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* Department of Epidemiology, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205; Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205; and Janeway Child Health Centre, St. John's, Newfoundland, A1A 1R8, Canada
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED
ABSTRACT 
We conducted a nonconcurrent prospective cohort study to examine associations between serum concentrations of vitamin B-6, vitamin B-12 and folate and the risk of progression to first acquired immunodeficiency syndrome (AIDS) diagnosis and CD4+ cell decline to < 2 × 108 cells/L. The study population was drawn from a cohort of homosexual and bisexual men in the Baltimore-Washington, DC, area. Eligible subjects were human immunodeficiency virus type 1 (HIV-1)-seropositive at study entry and had serum available in the serum repository from their 1984 baseline study visit. Serum micronutrient levels were assessed in 310 subjects. The follow-up period (April 1984 through December 1993) was approximately 9 y. In Kaplan-Meier analyses, participants with low serum vitamin B-12 concentrations (< 120 pmol/L) had significantly shorter AIDS-free time than those with adequate vitamin B-12 concentrations (median AIDS-free time = 4 vs. 8 y, respectively, P = 0.004). This effect persisted in Cox proportional hazards models after adjusting for HIV-1-related symptoms, CD4+ cell count, age, serum albumin, use of antiretroviral therapy before AIDS, frequency of alcohol consumption and serum folate concentration [relative hazard (RH) = 1.89, 95% confidence interval (CI) = 1.15-3.10). To further explore the temporal relation between low serum vitamin B-12 concentrations and disease progression, additional analyses were performed excluding subjects with more advanced disease at baseline. In these analyses, the increase in risk of progression to AIDS for those with low serum vitamin B-12 concentrations remained significant (RH = 2.21, 95% CI = 1.13-4.34), providing further evidence that low vitamin B-12 concentrations preceded disease progression. In contrast, low serum concentrations of vitamin B-6 and folate were not associated with either progression to AIDS or decline in CD4+ lymphocyte count. Intervention studies are needed to determine whether correction of low serum vitamin B-12 concentrations in early HIV-1 infection will influence the natural history of disease progression.
Key words: AIDS, folate, HIV-1 infection, vitamin B-6, vitamin B-12, humans.INTRODUCTION
Many B-group vitamins (vitamin B-6, vitamin B-12 and folate, in particular) play crucial roles in protein and nucleic acid synthesis. Deficiencies of these micronutrients can lead to dramatic reductions in host resistance and lymphocyte function (Beisel 1982
, Chandra and Newberne 1977, Rall and Meydani 1993). Abnormally high prevalences of low serum vitamin B-6 and vitamin B-12 have been reported in populations of humans infected with human immunodeficiency virus type 1 (HIV-1),4 whereas the evidence for folate deficiency has been less consistent (Baum et al. 1991b, Beach et al. 1992a, Bogden et al. 1990, Burkes et al. 1987, Coodley et al. 1993, Herbert et al. 1989, Mantero-Atienza et al. 1991). Low serum concentrations of these micronutrients, although not necessarily indicators of overt deficiency states, have been associated with poor outcomes in both acquired immunodeficiency syndrome (AIDS) patients and asymptomatic HIV-1-infected individuals (Baum et al. 1991b and 1995, Herzlich et al. 1990, Kieburtz et al. 1991, Remacha et al. 1991).
Vitamin B-12 has been the most extensively studied of the three micronutrients in the context of HIV-1 infection. Low serum vitamin B-12 concentrations have been associated with peripheral neuropathy and myelopathy in HIV-1-infected patients (Kieburtz et al. 1991), diminished performance on specific measures of information processing speed and visuospatial problem-solving skills (Beach et al. 1992b), CD4+ cell decline (Baum et al. 1995, Remacha et al. 1990), increased mortality (Remacha et al. 1991) and increased zidovudine-related bone marrow toxicity (Herzlich et al. 1990). Few studies have examined the consequences of low serum vitamin B-6 and folate concentrations in HIV-1 infection. Low vitamin B-6 status in HIV-1-seropositive subjects was significantly associated with a reduced response of peripheral blood lymphocytes to mitogens and decreased natural killer cell cytotoxicity (Baum et al., 1991b). In a more recent study, development of low vitamin B-6 status over 18 mo was associated with a significant decline in surrogate markers of HIV disease stage (Baum et al. 1995). Folate deficiency, like vitamin B-12 deficiency, may have adverse effects on the immune system and neurological functions (Smith et al. 1987) in HIV-1 infection.
We measured serum concentrations of vitamin B-6, vitamin B-12 and folate in a cohort of HIV-1 seropositive homosexual and bisexual men using sera collected and stored in 1984. These individuals have been followed semiannually for more than 10 y, providing us with a unique opportunity to examine the effect of these serum micronutrients on the natural history of HIV-1 infection using a longitudinal study design.
SUBJECTS AND METHODS
). The HIV-1 serostatus was determined by enzyme immunoassay (Genetic Systems, Seattle, WA) and a Western blot (Du Pont, Co. Wilmington, DE, and Bio-Rad, Hercules, CA) with interpretation using standard criteria. Details on the objectives, design and recruitment protocol of this study have been described elsewhere (Kaslow et al. 1987).
). Normal range for serum B-6 concentrations was defined as 88 to 390 nmol/L based on laboratory standards. The AccessR (Sanofi Pasteur, Chicago, IL) competitive-binding immunoenzymatic assay was used to determine serum vitamin B-12 and serum folate concentrations. The normal ranges for vitamin B-12 and folate, as determined by laboratory standards, were defined as 120 to 720 pmol/L and 3.4 and 4.5 nmol/L, respectively.
). Because serum albumin concentrations are influenced by factors such as physiologic stress and specific disease conditions, we also assessed C-reactive protein concentrations, an indicator of acute physiologic stress. C-reactive protein concentrations were determined using the RapitexR (Rapitex, London, UK) turbidimetric assay based on immunochemical reaction between C-reactive protein and antibodies to C-reactive protein bound to latex particles; there is a correlation between C-reactive protein concentrations and extent of agglutination of the latex particles. Levels above 8.0 mg/L (as determined by the laboratory) were considered to be elevated, indicating the presence of an acute, underlying infection.
). Subjects were asked to estimate their frequency of consumption of each food item over the previous 12 mo, in terms of a typical serving size. Nine frequency categories were available to choose from, ranging from "never or less than once per month" to "6+ per day." Also included on the form were sections for recording the brand name, frequency and amount of any nutritional supplements taken, a write-in section for foods not listed, and a series of questions on the brands and types of fat used for frying, cooking and baking. Completed questionnaires were reviewed and coded by a trained researcher and entered into computer data files. Estimates of food and supplemental intake of vitamin B-6, vitamin B-12 and folate were determined using the nutrient analysis program developed specifically for the questionnaire (Willett et al. 1988).
Independent covariates.
Most of the independent variables included in this study were based on data collected at the baseline clinic visit. Frequency of alcohol consumption was based on the question "how often do you drink now?" asked at the baseline study visit. This variable was originally coded into eight categories ranging from "one to five times per year" to "at least once per day." For this study, frequency of alcohol consumption was collapsed into two categories: 
2 times per week and >2 times per week. Twenty-three subjects were missing information on this variable from their baseline visit. To avoid excluding these individuals from the analyses, values from their next study visit (approximately 6 mo later) were substituted. Information on cigarette smoking was based on the question "do you smoke cigarettes now?" asked on the baseline questionnaire. Subjects were categorized either as nonsmokers or as regular or occasional smokers according to their response. Body mass index was calculated as weight divided by height squared (kg/m2). The HIV-1 related symptoms variable was defined as the presence of one or more of the following most commonly reported symptoms lasting two or more weeks: persistent or recurring fever >37.8°C, persistent diarrhea, oral thrush, persistent fatigue, or unintentional weight loss of more than 4.5 kg. Categories of CD4+ T-lymphocyte count were defined as <5 × 108, 5 × 108 to 7.5 × 108, and >7.5 × 108 cells/L in order to give approximately equal numbers of men in each category. CD4+ T cell counts were not available at the baseline visit for 32 (10.3%) of the subjects. These subjects were assigned to one of the three categories based on data from subsequent adjacent visits. Because the mean decline of CD4+ cell counts in this cohort was only approximately 3 × 107 cells/L per 6 mo, we believe it was possible to estimate the appropriate CD4+ cell category for these individuals with a high degree of accuracy.
). Median AIDS-free times were estimated based on the Kaplan-Meier curves. The logrank test was used to assess the association of the micronutrients and independent variables with both outcomes.
) were used to adjust for covariates that were associated with either serum micronutrient concentrations, disease progression or both (P < 0.15) in the univariate analyses. A step-down procedure was used to eliminate variables that did not produce significant changes in the model estimates. Separate Cox models were fit for each B-group vitamin. Serum nutrient concentrations were entered into the models as binary variables (indicating adequate vs. low levels) and as quartiles. All independent covariates were entered into the models as categorical variables.
) were used to examine the association between serum nutrient concentrations and nutrient intake. The Wilcoxon rank-sum test (Snedecor and Cochran 1989) was used to compare median daily nutrient intakes between subjects with adequate vs. low serum nutrient concentrations and to compare median serum nutrient concentrations between current and past or never users of multivitamin and single-vitamin supplements.
RESULTS
8.0 mg/L), and 71 (23%) had low serum albumin concentrations (<35 g/L).
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Table 1. Characteristics of the study population1 |
Table 2.
Summary of serum micronutrient levels and total nutrient intake (from food and supplements) in 310 HIV-1-seropositive homosexual and bisexual men1,2
88 nmol/L). Subjects with low serum vitamin B-12 concentrations (<120 pmol/L) were slightly older (chi-square test, P = 0.12), tended to drink more frequently (chi-square test, P = 0.13), had lower CD4+ cell counts (chi-square test, P = 0.14), lower serum albumin concentrations (t test, P < 0.001) and were less likely to have started antiretroviral therapy before AIDS (chi-square test, P = 0.12). Low serum folate concentrations (<6.8 nmol/L) were associated with younger age (chi-square test, P = 0.04), lower education level (chi-square test, P = 0.13), lower body mass index (t test, P = 0.05) and lower serum albumin concentrations (t test, P < 0.001).
120 pmol/L) was 8.4 y. The logrank test of the difference between these two curves was highly significant (P = 0.004). No significant differences were found between low vs. adequate serum concentrations for any of the three micronutrients in Kaplan-Meier analyses using CD4+ cell decline to <2 × 108 cells/L as the outcome (logrank P = 0.46, 0.24 and 0.70 for vitamin B-6, vitamin B-12 and folate, respectively).
Multivariate analyses. To examine the independent effect of serum nutrient concentrations on HIV-1 disease progression, Cox proportional hazards models were used to adjust for potential confounders. Variables that were significantly associated with AIDS or CD4+ cell decline (P < 0.15) were entered as covariates in the Cox models. These included HIV-related symptoms, CD4+ cell count, age, serum albumin concentration, serum C-reactive protein concentrations, antiretroviral therapy before AIDS, P. carinii prophylaxis before AIDS and frequency of alcohol consumption. Serum C-reactive protein concentration and use of P. carinii prophylaxis before AIDS were subsequently dropped from the multivariate Cox models because they did not produce significant changes in the model estimates.
Table 3.
Relative risk of AIDS progression in 310 HIV-1-seropositive homosexual and bisexual men1,2
120 pmol/L). After adjustment for serum folate and several other covariates, the RH increased slightly to 1.89 (95% CI = 1.15-3.10). When serum vitamin B-12 concentrations were entered into the Cox models as quartiles, a threshold effect was observed where subjects in quartiles 2 through 4 showed a significant decrease in risk of progression to AIDS compared with those in the lowest quartile. There was little evidence for changing risk after quartile 2 in the crude model or quartile 3 in the adjusted model.
Table 4.
Median serum micronutrient concentrations by use of oral vitamin supplements in 272 HIV-1-seropositive
homosexual or bisexual men1
DISCUSSION
, Remacha et al. 1991, Rule et al. 1994).
found that HIV-1-infected patients that had lower serum vitamin B-12 concentrations had lower hemoglobin, leukocytes, CD4+ lymphocytes and CD4+/CD8+ lymphocyte ratios than HIV-1-infected patients with normal serum vitamin B-12 concentrations. Ninety percent of the patients with low serum vitamin B-12 concentrations had AIDS compared with only 66% of patients with adequate vitamin B-12 concentrations. In a 30-mo follow-up study, Rule et al. (1994) reported that of nine subjects whose disease progressed to AIDS or AIDS-related complex (ARC), seven had falling serum vitamin B-12 concentrations before the onset of AIDS or ARC, and the remaining two experienced drops in serum vitamin B-12 concentration just after their first AIDS diagnosis. These findings were corroborated in a larger study by Baum et al. (1995), who found that development of a low serum vitamin B-12 concentration over an 18-mo follow-up period was associated with more rapid disease progression, as indicated by declines in CD4+ cell counts and an AIDS index (composite measurement of CD4+ cell count and 
2-microglobulin). Subjects in the lowest tertile of plasma vitamin B-12 concentration (195 pmol/L) showed a significantly greater decline in CD4+ cell count over time than those in the highest tertile (>349 pmol/L). We found that the associations of low serum vitamin B-12 concentration with short-term changes in immune variables continued to hold true for clinical outcomes over the long term, as evidenced by an 89% increase in risk of progression to clinical AIDS in our study population over an approximate 10-y follow-up period.
reported that plasma vitamin B-6 concentrations were highly stable in plasma frozen at 
20°C for at least 2 y, with a rate of decline of only about 2.2% per year. Ocke et al. (1995) noted no decline in serum vitamin B-6 and B-12 concentrations after 4 y of storage at 20°C. Because serum nutrient concentrations in our population were not analyzed when the sera were first collected, we could not document the actual amount of degradation that occurred. However, mean serum and plasma concentrations of these vitamins have been reported in many studies of HIV-1-infected individuals, at various stages of infection and from various parts of the world (Baum et al. 1991a and 1995, Beach et al. 1992a, Bogden et al. 1990, Boudes et al. 1990, Coodley et al. 1993, Dowling et al. 1993, Harriman et al. 1989, Herzlich et al. 1992, Mantero-Atienza et al. 1991, Remacha et al. 1993, Revell et al. 1991, Robertson et al. 1993, Rule et al. 1994, Veilleux et al. 1995). Mean serum vitamin B-6 and B-12 concentrations in our study fell within the ranges reported in these other studies. The mean folate concentration in our subjects was slightly lower than those reported in the above populations; however, the prevalence of low serum folate concentrations in our population was comparable to the prevalences reported in these studies. Because all the sera used in our study underwent the same freezing, storage and thawing processes, however, conclusions drawn from internal comparisons should not be affected by the possibility of slight folate degradation during the follow-up period.
). Therefore, we are confident that vitamin B-6 intake on the day of blood sampling did not greatly affect serum vitamin B-6 concentrations.
and Baum et al. (1995) that declining serum vitamin B-12 concentrations were associated with declining CD4+ cell counts. This may explain the lack of association with long-term decline in CD4+ cell count that we observed with only a single measurement of serum vitamin B-12 concentrations.
FOOTNOTES
Manuscript received 28 May 1996. Initial reviews completed 29 July 1996. Revision accepted 1 November 1996.
LITERATURE CITED
20 degrees C: consequences for epidemiologic research.
J. Clin. Epidemiol.
1995;
48:1077-1085
[Medline]
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