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1 From the School of Physiology, Nutrition, and Consumer Sciences, Faculty of Health Sciences, North-West University Potchefstroom Campus, Potchefstroom, Republic of South Africa
2 Supported by grants from the National Research Foundation, South African Medical Research Council, South African Sugar Association, The Dry Bean Producers Organization, and North-West University. 3 Reprints not available. Address correspondence to W Oosthuizen, Private Bag X6001, School of Physiology, Nutrition, and Consumer Sciences, North-West University Potchefstroom Campus, Potchefstroom, 2520, Republic of South Africa. E-mail: vgewo{at}puk.ac.za.
ABSTRACT
Background: Dietary fat intake in the South African population is increasing. This population also has a high prevalence of HIV infection. However, information about metabolic effects of dietary fatty acids on HIV-infected subjects is lacking.
Objective: Our objective was to investigate the relation between dietary fatty acid intake and liver function in HIV-infected compared with HIV-uninfected subjects.
Design: This cross-sectional epidemiologic survey included a representative sample of 1854 apparently healthy black volunteers aged 15 y, who were recruited from 37 randomly selected sites throughout the North West province of South Africa. Data from 216 asymptomatic HIV-infected and 1604 HIV-uninfected subjects were used.
Results: Intakes of polyunsaturated fatty acids (PUFAs), linoleic acid (n–6), and the ratio of PUFAs to saturated fatty acids (SFAs) were positively associated with all the liver enzymes measured in HIV-infected subjects (R = 0.16–0.65). Most of these R values differed significantly from the R values for HIV-uninfected subjects. No associations were seen between liver enzymes and intakes of SFAs and monounsaturated fatty acids. Vitamin E intake was positively associated with serum -glutamyl transpeptidase (R = 0.23), alanine aminotransferase (R = 0.37), and aspartate aminotransferase (R = 0.58) in HIV-infected subjects; these correlations differed significantly from those of the HIV-uninfected subjects because PUFA sources are the main carriers of vitamin E.
Conclusions: The results suggest that n–6 PUFA intakes may be related to liver damage in these HIV-infected asymptomatic subjects. The reasons or mechanisms responsible are not clear, and further research is necessary to determine the optimal safe amounts for intake of n–6 PUFAs by HIV-infected subjects, especially in countries with traditionally high intakes of n–6 PUFA–rich vegetable oils.
Key Words: Polyunsaturated fatty acids • HIV • liver enzymes • THUSA study
INTRODUCTION
The global HIV and AIDS pandemic is assuming proportions in sub-Saharan African countries that are threatening to neutralize gains in health, reducing life expectancy in some of these countries to <40 y (1), and paralyzing health care services and economies (2). The South African HIV household survey in 2002 indicated that 11.4% of South Africans aged >2 y (4.5 million people) are living with HIV and AIDS (3). According to the Joint United Nations Programme on HIV/AIDS (UNAIDS), South Africa continues to have the largest number of people living with HIV in the world. The adult prevalence rate between ages 15 and 49 y is estimated at 21.5%, and the total number of persons infected is estimated at 5.3 million. AIDS deaths among adults and children is estimated at 370 000 (4).
Optimizing nutritional status is a key objective in the South African government's 2003 plan for the care, management, and treatment of persons infected with the virus and with AIDS (5). "Optimum" nutrition could improve quality of life for persons living with HIV and AIDS, it may slow the progression of HIV infection to AIDS, and it may improve tolerance to antiretroviral therapy (6, 7). The World Health Organization (WHO) and the Food and Agriculture Organization also recognize the importance of nutrition in persons living with HIV and AIDS (8). Because asymptomatic HIV-infected persons will not have access to antiretroviral therapy, the role of nutrition is important at this stage of the infection (9). According to the WHO nutrition guidelines for persons living with HIV and AIDS, fat and fatty acid intakes do not differ from those of healthy persons (10). The South African national guidelines on nutrition for persons living with HIV and AIDS recommend increased intake of fat after periods of weight loss as an important source of energy (11). It is clear that information in the literature about the effects of dietary fat and fatty acids in HIV-infected subjects is lacking.
During an investigation of the relations between food (nutrient) intakes and biochemical markers of nutritional status in asymptomatic HIV-infected subjects compared with HIV-uninfected subjects who participated in the THUSA survey, positive correlations between consumption of polyunsaturated fatty acids (PUFAs) and serum liver enzymes were reported (9). THUSA means help in the Setswana language and is also an acronym for Transition in Health during Urbanization of South Africans. This possible adverse effect prompted us to do a more in-depth analysis of the data about the role of fat and different types of fat in the diets of HIV-infected subjects in relation to liver function, to make recommendations for the intake of dietary fat by HIV-infected subjects, and to give direction with regard to further research in this field. This study is unique in the sense that it involves asymptomatic HIV-infected subjects, and it investigates the effect of fatty acids in these subjects, not investigated before.
The aim of this study was therefore to investigate the relation between dietary intake of fatty acids and liver function in HIV-infected compared with HIV-uninfected black South Africans who participated in the THUSA survey. The main aim of the THUSA study, a cross-sectional epidemiologic study, was to monitor the effect of urbanization on the health determinants of black South Africans in transition to provide information for appropriate health interventions.
SUBJECTS AND METHODS
Study design and subjects
The methods of sample selection, all measurements, and analyses were previously described (12). Briefly, 1854 "apparently healthy" black volunteers, aged 15 y, were recruited from 37 randomly selected sites throughout the North West Province of South Africa in 1996 and 1998. Subjects were stratified into 5 levels of urbanization, based on the area of residence and type of employment. For data analysis of the current investigation, subjects in different strata of urbanization were grouped together, but statistical analyses controlled for the level of urbanization. Exclusion criteria included pregnant and lactating women, known diagnosed diseases, use of chronic medication, oral temperatures >37 °C, and inebriated subjects. For this investigation complete data were available for 216 asymptomatic HIV-infected and 1604 HIV-uninfected subjects.
Measurements and biochemical analyses
Demographic information, physical activity, and dietary intake data were obtained during individual interviews by specially trained, multilingual fieldworkers in the language of the subject's choice, by using questionnaires specially designed or adapted and validated for this population. A validated quantitative food-frequency questionnaire was used to obtain habitual dietary intake data (13). Nutrient intakes were calculated by using the FOODFINDER computer program (Medical Research Council of South Africa, Tygerberg, South Africa) based on the South African food-composition tables (14). Physical activity information was obtained by using a quantitative questionnaire, measuring the physical activity index, especially adapted and validated for this population (15). Anthropometric measurements were measured in triplicate by postgraduate anthropometry students from the Department of Human Movement Science, North-West University, Potchefstroom, South Africa, standardized by a level II anthropometrist. Body mass index (BMI; in kg/m2) was calculated. Two experienced researchers measured blood pressures after subjects had been seated for at least 10 min. Two specially trained nursing sisters assessed clinical signs of malnutrition. Fasting blood samples were drawn from the vena cephalica by using a sterile butterfly infusion set and sterile syringes. Blood was left to clot, and serum was prepared in the field with the use of a Universal 16R Hettich centrifuge (Tuttlingen, Germany) with cooling facilities. Aliquots of serum were stored at –20 °C in the field for 2–4 d and then at –84 °C in the laboratory until analysis was done. Serum protein, albumin, globulin, -glutamyl transpeptidase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, lactate dehydrogenase, and lipids were measured with the DAX system (discrete analyzer; Technicon DAX48; Miles Inc, Diagnostic Division, Tarrytown, NY). HIV status was determined with an enzyme-immunologic method (Enzymum-Test, anti-HIV 1 + 2+ subtype; Boehringer Mannheim, Mannheim, Germany; catalog no. 1557319).
Statistical analysis
The data were analyzed by using the SPSS package (version 9; SPSS Inc, Chicago, IL). Data are presented as adjusted means (95% CIs) for HIV-infected and HIV-uninfected subjects. Data that were not normally distributed were normalized by logarithmic transformations. Multivariate analysis, adjusted for age, year of study, urbanization, sex, and alcohol intake, was used to compare biochemical variables of HIV-infected and HIV-uninfected subjects. Partial correlations, controlling for age, sex, year of study, urbanization, and alcohol intake, were used to determine the associations between the intake of dietary fat and vitamin E and the biochemical variables. The Fisher z values were calculated for each correlation to test for significant differences in R values between HIV-infected and uninfected subjects. In cases when the test for equal correlations was not significant, a combined R value for HIV-infected and HIV-uninfected subjects was calculated by using the weighted average of the Fisher's z transform of the 2 independent R values and taking the inverse transformation of the result (16).
Ethical considerations
The study was conducted with the full cooperation of the North West Department of Health and Social Services and with the permission of the various communities from which the subjects were recruited. It was also approved by the ethics committee of the former Potchefstroom University (now North-West University; approval no. 4M5-95). All subjects were fully informed about the objectives and procedures of the study in their home language, and all signed an informed consent form. Illiterate subjects signed with a cross. After completion of the study, additional approval from the same ethics committee was obtained to test anonymously for HIV status. Subjects identified with hypertension, diabetes mellitus, and anemia or who complained of not feeling well were referred to local clinics, hospitals, or their physicians. The subject's travel expenses were paid. Volunteers who did not meet the inclusion criteria were screened for hypertension and diabetes mellitus and were referred for treatment if necessary. Subjects who requested HIV testing were informed that this testing could be obtained from health facilities, free of charge, with pretest and posttest counseling capacity.
RESULTS
Of the apparently healthy asymptomatic subjects, 12% (12.3% of the men and 11.5% of the women) tested positive for HIV (Table 1). HIV-infected subjects were slightly but significantly younger, and more of these subjects lived in urban environments than did uninfected subjects. Concentrations of total serum protein and globulin were significantly higher, whereas concentrations of total serum cholesterol, HDL cholesterol, and albumin were significantly lower in HIV-infected subjects than in uninfected subjects. Alcohol intake tended (P = 0.08) to be higher in HIV-infected subjects. HIV-infected and HIV-uninfected subjects did not differ significantly with regard to BMI, education, smoking or snuff taking, blood pressure, and physical activity.
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TABLE 1. Characteristics of the HIV-infected and HIV-uninfected subjects
Three of the measured liver enzymes—ALT, AST, and lactate dehydrogenase—in these asymptomatic HIV-infected subjects were significantly higher than in the HIV-uninfected subjects (Table 2), indicative of cell and tissue damage and probably an inflammatory response (17). The mean liver enzyme concentrations were still within the recommended reference ranges (18).
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TABLE 2. Serum liver enzymes of the HIV-infected and HIV-uninfected subjects1
Mean (95% CI) intakes of energy and fat, including total fat, saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and PUFAs, in the HIV-infected and uninfected subjects did not differ significantly (Table 3). PUFA intake was mainly linoleic acid (18:2n–6). The intake of n–3 PUFA (-linolenic acid) was low in both HIV-infected and HIV-uninfected subjects, whereas the intakes of eicosapentaenoic and docosahexaenoic acids were negligible. No statistically significant differences in the mean intake of other nutrients were observed between HIV-infected and uninfected subjects (9). When compared with dietary guidelines, it seems that both groups followed a low-fat (prudent) diet, with total fat providing <30% energy. However, intakes of micronutrients such as vitamin A, vitamin E, ascorbic acid, and zinc in both groups were inadequate (Table 3).
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TABLE 3. Daily intakes of energy, dietary fat, and selected micronutrients in HIV-infected and HIV-uninfected subjects1
Partial correlation coefficients between dietary fat intake and liver enzymes showed consistent significant positive associations between PUFAs (in g or % of energy), intake of linoleic acid, and PUFA:SFA and all liver enzymes in HIV-infected subjects but not in HIV-uninfected subjects (Table 4). Most of the correlation coefficients differed significantly between HIV-infected and HIV-uninfected subjects. Intake of vitamin E, which was highly correlated with intake of PUFAs in this study population (R = 0.57, P < 0.01 for HIV-infected and HIV-uninfected subjects combined), was positively associated with serum -glutamyl transpeptidase, ALT, and AST in HIV-infected subjects, and these correlations differed significantly from the correlations in HIV-uninfected subjects. The above correlations were probably not due to chance because other known correlations were also seen in these subjects, eg, between the concentration of total serum cholesterol and intake of SFA (HIV-uninfected subjects: R = 0.10, P = 0.00; HIV-infected subjects: R = 0.13, P = 0.09). No associations were seen between intake of PUFAs and other variables that differed significantly between HIV-infected and uninfected subjects, namely protein, albumin, globulin, and HDL cholesterol (data not shown).
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TABLE 4. Partial correlation coefficients between intakes of dietary polyunsaturated fat (% of energy) and vitamin E and liver enzymes in HIV-infected and HIV-uninfected subjects1
DISCUSSION
The main observation of this investigation was that the n–6 PUFA intake was adversely related to liver function in asymptomatic HIV-infected subjects compared with HIV-uninfected subjects. Because the THUSA study was not designed to investigate this specific relation, the following discussion about these observations and the possible mechanisms involved are therefore speculative but nevertheless provide important direction for urgent research in this regard.
The HIV-infected subjects in this study were apparently healthy and asymptomatic. Most were probably at an early stage of infection, indicated by the absence of significant differences between HIV-infected and uninfected subjects with regard to BMI, total sum of skinfold thickness (9), normal body temperatures, no use of chronic medication, and absence of enlarged glands as determined by 2 trained nursing sisters during a brief clinical examination.
The significantly lower concentrations of serum albumin, total cholesterol, and HDL cholesterol and higher concentration of total protein in HIV-infected subjects are in accordance with the literature (19, 20). The concentrations were, however, within desirable reference ranges, further reflecting an early stage of the disease. HIV infection is accompanied by persistent chronic inflammation, oxidative stress (21, 22), and a weakened antioxidant defense system (21).
PUFA is susceptible to attack by free radicals and oxidation into lipid peroxides (23) and consequently contributes to inflammation and oxidative stress. Decreased concentration of red blood cell PUFAs, resulting from increased lipid peroxidation, was shown in HIV-positive patients compared with healthy control subjects (24). Studies on rats have shown the role of PUFAs in promoting ethanol-induced liver injury through increased oxidative stress (25). It can, therefore, be speculated that, although the HIV-infected subjects in this study population were asymptomatic, they were more vulnerable to liver damage by PUFAs, probably because of increased oxidative stress, than were uninfected subjects. Because these subjects were at an early stage of the disease, the observed associations may have been underestimated, and the effect of PUFA intake may even be worse at a later stage of the disease or during concurrent infection. This, however, needs to be confirmed.
The reuse of abused fats (ie, fats that have been overheated and reused several times and that are unstable and highly oxidized) should also be considered. Despite regulations in South Africa since 1996 that prohibit the use of unstable highly oxidized abused fats, frying establishments still use these fats (mainly sunflower oil) in frying processes. In addition, these unstable fats, frequently containing breakdown products far above the regulatory limits (<25% polar compounds or <16% polymers), are still being distributed by these businesses to the uninformed poor for reuse in food preparation (26). Extensive animal studies have shown that ingesting oxidized fats can cause oxidative stress and a variety of adverse biological effects such as elevated concentrations of liver enzymes (alkaline phosphatase, AST, and ALT) (27, 28), growth retardation, diarrhea, tissue damage and increased mass of the liver and kidneys, increased peroxidation of membrane and tissue lipids, induction of cytochrome P-450 activities in the liver and colon, increased cell proliferation in the esophagus, and even death. In humans, mildly thermally oxidized fats when ingested or inhaled resulted in elevated concentrations of plasma lipid peroxides [summarized by Kock et al (26)].
Micronutrient deficiencies may exacerbate the oxidative stress induced by HIV (21). In this regard, the inadequate intake of micronutrients (specifically vitamin A, vitamin C, vitamin E, and zinc) by these subjects could have contributed to decreased antioxidant capacity and liver damage by PUFA intake. Reduced oxidative stress was shown in HIV-infected subjects after supplementation with antioxidant vitamins (29, 30).
The positive associations seen between the intake of vitamin E and the liver enzymes can probably be explained by the significant positive correlation seen between the intake of PUFAs and the intake of vitamin E. The main dietary sources of PUFAs (eg, sunflower oil) were probably also the main carriers of vitamin E in these subjects. The amount of vitamin E in the PUFA-rich dietary sources was therefore probably insufficient to protect against the harmful effects of PUFA intake on liver enzymes in HIV-infected subjects. It is therefore important for further research that, when vitamin E status is improved, is done by means of other sources than sources high in PUFAs.
Because HIV-infected patients are already vulnerable to lipid peroxidation and oxidative stress, which are related to the progression of HIV and AIDS (24, 31), it is extremely important to minimize their exposure to harmful substances. In South Africa, as well as other countries that have traditionally high intakes of n–6 PUFA-rich vegetable oils, it is imperative and urgent that the findings in this study be further investigated to make recommendations for HIV-infected patients about the use of PUFA-rich fats.
The hypothesis that PUFA intake promotes liver damage because of increased oxidative stress in HIV-infected subjects needs to be confirmed in appropriate and well-designed trials. The following questions also need to be addressed: Is PUFA intake, as such, or the intake of abused PUFAs to blame for the adverse effects? What is the optimal safe intake of n–6 PUFAs? Could an improved micronutrient status, especially dietary antioxidants, in conjunction with a diet rich in PUFAs protect against oxidative damage to tissues? Finally, should the PUFA-rich edible fats in the diets of HIV-infected patients be replaced with other types of fats, and what type of fat will be the most appropriate to meet the minimum PUFA requirements and is affordable and readily available?
ACKNOWLEDGMENTS
WO was the main author of the paper and planned, collected, and analyzed the data. AvG helped write the manuscript. AK planned and organized the study, collected the data, and provided statistical analysis of the data. HHV was the study leader, was responsible for all aspects of the study (planning, organization, data collection), and initiated the current investigation. None of the authors had a conflict of interest.
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