient, were not part of the prison-based patient population, and were willing to participate. Data including age, race, sex, smoking status, and history of current medications including multivitamin and vitamin C supplementation were obtained for each subject. Dietary histories were not obtained. In addition, laboratory data, including absolute CD4+ and CD8+ lymphocyte counts by flow cytometry and viral load by bDNA assay (Versant HIV-1 RNA 3.0 assay; Bayer), were also recorded for patients. A research nurse encoded all patient data to protect patient confidentiality. All subjects gave written informed consent before participating in the study, and the University Health Sciences Human Subjects Committee approved all protocols.
Blood samples from patients and healthy subjects were collected concurrently throughout the study to control for seasonal changes in plasma ascorbate. 35 Twenty milliliters of whole blood was collected into 2 10-mL heparinized tubes. Tubes were placed on ice immediately, and 10 mL of the blood was treated within 1 minute of phlebotomy with 150 μL of monobromobimane (180 mM) in acetonitrile (diluted to 1 mL in phosphate-buffered saline before addition to avoid erythrocyte (RBC) lysis from the acetonitrile). 36 This sample, treated with monobromobimane, was used for cysteine and GSH (thiol) measurements. The second untreated 10-mL sample of blood was used for ascorbate assays. Blood was centrifuged at 14,000 g at 4°C for 1 minute to harvest plasma, buffy coat, and packed RBCs. Plasma was harvested on ice and analyzed immediately, or frozen at −80°C, for ascorbate and cysteine determinations. RBCs were processed as described below for GSH determinations.
Plasma Ascorbate and Dehydroascorbate Assays
Plasma samples were prepared for ascorbate and dehydroascorbate measurements via a modification of the method of Lykkesfeldt. 37 Plasma was analyzed the same day as sampling or after storage at −80°C overnight; all frozen samples were thawed on ice. Total ascorbate was measured by adding 15 μL of cold 50% metaphosphoric acid to 150 μL of heparinized plasma to precipitate proteins. After vortexing and centrifugation at 14,000 g for 1 minute, 100 μL of the supernatant was transferred to microfuge tubes containing 50 μL of 3 mM tris[2-carboxyethyl] phosphine as a reducing agent. Samples were vortexed and left at room temperature for 6 minutes to allow reduction. Two hundred microliters of cold 5% meta-phosphoric acid was then added to stabilize the ascorbate followed by centrifugation, and the supernatant was analyzed for total ascorbate by HPLC. Reduced ascorbate was measured identically, except that DW was substituted for tris[2-carboxyethyl] phosphine. Dehydroascorbate was measured as the difference between total and reduced ascorbate. Using this method, tris[2-carboxyethyl] phosphine reduction led to >95% recovery of dehydroascorbate as ascorbate in spiked normal plasma, even after storage at −80°C overnight. Ascorbate was stable in whole blood or plasma kept on ice for >3 hours and in plasma at −80°C for at least 1 week; all patient and control samples were separated, processed, and analyzed within these time frames.
Samples for ascorbate analysis were loaded into pre-chilled autosampler vials and were analyzed in a refrigerated autosampler unit (Beckman Model 508). Ascorbate was quantitated by HPLC using a C18 Ultrasphere ODS column (4.6 mm × 25 cm; Beckman Coulter, Fullerton, CA) and ultraviolet detection at 254 nm. Gradient elution was performed with 100% mobile phase A (0.05% triethylamine and 1.0% glacial acetic acid in water), changing to 80% mobile phase B (acetonitrile) over 20 minutes, at 2 mL/min, yielding a retention time for ascorbate of 2.1 minutes. The gradient was introduced so that the other compounds of interest (thiols) could be measured using the same method. The limit of quantitation for ascorbate was 1 μM, with an intraassay CV of 1.2%–9.1% and an inter-assay CV of 2.7%–8.1% within the range of concentrations relevant to this study.
Plasma Cysteine Assay
Plasma cysteine was assayed using a modification of the methods of Naisbitt et al 36 and Jahoor et al. 38 Five hundred microliters of bromobimane-treated plasma was thawed and combined with 7.5 μL of monobromobimane (180 mM) in acetonitrile diluted with 42.5 μL of phosphate-buffered saline. Proteins were precipitated with 55 μL of 50% 5-sulfosalicyclic acid in 500 μM dithiothreitol. After centrifugation, 250 μL of the supernatant was mixed with 3 μL of 50% 5-sulfosalicyclic acid in dithiothreitol, 6.4 μL of N-ethylmorpholine, and 10 μL of acetonitrile. After incubation in the dark for 5 minutes at 37°C, 10 μL of trichloroacetic acid was added, and the samples were assayed for cysteine by HPLC using the same column and mobile phase gradient as described for ascorbate, except for the use of fluorescence detection (ex, 394 nm; em, 480 nm) (FP 1520; Jasco, Inc., Easton, MD). The retention time of cysteine using this method was 5.2 minutes. The limit of quantitation for cysteine was 1 μM, with an intraassay CV of 0.9%–7.1% and an interassay CV of 7.6%–14.6%. Cysteine was stable in plasma at −80°C for at least 1 week.
RBC GSH Assay
RBC GSH was assayed using a modification of the methods of Naisbitt et al 36 and Jahoor et al. 38 Briefly, 500 μL of packed RBCs was harvested from monobromobimane-treated heparinized blood and placed in an amber tube containing an additional 37.5 μL of 180 mM monobromobimane plus 712.5 μL of phosphate-buffered saline. The mixture was incubated in the dark at 37°C for 10 minutes. RBCs were then lysed with 125 μL of 9.1 M perchloric acid. The supernatant (RBC lysate) was aliquoted and frozen at −80°C until assay. On the day of the assay, a 20-μL aliquot of thawed RBC lysate was diluted with 480 μL of phosphate-buffered saline. Proteins were precipitated with 55 μL of 50% 5-sulfosalicylic acid in 500 μM dithiothreitol; 190 μL of the resulting supernatant was incubated with 30 μL of 5% 5-sulfosalicylic acid in dithiothreitol plus 50 μL of 1 M N-ethylmorpholine and 10 μL of acetonitrile in the dark at 37°C for 5 minutes. After the addition of 10 μL of trichloroacetic acid, the RBC lysate was assayed for GSH by HPLC as described for cysteine. The limit of quantitation for RBC GSH was 0.030 mM, with an intraassay CV of 2.4%–8.5% and an interassay CV of 8.7%–13.1%. GSH was stable in bromobimane-treated RBCs for at least 1 week at −80°C.
Plasma SMX-NO Reduction
SMX-NO (20 μM) in dimethyl sulfoxide (2% final dimethyl sulfoxide concentration) was added to plasma and mixed without incubation. Trichloroacetic acid (1% final volume) was added to precipitate proteins. Reactions were analyzed for SMX, SMX-HA, and SMX-NO using HPLC, 30 with ultraviolet detection at 274 nm and a modification to gradient elution to detect SMX-NO. Briefly, solvent A (1% acetic acid with 0.05% triethylamine in water) and solvent B (acetonitrile) were run at 80% A/20% B for 12 minutes, followed by a gradient over 3 minutes to 20% A/80% B, and then elution at 20% A/80% B for 4 minutes. Retention times for SMX-HA, SMX, and SMX-NO were 6.1, 7.6, and 16.2 minutes, respectively. A dimerized form of SMX-NO (confirmed by mass spectrometry) was also eluted at 16.6 minutes, which represented <10% of SMX-NO standard in solution. For patients taking SMX at the time of blood sampling, baseline SMX and HA metabolite levels measurable in plasma before addition of SMX-NO were subtracted from those generated after incubation with SMX-NO. The limit of quantitation for SMX-HA was 500 nM, with an intraassay CV of 2.1%–11.0% and an interassay CV of 5.2%–13.8% in the range of concentrations relevant to this study.
Data among groups was compared by the unpaired t test or analysis of variance followed by the FLSD test, as appropriate, using a commercial software program (Statview, Berkeley, CA). The effect of smoking on plasma ascorbate was analyzed with a 2-way analysis of variance. Correlations were made using a correlation z test. Significance was set at P < 0.05; all data are reported as mean ± SD.
In preliminary experiments, we found that ascorbate in HEPES buffer (pH 7.4) efficiently and nonenzymatically reduced SMX-NO to its HA in a concentration-dependent manner without generation of SMX and without a change in the amount of SMX-NO dimer present (Fig. 2). The stoichiometry of the maximal reduction was 1:1, consistent with 2-electron transfer from ascorbate to the NO. 39 In a time course of SMX-NO reduction in buffer at 37°C in the presence of an equimolar concentration of ascorbate, reduction of SMX-NO monomer to SMX-HA was immediate and complete; no SMX was generated, and the dimer was again unchanged over time. No other peaks appeared, although we did not have a standard for SMX-nitro. After 15 minutes, SMX-NO concentrations increased again, with a coordinate loss of SMX-HA. This was presumably due to the instability of ascorbate in buffer at 37°C over time and reoxidation of SMX-HA to SMX-NO. Because of the facile reduction of SMX-NO by ascorbate in vitro, we set out to examine the relationship between ascorbate and SMX-NO reduction in the plasma of healthy subjects and patients with HIV infection.
Fifty-one HIV-positive patients and 26 healthy controls were recruited for the study. Patients were assayed in 2 phases: initially, 30 HIV-positive patients and 15 controls were recruited for determination of plasma ascorbate and SMX-NO reduction. Subsequently, an additional 21 patients and 17 controls (6 healthy subjects were used again in the second phase) were recruited for determination of plasma ascorbate, dehydroascorbate, and cysteine and RBC GSH concentrations. At the time of sampling, 41% of patients were taking protease inhibitors and 63% were taking nucleoside or nonnucleoside reverse transcriptase inhibitors.
Patient and control groups were of comparable race and age distribution: white, 86.3% versus 88.5%; and mean age, 41.0 ± 6.5 years versus 35.7 ± 11.7 years; respectively. There were significantly more males in the patient group (92.2%) than in the control group (53.8%; P < 0.0001). Because of this, after initial analyses were performed, we reanalyzed our data for ascorbate, SMX-NO reduction, and thiols using a subgroup of males only. Similar results were found using either approach; therefore, results are reported for all subjects.
Our patient group also differed significantly in the number of smokers (39.2%) compared with the control group (5.6% smokers; P = 0.008). Because smoking has been associated with ascorbate deficiency, 40,41 we performed 2-way analysis of variance to examine plasma ascorbate in relation to both smoking status and supplementation. We found no significant effect of smoking in our population (P = 0.49) and no interaction between smoking and supplementation (P = 0.84). We also reanalyzed our data using a subgroup of nonsmokers only. Similar results were found using either approach; therefore, results are reported for all subjects.
Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 ± 22.3 μM) than in healthy subjects (54.8 ± 22.3 μM) and patients taking 500 or 1000 mg of ascorbate daily (82.5 ± 26.3 μM; P < 0.0001) (Fig. 3). HIV-positive patients taking only a multivitamin supplement (~60 mg of ascorbate daily) had plasma ascorbate concentrations (38.2 ± 23.6 μM) similar to those of patients not taking any supplementation (P = 0.24).
Overall, plasma from HIV-positive patients showed an impaired ability to reduce SMX-NO (20 μM) to its HA in vitro (5.2 ± 3.6 μM SMX-HA) compared with plasma from healthy controls (10.6 ± 2.4 μM; P < 0.0001). Not all of the NO was recovered as HA in plasma due to rapid binding of the NO to plasma proteins (data not shown). Very little SMX-NO was recovered as SMX in any subjects (0.62 ± 0.53 μM SMX after addition of 20 μM SMX-NO). Plasma ascorbate deficiency was strongly associated with impaired reduction of SMX-NO to the HA in plasma (r = 0.60, P < 0.0001;Fig. 4). Patients not taking supplements, or taking only multivitamins, had a lower capacity to reduce SMX-NO than both healthy subjects and HIV-positive patients taking ascorbate (P < 0.005–0.0001;Fig. 5). Furthermore, defective NO reduction to the HA in ascorbate-depleted patients was completely normalized by the addition of 40 μM ascorbate to plasma ex vivo (Fig. 5). Finally, during the reduction of SMX-NO in vitro , ascorbate was depleted in direct relationship to the amount of HA generated (r = 0.70, P < 0.0001;Fig. 6), further implicating the primary role of ascorbate in the reduction of SMX-NO in plasma.
Like ascorbate, cysteine can also reduce SMX-NO non-enzymatically in vitro, but conversion is primarily to SMX, not SMX-HA, at equimolar concentrations (data not shown). In preliminary studies with a small number of healthy (n = 7) and HIV-positive (n = 10) subjects, plasma cysteine concentrations were not significantly depleted in either group by the presence of 20 μM SMX-NO (data not shown). In addition, plasma cysteine concentrations did not differ significantly between HIV-positive patients and normal subjects in this sample size (P = 0.16), although the difference may have been significant in a larger number of subjects. Plasma cysteine concentrations were not different in patients taking ascorbate compared with patients taking no supplements or multivitamins only (Table 1).
Plasma ascorbate is regenerated in part from its oxidized form, dehydroascorbate, by RBC GSH. 42 Therefore, we also examined RBC GSH and plasma dehydroascorbate concentrations in our subjects. RBC GSH concentrations were significantly lower in HIV-infected patients than in healthy subjects (P = 0.001) but were not increased in those patients taking ascorbate (Table 1). For subjects not taking ascorbate supplementation, RBC GSH concentration was correlated with plasma ascorbate concentration (r = 0.53, P = 0.004;Fig. 7). RBC GSH concentration did not correlate with dehydroascorbate concentration (data not shown), and neither total nor percent plasma dehydroascorbate concentration was significantly increased in HIV-positive subjects, including those taking ascorbate supplements (Table 1).
In all patients regardless of supplementation, plasma ascorbate deficiency was significantly correlated with decreased CD4 cell counts (r = 0.36, P = 0.015) and decreased CD4/CD8 cell ratios (r = 0.46, P = 0.001;Fig. 8). In addition, patients with detectable HIV type 1 load had lower plasma ascorbate concentrations (36.6 ± 23.7 μM) than patients with undetectable (<50 copies/mL) viral load (ascorbate concentration, 58.1 ± 39.9 μM; P = 0.028). There were no differences in SMX-NO reduction and concentrations of plasma ascorbate, plasma cysteine, or RBC GSH between patients taking either protease inhibitors or reverse transcriptase inhibitors and those not taking either class of drug.
Ascorbate is an essential dietary nutrient in humans, due to an absence in primates of the enzyme l-gulonolactone oxidase, necessary for ascorbate synthesis. 39 Ascorbate is essential for collagen synthesis, recycling of vitamin E, prevention of lipid peroxidation, and stabilization of endogenous products, such as thyroid hormones, from oxidative degradation. 43,44 Although ascorbate is a 2-electron donor and is capable of reduction of free radicals, its role in drug detoxification has not been well characterized.
Ascorbate deficiency has been described in HIV-infected patients in several studies, 33,34,45 although the underlying mechanism has not been well characterized. Poor dietary intake, intestinal malabsorption, and altered redox status due to inflammatory cytokines have been postulated. 33 Other possible causes of ascorbate deficiency in HIV infection include impaired enzymatic or nonenzymatic recycling of ascorbate from dehydroascorbate or decreased synthesis of GSH (which contributes to the nonenzymatic reduction of dehydroascorbate). 42 Decreased plasma ascorbate has also been associated with advanced age (older than 60 years), 35 sampling during the winter months, 35 and smoking. 46 Our patient and control populations were of comparable age, and control samples were collected throughout the year concurrently with patient samples to control for seasonal variations in ascorbate. However, smoking was a possible confounding variable, in that significantly more HIV-positive patients than controls were smokers. We addressed this by performing 2-way analysis of variance to evaluate for a direct effect of smoking or for an interaction between smoking and supplementation. However, because other researchers have found lower ascorbate concentrations in smokers, we cannot conclude that our patients were ascorbate-deficient solely due to HIV infection. We can conclude that those patients with ascorbate deficiency showed defective drug detoxification and that those taking ascorbate supplements did not.
NO metabolites are generated by the spontaneous oxidation of HAs or by the reduction of nitro groups. 47 NO metabolites are thought to be involved in idiosyncratic toxicity from a number of drugs, including primary arylamines (SMX, sulfadiazine, dapsone, procainamide, and nomifensine) and aromatic nitro compounds (chloramphenicol and dantrolene). 47 For sulfonamides, SMX-NO, but not the parent compound, is toxic to lymphocytes and other cells in vitro 18,19 and can bind to and haptenize lymphocytes. 15–17 In addition, SMX-NO, when given to rats, elicits a strong humoral and T-cell–mediated response, but the parent drug and the HA do not. 16,18,20 NO is hypothesized to be the proximate toxin in hypersensitivity responses to sulfonamides and related drugs, and reduction of NO metabolites is therefore likely to be important in the modulation of these hypersensitivity reactions.
GSH protects against toxicity from SMX-NO primarily by preventing the auto-oxidation of SMX-HA to SMX-NO. 27 Thiols such as GSH and cysteine can also nonenzymatically reduce NO metabolites once they are formed. 27,48,49 GSH forms a labile semimercaptol conjugate with SMX-NO, which, in the presence of excess GSH, is reduced back to SMX-HA. When GSH is limited, the semimercaptol rearranges to a sulfinamide, which can degrade to SMX, particularly under acidic conditions. 27 A deficiency in thiols, therefore, could contribute to enhanced oxidation to the NO and subsequent defective NO reduction. GSH has been shown to be deficient in HIV-positive patients in several studies, 50–52 although a recent study using techniques that immediately stabilized GSH after phlebotomy (techniques also used in our study) found no difference. 53 In addition, GSH and/or cysteine supplementation in the form of N-acetylcysteine has not been shown to be of benefit in reducing the incidence of sulfonamide hypersensitivity. 11
Plasma cysteine concentrations have also been shown to be deficient in some patients with HIV infection, 36,51,54 although other studies have found no difference. 55 Although cysteine is very likely to contribute to the extracellular reduction of SMX-NO, it does not appear to be the primary reductant in plasma. Our data indicate that plasma ascorbate was depleted during reduction of SMX-NO (20 μM), while cysteine was not. This is consistent with the findings of Naisbitt et al, 36 who showed a significant difference in plasma cysteine concentrations between HIV-infected patients and controls but did not see a depletion of cysteine until SMX-NO concentrations were “supratherapeutic” (ie, ≥100 μM). They surmised that other reductants could also be involved in NO reduction in plasma , and our ascorbate data are consistent with this hypothesis.
Although we did not see a statistically significant difference in plasma cysteine concentrations in our study, plasma cysteine concentrations were somewhat lower in HIV-infected patients than in controls and may have reached a significant difference in a larger group of patients. We did not measure total cysteine or oxidized cysteine (cystine) in this study, although we acknowledge that these measurements may also have been informative. However, we were somewhat limited by the volume of plasma available from each patient relative to the number of assays that we performed. We focused on cysteine for the following reasons: Cribb et al 27 showed that stable thiol conjugates did not contribute significantly to the reduction of SMX-NO by thiols. Therefore, total (or oxidized) thiols would not be expected to be significantly depleted during reduction. In addition, Naisbitt et al 36 showed no difference in oxidized, protein-bound, or total cysteine concentrations between HIV-infected patients and controls. Only reduced cysteine was significantly different.
In this study, we demonstrated that ascorbate extensively and nonenzymatically reduces SMX-NO and that this reduction is defective in HIV-positive patients with low plasma ascorbate concentrations. We further showed that plasma ascorbate concentrations correlate across subjects with the ability to reduce SMX-NO and that HIV-positive patients who were taking ascorbate supplements (500 or 1000 mg/d) did not show this defect. The concentration of SMX-NO used in these studies (20 μM) was based upon concentrations of SMX-HA, the immediate precursor of NO, which have been measured in human plasma after a therapeutic dosage of SMX. 56 Our observations that ascorbate was depleted during SMX-NO reduction and that the reduction defect in ascorbate-deficient patients could be reversed by ascorbate supplementation ex vivo provide good evidence that ascorbate deficiency was the primary cause for defective NO reduction in plasma. One limitation of this study is its cross-sectional study design, without randomization of patients to supplementation groups. It is therefore difficult to completely exclude possible confounding factors, aside from ascorbate, that could influence drug detoxification. Another limitation is that we did not measure intracellular concentrations of ascorbate, because ascorbate is mainly located within the cell. However, we believe that the plasma compartment assessed in this study is quite important for NO detoxification, because HAs are stable enough to circulate in the plasma and some oxidation to NO would be expected to occur in this compartment.
Ascorbate supplementation has previously been shown to increase plasma ascorbate concentrations in HIV infection, even when deficiency is not present. 57 Plasma ascorbate concentrations in some of the patients in our study were markedly decreased, in the range (<10 μM) associated with scorbutic signs in humans. 58,59 Plasma ascorbate deficiency was significantly correlated with both decreased CD4+ lymphocyte counts and decreased CD4/CD8 cell ratios and was also more pronounced in patients with detectable viral loads, regardless of ascorbate supplementation. Although we cannot conclude a cause-and-effect relationship from this study, it is interesting that the association was independent of supplementation, suggesting an influence of ascorbate on CD4 cell counts rather than solely an influence of immunodeficiency on ascorbate concentrations. These observations are consistent with other studies that have shown that ascorbate supplementation, along with vitamin E or N-acetylcysteine, may decrease viral load and increase CD4+ lymphocyte counts in HIV infection. 57,60 Ascorbate at relatively high concentrations (>300 μM) inhibits HIV replication in vitro, via an inhibitory effect on reverse transcriptase activity. 61,62 Ascorbate also inhibits HIV replication via inhibition of NF-κB–dependent activation of the HIV type 1 promoter, although this requires millimolar concentrations (which, although not found in plasma, can be achieved intracellularly). 63
RBC GSH concentrations were significantly lower in HIV-positive patients than in healthy volunteers in our study population. Several studies have found a deficiency of GSH in HIV-positive patients, 38,50,52,64 although other investigators have found no difference. 53,65 GSH deficiency in HIV infection has been attributed to decreased GSH synthesis, 38 possibly due to decreased activity of GSH synthetase and down-regulation of a subunit of γ-glutamylcysteine ligase. 66 We were interested in looking at RBC GSH in the context of plasma ascorbate and ascorbate supplementation, because most of the regeneration of ascorbate from dehydroascorbate in whole blood takes place in RBCs 67 and high intracellular GSH concentrations can mediate this reduction. 44,68 Furthermore, GSH depletion impairs this recycling and can lead to secondary ascorbate deficiency. 42,69 These observations are consistent with our finding that, among patients not taking ascorbate supplements, plasma ascorbate deficiency was associated with RBC GSH deficiency (Fig. 7). However, it is interesting that RBC GSH concentrations were not increased in those patients taking ascorbate supplements, because studies of HIV-negative subjects have shown that ascorbate supplementation can increase intracellular GSH levels, even in patients with inherited defects in GSH synthesis. 70,71 Our findings suggest that ascorbate deficiency in HIV-positive patients may be secondary to GSH deficiency, but not vice versa; however, this hypothesis requires further evaluation.
We had hypothesized that if impaired ascorbate recycling (due to GSH deficiency and/or other mechanisms) were contributing to plasma ascorbate deficiency, then dehydroascorbate concentrations might be higher in HIV-positive patients than in controls. Contrary to what we expected, HIV infection was not associated with high dehydroascorbate concentrations, even in those patients taking ascorbate supplements. However, we had relatively few patients in the ascorbate supplement group for whom dehydroascorbate was measured. In addition, dehydroascorbate is quite unstable and oxidizes both in vivo and ex vivo into ring-opened metabolites that cannot be recovered as ascorbate-using reducing agents. 72,73 Therefore, we cannot completely rule out the possibility that HIV-positive patients have impaired reduction of dehydroascorbate as a primary mechanism of ascorbate deficiency. Additional studies are under way to evaluate this pathway in HIV infection.
In conclusion, ascorbate deficiency is associated with defective detoxification of SMX-NO in HIV infection. Patients taking ascorbate supplements (500–1000 mg/d) showed high plasma ascorbate concentrations with no differences in cysteine or GSH concentrations and did not show this drug detoxification defect. We are now working to determine the mechanistic basis of ascorbate deficiency in HIV infection and to further explore the relationship between ascorbate deficiency and the outcome of sulfonamide hypersensitivity in HIV-positive patients.
The authors thank Kaoru Murayama, Steven Williams, and Yafan Li for initial work on assay development; Angie Ladwig for preliminary data on nonenzymatic SMX-NO reduction; Christian Kastman, Eli Yoder, and Jennifer Maki for technical assistance; and Nick Keuler and Dr. Rick Nordheim for statistical advice.
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