Effect of Dietary Intake and Protease Inhibitors on Serum Vitamin B 12 Levels in a Cohort of Human Immunodeficiency Virus Positive Patients

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1 SUPPLEMENT ARTICLE Effect of Dietary Intake and Protease Inhibitors on Serum Vitamin B 12 Levels in a Cohort of Human Immunodeficiency Virus Positive Patients Margo N. Woods, 1 Alice M. Tang, 1 Janet Forrester, 1 Clara Jones, 1 Kristy Hendricks, 1 Bei Ding, 1 and Tamsin A. Knox 2 1 Division of Nutrition and Infection, Department of Family Medicine and Community Health, Tufts University School of Medicine, and 2 Tufts New England Medical Center, Boston, Massachusetts The dietary intake of micronutrients and serum micronutrient status have been topics of concern in relation to human immunodeficiency virus (HIV) progression. Most data, however, were collected prior to the introduction of protease inhibitors (PIs). We analyzed dietary intake and serum values of vitamin B 12, including the effect of PIs, in a cohort of persons with HIV infection. During intervals with no PI use, each 1 mg/day increase in B 12 intake was associated with a 1.06 pg/ml increase in serum B 12 levels. However, during intervals with PI use, each 1 mg/day increase in intake was associated with only a 0.12 increase in serum B 12 levels. Adequate serum B 12 levels (1350 pg/ml) cannot be assumed even in the presence of PIs, and dietary supplementation may not be adequate to significantly increase serum B 12 levels. Serum B 12 levels should be determined yearly in persons with HIV infection, regardless of whether they are receiving PI treatment. Initial reports on the nutritional status of persons living with HIV infection indicated that, despite adequate or increased energy and nutrient intake, serum levels of a number of micronutrients were often low [1 4]. The micronutrients of particular concern were vitamins A, E, B 6, and B 12, b-carotene, selenium, and zinc [5 7]. The results of further studies indicated that low serum levels of vitamins A [8, 9], E [10], and B 12 [11] and selenium [12] were associated with more rapid disease progression and death. Because illness often invokes an Financial support: National Institute of Diabetes and Digestive and Kidney Diseases (1P01DK ); General Clinical Research Center, funded by the National Center for Research Resources of the National Institutes of Health (M01- RR00054); Lifespan/Tufts/Brown Center for AIDS Research (P30 AI42853); National Institute on Drug Abuse (DA11598). Reprints or correspondence: Dr. Margo Woods, Dept. of Family Medicine and Community Health, Tufts University School of Medicine, 150 Harrison Ave., Boston, MA (margo.woods@tufts.edu). Clinical Infectious Diseases 2003; 37(Suppl 2):S by the Infectious Diseases Society of America. All rights reserved /2003/3705S2-0015$15.00 acute-phase response that can result in a redistribution of micronutrients in the body, serum values do not necessarily reflect nutritional status. However, Tang et al. [10, 11] adjusted for acute physiological stress by using C-reactive protein levels in their analysis and still found that low serum levels of vitamins B 12 and E were associated with a more rapid progression to AIDS. Data from Baum et al. [3] indicated that a normalization of serum levels of vitamins A and B 12 and of zinc (i.e., going from low to normal serum levels over an 18-month period of observation) was associated with significantly higher CD4 + cell counts. Still, the mechanisms underlying multiple deficiencies or marginally low serum levels in HIVpositive patients remain unclear. The dissemination of these results led many patients to begin taking vitamin/mineral supplements of various formulations and dosages [12]. However, because of the introduction of protease inhibitors (PIs) into the medical treatment regimen of HIV-positive patients in the mid-1990s, which resulted in substantial clinical S124 CID 2003:37 (Suppl 2) Woods et al.

2 and virological improvements, fewer studies have been published that have examined the potential benefit or harm of micronutrient status and supplementation with respect to HIV disease progression. Low and decreasing serum B 12 levels are of special concern, because they are associated with dementia and dementia was reported early during the AIDS epidemic [13, 14], along with reports of B 12 malabsorption [15]. Even during the era of HAART, the presence of AIDS dementia complex (ADC) is reported with reports of diagnosis at higher levels of CD4 cell counts than those seen prior to the HAART era [16, 17]. The role of vitamin B 12 in the etiology of ADC is still in doubt, but the symptoms observed in AIDS chemiopathological syndrome of dementia, vacuolar myelopathy, peripheral neuropathy, and a deficiency of S-adenosylmethionine (SAM) are the same as those seen in vitamin B 12 deficiency [18]. The objectives of the present article are (1) to report dietary intake and serum values of vitamin B 12 in a cohort of HIV-positive subjects and (2) to evaluate the influence of PIs on changes in serum B 12 levels. SUBJECTS AND METHODS Study population. The Nutrition for Healthy Living (NFHL) study is an ongoing longitudinal study that has recruited 1600 HIV-positive subjects at different stages of disease. Eligible participants included HIV-positive adults (aged 18 years) living in the greater Boston area or in Rhode Island. Individuals were excluded if they had any of the following conditions at enrollment: pregnancy, thyroid disease, or malignancies other than Kaposi sarcoma. Participants were also excluded if they were not fluent in English. Special efforts were made to recruit women and nonwhite minorities. Further details of the study have been published elsewhere [19, 20]. In brief, participants were seen in the clinic every 6 months. At each visit, the data collected included self-reports of sociodemographic information, clinical status, use of medications, and dietary intake. Blood was collected and stored for immunological, biochemical, and nutritional testing. d-xylose tests for carbohydrate absorption were done annually. The present analysis includes data collected from all study visits between February 1995 and 1 November To be eligible, participants had to have at least 2 consecutive visits with data available on vitamin B 12 intake and serum B 12 levels and no reports of ever receiving B 12 injections. Vitamin B 12 intake. Baseline data were collected over 2 study visits spaced 7 14 days apart. At the first baseline visit, trained dietitians instructed participants on how to keep a 3- day food record using a sample demonstration day of recorded food intake. The participants were each given a food scale (Sunbeam) and a ruler to take home with them, to accurately document portion sizes. At the second baseline visit, 1 of 2 dietitians reviewed the food record with the participant and obtained additional details, if necessary. At that time, standard templates of shapes were used to help quantify portion sizes. Participants were also encouraged to bring in bottles or labels of any vitamin/mineral supplements they consumed, to accurately document intake. Participants completed a 3-day food record every 6 months. The food records were analyzed using Nutrition Data System software (Food Database 9, version 13A, and Nutrient Database, version 28; Nutrition Coordinating Center). Vitamin B 12 intake, from food alone and from food plus supplements, was among the nutrients estimated. Serum B 12 level determination. At each study visit, blood was collected after a 5-h fast, and serum was sent to the Clinical Chemistry Laboratory of Tufts New England Medical Center. Vitamin B 12 levels were determined using the ACS-180 B 12 automated chemiluminescence test (Chiron Diagnostics). The HIV RNA level was measured with the Roche Amplicor Monitor RT-PCR assay (Roche Molecular Systems), which has a lower detection limit of 400 copies/ml. CD4 + lymphocyte counts were determined using a specific monoclonal antibody and fluorescence-activated cell sorter analysis (FACScan; Becton-Dickinson). d-xylose absorption tests were conducted yearly by administering a 25-g oral dose of d-xylose and obtaining a blood sample after 2 h. Analysis of serum d-xylose levels was done at American Medical Laboratory (Boston, MA), with a laboratory normal range of 120 mg/dl. Statistical analysis. To determine the effect of dietary B 12 intake on changes in serum B 12 levels, data from all study visits (baseline and follow-up) were eligible. The data for analysis included only data from those persons who had at least 2 consecutive visits spaced within 4 8 months of each other (average interval, 6 months) from which both dietary intake and serum B 12 values were available. The unit of analysis was personintervals (hereafter called intervals ) rather than individuals. Each participant could contribute anywhere from 1 to 4 intervals to the analysis, depending on the number of eligible study visits they had completed. The visit preceding each interval was considered to be the baseline for the interval. Regression models, including restricted cubic spline functions [21], were used to assess the linearity of the association between B 12 intake and any change in serum B 12 over the entire range of B 12 intake levels. These models showed a significant linear association between B 12 intake and change in serum B 12 levels at intakes of!150 mg/day. Above that level, the association appeared to be nonlinear. Because there were very few intervals in this upper range of intake ( n p 42 intervals), however, it was decided to truncate the data set to include only those intervals during which the mean B 12 intake level was!150 mg/ day. This encompassed 96% of all eligible intervals (1073 intervals from 412 participants). Dietary Intake and Protease Inhibitors CID 2003:37 (Suppl 2) S125

3 The outcome of interest for this analysis was a change in serum B 12 levels from the beginning of an interval to its end. Multivariate linear regression models using generalized estimating equations (GEEs) were used to correct SEs for the use of multiple intervals per participant. GEEs do not require the assumption of normality of the data. B 12 intake was calculated as the mean intake over an interval. PI use was coded as yes if any was reported at either of the 2 visits bounding the interval. Because the initiation of HAART (specifically the addition of a PI to a therapeutic regimen) can result in a 2-log decrease in virus load within 2 months [22], the use of PIs at either of the 2 visits bounding the interval was considered to be adequate to observe an effect of PIs. In the total interval set (1073), 615 intervals (57%) were classified as PI intervals, and 420 (68%) of those intervals reported PI use at both visits bounding the interval. An evaluation of medication use within the intervals indicated that, of the 615 intervals in which a PI was received 583 (96%) were intervals in which nucleoside reverse-transcriptase inhibitors (NRTIs) were received. Of the 454 intervals in which a PI was not received, 291 (63%) were intervals in which NRTIs were received; in 163 (36%) of those 454 intervals, there was no PI and no NRTI use. These 3 groups were collapsed into 2 groups: one group (615 intervals) during which almost all patients were receiving triple therapy with PIs, and a second group (454 intervals) during which patients were receiving no therapy or dual therapy or monotherapy that did not include a PI. The data set for evaluating the change in serum B 12 levels used participant intervals; therefore, data for a participant could be used multiple times. In the 1069 intervals involving 410 participants, 193 participants were always receiving a PI, 137 were never receiving a PI, and 80 were represented in both groups of intervals. Persons always receiving PI had CD4 cell counts (381 cells/mm 3 ) between those of persons never receiving PIs (460 cells/mm 3 ) and those represented in both groups of intervals (290 cells/mm 3 ). Baseline virus loads were lowest in those participants always in PI intervals and were similar in those never in PI intervals or in both groups of intervals. There was no difference in age or body mass index (BMI) among these 3 groups of patients. Sex, race, and baseline CD4 cell counts, virus load, neutrophil counts, and hemoglobin levels for each interval were evaluated as potential confounders and effect modifiers [23, 24]. We did not adjust for virus load or CD4 cell counts, because they are part of the mechanistic pathway of the interaction of PIs and HIVonB 12 absorption and metabolism; it would therefore be inappropriate to correct for them. To examine the influence of PIs on the relationship between B 12 intake and serum B 12 level, an interaction term between B 12 intake and PI use was included in the model. Multivariate regression models, including interaction terms, were used to determine whether B 12 malabsorption, as indicated by d-xylose absorption, played any role in the association between B 12 intake and changes in serum B 12 levels. The analysis of responses to dietary intake in those intervals during which the serum B 12 level at baseline was!350 pg/ml was of interest, both overall and within the PI group. However, because ours was an observational study, we would not have been able to distinguish differences in responses to dietary intake between those with baseline levels above and below 350 pg/ml from regression to the mean; therefore, the analyses were not done. RESULTS Of the 686 participants who were enrolled in the NFHL study between February 1995 and November 2000, 63 (9%) were excluded from further analysis because they reported receiving ab 12 injection while enrolled in the study. Of the remaining 623 participants, 422 (68%) had at least 2 consecutive study visits that were within a 4 8 month window. Of these, 412 participants had intervals with mean B 12 intakes!150 mg/day. These 412 participants contributed 1073 intervals for the analysis. Table 1 compares baseline characteristics between the complete NFHL cohort ( N p 686) and the 412 participants eligible for the present analysis. No important differences were seen between the 2 groups, which indicates that the analysis subset was representative of the total cohort. Men constituted 73% of the study population. Sixty-two percent were white, 25% were black, and 8% were Hispanic. The mean age of the study population was 40 years. The average BMI was within the normal range for men and was slightly above normal for women. Mean hemoglobin levels were on the low end of the normal range for both men and women (4 18 g/dl for men and g/dl for women). The mean CD4 + lymphocyte and neutrophil counts at study entry were 353 and 3093 cells/mm 3, respectively. Participants had a mean HIV log 10 RNA level of 3.8 copies/mm 3. Figure 1A shows the baseline distribution of dietary B 12 intake in participants ( n p 623) using and not using B 12 supplements. The dietary intake graph clearly shows the extremely high B 12 supplement intake (140 times the dietary reference intake [DRI]) in 10% of the study population. Dietary intakes from food alone reached a maximum at 20 mg/day. Serum B 12 levels were higher in participants who used B 12 supplements than in those who did not (figure 1B). Median serum values were 546 pg/ml in supplement users versus 420 pg/ml in non supplement users ( P!.0001). The figure indicates that 30% of non supplement users had serum values!350 pg/ml. However, even among supplement users, 15% had serum values!350 pg/ml. Although the standard normal range for serum B 12 levels is pg/ml, some researchers consider values S126 CID 2003:37 (Suppl 2) Woods et al.

4 Table 1. Baseline characteristics of all Nutrition for Healthy Living study participants, compared with those of the analysis subset. Characteristic Total cohort (n p 686) Analysis subset (n p 412) Sex Male 492 (72) 301 (73) Female 194 (28) 111 (27) Race/ethnicity White 411 (60) 255 (62) Black 186 (27) 108 (26) Latino/Hispanic 56 (8) 33 (8) Other 33 (5) 16 (4) Age, years Weight, kg Men Women Body mass index Men Women Hemoglobin, g/dl Men Women CD4 + cells/mm HIV RNA copies/mm Neutrophil count, cells/mm NOTE. Data are presented as no. (%) or mean SD.!350 pg/ml to be deficient [25], whereas levels!500 pg/ml are considered to be low to marginally low [26]. Table 2 reports the distributions of B 12 intake and serum B 12 levels at study entry in the 412 participants in the analysis subset. Forty-three percent reported using B 12 supplements, and the median intake of vitamin B 12 (food plus supplements) in the subset was 8.1 mg/day, which is 13 times the DRI (2.4 mg/ day). Data from the National Health and Examination Survey (NHANES) III reported a median vitamin B 12 intake of 3 5 mg/day [27] (women and men, respectively) in the US population. Twenty-five percent of participants had total B 12 intake levels 116 mg/day, well over 6 times the DRI. The upper quartile were taking B 12 supplements at levels 16.0 mg/day, which is more than is found in a standard one-a-day pill. The median intake from food alone was 5.3 mg/day. Malabsorption, as measured by d-xylose levels!20 mg/dl, was not significantly associated with changes in serum B 12 levels in any of our analyses, nor did it affect the association between B 12 intake and changes in serum B 12 levels. Table 2 shows that 40% of the analysis subset reported the use of PIs at baseline. During the followup period, 58% of the intervals showed PI use (data not shown). Table 3 shows data on B 12 intake and serum B 12 levels in intervals with and without PI use. Intervals during which participants reported PI use had, on average, higher B 12 intakes ( P p.0002) and higher serum B 12 ( P p.0077) levels at the baseline for each interval. The interquartile ranges for serum values indicated that 21.8% (99/454) of those intervals with no PI use had serum values!350 pg/ml, whereas 17.4% (107/ 615) of those intervals with PI use had serum values!350 pg/ ml ( P p.06). Baseline CD4 cell counts for each interval were not significantly different between the 2 groups ( P p.58). As had been expected, interval baseline values of log 10 virus load were significantly higher in those intervals during which PIs were not used ( P p.0001). Figure 2 shows a graph that uses the final regression model. The association of total B 12 intake and change in serum B 12 depended on PI use, as was indicated by the difference in the slopes of the lines describing the relation between B 12 intake level and change in serum B 12 levels during intervals with PI use and those without (difference in slopes, P p.05). There are 2 things to note about this figure. The first is that B 12 intake levels as high as 4 times the DRI were associated with negative changes in serum B 12 levels during intervals in which the participants were not receiving PIs. As B 12 intake increased, changes in serum B 12 levels became positive during the interval. As an example, during an interval when a participant was not receiving a PI, a dietary intake of 2.4 mg/day of B 12 (the DRI for B 12 ) was associated with a decrease of 12.2 pg/ml in serum B 12 levels during that interval. Even at an intake of 48 mg/day of B 12 (20 times the DRI), there would be an increase of serum B 12 of only 36.2 pg/ml during that interval. This is not a clinically important increase during a 6-month time period. The second point to note from figure 2 is that, although data for intervals with PI use show a positive slope and intercept, the corresponding group of patients showed little responsiveness of serum B 12 changes to increasing dietary intake levels of B 12 over a wide range of B 12 intake. Race, sex, and baseline CD4 cell counts, virus load, and neutrophil counts for each interval were not confounders of this association. DISCUSSION In our longitudinal study of HIV-positive men and women living in the greater Boston area and Rhode Island, we found that the effect of vitamin B 12 intake on changes in serum B 12 levels over a 6-month period differed depending on whether the participant was using PI therapy during the interval or not. Although studies in the past have shown significant correlations between the intake of a micronutrient and its absolute serum levels, to our knowledge, ours is the first study to demonstrate an association between the intake of a micronutrient and a change in its serum level over a 6-month period that is modified by PI use. Our results showed that very high intake levels of vitamin Dietary Intake and Protease Inhibitors CID 2003:37 (Suppl 2) S127

5 Figure 1. A, The distribution of intake levels of vitamin B 12 from food only (dotted line) and from food and supplements (solid line), representing 5%, 10%, 25%, 50%, 75%, 90%, and 95% of the population, are shown. B, Distribution of serum B 12 levels in non supplement users (dotted line) and supplement users (solid line), representing 5%, 10%, 25%, 50%, 75%, 90%, and 95% of the population, are shown. *Serum B 12 levels were!350 pg/ml in 19.4% of supplement users and in 32.21% of non supplement users. DRI, dietary reference intake. B 12 were needed to demonstrate increases, not decreases, in serum B 12 levels when subjects were not receiving PIs. These intake levels of dietary vitamin B 12 could only be achieved with significant supplement use. During the intervals in which participants were receiving PIs, decreases in serum B 12 were not seen, even at low levels of dietary intake, but there was only a 0.12 pg/ml increase in serum B 12 levels with every 1-mg intake of vitamin B 12 per day. This increase in serum B 12 is a very low level of responsiveness to dietary intake. The median serum values of B 12 during intervals with PI use were significantly higher than in intervals without PI use but were not very different from a clinical point of view (median value, 491 vs. 462 pg/ml, respectively). The incidence of intervals with serum B 12 levels!350 pg/ml with PI use versus no PI use was 17% versus S128 CID 2003:37 (Suppl 2) Woods et al.

6 Table 2. Dietary B 12 intake, serum B 12 levels, d-xylose levels, and protease inhibitor (PI) use at baseline for the analysis subset ( N p 412). Characteristic Analysis subset (n p 412) B 12 intake, mg/day Total 8.1 ( ) Food only 5.3 ( ) Supplement only 0.0 ( ) Serum B 12 level, pg/ml 457 ( ) Supplement users 546 ( ) Supplement nonusers 420 ( ) d-xylose levels, mg/dl 35 (28 43) B 12 supplement use, % of 43.4 subjects B 12 shots, % of subjects 0 a PI use, % of subjects 40.8 NOTE. Data are given as median (25% and 75% interquartile range), unless otherwise specified. a Exclusion criterion. 22%, respectively, and approached significance ( P p.06). A greater prevalence of marginal or deficient micronutrient status can effect responsiveness to dietary intake of a micronutritent. However, the median values of our 2 groups were not in the marginal range and the values of the lower quintiles in the 2 groups were very similar, so we do not believe that a greater deficiency status in the group not receiving PIs is an explanation for their greater increase in serum B 12 per unit intake of dietary B 12. A recent article by Remacha et al. [28] also reported on serum B 12 levels in HIV-infected patients receiving or not receiving HAART. They observed values of 402 pmol/l (545 pg/ ml) in those receiving HAART versus 330 pmol/l (447 pg/ ml) in those not receiving HAART ( P p.013) but did not investigate the change in serum B 12 levels during HAART. Population studies in healthy people have reported percentages of people with serum B 12 levels!350 pg/ml that are equal to or greater than those we have observed in our cohort of persons with HIV. One study, using the NHANES III data, reported 20% of the population with serum B 12 levels!350 pg/ ml (ages years) [29]. Median serum values of 459 and 469 pg/ml were reported in that study for men and women, respectively, which are similar to our values in this HIV population. Tucker et al. [30] reported a 38% prevalence of serum B 12 values!350 pg/ml in a group of older but healthy Americans aged years. Tucker et al. [30], using a cross-sectional regression analysis of plasma B 12 in relation to log of B 12 intake, reported a difference in plasma B 12 concentrations of 45.5 pmol/l for each doubling of vitamin B 12 intake in a healthy US population [30]. Our data indicates that had our non-pi HIV population doubled their dietary intake of B 12 from 4.8 to 9.6 mg/day, their serum levels would only have increased by 5 pg/ml ( 4 pmol/ L), which is 10% of that reported by Tucker et al. [30] and indicates a decreased responsiveness in this HIV population. An evaluation of the mechanism of ADC has suggested that B 12 deficiency may exacerbate SAM deficiency but may not be the primary cause of dementia, which may be more closely related to the role of products and cytokines derived from Table 3. Levels of vitamin B 12 intake and serum B 12 relative to protease inhibitor (PI) use in the analysis subset of patients. Variable All intervals Intervals with PI use Intervals without PI use P a Dietary B 12 intake No. of intervals b Median mg/day (IQR) 9.67 ( ) 11.9 ( ) 7.6 ( ).0002 Interval baseline values Serum B 12 No. of intervals b Median pg/ml (IQR) 479 ( ( ) 462 ( ) CD4 cell count No. of intervals b Median cells/mm 3 (IQR) 346 ( ) 308 ( ) 408 ( ).5761 HIV load No. of intervals b Median log 10 copies/ml (IQR) 3.47 ( ) 3.07 ( ) 3.8 ( )!.0001 NOTE. IQR, interquartile range (25% 75%). The questionable PI status of some subjects at the start of some intervals results the sum of PI and non-pi use not equalling the total value. a During intervals of PI use vs. during intervals with no PI use. b Intervals during which subjects did or did not receive a PI; for the full definition of this term, see Statistical analysis in Subjects and Methods. Dietary Intake and Protease Inhibitors CID 2003:37 (Suppl 2) S129

7 Figure 2. Changes in serum B 12 levels with dietary intake of B 12 plus protease inhibitor (PI) use are shown. The plot of the final regression model depicting the significant interaction between total B 12 intake and PI use shows the change in serum B 12, according to the formula b 0 b (B intake) + b (PI use) + b (B intake PI use) + E. Pp.05 for difference in slope (or interaction term, b 3 ) activated macrophages or microglea in HIV disease [18]. The literature on the association of serum B 12 and ADC is unclear. Several studies prior to PI introduction cited that serum B 12 levels were normal in the presence of ADC, but values for serum B 12 were often not included, the definitions of normal or deficiency were not given, either 150 or 250 pg/ml was used as the lower range of normal [13, 31 33]. Data are available that have indicated that, at a serum B 12 level!350 pg/ml, there is a significant increase in the percentage of samples that show increases in methylmalonic acid, a sensitive marker of tissue B 12 deficiency [34, 35]. In elderly persons, dementia has been observed in persons with serum B 12 levels!350 pg/ml who have been responsive to B 12 treatment [36, 36]. The finding that PI users showed little increase in their serum B 12 levels through dietary intake was unexpected. PI use is known to increase diarrhea, and this may explain some of the effect. Other possible areas of interference are absorption (related to intrinsic factor), passive absorption, or decreased reabsorption of B 12 during its enterohepatic circulation [25]. The absorption of B 12 via intrinsic factor is generally limited to a maximum of 3 mg/meal, whereas the passive absorption of B 12 by dietary intake is 1% of the ingested amount [38]. Adams et al. [39] showed decreasing fractional absorption estimates with increasing doses of B 12, to a low of 5% absorption at a B 12 dose of 25 mg, which supports a decrease in benefits from high levels of supplemental B 12 intake. There were a few limitations to our study. Because it was an observational study, the most relevant concern was the impact of confounders. We included the confounders that we felt might affect the study end points in our models. The inclusion of these confounders did not alter our conclusions. Because a large percentage of our participants receiving PIs or not were also receiving NRTIs, we cannot separate an effect modification by NRTI use in our study design. However, the drop in virus load and improvement in CD4 cell counts, which could be expected to alter B 12 absorption or metabolism, were only seen with the introduction of PIs to the HIV treatment regimen. The clinical message of importance from these data is more straightforward. The incidence of subjects and intervals in which serum B 12 values were!350 pg/ml was 20%, whether a person was receiving PI or not. Because levels!350 pg/ml are not recommended and can result in neurological changes that have been described in the HIV population, we would recommend yearly determinations of serum B 12. The recommended treatment would be B 12 injections if serum levels were!350 pg/ml, because dietary supplementation may not be effective, especially in those receiving PIs. Therefore, during this era of PI use, the observation of low levels of serum B 12 is still pertinent, and the difficulty in increasing serum B 12 levels is still present. Our next step is to analyze our cohort data longitudinally to explore the effect of serum B 12 levels on the progression of HIV and the effect of PIs. References 1. Mantero-Atienza E, Baum MK, Morgan R, et al. Vitamin B 12 in early human immunodeficiency virus-1 infection. Arch Intern Med 1991; 151: Baum MK, Mantero-Atienza E, Shor-Posner G, et al. Association of vitamin B6 status with parameters of immune function in early HIV- 1 infection. J Acquir Immune Defic Syndr 1991; 4: Baum MK, Shor-Posner G, Lu Y, et al. Micronutrients and HIV-1 disease progression. AIDS 1995; 9: Smit E, Graham NMH, Tang A, Flynn C, Solomon L, Vlahov D. Dietary intake of community-based HIV-1 seropositive and seronegative injecting drug users. Nutrition 1996; 12: Beach RS, Mantero-Atienza E, Shor-Posner G, et al. Specific nutrient abnormalities in asymptomatic HIV-1 infection. AIDS 1992; 6: Coodley GO, Coodley MK, Nelson HD, Loveless MO. Micronutrient concentrations in the HIV wasting syndrome. AIDS 1993; 7: Tang AM, Smit E. Selected vitamins in HIV infection: a review. AIDS Patient Care STDS 1998; 12: Semba RD, Graham NMH, Caiaffa WT, Margolick JB, Clement L, S130 CID 2003:37 (Suppl 2) Woods et al.

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