Vitamin D status in adolescents and young adults with HIV infection 1 3

Transcription

1 Vitamin D status in adolescents and young adults with HIV infection 1 3 Charles B Stephensen, Grace S Marquis, Laurie A Kruzich, Steven D Douglas, Grace M Aldrovandi, and Craig M Wilson ABSTRACT Background: Vitamin D status affects immune function and thus may affect the progress of HIV infection. Objectives: Our goals were to assess vitamin D intake and status in subjects with HIV infection and in matched control subjects and to determine whether HIV infection was associated with vitamin D insufficiency. Design: Plasma 25-hydroxyvitamin D [25(OH)D] concentrations and vitamin D intake were measured in a cross-sectional study of members of the Reaching for Excellence in Adolescent Health (REACH) cohort. Results: The subjects were aged y; 74% were female, and 72% were black. Mean ( SE) vitamin D intake from food was 30% greater (P 0.023) in HIV-positive subjects ( IU/d; n 237) than in HIV-negative subjects ( IU/d; n 121). The prevalence of vitamin D supplement use was 29% (104 of 358 subjects) and did not differ significantly by HIV status (P 0.87). Mean plasma 25(OH)D did not differ significantly (P 0.62) between the HIV-positive ( nmol/l; n 238) and HIVnegative ( nmol/l; n 121) subjects, nor was HIV status a significant predictor of plasma 25(OH)D when multiple regression analysis was used to adjust for other variables. The prevalence of vitamin D insufficiency [plasma 25(OH)D 37.5 nmol/l] in the subjects was 87% (312 of 359 subjects). Conclusions: HIV infection did not influence vitamin D status. The prevalence of vitamin D insufficiency in both HIV-positive and HIV-negative REACH subjects was high, perhaps because these disadvantaged, largely urban youth have limited sun exposure. Am J Clin Nutr 2006;83: Vitamin D, HIV infection, dietary intake, ad- KEY WORDS olescents, race INTRODUCTION Subjects with HIV infection are at increased risk of osteopenia and osteoporosis (1 3). Immune activation and antiretroviral therapy may underlie this phenomenon. Changes in vitamin D metabolism due to HIV infection may also be a factor (4), because vitamin D is required for bone health maintenance (5) and for adequate immune function (6). Vitamin D affects the development and function of cells of the immune system that help control HIV infection, including macrophages and T lymphocytes. However, few studies have examined the vitamin D status of subjects living with HIV infection (1, 4). The Reaching for Excellence in Adolescent Health (REACH) cohort study examined many aspects of HIV infection in subjects aged y who were recruited in 13 US cities (7, 8). Dietary quality in many REACH subjects did not meet current recommendations (9), which raises questions about their vitamin D intake. This question is particularly timely, because recent studies suggest that vitamin D insufficiency in the United States is more widespread than was previously appreciated (10). Another reason for concern about the vitamin D status of REACH subjects is that approximately three-fourths of the REACH subjects are black, which itself is a risk factor for the development of vitamin D insufficiency (11). Sunlight is a principal source of vitamin D because UV irradiation converts 7-dehydrocholesterol to previtamin D 3 in the skin, but skin pigmentation decreases this conversion (12, 13). For these reasons, we examined the dietary intake and plasma concentrations of 25-hydroxyvitamin D [25(OH)D] in members of the REACH cohort. We also examined the association between HIV status and markers of immune activation and plasma 25(OH)D to determine whether HIV infection is associated with poor vitamin D status. SUBJECTS AND METHODS Study population The REACH study was a prospective, observational study of HIV infection in adolescents conducted at 15 US clinical sites 1 From the US Department of Agriculture, Agricultural Research Service, Western Human Nutrition Research Center at the University of California, Davis, CA (CBS); the Department of Nutrition, University of California, Davis, CA (CBS); the Department of Food Science and Human Nutrition, Iowa State University, Ames, IA (GSM and LAK); the Division of Allergy and Immunology, Joseph Stokes Jr Research Institute at The Children s Hospital of Pennsylvania, Philadelphia, PA (SDD); the Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA (SDD); the Saban Research Institute at the Children s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA (GMA); and the Department of Pediatrics and Medicine, University of Alabama at Birmingham, Birmingham, AL (CMW). 2 Supported by NIH grants R01 AI46183 and P60 MD and by USDA-ARS Project D. The Reaching for Excellence in Adolescent Health study was funded by grant U01-HD32830 from the National Institute of Child Health and Human Development with cofunding from the National Institute on Drug Abuse, the National Institute of Allergy and Infectious Diseases, and the National Institute of Mental Health. 3 Reprints not available. Address correspondence to CB Stephensen, Nutrition Department, 3243 Meyer Hall, University of California, Davis, CA cstephen@whnrc.usda.gov. Received October 12, Accepted for publication January 23, Am J Clin Nutr 2006;83: Printed in USA American Society for Nutrition 1135

2 1136 STEPHENSEN ET AL (7, 8). A standardized protocol was developed through the Adolescent Medicine HIV-AIDS Research Network. Between March 1996 and November 1999, 325 adolescents aged y who had acquired HIV infection through sexual activity or intravenous drug use were recruited. In addition, 171 HIV-negative adolescents were recruited from the same sites by using selection criteria to make the HIV-negative and HIV-positive groups comparable with regard to risk-behavior profiles and demographic characteristics (including age, sex, race, and ethnicity). The present study describes the results of a supplemental study that was conducted during one study visit between January and October One site did not participate in the present study due to logistical difficulties. Of the 436 subjects active in the 14 REACH network sites, 391 agreed to participate in the substudy conducted between January and October 2000 (264 HIVpositive and 127 HIV-negative subjects). Of these, samples from 359 subjects (238 HIV-positive and 121 HIV-negative subjects) were available for vitamin D analysis. The 14 clinical sites were located in Miami, Fort Lauderdale, New Orleans, Birmingham, Los Angeles, Memphis, Washington DC, Baltimore, Philadelphia, Newark, New York (3 sites), and Chicago. The study received approval from the human subjects review boards at the University of California, Davis, the Iowa State University, the University of Alabama at Birmingham, and from each clinic site. All subjects provided informed written consent. Data collected by the REACH study Data were collected with face-to-face interviews, interactive computer interviews, medical record abstractions, and physical and laboratory examinations. The subjects were gowned and weighed at each visit with digital scales that were accurate to 0.1 kg. Heights were measured with calibrated stadiometers that were installed at each study site. Body mass index (BMI) was calculated as weight (in kg)/height 2 (in m) for each subject. Latitudes of cities were taken from the Information Please Database (2005 version; Pearson Education Inc, Upper Saddle River, NJ; The date of the blood drawing for 25(OH)D analysis was used to group the subjects by the following seasons: winter (December- February), spring (March-May), summer (June-August), and fall (September-November). Laboratory tests were performed at local clinic sites according to the REACH protocol, which was described elsewhere (7, 8, 14, 15). Activated CD8 T-cells were measured as previously described (16) by using CD38 and human leukocyte antigen-dr as markers of activation. Absolute CD4 T-cell counts for the HIVpositive subjects were stratified based on the following Centers for Disease Control and Prevention criteria for HIV and AIDS classifications: 499, , and 200 cells/mm 3. A quantitative HIV-1 RNA viral load in plasma was measured in a centralized laboratory on frozen specimens by using either nucleic acid sequence-based amplification or NucliSens assays (Organon Teknika, Durham, NC) as previously described (17). Antiretroviral therapy was coded as a dichotomous variable (receiving therapy or not receiving therapy); descriptive data on antiretroviral therapy use and compliance was previously reported (18). Variables collected for the present study Nonfasting blood samples were collected during a regularly scheduled REACH study visit in The samples were stored at 80 C. Site-to-site variation within biochemical variables was minimized by providing all sites with blood collection and processing tubes obtained from the same central source and by processing and analyzing all samples collected for the present study in batch at a central laboratory. The Block Food Frequency Questionnaire (version 98.2; Block Dietary Data Systems, Berkeley, CA) was used to estimate usual dietary intake patterns and the use of vitamin supplements over the preceding year as previously described (9). The Block Food Frequency Questionnaire was administered in an interview format by trained clinic staff within days of the clinic visit during which blood was drawn. Serving-size pictures, plates, and cups were used to help the subjects estimate portion sizes. Information on supplement use, which was principally the use of multivitamins, was also collected. A registered dietitian reviewed the questionnaires, and interviewers were contacted regarding missing information, unusual responses, or discrepancies before data entry. Nutrient intake was determined with a dietary analysis software package (Block Dietary Data Systems). 25-hydroxyvitamin D analysis Plasma 25(OH)D was measured in 2004 with a 25(OH)D enzyme-based protein binding assay from Alpco Diagnostics (American Laboratory Products Company, Windham, NH; catalog # ). This competitive binding assay detects 25(OH)D 3, 24,25(OH) 2 D 3, and 25(OH)D 2 by competing with labeled 25(OH)D for binding to the vitamin D binding protein. Nonfasting plasma samples were assayed in duplicate by using standards and reference sera that were provided with the kit. Some samples were thawed and refrozen before 25(OH)D analysis. Samples that appeared lipemic on visual inspection (n 12) were not analyzed per the manufacturer s recommendation, because excess lipid can interfere with the assay. Laboratory reference sera and 14 plasma samples collected between 27 January and 15 February 2000 from UC Davis students and staff (6 women and 8 men, aged y) were also analyzed at the same time. Data on race and ethnicity were not collected from the UC Davis subjects. The calibration curve ranged from 6.4 to 250 nmol/l. Samples with values greater than the negative control but 6.4 nmol/l were assigned values based on an extrapolation of the standard curve. No samples had 25(OH)D concentrations greater than the highest standard, but 7 samples had no detectable 25(OH)D concentrations after remeasurement and were assigned a value of 0.05 nmol/l for statistical analysis. Samples with undetectable 25(OH)D on the first analysis or with a difference between duplicates of 20% and 10 nmol/l were reanalyzed. Standards were within the expected ranges. The mean ( SD) value of duplicate measurements on 13 different days for a standard with a known value of 3 nmol/l was 7 5 nmol/l, whereas for a standard with a known value of 24 nmol/l the corresponding value was 27 7 nmol/l. The intraassay CV reported by the manufacturer on 24 replicate determinations of one sample was 11%. The mean CV of duplicate measures from 488 samples in the present analysis was 21%. The mean ( SE) 25(OH)D concentration of the UC Davis samples was nmol/l (range: nmol/l). The mean 25(OH)D concentration of the REACH samples was nmol/l (n 359; range: undetectable 76 nmol/l).

3 VITAMIN D STATUS AND HIV INFECTION 1137 STATISTICAL ANALYSIS Software All analyses were performed with SIGMASTAT for Windows, version 3.01 (Jandel Scientific, San Rafael, CA). Unless otherwise indicated, data are presented as means SEs, and statistical significance was considered to be P Bivariate analysis Prevalence rates were compared between groups by the chisquare test. Means were compared between 2 groups by Student s t test and between multiple groups by one-way analysis of variance (ANOVA) with the Tukey test for all-pairwise post hoc comparisons and the Holm-Sidak test for comparisons with a single group. When distributions were not normal, continuous variables were compared between 2 groups by the Mann- Whitney rank-sum test or between 3 or more groups with a nonparametric ANOVA. A 2-way ANOVA was used to compare differences by sex and HIV status simultaneously on normally distributed variables. Regression analysis Plasma 25(OH)D data were not normally distributed, but use of the square root of these values produced normality and equal variance for these analyses. A backwards, stepwise regression analysis was used to identify significant predictors of the plasma 25(OH)D concentration from among several types of independent variables that were described previously (18). These included the following: 1) demographic and behavior variables, such as age, sex, race (black or other), ethnicity (Hispanic or other), BMI, smoking (yes or no), alcohol use (intensity), marijuana use (intensity), and illicit drug use (intensity); 2) intake of vitamin D from food and supplements; 3) HIV variables, such as HIV status, CD4 T-cell count, plasma virus load, and use of antiretroviral therapy (yes or no); 4) immune activation variables, such as activated CD8 T-cells, neutrophil count, and plasma neopterin, C-reactive protein, and ceruloplasmin concentrations; and 5) environmental variables, such as latitude and season. Intensity of alcohol and drug use was scored as follows: 0 never; 1 1time/mo; 2 1time/mo and 1 time/wk; 3 1times/wk, but not every day; and 4 every day. These variables were selected for analysis because of their known effect on vitamin D status (eg, sex, race, BMI, intake, latitude, and season) and to examine the effect of HIV infection on vitamin D status (eg, HIV variables and immune activation variables). Additional behavior variables were also included (eg, smoking status and drug and alcohol use) because these may affect vitamin D intake, sun exposure, or vitamin D metabolism. Stepwise regression was performed on all subjects together, HIV-positive subjects only, and HIV-negative subjects only. Significant variables (P 0.05) identified in this manner were included in a model for all subjects and were tested for interaction with HIV status by using interaction terms. Variables identified in preliminary analysis by using HIV-positive and HIV-negative subjects separately were not included in the final model if the interaction term was not significant at P The terms of the final equation were also examined for logical interactions by visual inspection of data plots and by a 2-way ANOVA. TABLE 1 Demographic characteristics of subjects in the Reaching for Excellence in Adolescent Health cohort Variable RESULTS HIV-positive subjects (n 238) HIV-negative subjects (n 121) P 1 Female [n (%)] 171 (72) 93 (77) 0.37 Black [n (%)] 178 (75) 80 (66) 0.11 Hispanic [n (%)] 41 (17) 26 (21) 0.40 Age (y) Pregnant females [n (%)] 16 (9) 11 (12) 0.67 Season of blood drawing 0.59 Winter [n (%)] 29 (12) 14 (12) Spring [n (%)] 107 (45) 63 (52) Summer [n (%)] 97 (41) 41 (34) Fall [n (%)] 5 (2) 3 (2) 1 Comparison of the prevalence between HIV-positive and HIVnegative subjects (chi-square test or Student s t test). 2 x SE (all such values). Subject characteristics Members of the REACH cohort were aged between 14 and 23 y at the time of analysis. They were predominately female and black (Table 1). Of the 359 subjects in the present analysis, 258 were black (71.9%) and 8 also reported Hispanic ethnicity (2.2%). Of the 101 nonblack subjects, 59 (58.4%) were Hispanic. Demographic data were reported previously in greater detail (8, 9), including the observation that only 4.1% of REACH subjects reported their ethnicity and race as non-hispanic and white. Blood samples and data on vitamin D intake from food and supplements were collected between January and October, with most subjects being recruited in the spring and summer months (Table 1). Dietary vitamin D intake and supplement use The mean ( SE) intake of vitamin D from food for all subjects (n 358) was IU/d, whereas the median intake was 179 IU/d (25th/75th percentiles: 98/334 IU/d). The corresponding values for intake from food plus supplements were IU/d and 296 IU/d (127/554 IU/d). Intake of vitamin D from food did not differ significantly by sex, but intake was 30% greater for HIV-positive subjects ( IU/d) than it was for HIVnegative subjects ( IU/d; P by 2-way ANOVA comparing sex and HIV status; Table 2). Of all subjects, 29% used supplements (104 of 358), with the concentration of vitamin D ranging from 114 to 400 IU/d. No significant difference in supplement use was seen between sexes or by HIV status (Table 2). When intakes of vitamin D from food and supplements were considered together, 39% of the subjects consumed less than the recommended intake ( 200 IU vitamin D/d). No significant difference in the percentage of subjects with adequate intake was seen by sex or HIV status (Table 2). Plasma 25-hydroxyvitamin D The mean ( SE) plasma 25(OH)D concentration for all subjects was nmol/l (n 359). The median concentration was 17.7 nmol/l (25th/75th percentiles: 8.0/26.3 nmol/l). Plasma 25(OH)D concentrations did not differ significantly by

4 1138 STEPHENSEN ET AL TABLE 2 Intake of vitamin D from food and supplements HIV-positive subjects HIV-negative subjects P Females (n 170) Males (n 67) Females (n 93) Males (n 28) HIV Sex Interaction Intake from food (IU/d) Supplement use [n (%)] 3,4 59 (35) 17 (25) 27 (29) 10 (36) Total intake (IU/d) Intake 200 IU/d [n (%)] 4,5 61 (36) 24 (36) 42 (45) 11 (39) Two-way ANOVA on log 10 values. 2 x SE (all such values). 3 Of the 113 subjects who used supplements, 86 consumed 400 IU/d, 10 consumed 286 IU/d, and 17 consumed 114 IU/d. 4 P values from chi-square test comparing differences by sex and HIV status for all subjects (n 358). No significant differences were seen when HIV status was compared within sexes, P Adequate vitamin D intake is 200 IU/d. HIV status or sex, and the median values in all 4 groups were quite low ( 20 nmol/l; Figure 1). Eighty-seven percent (312 of 359) of all subjects had values indicative of vitamin D insufficiency ( 37.5 nmol/l). The prevalence did not differ significantly by either sex (P 0.98) or HIV status (P 0.91). The subjects who consumed vitamin D above the adequate intake (ie, 200 IU/d) had a slightly lower prevalence (84%; 185 of 220) of vitamin D insufficiency than did the subjects who consumed less than this amount (92%; 27 of 138; P 0.043). The mean ( SE) plasma 25(OH)D concentration was 33% higher in those who consumed at least the adequate intake of vitamin D ( nmol/l; n 220) than in those who did not consume FIGURE 1. Distributions of plasma 25-hydroxyvitamin D [25(OH)D] concentrations in Reaching for Excellence in Adolescent Health subjects by sex and HIV status. Box plots show median (horizontal line in center of each box), 25th and 75th percentiles (bottom and top of each box), 10th and 90th percentiles (bottom and top of error bars), and 5th and 95th percentiles (F). Data are shown separately for HIV-positive females (n 171) and males (n 67) and for HIV-negative females (n 93) and males (n 28). Plasma values did not differ significantly between the groups by 2-way ANOVA (P 0.74 for sex; P 0.62 for HIV status; P for interaction 0.68). this amount ( nmol/l; n 138; P 0.001). Many of the subjects who used supplements consumed concentrations of vitamin D from supplements alone that were greater than the recommended adequate intake (Table 2). Not surprisingly, supplement use was associated with an 18% increase in plasma 25(OH)D concentrations [ nmol/l (n 113) for supplement users compared with nmol/l (n 245; P 0.049) for those who did not use supplements]. Supplement users did not have a significantly different prevalence of vitamin D insufficiency than did nonusers (P 0.18). The subjects who were not vitamin D insufficient by measurement of plasma 25(OH)D (n 46) had a 58% greater median intake (431 IU/d; 25th/75th percentile: 203/751 IU/d) of vitamin D from food and supplements than did insufficient subjects (272 IU/d; 116/542 IU/d; n 312; P 0.002). Plasma 25(OH)D concentrations vary by season and are typically greater in the summer and fall than in the winter and spring due to variation in sun exposure. This seasonal effect was also observed in the present study. The mean ( SE) 25(OH)D value for samples collected in the summer or fall was nmol/l (n 146) compared with nmol/l (n 213; P 0.001) for samples collected in the winter and spring, a difference of 36%. The risk of insufficiency was also greater in the winter and spring (median: 92%; 25th/75th percentile: 195%/ 213%) than in the summer and fall (80%; 117%/146%; P 0.003). The intensity of sun exposure is greater in the southern than in the northern United States, which can also affect vitamin D status. Because our study sites ranged from Miami in the south (25 46 ) to Chicago in the north (41 50 ), we compared plasma 25(OH)D concentrations by the different study sites (Table 3). Using Miami as the comparison site, we found that subjects from all sites except Fort Lauderdale, New Orleans, Los Angeles, and one site in New York City had significantly lower mean plasma 25(OH)D concentrations than did the subjects in Miami. The prevalence of insufficiency was also greater in the more northern latitudes. Vitamin D intake from food and supplements did not differ significantly by study site. Self-report of race (eg, black or white) may also be associated with plasma 25(OH)D concentrations either because of its association with dietary patterns or because of the effect of skin pigmentation on vitamin D production after sun exposure. In the present study, black subjects had 20% lower plasma 25(OH)D

5 VITAMIN D STATUS AND HIV INFECTION 1139 TABLE 3 Latitude, plasma 25-hydroxyvitamin D [25(OH)D] concentrations, and percentage of vitamin D deficient subjects by study site and mean plasma 25(OH)D concentrations and percentage of vitamin D deficient subjects from the third National Health and Nutrition Examination Survey (NHANES III) Plasma 25(OH)D Total vitamin D intake (IU/d) 1 Study site Latitude Subjects Black Mean SE Median (25th/75th percentile) n % nmol/l % IU/d Miami (197/610) Fort Lauderdale (163/640) New Orleans (105/402) Birmingham (91/492) Los Angeles (118/477) Memphis (88/418) Washington, DC (154/399) Baltimore (153/533) Philadelphia (148/641) Newark (234/574) New York Bronx (113/527) Manhattan (191/609) Brooklyn (40/674) Chicago (73/281) Total (127/554) NHANES III Black females aged y Black males aged y Total vitamin D intakes did not differ significantly by site, P 0.91 (rank-sum test). 2 Plasma 25(OH)D concentrations 37.5 nmol/l are considered as vitamin D deficiency. 3 n 50 for dietary intake data. 4 Significantly different from mean plasma 25(OH)D concentrations in Miami, P 0.05 (one-way ANOVA with Holm-Sidak post hoc test). 5 These data from NHANES III were published previously (19). concentrations (x SE: nmol/l; n 258) than did other subjects ( nmol/l; n 101; P 0.001). However, the risk of vitamin D insufficiency did not differ significantly by race (P 0.66). Mean intake of vitamin D from food did not differ significantly by race (P 0.87), nor did the percentage of subjects who used supplements (P 0.39) or the mean total vitamin D intake (P 0.50). Some of the site-to-site variation in plasma 25(OH)D (Table 3) appeared to be due to race, in addition to latitude. For example, Birmingham, Los Angeles, and Memphis are all at very similar latitudes (Table 3), but the concentrations of plasma 25(OH)D were significantly higher in the subjects in Los Angeles than in the subjects in Birmingham or Memphis. Additionally, the percentage of black subjects was lower in the Los Angeles site than in the Birmingham (P 0.002) or Memphis (P 0.002) sites. Because the increased immune activation of HIV infection can increase requirements for some nutrients (20), we reexamined the question of whether HIV status affected plasma 25(OH)D concentrations in the REACH subjects. Using multiple regression analysis, we adjusted for sex, latitude, summer or fall season, total vitamin D intake, and race to determine whether HIV status was a significant predictor of plasma 25(OH)D concentrations. Although the overall equation explained 25% of the variation in plasma 25(OH)D concentrations (R ; n 358), the coefficient for HIV status was not significantly different from zero (P 0.94), which indicated that HIV status had no significant association with plasma 25(OH)D concentrations. We hypothesized that behavior variables or markers of immune activation may also be associated with plasma 25(OH)D. For these reasons, we performed a stepwise, multiple regression analysis to identify significant predictors of plasma 25(OH)D (Table 4). The quantitative contribution of each variable to prediction of plasma 25(OH)D is represented by the standardized coefficients shown in Table 4. The relative contribution of each variable is indicated by the rank order. In addition to the variables described above, BMI, alcohol use, and plasma neopterin, which is a marker of macrophage activity, were associated with plasma 25(OH)D. TABLE 4 Prediction of plasma 25-hydroxyvitamin D concentrations (square root nmol/l) by multiple regression analysis for all subjects in the Reaching for Excellence in Adolescent Health cohort 1 Variables Coefficient SE P Rank order Environment Latitude Summer and fall seasons Demographics Black subjects BMI Diet and behavior Total vitamin D intake Alcohol intake Immune function Log 10 neopterin n 330. R Variables were selected by backward stepwise regression analysis, as described in Methods. Standardized coefficients (Coefficient), SEs, and P values for each coefficient are shown. The rank order of each variable based on the absolute value of the coefficient is also shown.

6 1140 STEPHENSEN ET AL Vitamin D affects macrophage development and function (6) and may thus affect plasma neopterin concentrations. We thus compared log 10 neopterin concentrations in subjects with and without vitamin D insufficiency. However, because plasma neopterin is elevated by HIV infection (18), we compared neopterin concentrations in subjects with and without vitamin D insufficiency using a 2-way ANOVA to control for the effect of HIV status. Plasma neopterin was higher in subjects with HIV infection than in subjects without HIV infection (P 0.001), as was previously shown for the REACH subjects (18), and was significantly lower (P 0.022) in the subjects with vitamin D insufficiency (x SD: g neopterin/l; n 295) than in the subjects without insufficiency ( g neopterin/l; n 47; P for interaction 0.50). DISCUSSION The prevalence of vitamin D insufficiency was 87% in the REACH subjects, which is remarkably high. The REACH subjects were recruited at 14 clinical sites in 12 US cities ranging from Miami in the south to Chicago in the north. Vitamin D insufficiency was highly prevalent at all sites, indicating that the risk of insufficiency is not a localized phenomenon. Previous studies conducted in adolescent and young adult African Americans found similar, though less extreme, risks of vitamin D insufficiency. Among healthy adolescents in Boston, 36% of black adolescents had a vitamin D insufficiency compared with 22% of Hispanic and 6% of white adolescents (21). In the National Health and Nutrition Examination Survey (NHANES) III study, the prevalence of vitamin D insufficiency in subjects aged y was 32% in non-hispanic blacks compared with 16% in Mexican Americans and 5% in whites (19). In women aged y from the same survey, the prevalence of insufficiency was 42% in blacks and was 4% in whites (22). In a second study from Boston conducted in black women aged 20 to 40 y, the mean plasma 25(OH)D concentration was 30.2 nmol/l in the winter compared with 60.0 nmol/l in white women (23). Thus, it is not surprising to find biochemical evidence of vitamin D insufficiency in the predominantly black and Hispanic REACH subjects, but the magnitude of the problem was unexpected. Several factors contribute to the high level of vitamin D insufficiency in the REACH subjects. First, vitamin D insufficiency is more prevalent in the winter and spring than in the summer and fall (12, 23) and, by chance, we collected most of our samples (59%) during the winter and spring months. However, the prevalence of vitamin D insufficiency in the REACH subjects during the summer and fall was still 80%; thus, seasonality was only a minor factor in boosting the prevalence of insufficiency, as indicated by its relatively low rank order of 6 of the 7 variables in the multiple regression analysis (Table 4). A second factor that contributed to vitamin D insufficiency was latitude. Latitude was the strongest predictor of plasma 25(OH)D, as indicated by its rank order of 1 in the significant variables. Many REACH subjects lived in the northern United States where less intense sunlight presumably contributed to the higher prevalence of vitamin D insufficiency compared with subjects in the southern REACH study sites (Table 3). Still, the high prevalence of vitamin D insufficiency in the southern sites indicated that other factors also contribute to the insufficiency. A low intake of vitamin D was also a significant contributor to vitamin D insufficiency in the REACH subjects, and total vitamin D intake from food and supplements ranked as the second most important variable in our analysis (Table 4). Overall, 39% of the REACH subjects consumed 200 IU vitamin D/d, which is the recommended adequate intake for this age group. However, this was not lower than the intake reported for y-old subjects from the NHANES III study (24). Because the prevalence of vitamin D insufficiency was lower in the NHANES III subjects (21) than in the REACH subjects (Table 3), this indicates that factors other than vitamin D intake contributed to the higher level of insufficiency in the REACH subjects. HIV infection was not significantly associated with vitamin D status in the REACH subjects. This is consistent with the limited data available from earlier studies (1, 4). However, HIV affects plasma concentrations of the active metabolite of vitamin D, 1,25-dihydroxyvitamin D (1, 4). Thus, an apparent lack of effect of HIV infection on plasma 25(OH)D concentrations does not mean that HIV infection has no effect on vitamin D metabolism and, consequently, on vitamin D related physiologic functions, including bone metabolism and the immune response. BMI was a negative predictor of plasma 25(OH)D in the REACH subjects. This association is usually attributed to sequestration of vitamin D in adipose tissue (22). Because approximately one-half of REACH subjects were obese (9), the significance of this variable is not surprising. A multiple regression analysis uncovered an unexpected, positive association between alcohol intake and plasma 25(OH)D. Inspection of the data did not uncover any significant association between alcohol intake and factors known to affect vitamin D status (eg, dietary vitamin D intake, race, latitude, or season), nor did examination of the literature offer a direct, mechanistic explanation for this association (eg, increased hepatic hydroxylation of vitamin D to 25(OH)D as a result of alcohol consumption). Because this is a cross-sectional, observational study with a relatively small sample size, it is possible that this association is spurious and it should be viewed with caution. Plasma neopterin, a marker of macrophage activation, had a positive association with plasma vitamin D in a multiple regression analysis. Reverse causality may offer an explanation for this association. Because vitamin D affects macrophage development (6), it is possible that the positive association between 25(OH)D and neopterin is because of the ability of subjects with better vitamin D status to produce a more robust macrophagemediated immune response (and hence have higher plasma neopterin concentrations) than that of the subjects with poorer vitamin D status. Such an explanation would explain why subjects with vitamin D insufficiency had lower plasma neopterin concentrations than did subjects with better vitamin D status. An intervention study would be needed to test this hypothesis. The much higher prevalence of vitamin D insufficiency seen in the REACH subjects compared with subjects of previous studies raises questions about the ability to compare study populations. The REACH study was designed to sample adolescents and young adults with or at risk of acquiring HIV infection by sexual contact or drug use. Various indicators suggest that the REACH subjects are drawn from urban families of lower socioeconomic status (8). For example, 85% of REACH subjects had no health insurance or received health care provided by public programs, 27% reported having dropped out of high school, and 26% reported being homeless at some point in their life. In

7 VITAMIN D STATUS AND HIV INFECTION 1141 addition, many REACH subjects have had troubled lives, with 20% reporting that they have spent 2 nights in a detention facility. Twenty-nine percent of REACH subjects are parents themselves. This information suggests that the REACH subjects represent a poorer, more urban, and more troubled group of youth than has previously been studied. These social and economic factors may contribute directly to their risk of vitamin D insufficiency by affecting food choices and, perhaps, outdoor activity and related sun exposure. In conclusion, the prevalence of vitamin D insufficiency was alarmingly high in the adolescent and young adult members of the REACH cohort. Low plasma 25(OH)D concentrations were associated with race, obesity, season (winter or spring), living in a northern city, and low vitamin D intake. These risk factors have been reported previously, but the prevalence of vitamin D insufficiency in these subjects appears to be much greater than expected even considering these factors. Because vitamin D intake in the REACH subjects was similar to a nationally representative sample, it may be that limited sun exposure in a poor, urban environment is the factor that accounts for the higher than expected prevalence of vitamin D insufficiency in REACH subjects. Additional studies are clearly needed to identify the reasons for vitamin D insufficiency in these subjects to implement appropriate interventions to improve vitamin D status. We thank the investigators and staff of the Adolescent Medicine HIV- AIDS Research Network ( ), as well as the participants for their valuable contributions. The Reaching for Excellence in Adolescent Care and Health project was supported by grant U01 HD32830 from the National Institute of Child Health and Human Development, with additional funding from the National Institute on Drug Abuse, the National Institute of Allergy and Infectious Diseases, and the National Institute of Mental Health. Support for the vitamin D studies came from the Adolescent Medicine Trials Network for HIV-AIDS Interventions (ATN), which is supported by National Institute of Child Health and Human Development (U01 HD40533) with additional support from the National Institute of Drug Abuse, the National Institute of Mental Health, and the National Institute of Alcohol Abuse and Alcoholism. We acknowledge Tammy Freytag of the US Department of Agriculture Western Human Nutrition Research Center at University of California Davis for performing the plasma 25(OH)D assays. CBS, GSM, GMA, and CMW participated in the design and implementation of the study and in the analysis of the data. LAK and SDD participated in the implementation and analysis. None of the authors had conflicts of interest with this research. REFERENCES 1. O Brien KO, Razavi M, Henderson RA, Caballero B, Ellis KJ. Bone mineral content in girls perinatally infected with HIV. Am J Clin Nutr 2001;73: Madeddu G, Spanu A, Solinas P, et al. Bone mass loss and vitamin D metabolism impairment in HIV patients receiving highly active antiretroviral therapy. Q J Nucl Med Mol Imaging 2004;48: Mondy K, Powderly WG, Claxton SA, et al. Alendronate, vitamin D, and calcium for the treatment of osteopenia/osteoporosis associated with HIV infection. J Acquir Immune Defic Syndr 2005;38: Haug CJ, Aukrust P, Haug E, Morkrid L, Muller F, Froland SS. Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis. J Clin Endocrinol Metab 1998; 83: Reginster JY. The high prevalence of inadequate serum vitamin D levels and implications for bone health. Curr Med Res Opin 2005;21: Griffin MD, Xing N, Kumar R. Vitamin D and its analogs as regulators of immune activation and antigen presentation. Annu Rev Nutr 2003; 23: Rogers AS, Futterman DK, Moscicki AB, Wilson CM, Ellenberg J, Vermund SH. The REACH Project of the Adolescent Medicine HIV/ AIDS Research Network: design, methods, and selected characteristics of participants. J Adolesc Health 1998;22: The Adolescent Medicine HIV/AIDS Research Network. Listing of Investigators and Research Staff. J Adolesc Health 2001;29: Kruzich LA, Marquis GS, Wilson CM, Stephensen CB. HIV-infected US youth are at high risk of obesity and poor diet quality: a challenge for improving short- and long-term health outcomes. J Am Diet Assoc 2004;104: Hanley DA, Davison KS. Vitamin D insufficiency in North America. J Nutr 2005;135: Dawson-Hughes B. Racial/ethnic considerations in making recommendations for vitamin D for adult and elderly men and women. Am J Clin Nutr 2004;80(suppl):1763S 6S. 12. Holick MF. Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease. Am J Clin Nutr 2004;80(suppl):1678S 88S. 13. Norman AW. Sunlight, season, skin pigmentation, vitamin D, and 25- hydroxyvitamin D: integral components of the vitamin D endocrine system. Am J Clin Nutr 1998;67: Douglas SD, Rudy B, Muenz L, et al. T-lymphocyte subsets in HIVinfected and high-risk HIV-uninfected adolescents: retention of naive T lymphocytes in HIV-infected adolescents. The Adolescent Medicine HIV/AIDS Research Network. Arch Pediatr Adolesc Med 2000;154: Douglas SD, Rudy B, Muenz L, et al. Peripheral blood mononuclear cell markers in antiretroviral therapy-naive HIV-infected and high risk seronegative adolescents. Adolescent Medicine HIV/AIDS Research Network. AIDS 1999;13: Rudy BJ, Crowley-Nowick PA, Douglas SD. Immunology and the REACH study: HIV immunology and preliminary findings. Reaching for Excellence in Adolescent Care and Health. J Adolesc Health 2001; 29: Holland CA, Ellenberg JH, Wilson CM, et al. Relationship of CD4 T cell counts and HIV type 1 viral loads in untreated, infected adolescents. Adolescent Medicine HIV/AIDS Research Network. AIDS Res Hum Retroviruses 2000;16: Stephensen CB, Marquis GS, Douglas SD, Wilson CM. Immune activation and oxidative damage in HIV-positive and HIV-negative adolescents. J Acquir Immune Defic Syndr 2005;38: Looker AC, Dawson-Hughes B, Calvo MS, Gunter EW, Sahyoun NR. Serum 25-hydroxyvitamin D status of adolescents and adults in two seasonal subpopulations from NHANES III. Bone 2002;30: Evans P, Halliwell B. Micronutrients: oxidant/antioxidant status. Br J Nutr 2001;85(suppl):S Gordon CM, DePeter KC, Feldman HA, Grace E, Emans SJ. Prevalence of vitamin D deficiency among healthy adolescents. Arch Pediatr Adolesc Med 2004;158: Nesby-O Dell S, Scanlon KS, Cogswell ME, et al. Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age: third National Health and Nutrition Examination Survey, Am J Clin Nutr 2002;76: Harris SS, Dawson-Hughes B. Seasonal changes in plasma 25- hydroxyvitamin D concentrations of young American black and white women. Am J Clin Nutr 1998;67: Moore C, Murphy MM, Keast DR, Holick MF. Vitamin D intake in the United States. J Am Diet Assoc 2004;104:980 3.