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Changes in antioxidant profile among HIV-infected individuals on generic highly active antiretroviral therapy in southern India
International Journal of Infectious Diseases (2008) 12, e61—e66
http://intl.elsevierhealth.com/journals/ijid
Changes in antioxidant profile among HIV-infected individuals on generic highly active antiretroviral therapy in southern India Muthu Sundaram a, Suneeta Saghayam a, Bhaskar Priya a, Kartik K. Venkatesh b, Pachamuthu Balakrishnan a, Esaki Muthu Shankar a, Kailapuri G. Murugavel a, Suniti Solomon a, Nagalingeswaran Kumarasamy a,* a
Infectious Diseases Laboratory, YRG Centre for AIDS Research and Education (YRG CARE), VHS Hospital Campus, Rajiv Gandhi Salai, Taramani, Chennai 600 113, India b Brown University Medical School, Providence, Rhode Island, USA Received 10 October 2007; received in revised form 26 March 2008; accepted 14 April 2008 Corresponding Editor: William Cameron, Ottawa, Canada
KEYWORDS Antioxidants; Oxidative stress; HIV; Generic HAART; India
Summary Objective: The role of oxidative stress in disease progression has been shown to be more complicated in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) compared to those who remain treatment-naı¨ve. This study examined the changes in the antioxidant profile of HIV-infected subjects who remained HAART-naı¨ve due to a high CD4 cell count and HIV-negative controls, over a 12-month follow-up period at YRG CARE, a tertiary HIV referral centre in southern India. Methods: We prospectively studied 35 HIV-infected participants (18 on d4T+3TC+EFV (stavudine + lamivudine + efavirenz), eight on AZT+3TC+EFV (zidovudine + lamivudine + efavirenz), and nine who were antiretroviral therapy-naı¨ve) and 20 HIV-negative controls. Antioxidant profile (total antioxidant status, glutathione reductase, glutathione peroxidase, uric acid, ceruloplasmin, zinc, and albumin), CD4 cell count, plasma viral load, dietary intake, and history of smoking and alcohol use were determined at baseline and at twelve months. Results: At 12 months, participants on HAART showed a significant increase in glutathione peroxidase (baseline: 1765 vs. 12 months: 2850 U/l; p < 0.001) and albumin (3.6 vs. 4.4 g/dl; p < 0.001), and a significant decrease in glutathione reductase (52.6 vs. 50.5 U/l; p = 0.054) and uric acid (5.4 vs. 4.8 mg/dl; p = 0.027) compared to baseline. Also HAART-naı¨ve participants had a significant increase in albumin (baseline: 3.7 vs.12 months: 4.3 g/dl; p = 0.023) and a significant decrease in zinc levels (baseline: 79.0 vs.12 months: 74.5 mg/dl; p = 0.052) from baseline to 12 months. HIV-negative subjects had a significant increase in glutathione reductase at 12 months from baseline (baseline: 37 vs.12 months: 39 U/l; p = 0.002). No significant difference in total
* Corresponding author. Tel.: +91 44 22542929; fax: +91 44 22542939. E-mail address: [email protected] (N. Kumarasamy). 1201-9712/$32.00 # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2008.04.004
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M. Sundaram et al. antioxidant status, ceruloplasmin, and zinc levels were observed in HAART-experienced subjects and negative controls over the 12-month follow-up period. Conclusion: This study documents changes in antioxidants over a period of time in HAART-experienced subjects in a southern India setting. # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Introduction HIV disease results in considerable morbidity and mortality due to opportunistic infections (OIs) that can be effectively managed with the prompt initiation of highly active antiretroviral therapy (HAART) in HIV-infected individuals.1 However, recent studies have documented the possibility of increasing toxicities and metabolic changes with the longterm use of HAART.2—4 Cells contain several enzymatic and non-enzymatic antioxidants to prevent or limit reactive oxygen species (ROS)-associated damage.5 The defense system involves many compounds with vastly different properties: enzymes (superoxide dismutase, catalase, glutathione peroxidase), macromolecules (albumin, ceruloplasmin), and small molecules (vitamin C, vitamin E, b-carotene, reduced glutathione, and uric acid). ROS may enhance viral replication by activating nuclear transcription factors such as nuclear factor-kB (NF-kB), which might ultimately lead to an increased rate of viral gene expression.6 In the pre-HAARTera, several studies found that both asymptomatic and symptomatic HIV-infected individuals are at increased risk of oxidative stress, including raised plasma metabolites of lipid peroxidation (LPO) and reduced antioxidant levels, compared with non-HIV-infected control participants.7,8 Certain micronutrients may play a key role in maintaining normal immune functions that may protect immune effector cells from oxidative damage.9 As the natural history and disease progression of HIV can vary across ethnic and racial groups, it is necessary to identify the exact role of oxidative stress and changes in the antioxidant profile among the HIV-infected on HAART in southern India. The present study was undertaken to examine the changes in antioxidant profile among HIV-infected participants on generic HAART, participants who remained HAARTnaı¨ve, and HIV-negative controls.
Patients and methods Setting and subjects This prospective study was conducted at YR Gaitonde Centre for AIDS Research and Education (YRG CARE), a non-profit HIV referral centre in southern India, providing medical and psychosocial care to over 10 000 individuals with HIV infection. A total of 35 HIV-infected subjects (26 on HAART, nine HAARTnaı¨ve) and 20 HIV-negative controls were included in the study. All the participants were involved in other clinic-based research studies. Study participants from prior studies were selected due to limitations in funding. All the laboratory investigations were done at baseline and at 12 months. Dietary data were collected using a 24-h recall, and the nutritive value was calculated using indigenous software ‘DIGEST’.
HIV-infected participants met the following inclusion criteria: >18 years of age, HIV-positive, antiretroviral therapy (ART)-naı¨ve prior to initiating HAART, and without any active OIs. HIV-infected participants who remained naı¨ve did not require HAART due to their high CD4 count. HIV-positive participants were either teetotalers or had quit smoking and drinking on knowing their HIV status. All HIV-infected participants were on vitamin and mineral supplementation. HIV-negative control participants had no serological evidence of HIV and/or HCV-infection, were not smokers or drinkers, had no abnormal laboratory findings (complete blood count, AST and ALT levels within normal range), and had normal blood pressure (120/80 mmHg), a body mass index (BMI) in the range 18.6—25.2 kg/m2, normal chest Xray, and normal electrocardiogram. The YRG CARE institutional review board approved the study protocol. Written informed consent was obtained from all the individuals enrolled in the study.
Specimens To analyze serum albumin, uric acid, total antioxidant status (TAS), ceruloplasmin, and glutathione reductase (GR), 5 ml of peripheral blood was drawn after an overnight fast. Additionally, 3.5 ml of blood was collected into EDTA-containing tubes (BDVacutainer1, BD, Franklin Lakes, NJ, USA) to determine glutathione peroxidase (GPx). Two aliquots of serum and plasma were stored at 80 8C, protected from light, and thawed only once.
Immunological and virological markers Plasma viral load (PVL) was determined by the Cobas Amplicor HIV-1 Monitor Assay version 1.5 (Roche Molecular Systems, Branchburg, NJ, USA); results below the limit of detection were assigned a value of <400 copies/ml. Absolute CD4 T-lymphocyte counts were determined using the Guava Personal Cell Analyzer (Guava Technologies, Inc., USA).
Estimation of antioxidant levels TAS, GR, GPx, and ceruloplasmin levels were measured using commercial kits procured from Randox Laboratories, UK, and Aptec Diagnostics, Belgium. The TAS test measures the total antioxidant effect; ABTS (2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonate)) reacts with all of the defense systems in the circulation, namely superoxide dismutase (SOD), GPx and metal-binding protein (e.g., ceruloplasmin), uric acid, albumin, and DNA repair enzymes. For TAS quantitation, Randox kit Cat. No. NX2332 (Randox Laboratories Ltd, Crumlin, UK) was used.
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Changes in antioxidant profile and HAART, India Evaluation of GPx activity was determined using Randox kit Cat. No. RS504 (Randox Laboratories Ltd). GPx reduces H2O2 to H2O by oxidizing glutathione (GSH). Re-reduction of the oxidized form of glutathione (GSSG) is then catalyzed by glutathione reductase. The decrease in absorbance at 340 nm was measured.10 The activity of GR was determined by using Randox diagnostic reagents (GR 2368; Randox Laboratories Ltd). The level of ceruloplasmin was determined by immunoturbidometric method using an Olympus AU 400 autoanalyzer (Olympus Optical, Japan). It has been shown that ceruloplasmin, a copper-dependent acute phase protein, has antioxidant functions that can prove beneficial in several pathological conditions. The level of uric acid was determined using Raichem diagnostic reagents (Hemagen Diagnostics Inc., San Diego, CA, USA) by uricase method. It has been shown that albumin levels <3.0 g/dl are associated with decreased survival in HIV-infected individuals. Serum albumin was estimated using Olympus diagnostic reagents (Olympus Diagnostica, GmbH, Ireland) by bromocresol green (BCG) method. Low levels of plasma zinc predict a three-fold increase in HIV-related mortality. Zinc acts as an antioxidant or helps such functions, which not only regulate the immune responses of the host, but may also alter the genome of the viruses. Zinc was estimated using Chema diagnostic reagents (Chema Diagnostica, Italy) by photometric method.
Statistical analysis Descriptive statistics, Chi-square statistics, and the Mann— Whitney U-test were used. Statistical analyses were performed with Statistical Package for Social Sciences software
Table 1
(SPSS, version 13.0, Chicago, IL, USA). A p value of less than 0.05 was considered statistically significant.
Results Among the 35 HIV-infected subjects investigated, 26 were treated with HAART and nine were HAART-naı¨ve; there were 20 HIV-negative controls. The mean age in the HAART-experienced, HAART-naı¨ve, and the control groups was 32 7.1, 29 6.3, and 29 6.0 years, respectively. Over two-thirds (65%) of HAART-experienced participants, 44% of HAARTnaı¨ve participants, and 50% of controls were male. The baseline characteristics of the study population are shown in Table 1. At baseline, the median BMI was 16.9, 19.9, and 21.2 kg/ m2 in the HAART-experienced, HAART-naı¨ve, and the control groups, respectively. HAART-experienced participants had a median CD4 count of 167 cells/ml (interquartile range (IQR) 116—219) and a higher viral load of 286 500 copies/ml (IQR 117 500—696 710) compared to HAART-naı¨ve participants who had a median CD4 of 418 cells/ml (IQR 293—624) and viral load of 22 400 copies/ml (400—193 000). Also at baseline, HAART-experienced participants had a higher mean calorie intake of 2879 kcal and a protein intake of 94.1 g than the HAART-naı¨ve participants whose calorie intake was 2320 kcal and protein intake 75.1 g. Among the HAARTexperienced group, 69% of participants received d4T+3TC+EFV (stavudine + lamivudine + efavirenz) and 31% received AZT+3TC+EFV (zidovudine + lamivudine + efavirenz). The antioxidant profiles of all three groups are shown in Table 2. At baseline, albumin (3.6 vs. 3.7 vs. 4.8 g/dl) and zinc (71.0 vs. 79.0 vs. 92.5 mg/dl) were significantly lower and GR (52.6 vs. 46.0 vs. 37.0 U/l), uric acid (5.4 vs. 4.8 vs.
Demographic and laboratory characteristics of study participants
Characteristics
HAART-experienced (N = 26)
HAART-naı¨ve (N = 9)
Control (N = 20)
Mean age (SD) in years
32.3 (7.1)
29.4 (6.35)
29 (6.0)
Sex Male Female Median BMI kg/m2 (IQR)
17 (65.4) 9 (34.6) 16.86 (15.3—19.7)
4 (44.4) 5 (55.6) 19.95 (16.23—26.4)
10 (50) 10 (50) 21.2 (18.6—25.2)
Median CD4 cells/ml (IQR) Baseline 12 months Median PVL copies/ml (IQR) Baseline 12 months Energy (kcal) Baseline 12 months Protein (g) Baseline 12 months
167 (116—219) 338 (281—429) 286 500 (117 500—696 710) <400 2879 (845) 2605 (619) 94.1 (41.1) 80.9 (22.8)
418 (293—624) 416 (246—668)
NA
22 400 (400—193 000) 22 900 (400—115 000)
NA
2320 (864) 2397 (498) 75.1 (33.8) 73.0 (28.3)
Not collected
Not collected
HAART, highly active antiretroviral therapy; SD, standard deviation; BMI, body mass index; IQR, interquartile range; PVL, plasma viral load; NA, not applicable.
0.027 <0.001 0.675 4.8 (3.4—6.3) 4.4 (4.1—4.6) 75.0 (66.7—80) HAART, highly active antiretroviral therapy; TAS, total antioxidant status; GR, glutathione reductase; GPx, glutathione peroxidase.
5.4 (4.8—7.1) 3.6 (3.5—4.0) 71.0 (53.5—86.2) 0.229 0.023 0.052 4.6 (3.9—5.6) 4.3 (4—4.4) 74.5 (64.3—84.8) 4.8 (3.8—6) 3.7 (3.45—3.95) 79.0 (73.5—84.5) 0.038 0.410 0.111 4.1 (3.4—5.7) 4.7 (4.5—4.9) 91.5 (89.0—99.5) 4.1 (3—5.6) 4.8 (4.6—5) 92.5 (86.2—98.7)
1.3 (1.1—1.5) 50.5 (45.2—57.2) 2850 (1825—3512) 31 (29—32) 1.3 (1.2—1.5) 52.6 (46.7—67.7) 1765 (1450—2275) 31 (28—33) 1.30 (1.2—1.5) 47.0 (44—54) 2672 (1610—3600) 26 (23—31)
TAS (mmol/l) GR (U/L) GPx (U/L) Ceruloplasmin mg/dl Uric acid mg/dl Albumin g/dl Zinc mg/dl
1.5 (1.4—1.6) 37.0 (34.2—44.5) 3595 (2787—4322) 26 (23—30)
1.5 (1.4—1.6) 39.0 (35.0—48) 3660 (2905—4195) 25 (21—29)
0.983 0.002 0.765 0.961
1.20 (1.2—1.4) 46.0 (39—52.5) 1650 (1280—2440) 25 (21—26)
0.206 0.260 0.227 0.354
12 months Baseline 12 months Baseline 12 months
p-Value Baseline
p-Value
HIV HAART-experienced (N = 26) HIV HAART-naı¨ve (N = 9) HIV-negative controls (N = 20) Antioxidant
Antioxidant profiles of the HAART-experienced, HAART-naı¨ve, and control subjects Table 2
0.742 0.054 <0.001 0.439
M. Sundaram et al. p-Value
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4.1 mg/dl), and ceruloplasmin (31 vs. 25 vs. 26 mg/dl) were significantly higher among the HIV-infected participants who were at a more advanced stage of HIV disease and about to initiate HAART as compared to the HIV-infected participants who would remain ART-naı¨ve and the HIV-negative controls. Participants in the three groups did not experience significant changes in TAS from baseline to 12 months. From baseline to 12 months, GPx significantly increased (1765 U/l to 2850 U/l) in the HAART-experienced group ( p 0.001). Additionally from baseline to 12 months, HAART-experienced participants underwent a significant decrease in their uric acid levels (5.4 mg/dl to 4.8 mg/dl; p = 0.027). From baseline to 12 months, HAART-experienced participants experienced a significant increase in their median albumin levels (3.6 g/dl to 4.4 g/dl; p < 0.001). Similarly HAART-naı¨ve participants experienced a significant increase in their median albumin levels (3.7 g/dl to 4.3 g/dl; p = 0.002) and significant decrease in their median zinc levels from baseline to 12 months (baseline: 79.0 vs. 12 months: 74.5 mg/dl; p = 0.052). At 12 months, GR significantly increased in the control group (37 U/l to 39 U/ l; p = 0.002) and significantly decreased in the HAARTexperienced group from baseline (52.6 U/l to 50.5 U/l; p = 0.054). HAART-experienced participants and HIV-negative controls did not experience significant changes in their median zinc and ceruloplasmin levels from baseline to 12 months. There was also no significant difference in the dietary intake of the HIV-infected subjects from baseline to 12 months.
Discussion This study documents alterations in the levels of GPx, albumin, GR, and uric acid between baseline and 12 months among HAART-experienced participants. Studies from the developed world have documented biochemical changes that could be a result of greater utilization of antioxidant micronutrients subsequent to increased oxidative stress rather than inadequate dietary intake11 or malabsorption,12 inadequate nutrient release from the liver, acute infections, and/or inadequate availability of carrier molecules that may influence circulating antioxidant concentrations.13—15 This study suggests that enhanced oxidative stress and disturbed glutathione metabolism occur after initiating HAART. These effects appear to be related to persistent tumor necrosis factor-a (TNF-a) activation in HIV-infected patients. HIV-infected individuals have been shown to have decreased antioxidant concentrations, disruption in glutathione metabolism, and enhanced spontaneous generation of ROS.16 Furthermore, a significant decrease in GR levels was documented in treated patients at 12 months. To be cognizant, lower GR levels and oxidative stress in general are known to upregulate inflammatory cytokine activities in patients receiving HAART. HIV-infected individuals can have significantly higher GPx activity than HIV-uninfected individuals.17 We found that HIV-infected individuals receiving HAART experienced increased levels of GPx over time. GPx plays an important role in the metabolism of ROS as a defense mechanism against oxidative damage by catalyzing the reduction of a variety of hydroperoxides via glutathione as
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Changes in antioxidant profile and HAART, India the reducing substrate. In addition to its role as a substrate in the GSH redox cycle, glutathione also acts as a direct endogenous scavenger of hydroxyl radicals, and is involved in the detoxification and metabolism of a number of chemicals and drugs in the liver. The changes observed in the current study therefore reveal that antioxidant supplementation, including glutathione, might have an effect on oxidative stress in HIVinfected individuals. Antioxidant enzyme levels are sensitive to oxidative stress; alterations were observed in the present study, which prove the cell damage and weakened antioxidant defense in HIV-infected individuals. The current study found that HIV infection can increase the oxidative stress process, which is then further increased by HAART usage; this finding is in line with other studies.18 In HIV infection, ROS species may enhance viral replication by activating nuclear transcription factors, which ultimately could lead to viral gene expression.19 The deficiency of total antioxidant status might markedly increase oxidative stress, possibly adversely affecting the immune response and predisposing to drug toxicity.20 The current study found significant changes in antioxidants such as GR, GPx, uric acid, and albumin among HAART-experienced participants over a period of time. Supplementation with glutathione or antioxidants may improve immunological and virological indices in HIVinfected individuals.16 These agents may positively act on the individuals’ immunological status and decrease HIV replication. Further, a greater understanding of the formation of ROS and the subsequent oxidative stress is important for effective clinical care of HIV-infected individuals. Clinical guidelines that indicate cellular damage due to oxidative stress need to be established and monitored during HAART treatment. Current drug therapies and other therapeutic measures such as administration of N-acetylcysteine supplements, may improve the quality of patient care, in addition to diet counseling. This study demonstrates that changes in antioxidants are observed among HAART-experienced participants in the southern India setting. Further long-term prospective studies on a large population must be designed to substantiate and evaluate the relationship between antioxidants and disease progression in persons receiving HAART among HIV-infected individuals, especially in resourcelimited settings. This study had certain limitations viz. limited funding, small sample size, and inadequacy of follow-up (for 12 months). Furthermore, there were certain constraints with regard to including more parameters related to oxidative stress, largely due to lack of funds. The analytical stability of the kits and pre-analytical variables were also a challenge. In addition, the study was probably underpowered to detect some of the parameter changes over a period of time.
Acknowledgements The authors gratefully acknowledge the statistical assistance of Ms Anitha J. Cecelia, and the clinical and laboratory staff of YRG CARE. This paper was presented at the 8th International Congress on AIDS in Asia and the Pacific (ICAAP), Colombo, Sri Lanka, August 2007 (Abstract No. 1602).
Conflict of interest: The authors declare no conflict of interest for the present study.
References 1. Kumarasamy N, Solomon S, Flanigan TP, Hemalatha R, Thyagarajan SP, Mayer KH. Natural history of human immunodeficiency virus disease in Southern India. Clin Infect Dis 2003;36: 79—85. 2. Young J, Weber R, Rickenbach M, Furrer H, Bernasconi E, Hirschel B, et al. Lipid profiles for antiretroviral-naı¨ve patients starting PI- and NNRTI-based therapy in the Swiss HIV cohort study. Antivir Ther 2005;10:585—91. 3. Friis-Møller N, Weber R, Reiss P, Thie ´baut R, Kirk O, d’Arminio Monforte A, et al. Cardiovascular disease risk factors in HIV patients–—association with antiretroviral therapy: results from the DAD study. AIDS 2003;17:1179—93. 4. Jones R, Sawleshwarkar S, Michailidis C, Jackson A, Mandalia S, Stebbing J, et al. Impact of antiretroviral choice on hypercholesterolaemia events: the role of the nucleoside reverse transcriptase inhibitor backbone. HIV Med 2005;6:396—402. 5. Orem A, Yandi YE, Vanizor B, Cims¸it G, Uydu HA, Malkoc¸ M. The evaluation of autoantibodies against oxidatively modified low density lipoprotein (LDL), susceptibility of LDL to oxidation, serum lipids and lipid hydroperoxide levels, total antioxidant status, antioxidant enzyme activities, and endothelial dysfunction in patients with Behcet’s disease. Clin Biochem 2002;35:217—24. 6. Greenspan HC, Aruoma O. Could oxidative stress initiate programmed cell death in HIV infection? A role from plant derived metabolites having synergistic antioxidant activity. Chem Biol Interact 1994;143:145—8. 7. Allard JP, Aghdassi E, Chau J, Salit I, Walmsley S. Oxidative stress and plasma antioxidant micronutrients in humans with HIV infection. Am J Clin Nutr 1998;67:143—7. 8. Favier A, Sappey C, Leclerc P, Faure P, Micoud M. Antioxidant status and lipid peroxidation in patients infected with HIV. Chem Biol Interact 1994;91:165—80. 9. Meydani SN, Beharka AA. Recent developments in vitamin E and the immune response. Nutr Rev 1998;56:s49—58. 10. Randox Radicales Libres. Crumlin, UK: Randox Laboratories Ltd; 1996, p. 1—16. 11. Dworkin BM, Wormser GP, Axelrod F, Pierre N, Schwarz E, Schwartz E, et al. Dietary intake in patients with AIDS, patients with AIDS-related complex and serologically positive HIV patients: correlations with nutritional status. J Parenter Enteral Nutr 1990;14:605—9. 12. Kapembwa M, Bridges C, Joseph AE, Fleming SC, Batman P, Griffin GE. Ileal and jejunal absorptive function in patients with AIDS and enterococcidial infection. J Infect 1990;21:43—53. 13. Rock CL, Jacob RA, Bowen PE. Update on the biological characteristics of the antioxidant micronutrients: vitamin C, vitamin E and the carotenoids. J Am Diet Assoc 1996;96:693—704. 14. Siez H, Stahl W. Vitamin E and C, b-carotene and other carotenoids as antioxidants. Am J Clin Nutr 1995;62:13158—218. 15. Das BS, Thurnham DI, Das DB. Plasma a-tocopherol, retinol and carotenoids in children with falciparum malaria. Am J Clin Nutr 1996;64:94—100. 16. Muller F, Svardal AM, Nordoy I, Berge RK, Aukrust P, Froland SS. Virological and immunological effects of antioxidant treatment in patients with HIV infection. Eur J Clin Invest 2000;30: 905—14. 17. Delmas-Beauvieux MC, Peuchant E, Couchouron A, Constans J, Sergeant C, Simonoff M, et al. The enzymatic antioxidant system in blood and glutathione status in human immunodeficiency virus (HIV)-infected patients: effects of supplementation with selenium or b-carotene. Am J Clin Nutr 1996;64:101—7.
e66 18. Ngondi JL, Oben J, Forkah DM, Etame LH, Mbanya D. The effect of different combination therapies on oxidative stress markers in HIV infected patients in Cameroon. AIDS Res Ther 2006;3:19. 19. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of
M. Sundaram et al. the NF-kB transcription factor and HIV-1. EMBO J 1991;10: 2247—58. 20. Walubo A, Smith PJ, Folb PI. Oxidative stress during antituberculosis therapy in young and elderly patients. Biomed Environ Sci 1995;8:106—10.
http://intl.elsevierhealth.com/journals/ijid
Changes in antioxidant profile among HIV-infected individuals on generic highly active antiretroviral therapy in southern India Muthu Sundaram a, Suneeta Saghayam a, Bhaskar Priya a, Kartik K. Venkatesh b, Pachamuthu Balakrishnan a, Esaki Muthu Shankar a, Kailapuri G. Murugavel a, Suniti Solomon a, Nagalingeswaran Kumarasamy a,* a
Infectious Diseases Laboratory, YRG Centre for AIDS Research and Education (YRG CARE), VHS Hospital Campus, Rajiv Gandhi Salai, Taramani, Chennai 600 113, India b Brown University Medical School, Providence, Rhode Island, USA Received 10 October 2007; received in revised form 26 March 2008; accepted 14 April 2008 Corresponding Editor: William Cameron, Ottawa, Canada
KEYWORDS Antioxidants; Oxidative stress; HIV; Generic HAART; India
Summary Objective: The role of oxidative stress in disease progression has been shown to be more complicated in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) compared to those who remain treatment-naı¨ve. This study examined the changes in the antioxidant profile of HIV-infected subjects who remained HAART-naı¨ve due to a high CD4 cell count and HIV-negative controls, over a 12-month follow-up period at YRG CARE, a tertiary HIV referral centre in southern India. Methods: We prospectively studied 35 HIV-infected participants (18 on d4T+3TC+EFV (stavudine + lamivudine + efavirenz), eight on AZT+3TC+EFV (zidovudine + lamivudine + efavirenz), and nine who were antiretroviral therapy-naı¨ve) and 20 HIV-negative controls. Antioxidant profile (total antioxidant status, glutathione reductase, glutathione peroxidase, uric acid, ceruloplasmin, zinc, and albumin), CD4 cell count, plasma viral load, dietary intake, and history of smoking and alcohol use were determined at baseline and at twelve months. Results: At 12 months, participants on HAART showed a significant increase in glutathione peroxidase (baseline: 1765 vs. 12 months: 2850 U/l; p < 0.001) and albumin (3.6 vs. 4.4 g/dl; p < 0.001), and a significant decrease in glutathione reductase (52.6 vs. 50.5 U/l; p = 0.054) and uric acid (5.4 vs. 4.8 mg/dl; p = 0.027) compared to baseline. Also HAART-naı¨ve participants had a significant increase in albumin (baseline: 3.7 vs.12 months: 4.3 g/dl; p = 0.023) and a significant decrease in zinc levels (baseline: 79.0 vs.12 months: 74.5 mg/dl; p = 0.052) from baseline to 12 months. HIV-negative subjects had a significant increase in glutathione reductase at 12 months from baseline (baseline: 37 vs.12 months: 39 U/l; p = 0.002). No significant difference in total
* Corresponding author. Tel.: +91 44 22542929; fax: +91 44 22542939. E-mail address: [email protected] (N. Kumarasamy). 1201-9712/$32.00 # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2008.04.004
e62
M. Sundaram et al. antioxidant status, ceruloplasmin, and zinc levels were observed in HAART-experienced subjects and negative controls over the 12-month follow-up period. Conclusion: This study documents changes in antioxidants over a period of time in HAART-experienced subjects in a southern India setting. # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Introduction HIV disease results in considerable morbidity and mortality due to opportunistic infections (OIs) that can be effectively managed with the prompt initiation of highly active antiretroviral therapy (HAART) in HIV-infected individuals.1 However, recent studies have documented the possibility of increasing toxicities and metabolic changes with the longterm use of HAART.2—4 Cells contain several enzymatic and non-enzymatic antioxidants to prevent or limit reactive oxygen species (ROS)-associated damage.5 The defense system involves many compounds with vastly different properties: enzymes (superoxide dismutase, catalase, glutathione peroxidase), macromolecules (albumin, ceruloplasmin), and small molecules (vitamin C, vitamin E, b-carotene, reduced glutathione, and uric acid). ROS may enhance viral replication by activating nuclear transcription factors such as nuclear factor-kB (NF-kB), which might ultimately lead to an increased rate of viral gene expression.6 In the pre-HAARTera, several studies found that both asymptomatic and symptomatic HIV-infected individuals are at increased risk of oxidative stress, including raised plasma metabolites of lipid peroxidation (LPO) and reduced antioxidant levels, compared with non-HIV-infected control participants.7,8 Certain micronutrients may play a key role in maintaining normal immune functions that may protect immune effector cells from oxidative damage.9 As the natural history and disease progression of HIV can vary across ethnic and racial groups, it is necessary to identify the exact role of oxidative stress and changes in the antioxidant profile among the HIV-infected on HAART in southern India. The present study was undertaken to examine the changes in antioxidant profile among HIV-infected participants on generic HAART, participants who remained HAARTnaı¨ve, and HIV-negative controls.
Patients and methods Setting and subjects This prospective study was conducted at YR Gaitonde Centre for AIDS Research and Education (YRG CARE), a non-profit HIV referral centre in southern India, providing medical and psychosocial care to over 10 000 individuals with HIV infection. A total of 35 HIV-infected subjects (26 on HAART, nine HAARTnaı¨ve) and 20 HIV-negative controls were included in the study. All the participants were involved in other clinic-based research studies. Study participants from prior studies were selected due to limitations in funding. All the laboratory investigations were done at baseline and at 12 months. Dietary data were collected using a 24-h recall, and the nutritive value was calculated using indigenous software ‘DIGEST’.
HIV-infected participants met the following inclusion criteria: >18 years of age, HIV-positive, antiretroviral therapy (ART)-naı¨ve prior to initiating HAART, and without any active OIs. HIV-infected participants who remained naı¨ve did not require HAART due to their high CD4 count. HIV-positive participants were either teetotalers or had quit smoking and drinking on knowing their HIV status. All HIV-infected participants were on vitamin and mineral supplementation. HIV-negative control participants had no serological evidence of HIV and/or HCV-infection, were not smokers or drinkers, had no abnormal laboratory findings (complete blood count, AST and ALT levels within normal range), and had normal blood pressure (120/80 mmHg), a body mass index (BMI) in the range 18.6—25.2 kg/m2, normal chest Xray, and normal electrocardiogram. The YRG CARE institutional review board approved the study protocol. Written informed consent was obtained from all the individuals enrolled in the study.
Specimens To analyze serum albumin, uric acid, total antioxidant status (TAS), ceruloplasmin, and glutathione reductase (GR), 5 ml of peripheral blood was drawn after an overnight fast. Additionally, 3.5 ml of blood was collected into EDTA-containing tubes (BDVacutainer1, BD, Franklin Lakes, NJ, USA) to determine glutathione peroxidase (GPx). Two aliquots of serum and plasma were stored at 80 8C, protected from light, and thawed only once.
Immunological and virological markers Plasma viral load (PVL) was determined by the Cobas Amplicor HIV-1 Monitor Assay version 1.5 (Roche Molecular Systems, Branchburg, NJ, USA); results below the limit of detection were assigned a value of <400 copies/ml. Absolute CD4 T-lymphocyte counts were determined using the Guava Personal Cell Analyzer (Guava Technologies, Inc., USA).
Estimation of antioxidant levels TAS, GR, GPx, and ceruloplasmin levels were measured using commercial kits procured from Randox Laboratories, UK, and Aptec Diagnostics, Belgium. The TAS test measures the total antioxidant effect; ABTS (2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonate)) reacts with all of the defense systems in the circulation, namely superoxide dismutase (SOD), GPx and metal-binding protein (e.g., ceruloplasmin), uric acid, albumin, and DNA repair enzymes. For TAS quantitation, Randox kit Cat. No. NX2332 (Randox Laboratories Ltd, Crumlin, UK) was used.
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Changes in antioxidant profile and HAART, India Evaluation of GPx activity was determined using Randox kit Cat. No. RS504 (Randox Laboratories Ltd). GPx reduces H2O2 to H2O by oxidizing glutathione (GSH). Re-reduction of the oxidized form of glutathione (GSSG) is then catalyzed by glutathione reductase. The decrease in absorbance at 340 nm was measured.10 The activity of GR was determined by using Randox diagnostic reagents (GR 2368; Randox Laboratories Ltd). The level of ceruloplasmin was determined by immunoturbidometric method using an Olympus AU 400 autoanalyzer (Olympus Optical, Japan). It has been shown that ceruloplasmin, a copper-dependent acute phase protein, has antioxidant functions that can prove beneficial in several pathological conditions. The level of uric acid was determined using Raichem diagnostic reagents (Hemagen Diagnostics Inc., San Diego, CA, USA) by uricase method. It has been shown that albumin levels <3.0 g/dl are associated with decreased survival in HIV-infected individuals. Serum albumin was estimated using Olympus diagnostic reagents (Olympus Diagnostica, GmbH, Ireland) by bromocresol green (BCG) method. Low levels of plasma zinc predict a three-fold increase in HIV-related mortality. Zinc acts as an antioxidant or helps such functions, which not only regulate the immune responses of the host, but may also alter the genome of the viruses. Zinc was estimated using Chema diagnostic reagents (Chema Diagnostica, Italy) by photometric method.
Statistical analysis Descriptive statistics, Chi-square statistics, and the Mann— Whitney U-test were used. Statistical analyses were performed with Statistical Package for Social Sciences software
Table 1
(SPSS, version 13.0, Chicago, IL, USA). A p value of less than 0.05 was considered statistically significant.
Results Among the 35 HIV-infected subjects investigated, 26 were treated with HAART and nine were HAART-naı¨ve; there were 20 HIV-negative controls. The mean age in the HAART-experienced, HAART-naı¨ve, and the control groups was 32 7.1, 29 6.3, and 29 6.0 years, respectively. Over two-thirds (65%) of HAART-experienced participants, 44% of HAARTnaı¨ve participants, and 50% of controls were male. The baseline characteristics of the study population are shown in Table 1. At baseline, the median BMI was 16.9, 19.9, and 21.2 kg/ m2 in the HAART-experienced, HAART-naı¨ve, and the control groups, respectively. HAART-experienced participants had a median CD4 count of 167 cells/ml (interquartile range (IQR) 116—219) and a higher viral load of 286 500 copies/ml (IQR 117 500—696 710) compared to HAART-naı¨ve participants who had a median CD4 of 418 cells/ml (IQR 293—624) and viral load of 22 400 copies/ml (400—193 000). Also at baseline, HAART-experienced participants had a higher mean calorie intake of 2879 kcal and a protein intake of 94.1 g than the HAART-naı¨ve participants whose calorie intake was 2320 kcal and protein intake 75.1 g. Among the HAARTexperienced group, 69% of participants received d4T+3TC+EFV (stavudine + lamivudine + efavirenz) and 31% received AZT+3TC+EFV (zidovudine + lamivudine + efavirenz). The antioxidant profiles of all three groups are shown in Table 2. At baseline, albumin (3.6 vs. 3.7 vs. 4.8 g/dl) and zinc (71.0 vs. 79.0 vs. 92.5 mg/dl) were significantly lower and GR (52.6 vs. 46.0 vs. 37.0 U/l), uric acid (5.4 vs. 4.8 vs.
Demographic and laboratory characteristics of study participants
Characteristics
HAART-experienced (N = 26)
HAART-naı¨ve (N = 9)
Control (N = 20)
Mean age (SD) in years
32.3 (7.1)
29.4 (6.35)
29 (6.0)
Sex Male Female Median BMI kg/m2 (IQR)
17 (65.4) 9 (34.6) 16.86 (15.3—19.7)
4 (44.4) 5 (55.6) 19.95 (16.23—26.4)
10 (50) 10 (50) 21.2 (18.6—25.2)
Median CD4 cells/ml (IQR) Baseline 12 months Median PVL copies/ml (IQR) Baseline 12 months Energy (kcal) Baseline 12 months Protein (g) Baseline 12 months
167 (116—219) 338 (281—429) 286 500 (117 500—696 710) <400 2879 (845) 2605 (619) 94.1 (41.1) 80.9 (22.8)
418 (293—624) 416 (246—668)
NA
22 400 (400—193 000) 22 900 (400—115 000)
NA
2320 (864) 2397 (498) 75.1 (33.8) 73.0 (28.3)
Not collected
Not collected
HAART, highly active antiretroviral therapy; SD, standard deviation; BMI, body mass index; IQR, interquartile range; PVL, plasma viral load; NA, not applicable.
0.027 <0.001 0.675 4.8 (3.4—6.3) 4.4 (4.1—4.6) 75.0 (66.7—80) HAART, highly active antiretroviral therapy; TAS, total antioxidant status; GR, glutathione reductase; GPx, glutathione peroxidase.
5.4 (4.8—7.1) 3.6 (3.5—4.0) 71.0 (53.5—86.2) 0.229 0.023 0.052 4.6 (3.9—5.6) 4.3 (4—4.4) 74.5 (64.3—84.8) 4.8 (3.8—6) 3.7 (3.45—3.95) 79.0 (73.5—84.5) 0.038 0.410 0.111 4.1 (3.4—5.7) 4.7 (4.5—4.9) 91.5 (89.0—99.5) 4.1 (3—5.6) 4.8 (4.6—5) 92.5 (86.2—98.7)
1.3 (1.1—1.5) 50.5 (45.2—57.2) 2850 (1825—3512) 31 (29—32) 1.3 (1.2—1.5) 52.6 (46.7—67.7) 1765 (1450—2275) 31 (28—33) 1.30 (1.2—1.5) 47.0 (44—54) 2672 (1610—3600) 26 (23—31)
TAS (mmol/l) GR (U/L) GPx (U/L) Ceruloplasmin mg/dl Uric acid mg/dl Albumin g/dl Zinc mg/dl
1.5 (1.4—1.6) 37.0 (34.2—44.5) 3595 (2787—4322) 26 (23—30)
1.5 (1.4—1.6) 39.0 (35.0—48) 3660 (2905—4195) 25 (21—29)
0.983 0.002 0.765 0.961
1.20 (1.2—1.4) 46.0 (39—52.5) 1650 (1280—2440) 25 (21—26)
0.206 0.260 0.227 0.354
12 months Baseline 12 months Baseline 12 months
p-Value Baseline
p-Value
HIV HAART-experienced (N = 26) HIV HAART-naı¨ve (N = 9) HIV-negative controls (N = 20) Antioxidant
Antioxidant profiles of the HAART-experienced, HAART-naı¨ve, and control subjects Table 2
0.742 0.054 <0.001 0.439
M. Sundaram et al. p-Value
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4.1 mg/dl), and ceruloplasmin (31 vs. 25 vs. 26 mg/dl) were significantly higher among the HIV-infected participants who were at a more advanced stage of HIV disease and about to initiate HAART as compared to the HIV-infected participants who would remain ART-naı¨ve and the HIV-negative controls. Participants in the three groups did not experience significant changes in TAS from baseline to 12 months. From baseline to 12 months, GPx significantly increased (1765 U/l to 2850 U/l) in the HAART-experienced group ( p 0.001). Additionally from baseline to 12 months, HAART-experienced participants underwent a significant decrease in their uric acid levels (5.4 mg/dl to 4.8 mg/dl; p = 0.027). From baseline to 12 months, HAART-experienced participants experienced a significant increase in their median albumin levels (3.6 g/dl to 4.4 g/dl; p < 0.001). Similarly HAART-naı¨ve participants experienced a significant increase in their median albumin levels (3.7 g/dl to 4.3 g/dl; p = 0.002) and significant decrease in their median zinc levels from baseline to 12 months (baseline: 79.0 vs. 12 months: 74.5 mg/dl; p = 0.052). At 12 months, GR significantly increased in the control group (37 U/l to 39 U/ l; p = 0.002) and significantly decreased in the HAARTexperienced group from baseline (52.6 U/l to 50.5 U/l; p = 0.054). HAART-experienced participants and HIV-negative controls did not experience significant changes in their median zinc and ceruloplasmin levels from baseline to 12 months. There was also no significant difference in the dietary intake of the HIV-infected subjects from baseline to 12 months.
Discussion This study documents alterations in the levels of GPx, albumin, GR, and uric acid between baseline and 12 months among HAART-experienced participants. Studies from the developed world have documented biochemical changes that could be a result of greater utilization of antioxidant micronutrients subsequent to increased oxidative stress rather than inadequate dietary intake11 or malabsorption,12 inadequate nutrient release from the liver, acute infections, and/or inadequate availability of carrier molecules that may influence circulating antioxidant concentrations.13—15 This study suggests that enhanced oxidative stress and disturbed glutathione metabolism occur after initiating HAART. These effects appear to be related to persistent tumor necrosis factor-a (TNF-a) activation in HIV-infected patients. HIV-infected individuals have been shown to have decreased antioxidant concentrations, disruption in glutathione metabolism, and enhanced spontaneous generation of ROS.16 Furthermore, a significant decrease in GR levels was documented in treated patients at 12 months. To be cognizant, lower GR levels and oxidative stress in general are known to upregulate inflammatory cytokine activities in patients receiving HAART. HIV-infected individuals can have significantly higher GPx activity than HIV-uninfected individuals.17 We found that HIV-infected individuals receiving HAART experienced increased levels of GPx over time. GPx plays an important role in the metabolism of ROS as a defense mechanism against oxidative damage by catalyzing the reduction of a variety of hydroperoxides via glutathione as
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Changes in antioxidant profile and HAART, India the reducing substrate. In addition to its role as a substrate in the GSH redox cycle, glutathione also acts as a direct endogenous scavenger of hydroxyl radicals, and is involved in the detoxification and metabolism of a number of chemicals and drugs in the liver. The changes observed in the current study therefore reveal that antioxidant supplementation, including glutathione, might have an effect on oxidative stress in HIVinfected individuals. Antioxidant enzyme levels are sensitive to oxidative stress; alterations were observed in the present study, which prove the cell damage and weakened antioxidant defense in HIV-infected individuals. The current study found that HIV infection can increase the oxidative stress process, which is then further increased by HAART usage; this finding is in line with other studies.18 In HIV infection, ROS species may enhance viral replication by activating nuclear transcription factors, which ultimately could lead to viral gene expression.19 The deficiency of total antioxidant status might markedly increase oxidative stress, possibly adversely affecting the immune response and predisposing to drug toxicity.20 The current study found significant changes in antioxidants such as GR, GPx, uric acid, and albumin among HAART-experienced participants over a period of time. Supplementation with glutathione or antioxidants may improve immunological and virological indices in HIVinfected individuals.16 These agents may positively act on the individuals’ immunological status and decrease HIV replication. Further, a greater understanding of the formation of ROS and the subsequent oxidative stress is important for effective clinical care of HIV-infected individuals. Clinical guidelines that indicate cellular damage due to oxidative stress need to be established and monitored during HAART treatment. Current drug therapies and other therapeutic measures such as administration of N-acetylcysteine supplements, may improve the quality of patient care, in addition to diet counseling. This study demonstrates that changes in antioxidants are observed among HAART-experienced participants in the southern India setting. Further long-term prospective studies on a large population must be designed to substantiate and evaluate the relationship between antioxidants and disease progression in persons receiving HAART among HIV-infected individuals, especially in resourcelimited settings. This study had certain limitations viz. limited funding, small sample size, and inadequacy of follow-up (for 12 months). Furthermore, there were certain constraints with regard to including more parameters related to oxidative stress, largely due to lack of funds. The analytical stability of the kits and pre-analytical variables were also a challenge. In addition, the study was probably underpowered to detect some of the parameter changes over a period of time.
Acknowledgements The authors gratefully acknowledge the statistical assistance of Ms Anitha J. Cecelia, and the clinical and laboratory staff of YRG CARE. This paper was presented at the 8th International Congress on AIDS in Asia and the Pacific (ICAAP), Colombo, Sri Lanka, August 2007 (Abstract No. 1602).
Conflict of interest: The authors declare no conflict of interest for the present study.
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