A synergistic relationship between immune dysfunction and malnutrition has long been recognized(1), suggesting that inadequate nutritional status may be an important cofactor of HIV-1 disease progression. Our studies indicate that many of the nutrient deficiencies that are highly prevalent in asymptomatic HIV-1-infected individuals, including zinc and vitamins A, E, B6, and B12(2), are well documented to impair immunological function(3-5). Furthermore, development of nutrient deficiency has been associated with faster disease progression in vitamin A- or vitamin B12-deficient HIV-1-infected homosexual men(6), and with increased mortality in vitamin A-deficient HIV-1-seropositive drug users(7).
HIV-1-infected men and women who abuse drugs may be particularly vulnerable to nutritional deficiencies due to drug-nutrient interactions as well as relatively poor dietary intake. Addictive drugs of abuse have been shown to produce significant nutritional alterations(8,9), which may contribute to the immunodeficiency observed in HIV-1-infected individuals(4,5). A detailed nutritional profile of the HIV-1-infected drug user, however, has yet to be described. Moreover, although women and their infected children comprise rapidly growing groups of newly diagnosed cases(10-12), little information is available regarding the nutritional status of HIV-1-infected female drug users, whose nutritional requirements and dietary patterns may be different from those of HIV-1-infected male drug users. The present study was designed to evaluate the relationship between immune and nutritional status in HIV-1-infected men and women who abuse drugs.
MATERIALS AND METHODS
Study Population
Nutritional and immune measures were available for 125 subjects randomly selected from a cohort of 284 men and women being followed longitudinally for HIV-1 infection and cofactors of disease progression. Subjects were recruited from a street drug-using population, which has been followed for the past 9 years. The HIV antibody status of participants was determined by standard HIV enzyme-linked immunosorbent assay antibody testing, with Western blot confirmation. The 125 HIV-1-seropositive participants were examined every 6 months for 3.5 years at a community-based study clinic, where blood was drawn for evaluation of immune function and nutritional status, and a history and physical examination conducted.
Informed consent was obtained from all individuals, and the investigation was approved by the Human Studies Committee of the University of Miami School of Medicine.
Immunological Parameters
Immune assessment included measurements of lymphocyte subpopulations(CD4, CD8) and immune activation (β2-microglobulin). Flow cytometry and monoclonal antibodies(13) were used to quantitate numbers of circulatory T-helper/inducer (CD4) and T-suppressor/cytotoxic(CD8) cells. β2-Microglobulin was measured by radioimmunoassay(Pharmacia, Uppsala, Sweden), with the use of standards provided by the manufacturers.
Nutritional Laboratory Analysis
Fasting blood samples were obtained from all participants between 8:00 AM and 11:00 AM, to minimize the influence of either the last meal or diurnal variations on specific nutritional, metabolic, and endocrine parameters. After processing, all samples of blood were stored at-70°C, until ready for analysis.
Biochemical determinations of specific nutrient levels known to influence immune function and to be altered in HIV-1-infected individuals were evaluated, as previously described(2).
Briefly, total serum vitamin A and E levels were determined by high-performance liquid chromatography using a simultaneous determination method. Levels of vitamin B6 in all participants were determined by erythrocyte transaminase assay, and total plasma cobalamin levels (vitamin B12) were established by radioisotope dilution assay. Plasma levels of zinc were determined using atomic absorption spectrophotometry with graphite furnace attachment, and selenium levels were evaluated by a standardized fluorometric method, with selenium reference obtained from the National Institute of Standards and Technology(Standard Reference Material 1598), using bovine serum.
Overall nutritional status was evaluated by prealbumin and retinol binding protein measurements, which are considered to be sensitive indicators of subclinical malnutrition(14). Levels of prealbumin and retinol binding protein were determined by standard radial immunodiffusion, with measurements <15.0 and 3.0 mg/dl considered deficient for prealbumin and retinol binding protein, respectively.
Dietary Intake
Dietary intake was evaluated using a semiquantitative food frequency questionnaire(15), an instrument standardized for nutritional epidemiological studies.
In this cohort, dietary intake was assessed over a 6-month period. Food consumption was evaluated in terms of dietary intake and supplementation from pills, and then was compared with the U.S. Recommended Dietary Allowance (RDA) standards(16).
Statistical Analysis
Comparisons of the distribution of continuous nutritional measurements were made using the Wilcoxon rank-sum test. The statistical significance of observed differences between men and women in dichotomous parameters was examined with the use of the χ2 test. When cell counts were not balanced, the exact permutational method was used to calculate the exact p values. A paired Student's t test was used to compare dietary intake and RDA values. Spearman's correlation procedures were used to examine the relationship between dietary intake and plasma nutrient status.
RESULTS
Group Characteristics
The 125 subjects in this study included 82 men and 43 women, ranging in age from 27 to 61 years (mean age, 40.4 ± 7.5 years). As indicated in Table 1, the majority of the participants were African American (88%), with a smaller proportion of Hispanic (8%) and white (4%) men and women. Socioeconomic status and mean years of education were similar in the men and women. The majority of the population reported yearly income <$10,000 U.S. The major drug of abuse was cocaine, with positive urine toxicology noted in 85% of the men and 80% of the women. The immune status was similar in the men and women.
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TABLE 1: Group characteristics
Nutritional Status of HIV-1-Infected Men and Women Who Abuse Drugs
Biochemical determinations revealed widespread nutritional abnormalities in the HIV-1-infected drug users. Up to 86% (108 of 125) of the HIV-1-seropositive men and women had at least one nutritional deficiency, with multiple deficiencies observed in 56% of the group.
Table 2 indicates specific differences in plasma nutrient levels between the HIV-1-infected men and women. Overall nutritional status, as determined by prealbumin measurements, was significantly better in the HIV-1-infected men (20.9 ± 6.6 mg/dl), relative to the women(17.3 ± 8.2 mg/dl; p < .01). Moreover, a significantly higher proportion of overly low prealbumin levels (<15 mg/dl) was observed in the women (44%) as compared with 13% in men (p < .01). Measurements of retinol binding protein were similarly significantly lower in the women (3.3 ± 2.0 mg/dl) than in the men (4.1 ± 2.8; p=.02). HIV-1-infected women also exhibited lower levels of the trace element selenium (110 ± 20 µg/L) than the men (120 ± 20 µg/L; p < .01), and had a significantly higher proportion of selenium deficiency (<85 µg/L) relative to the men (16% vs. 3%; p < .01).
In both the male and female groups, micronutrient deficiencies, particularly of zinc and vitamins A and E, were widespread. As shown in Figure 1, 57% of the women and 45% of the men had zinc levels <0.75 mg/dl. Moreover, 49% of the HIV-1-seropositive men and 63% of the infected women exhibited low levels of vitamin A(<0.3 µg/ml), with plasma levels of vitamin E <5 µg/ml noted in 48% of the women and 46% of the men. Deficiency of vitamin B12 (<200 pg/ml) and vitamin B6 (activity coefficient [A/C] > 1.85) was considerably smaller, occurring in 9.3% to 11.6% of the women and 8.5% to 16.3% of the infected men.
TABLE 2: Plasma levels of nutrients
Dietary Intake Patterns in HIV-1-Infected Drug Users
Table 3 and 4 indicate dietary consumption patterns for HIV-1-infected men and women. Intake of most micronutrients was at or above the RDA values, although zinc intake was below the RDA for both the women (p <.01) and the men (p < .01).
The proportion of participants reporting use of supplements ranged from a low of 6% to 7% for zinc to a high of 24% to 29% for vitamins B6, B12, E, and A. A comparison of the proportion of deficiency between supplemented and nonsupplemented HIV-1-infected drug users revealed similar levels of deficiency of vitamins B6, B12, A, and zinc. The proportion of vitamin E deficiency, however, was significantly higher in the nonsupplemented(50%), relative to the supplemented group (26%;p < .05).
Immune and Micronutrient Status
Parameters of immune function were similar in the HIV-1-infected men and women. As shown in Table 1, mean CD4 counts were not significantly different between men (407 ± 291/mm3) and women (470 ± 376/mm3). Additionally, no significant differences in CD8 counts or β2-microglobulin measurements were noted in the HIV-1-seropositive men and women.
FIG. 1: Micronutrient deficiencies, particularly vitamins A (<0.3µg/ml), E (<5 µg/ml), and zinc (<0.75 µg/ml), were widespread in both HIV-1-infected men (solid bar) and HIV-1-infected women(shaded bar). A significantly higher proportion of selenium deficiency(<85 µg/L) was exhibited in the women relative to the men(**p < .01).
TABLE 3: Dietary intake in HIV-1-infected men
Stratification of the data (CD4 count <200/mm3, CD4 count >200/mm3), revealed that the nutritional status among men (n = 55) and women (n = 28) with CD4 counts >200/mm3 was similar. Women with advanced disease (CD4 count ≤200/mm3; n = 11), however, had significantly lower levels of vitamin A (0.22 ± 0.01 µg/ml), vitamin E (5.0 ± 1.6 µg/ml), and selenium (99 ± 19µg/L), as compared with levels in the men with CD4 count ≤200/mm3 (n = 20), which were 0.40 ± 0.20µg/ml for vitamin A (p = .009), 6.5 ± 2.1µg/ml for vitamin E (p = .05), and 117 ± 21 µg/L for selenium(p= .02). Levels of prealbumin (11.3 ± 5.5 mg/dl) and retinol binding protein (2.2 ± 0.93 mg/dl) were also significantly lower in the women with low CD4 counts, as compared with the men whose prealbumin and retinol binding levels were 20.8 ± 7.0 mg/dl, (p = .0008), and 4.6 ± 3.1 mg/dl (p = .0005), respectively.
TABLE 4: Dietary intake in HIV-1-infected women
Figure 2 illustrates the proportion of nutrient deficiency in men and women (n = 11) with CD4 counts≤200/mm3. Women with low CD4 counts exhibited significantly more vitamin A deficiency (90%) than men(30%) with CD4 counts <200/mm3 (p < .01). Vitamin E deficiency was also more prevalent in women with CD4 counts <200, occurring in 70% of this group, as compared with only 20% of the men (p < .02). In addition, poorer overall nutritional status, as indicated by overly low levels of prealbumin, was observed in 73% of the women with advanced disease, as opposed to 25% of the men with CD4 counts <200/mm3 (p < .05).
Nutritional Status in Comparison to Other HIV-1-Infected Cohorts
Specific nutrient deficiencies have been previously demonstrated in asymptomatic HIV-1 infection(2). In 100 HIV-1-infected homosexual men, ages 20 to 55 years(2), overtly low blood levels of vitamins A (11%), E (19%), B6 (30%), B12 (11%), and zinc (26%) were documented, whereas mean plasma nutrient levels were within the range considered to be adequate for these micronutrients.
FIG. 2: Women with advanced disease (n = 11; shaded bar) exhibited significantly more deficiency (<0.3 µg/ml) of vitamins A(**p < .01) and E (<5 µg/ml; *p < .02) compared with men with CD4 counts <200/mm3 (n = 20; solid bar).
Micronutrient concentrations below the normal laboratory range in HIV-1-infected subjects have also been reported in a small proportion of HIV-1-seropositive men and women participating in a study of heterosexual HIV-1 transmission(17). In this investigation, 4% of the participants had low zinc and vitamin E levels and 2% had low selenium and vitamin B6 levels.
DISCUSSION
In accord with our previous studies indicating abnormal nutrient measurements in HIV-1-infected homosexual men(2,6), the present findings demonstrate that nutritional alterations, known to affect immune function, are widespread among HIV-1-infected men and women who abuse drugs. Although nutritional deficiencies are common in both drug abusers and homosexual men, the prevalence of nutritional alterations varies. Severely decreased levels of antioxidants, vitamins A and E, which were observed in 40% to 50% of the HIV-1-infected men and women who abuse drugs, are not as evident in HIV-1-infected gay men, who exhibit only 10% to 15% deficiency of these nutrients(2), and appear to be rare in men and women participating in a heterosexual HIV-1 transmission study(17). Inadequate zinc status, however, was prominent in both the homosexual men and the drug users, supporting the importance of this nutrient as a sensitive marker of HIV-1 disease progression(18). The impaired antioxidant status of the HIV-1-infected drug user is of particular concern, in light of the importance of antioxidants in immune function, and evidence that antioxidant function may be a significant factor in AIDS(19-21).
This study also indicates that HIV-1-infected female drug users exhibit poorer overall nutritional status than male users, despite similar immune function, as measured by CD4 counts and β2- microglobulin, and comparable dietary intake. Furthermore, in women with advanced HIV-1 disease, nutritional alterations are especially prominent, with significantly lower plasma levels of vitamins A and E, selenium, retinol binding protein, and prealbumin noted in the women relative to the men. The causes for the low plasma levels and severe nutritional deficiencies exhibited in HIV-1-seropositive women with advanced disease remain unclear, and are most likely multifactorial in origin.
There is substantial evidence to indicate that nutritional status may be an important determinant of survival in AIDS. Severe malnutrition is frequently encountered in the later stages of infection, and marked wasting is one of the most prominent characteristics of advanced disease. Although nutritional factors are not likely to be the most important etiological determinants, they may influence initial susceptibility to HIV-1 infection and have a significant impact on the rate and form of disease expression. Inadequate visceral protein status, as measured by low serum albumin levels, has been demonstrated to have a significant prognostic influence on survival(22,23) and be associated with mortality in HIV-1-infected individuals(24,25). A number of studies suggest that antioxidant status is critical for the proper function of healthy immune cells, and that failure of antioxidant function may be an important factor in AIDS(19-21). A decline in plasma selenium levels, which is part of the antioxidant defense system, has been reported in HIV/AIDS patients(26-30), and is associated with a dramatic increased risk of mortality(31). Consistent with the present findings, studies by Semba et al. have reported widespread vitamin A deficiency in HIV-1-infected intravenous drug users, in association with decreased survival(7). In addition, the development of vitamin A or vitamin B12 deficiency in HIV-1-seropositive homosexual men has been associated with a decline in CD4 count(6). Normalization of vitamin A, vitamin B12, and zinc, on the other hand, has been linked to higher CD4 counts and slower disease progression(6), suggesting that normalization may increase symptom-free survival.
Women have been reported to have poorer survival after AIDS diagnosis than men in some(32-37) but not all(38-44) studies. Additionally, there is evidence to indicate that opportunistic infections (e.g., bacterial pneumonia) occur significantly more in HIV-1-infected women than in men(37,45). These findings may reflect differences in access to health care and experimental therapy, as well as differences in ethnicity and socioeconomic status(46-48), circumstances that could also affect nutritional status and contribute to HIV-1-related mortality. The present investigation reveals that HIV-1 disease in female drug users is associated with a marked, deficient nutritional status, which may be an important determinant of disease progression and survival. Further investigation will be required to determine the mechanisms by which the micronutrient profile is altered in HIV-1-infected male and female drug users, and whether correction can be of therapeutic value in reducing risk of opportunistic infection and slowing disease progression.
Acknowledgments: We thank Noaris Rodriguez for the editorial preparation of this article. This study was supported by National Institute of Mental Health grant number MH50239-05(M.K.B.) and National Institutes of Health and Fogarty International Center grant number 2D43TW00017-08 (M.K.B.).
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