Biochemical Alterations and Antioxidant Levels in Patients Living with HIV

Abstract

Acquired Immunodeficiency Syndrome (AIDS), is a contagious disease of the immune system caused by Human Immunodeficiency Virus (HIV). Present study aimed to find out biochemical parameters and antioxidant status of people living with HIV with or without co-morbidities. Total 110 patients enrolled in the study, maximum had no co-morbidities 56%, while 30% had tuberculosis. Majority of the patients were unemployed (35.5%) while 21.8% were unskilled workers. Total serum bilirubin (0.95 ± 0.51), total protein (7.22 ± 1.07) and alkaline phosphatase levels (134.52 ± 64.38) were found to be increased while serum albumin level was found to be decreased slightly (4.17 ± 0.79). Total antioxidant level was found to be reduced with or without co-morbidities in HIV patients. The study concluded for regular monitoring of biochemical parameters and antioxidant levels for better management of peoples living with HIV.

1. Introduction

HIV, as name suggests damages the immune system. As the lentivirus travels via the lymphatic system, it affects the CD4 T cells by replicating in them and ultimately destroying them [1]. Cellular CD4 immunodeficiency at an early stage is a hallmark of HIV infection. AIDS has evolved itself from a mysterious illness to a global pandemic [2]. Data suggests that in the year 2021, around 650,000 people died from HIV-related grounds while 1.5 million people acquired HIV [3]. Highly reactive antiretroviral therapy (HAART), have been choice for management of HIV positive patients. Infected T lymphocytes leads to immune compromised situation. The body is faced with a dilemma of promoting apoptosis to eliminate HIV, yet consequently lowering the body’s resistance to HIV.

Due to compromised immune system and the long-term ART therapy, people living with HIV may be at a higher risk of attaining co-morbidities related to cardio-vascular disease, chronic kidney disease, metabolic syndromes, hepatobiliary diseases etc. [4]. Moreover, HIV infection is complicated by various opportunistic infections (OIs) such as tuberculosis (TB), Hepatitis, candidiasis, herpes zoster, Pneumocystis jerovicii, cytomegalovirus (CMV) etc.

The involvement of the hepatobiliary system is a major concern in patients with HIV infection. Liver being the master organ for all the metabolic processes, approximately one third of the deaths in patients with HIV infection are in some way, related to liver disease. Opportunistic infections also add to the liver’s burden. To ensure that the liver maintains its physiological normality throughout both the chronic and acute management of HIV infection, it is extremely crucial that people with HIV take care of their liver health [5].

Oxidative stress (OS) induced by the production of reactive oxygen species may play a critical role in the stimulation of HIV replication and the development of immunodeficiency. OS may have a principal contribution to both the expression of HIV and the development of AIDS. Oxidative stress is closely linked to chronic human immunodeficiency virus (HIV) infection, both alone and in combination with highly active antiretroviral therapy (HAART). Level of oxidative stress therefore also becomes a matter of concern in HIV patients, as it increases with disease progression. Studies also suggest a role of antioxidants in inactivation of viral replication, thereby making them an important early biochemical marker [6].

2. Material and methods

2.1 Study place

Study was conducted in the Department of Biochemistry in collaboration with the Department of Medicine and ART Centre at a tertiary care hospital of central India.

2.2 Inclusion criteria

• Patients registered at ART Centre, willing to participate and giving consent.

• Irrespective of their treatment status.

• Patients having any deranged parameters will be included and categorized for further data analysis.

2.3 Exclusion criteria

• Patient on oral antioxidant or multivitamins

• Newly detected patients

2.4 Study procedure

The study was conducted after obtaining clearance from the regional NACO Center and Institutional Ethical Committee. An informed consent in bilingual language was given and obtained from the study participants.

2.4.1 Sample size

The study group consisted of 110 HIV positive individuals, above 18 years of either sex. (Sample size calculated by sample size formula using total registered patients at ART centre).

2.4.2 Sample processing

Overnight fasting, 5 ml blood samples were collected following the universal aseptic precautions. A sterile vial without any anti-coagulant was used to collect a sample. The serum was separated after centrifugation and if required stored at -15^o in deep freezer.

2.4.3 Biochemistry investigations

Following biochemical investigations were performed using Bio Systems BA400 analyzer.

• Serum bilirubin (Total and Direct) was measured by 2,4- Dichlorphenyl Diazonium (2,4- DCPD) method [7, 8];

• Alkaline phosphatase (ALP), Aspartate transaminase (AST) and Alanine transaminase (ALT) by modified IFCC method [7, 8].

• Serum albumin was assessed by (BCG) method [7],

• Total protein by Biuret method [8, 9] and

• Serum Globulin was calculated using the formula: Total protein - serum albumin.

Total antioxidant capacity (TAC) was measured by Colorimetric D Koracevic method [10].

CD4 count was done by flow cytometry.

2.4.4 Data collection and statistical analysis

Data was collected using MS Excel sheet. All the data was analyzed using Epi-info software. All variables were expressed as mean +/− SD (Standard Deviation). P value <0.05 was considered statistically significant.

3. Results and discussion

Present cross-sectional study of HIV positive patients, total 110 patients fulfilling all the inclusion and exclusion criteria were enrolled. Demographic profile showed diversity in socio-demography and HIV associated variables (Figure 1). The mean age of the patients reported was found to be 41.20 ± 10.44 years, out of them 62 of the subjects were males while 48 were females. Most of the subjects reported were unemployed (35.5%) followed by unskilled workers (21.8%) (Table 1). Study conducted by Amandeep Singh et al., showed similar results with maximum number of patients reported in their study that 73.8% patients belonged to the age group 41–60 years, 57.75% were males and 42.25% were females. They also reported predominance of patients from rural areas and from the lower middle and upper lower socio-economic classes [11].

Socio-demographic variables	HIV patients; (Mean ± S.D.) (Percentage) n = 110
Age (in years)	41.20 ± 10.44
Male: Female Sex	62 (56.36%): 48 (43.64%)
Duration of HIV infection (in years)	6.43 ± 9.36

Table 1.

Age and sex distribution of HIV patients.

Figure 1.

Occupation wise distribution of study subjects. (classification of occupation is according to Kuppuswamy’s classification).

Maximum number of HIV patients showed no co-morbidities (56%), while 30% had Tuberculosis. (Figure 2) This presents unique treatment challenges due to interactions between antitubercular and antiretroviral medications, overlapping drug toxicities, and the fact that HIV’s immune-suppression delays the diagnosis of tuberculosis. In present study, 1% were infected with Hepatitis B, 5% with Hepatitis C and 2% with both hepatitis C and TB as co-infections. According to WHO guidelines co-infections are common among PLHIV, regardless of immune status. Some of those coinfections can be silent and do not cause significant impact on HIV disease nor are affected by it. In contrast, certain co-infections can affect the natural history of HIV infection and vice-versa, with implications in diagnosis, susceptibility, clinical presentation, and care, including timing and choice of drug regimens for treatment and prevention. WHO has developed specific guidance to manage some of these co-infections, focusing on the screening, prophylaxis, treatment and timing of ART for these conditions. The study by A Treitinger, et al., reported significant associations between hepatitis serological status and lipid profile [12].

Figure 2.

Incidence of co-morbidities among HIV patients.

Table 2 shows levels of various biochemical parameters, levels of total bilirubin are significantly increased (0.95 ± 0.51) whereas levels of AST, ALT was deranged slightly. Total protein level was in normal range (7.22 ± 1.07) however serum albumin level was decreased (4.17 ± 0.79). According to Sunny Pathania et al., liver diseases account for almost 14–18% of all deaths in PLHA. They also reported hyperbilirubinemia (10.93%) and mild abnormalities in transaminases [13]. In a study by Sabir Kumar Dey et al., there was significant increase in total bilirubin levels of HIV positive patients as compared to normal [14].

Biochemical parameters	Values (mean ± S.D.); n = 110
Liver function test
Total bilirubin(mg/dl)	0.95 ± 0.51
AST(U/l)	35.13 ± 27.39
ALT(U/l)	30.94 ± 22.69
Serum albumin(g/dl)	4.17 ± 0.79
Total protein (g/dl)	7.22 ± 1.07
Serum globulin (g/dl)	3.05 ± 0.84
Alkaline phosphatase(IU/l)	134.52 ± 64.38

Table 2.

Mean values of common biochemical parameters in study subjects.

Deranged parameters were also corelated on the basis of co-morbidities (Table 3), no strong association was found. This indicates that each co-morbidity has a different mechanism and it alters biochemical parameters in different ways. Hence there is need of assessment of each co-morbidity separately and the associated biochemical alterations.

S.NO	Biochemical marker	Within range/deranged	Comorbidities		Pearson Chi square	Df	P value
			Present	Absent
1	Total bilirubin	Within range	33 (30%)	54 (49.09%)	3.911	1	0.041
		Deranged	14 (12.72%)	9 (8.18%)
2	AST	Within range	40 (36.36%)	57 (51.81%)	0.745	1	0.284
		Deranged	7 (6.36%)	6 (5.45%)
3	ALT	Within range	43 (39.09%)	60 (54.54%)	0.635	1	0.340
		Deranged	4 (3.63%)	3 (2.72%)
4	Total protein	Within range	37 (33.63%)	50 (45.45%)	0.007	1	0.559
		Deranged	10 (9.09%)	13 (11.81%)
5	Serum albumin	Within range	41 (37.27%)	55 (50%)	0.11	1	0.605
		Deranged	6 (5.45%)	8 (7.27%)
6	Serum globulin	Within range	35 (31.81%)	45 (49.09%)	0.125	1	0.447
		Deranged	12 (10.9%)	18 (16.36%)
7	Alkaline phosphatase	Within range	44 (40%)	50 (45.45%)	4.499	1	0.031
		Deranged	3 (2.72%)	13 (11.81%)

Table 3.

Percentage of biochemical parameters in HIV patients with or without comorbidities.

Out of all co-morbidities, Tuberculosis (TB) had the highest percentage among the subjects. This presents unique treatment challenges due to interactions between antitubercular and antiretroviral medications, overlapping drug toxicities, and the fact that HIV’s immune-suppression delays the diagnosis of tuberculosis. AST, ALT as well as ALP level was slightly elevated in patients with TB than without TB. Subir K et al., in their study of HIV patients with TB comorbidity reported elevated bilirubin, AST, ALT and ALP levels. But they reported low levels of albumin and globulin.

When oxidative stress was studied by measuring total antioxidant capacity of patients with co-morbidity, a clear correlation was seen (Table 4). It was seen that CD4 count was directly proportional to the total antioxidant capacity of patients. Similar study done by Suresh et al., reported strong association of oxidative stress with the level of CD4 count [15]. The discovery of HIV also led to a broadening of the view that ROS play a critical role in the expression of HIV and the development of AIDS. It has been reported that the virus induces OS by disturbing cellular antioxidant defense and initiating oxidative reactions. Advanced cases of HIV infection renders individuals susceptible to opportunistic infections, which take advantage of the progressive immunodeficiency caused by HIV [15]. Since cellular redox status is a normal physiological variable, any imbalance may elicit cellular response through proliferation, transcriptional activation, or apoptosis. For this reason, OS may be a principal mechanism in the progression of AIDS. Since OS can induce apoptosis, ROS may trigger apoptotic pathways responsible for the initial T-cell depletion upon HIV infection [15].

Biochemical parameter	Controls; (mean ± S.D.)	HIV symptomatic patients (mean ± S.D.); N = 69	HIV asymptomatic patients (mean ± S.D.); N = 41	P value
Total antioxidant capacity (expressed in mmol/l)	1.08 ± 0.08	0.68 ± 0.14	O.75 ± 0.12	<0.02

Table 4.

Comparison of total antioxidant capacity with or without comorbidities.

4. Conclusion

It can be concluded from the present study that HIV causes either primary or treatment associated, liver function anomalies as well as OS. Majority of patients belonged to low socioeconomic status and productive age groups of their husband.

Due to the unique treatment challenges that TB + HIV co-infection poses and especially in a country like India who already suffers from a high TB load and a lack of resources, there’s a need of careful monitoring of the interplay of anti-tubercular therapy with antiretroviral therapy, and that LFT and total antioxidant capacity are effective parameters that can be advised on a routine basis to assess any abnormalities. Total antioxidant capacity can be a novel early biomarker as it helps to assess the levels of oxidative stress in patients. Since present study has found direct correlation of OS with CD4 levels, the therapeutic effect of dietary anti-oxidant supplements and their role in delaying the progression of the disease comes into question. Further research will help to make ART regimens much better.