Vitamin B-12 metabolism in HIV-infected patients in the age of highly active antiretroviral therapy: role of homocysteine in assessing vitamin B-12 st
1 From the Departments of Hematology (AFR and PS) and Internal Medicine (JC, MB, and MF), Hospital de Sant Pau, Barcelona, Spain.\, http://www.100md.com
2 Supported by the SANTPAU-CITRAN program. \, http://www.100md.com
ABSTRACT\, http://www.100md.com
Background: Before the advent of highly active antiretroviral therapy (HAART), 20% and 10% of HIV-infected patients had low vitamin B-12 and red blood cell folate (RBCF) concentrations, respectively. However, few patients had real vitamin B-12 deficiency.\, http://www.100md.com
Objective: We evaluated the prevalence of low vitamin B-12 and RBCF concentrations in HIV-infected patients receiving HAART and the usefulness of serum homocysteine (sHcy) for differentiating patients with deficiency from those with harmlessly low vitamin B-12.\, http://www.100md.com
Design: The prevalence of low vitamin B-12 and RBCF was evaluated in 126 HIV-infected patients receiving HAART. Moreover, sHcy concentrations were evaluated in 40 HIV-infected patients with low vitamin B-12 and in 37 HIV-infected patients with low RBCF and were compared with those in 128 HIV-infected patients with normal vitamin B-12 and RBCF. sHcy was used to monitor treatment with vitamin B-12 and folic acid in 28 patients (24 with low vitamin B-12 and RBCF and 4 with hyperhomocysteinemia but normal vitamin B-12 and RBCF).
Results: The prevalence of low vitamin B-12 was significantly lower in patients receiving HAART than in previously studied patients who did not receive HAART (8.7% compared with 27%). Nine of the 40 patients (22.5%) with low vitamin B-12 ( 200 pmol/L) had hyperhomocysteinemia (> 17.5 µmol homocysteine/L). Nineteen (51.4%) of the 37 patients with low RBCF ( 580 nmol/L, percentile 10) had hyperhomocysteinemia. Among the 9 patients with an RBCF concentration 450 nmol/L (percentile 2.5), all had hyperhomocysteinemia. The treatment with vitamin B-12 and folic acid normalized sHcy concentrations.7k]])t0, 百拇医药
Conclusions: The prevalence of low vitamin B-12 decreased after the introduction of HAART. The study of sHcy is useful for detecting HIV-infected patients with low vitamin B-12 and real deficiency.7k]])t0, 百拇医药
Key Words: HIV • vitamin B-12 • folate • homocysteine • highly active antiretroviral therapy • HAART7k]])t0, 百拇医药
INTRODUCTION
Low concentrations of vitamin B-12 and red blood cell folate were found in 20% and 10% of HIV-infected patients, respectively (1). However, during the clinical evaluation of HIV-infected patients who have a low vitamin B-12 concentration, it is necessary to ascertain whether these patients have a real deficiency or merely a harmlessly low vitamin B-12 concentration. This situation resembles that of iron deficiency during pregnancy and in persons with multiple myeloma (2), in which a low vitamin B-12 concentration can be found without a real vitamin B-12 deficiency. Some tests can help to distinguish patients with a real deficiency from those without a deficiency.!{%(f0}, 百拇医药
The deoxyuridine suppression test (dUST) is used to determine megaloblastic alterations by measuring changes in the incorporation of labeled thymidine (3). Our group showed that among HIV-infected patients with low vitamin B-12, most did not have a deficiency, except when a low red blood cell folate concentration was observed. However, the dUST demands a bone marrow sample, which is time consuming to obtain; as a result, few laboratories currently use this test routinely (4).
Some authors have suggested that the study of abnormalities in vitamin B-12 metabolites and in folate metabolism is an alternative to the dUST (5). In this regard, methylmalonic acid and homocysteine (Hcy) were found to have excellent sensitivity for diagnosing vitamin B-12 or folate deficiency (5, 6).y, 百拇医药
Because Hcy concentrations increase in vitamin B-12 and folate deficiencies (6), Hcy measurement may be a good tool for detecting and managing low vitamin B-12 concentrations in HIV-infected patients. Moreover, Hcy evaluation is routinely used in many laboratories and could easily be used in HIV-infected patients with low vitamin B-12 (7–9) and in monitoring the response to treatment.y, 百拇医药
Almost all studies on vitamin B-12 and folate metabolism in HIV-infected patients were carried out before the advent of highly active antiretroviral therapy (HAART), a treatment with 3 antiretroviral drugs including a protease inhibitor (1, 10). Today, in the age of HAART, the prevalence of low serum vitamin B-12 concentrations and low red blood cell folate concentrations should be reappraised in HIV-infected patients undergoing this therapy because many HIV-infected patients develop macrocytosis (11). The present study was undertaken to ascertain whether the prevalence of low concentrations of these vitamins is lower now than it was before the advent of HAART and whether serum Hcy concentrations can be used to identify patients with a real vitamin B-12 deficiency and to monitor responses to treatment.
SUBJECTS AND METHODSu(+4t, 百拇医药
Evaluation of serum vitamin B-12 and red blood cell folate concentrations in HIV-infected patients receiving HAARTu(+4t, 百拇医药
To evaluate the prevalence of abnormalities in these variables, a group of HIV-infected patients receiving HAART (taking 3 antiretroviral drugs) was compared with a group of previously studied HIV-infected patients who did not receive HAART. The HAART group consisted of all the HIV-infected patients who received HAART in the SANTPAU-CITRAN (Centre d’Investigació, Tractament i Rehabilitació Adictes a Narcòtics) program for 11 mo (126 patients). The non-HAART group was made up of 109 patients who either received treatment with one antiretroviral drug or received no treatment with antiretroviral drugs. The subjects in the non-HAART group were recruited from 1989 to 1992. The characteristics of these patients were published elsewhere (12, 13). The study was performed in accord with the Helsinki Declaration of 1975 as revised in 1983, and all subjects provided written informed consent.
Assessment of serum Hcy concentrations in the diagnosis of HIV-infected patients with low serum vitamin B-12 or red blood cell folate concentrations3@, 百拇医药
Serum Hcy concentrations in HIV-infected patients with a low serum vitamin B-12 concentration (" 200 pmol/L; n = 40) or a low red blood cell folate concentration (" 580 nmol/L; n = 37) were compared with those in a group consisting of all the HIV-infected patients with a serum vitamin B-12 concentration > 200 pmol/L and a red blood cell folate concentration > 580 nmol/L who were recruited in the 11-mo period (128 HIV-infected patients who did or did not receive HAART). The patients with low concentrations of serum vitamin B-12 or red blood cell folate were recruited from patients who agreed to participate in the SANTPAU-CITRAN program from 1996 until 2001.3@, 百拇医药
In this study, the patients were considered to have a low concentration of serum vitamin B-12 and red blood cell folate if serum vitamin B-12 and red blood cell folate concentrations were below percentile 10 ( 200 pmol/L and 580 nmol/L, respectively). The rationale for using percentile 10 was based on the fact that a considerable percentage of patients below this cutoff had hyperhomocysteinemia (14). We investigated serum Hcy concentrations in a population of 230 patients with different disorders. Ninety-two percent of patients with a red blood cell folate concentration below percentile 2.5 (60 patients) had hyperhomocysteinemia, as did 47% of patients (n = 54) with a red blood cell folate concentration between percentiles 10 and 2.5 (AF Remacha, unpublished observation, 2000). Moreover, the patients with concentrations below percentile 2.5 of serum vitamin B-12 ( 150 pmol/L) and red blood cell folate ( 450 nmol/L) concentrations were also evaluated. The reference values for red blood cell folate were calculated from 159 healthy persons ( ± SD: 837 ± 235 nmol/L; percentile 2.5: 450 nmol/L; percentile 97.5: 1750 nmol/L).
Evaluation of treatment with vitamin B-12 and folic acid in a group of HIV-infected patients with low vitamin B-12 or red blood cell folate concentrations2'*kv, 百拇医药
Twenty-eight patients were recruited from the same 11-mo period. This group consisted of 22 of 24 patients receiving HAART who had low vitamin concentrations, 2 patients who had low vitamin concentrations but were not receiving HAART, and 4 of the 8 control subjects (receiving HAART or not) who had hyperhomocysteinemia and vitamin B-12 and red blood cell folate concentrations ">=" 200 pmol/L and 580 nmol/L, respectively. Twenty-eight of the 34 possible candidates were treated (2 patients rejected the treatment, one patient died, and 3 candidates were lost before starting the treatment).2'*kv, 百拇医药
One milligram vitamin B-12 (cyanocobalamin; intramuscular) per week and 5 mg folic acid (oral)/d were administered to all patients for 6 wk. Vitamin B-12, red blood cell folate, and serum Hcy concentrations were measured before and after treatment.
Methodsi9i?7, http://www.100md.com
Serum vitamin B-12 and red blood cell folate concentrations were measured by using CEDIA vitamin B-12 and folate in a Hitachi 911 automatic analyzer (Roche Diagnostics, Mannheim, Germany). In the historical group, a radioassay (Amersham, Buckinghamshire, United Kingdom) was used to measure concentrations of vitamin B-12 and folate (reference values: 450–1300 nmol/L) (12, 13).i9i?7, http://www.100md.com
Serum Hcy concentrations were measured by using 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) as the derivative and HPLC with fluorescence detection according to the method of Vester and Rasmussen (15). Homocysteic acid was used as the internal standard. This procedure was used for all serum samples, including those of a group of healthy persons who were used as a reference population.i9i?7, http://www.100md.com
For serum Hcy measurement, blood was collected in sterile, evacuated serum-separator tubes containing a gel and clot activator. The samples were kept at room temperature until centrifugation for 20 min at 2000 x g and room temperature. The interval between the phlebotomy and the centrifugation ranged from 1 to 3 h, after which the sample was maintained at -20 °C until use. The serum sample used in the Hcy measurement was also used for the vitamin B-12 measurement.
Several studies showed a difference in Hcy concentrations between samples maintained at room temperature and those maintained at 4 °C (15, 16). Thus, with the use of the same HPLC procedure, the reference range for serum Hcy was calculated in 71 healthy persons (hemoglobin concentration > 120 g/L, mean corpuscular volume (MCV) < 100 fL, serum vitamin B-12 concentration > 200 pmol/L, red blood cell folate concentration > 600 nmol/L, and serum creatinine concentration < 100 µmol/L; age range: 20–84 y). The mean (± SD) reference value for serum Hcy was 7.5 ± 4.5 µmol/L. Because the Hcy distribution had a long tail on the right side, percentiles were used for calculating the reference range (percentiles 2.5–97.5: 2–17.5 µmol/L), and values for serum Hcy > 17.5 µmol/L were considered to be elevated (hyperhomocysteinemia). This reference population was not related to the reference population used for red blood cell folate.xo;9, http://www.100md.com
Statistical procedures
Calculations were made by using SPSS 10.0 (SPSS Inc, Chicago). Quantitative variables were expressed as the mean ± SD, and qualitative variables were expressed as a percentage of positive cases. For comparisons involving quantitative variables, the paired or unpaired Student’s t test was used; CIs were calculated when the differences were significant. For comparisons involving qualitative variables, the chi-square test was used. A simple or multiple regression analysis was performed to study relations between variables.p|m, 百拇医药
RESULTSp|m, 百拇医药
Evaluation of serum vitamin B-12 and red blood cell folate concentrations in HIV-infected patients receiving HAARTp|m, 百拇医药
Among the 126 patients in the HAART group, a serum vitamin B-12 concentration 150 and 200 pmol/L was detected in 2 (1.6%) and 11 (8.7%) patients, respectively. In contrast, among the 109 patients in the non-HAART group who were receiving treatment with only one or no antiretroviral drugs, 20 (18%) and 30 (27%) patients had a serum vitamin B-12 concentration " 150 and 200 pmol/L, respectively. The 2 groups differed significantly in these percentages (chi-square test, P < 0.0001 for both concentrations of serum vitamin B-12).
A red blood cell folate concentration " 450 or 580 nmol/L was observed in 1 (0.8%) and 13 (10.3%) patients in the HAART group, respectively, and in 19 (17.4%) and 24 (22%) patients in the non-HAART group, respectively. There was a significant difference between the 2 groups in the percentage of subjects who had a red blood cell folate concentration " 450 nmol/L (chi-square test, P < 0.0001).jyk3z1], 百拇医药
The patients in the non-HAART group had significantly lower values for hemoglobin, MCV, leukocytes, CD4, and CD8 (Table 1) than did the patients in the HAART group. As shown previously in the non-HAART group (12, 13), there was a relation in the HAART group between serum vitamin B-12 concentrations and granulocytes (r = 0.33, P < 0.0001); this relation was independent of other variables (hemoglobin, MCV, age, sex, red blood cell folate, and serum homocysteine).jyk3z1], 百拇医药
fig.ommittedjyk3z1], 百拇医药
TABLE 1 Vitamin B-12 and folate metabolism in 2 groups of HIV-infected patients1
Role of serum Hcy in the diagnosis of HIV-infected patients with low serum vitamin B-12 or red blood cell folate concentrationsfj/k, 百拇医药
Evaluation of patients with a low serum vitamin B-12 concentrationfj/k, 百拇医药
Only 9 of 40 patients (22.5%) with a low vitamin B-12 concentration (ranging from 60 to 198 pmol/L) had hyperhomocysteinemia (Table 2). The red blood cell folate concentration was also low in 6 patients with a low serum vitamin B-12 concentration. Five of 6 patients with both a low red blood cell folate concentration (" 580 nmol/L) and a low serum vitamin B-12 concentration had hyperhomocysteinemia (mean serum Hcy: 32.8 ± 16.8 µmol/L; range: 12.5–54 µmol/L). Hcy concentrations were significantly higher (Student’s t test, P = 0.017) in these 6 patients than in the 34 HIV-infected patients with a low serum vitamin B-12 concentration and normal red blood cell folate concentration (mean serum Hcy: 8.7 ± 6.2 µmol/L; range: 1–24 µmol/L). Only 4 of 34 patients (11.8%) with a low vitamin B-12 concentration and a normal red blood cell folate concentration had hyperhomocysteinemia.
fig.ommitted*9z3./, http://www.100md.com
TABLE 2 Characteristics of HIV-infected patients with low serum vitamin B-12 or red blood cell folate (RBCF) concentrations1*9z3./, http://www.100md.com
Serum Hcy concentrations in the group with a low vitamin B-12 concentration without a low red blood cell folate concentration did not differ significantly from those of the control group (Student’s t test, P = 0.74). Four of 15 patients (26.7%) with a serum vitamin B-12 concentration " 150 pmol/L had a high Hcy concentration. However, 3 of 4 patients with an elevated serum Hcy concentration had a low red blood cell folate concentration. HIV-infected patients with a low vitamin B-12 concentration and hyperhomocysteinemia had neutrophil counts similar to those of patients with a low vitamin B-12 concentration and a normal Hcy concentration (Student’s t test, P = 0.152).*9z3./, http://www.100md.com
Evaluation of patients with a low red blood cell folate concentration*9z3./, http://www.100md.com
Nineteen (51.4%) of 37 patients with a low red blood cell folate concentration (ranging from 313 to 580 nmol/L; the latter concentration corresponded to percentile 10 of the reference population) had hyperhomocysteinemia. Among the 9 patients who had a red blood cell folate concentration 450 nmol/L (percentile 2.5 of the reference population), all of them (100%) had an elevated serum Hcy concentration.
In contrast with the patients with a low vitamin B-12 concentration, the 31 patients with a low red blood cell folate concentration without a low vitamin B-12 concentration had significantly higher (Student’s t test, P = 0.002) serum Hcy concentrations than did the patients in the control group (20 ± 19 compared with 8.3 ± 7.8 µmol/L). A negative relation was found between serum Hcy concentrations and red blood cell folate concentrations. Multiple regression analysis that included the variables age, sex, hemoglobin, MCV, red blood cell folate, vitamin B-12, and creatinine showed that red blood cell folate concentrations (r = -0.42) and creatinine concentrations (r = 0.3) were independent variables related to serum Hcy concentrations.4\0sm, 百拇医药
Effect of treatment with vitamin B-12 and folic acid in a group of HIV-infected patients with low vitamin B-12 or red blood cell folate concentrations4\0sm, 百拇医药
Among the 24 patients who had a low serum vitamin B-12 concentration and a low red blood cell folate concentration, serum Hcy concentrations in the 14 patients who had a high Hcy concentration decreased to normal values after treatment with vitamin B-12 and folic acid (Table 3). The treatment decreased Hcy concentrations > 50% in 4 of 10 patients whose starting serum Hcy concentration was normal.
fig.ommitted]5f2$, 百拇医药
TABLE 3 Effect of vitamin B-12 and folate on serum homocysteine (Hcy) concentrations in HIV-infected patients1]5f2$, 百拇医药
A decrease in Hcy concentrations was also observed in the 4 patients who had a high serum Hcy concentration and normal serum vitamin B-12 and red blood cell folate concentrations. The Hcy concentrations were normalized in 3 of the 4 patients. As expected, significant increases in serum vitamin B-12 (before and after treatment: 334 ± 183 and 437 ± 146 pmol/L, respectively; P = 0.0002) and red blood cell folate (before and after treatment: 880 ± 778 and 1881 ± 1115 nmol/L, respectively; P < 0.0001) occurred after treatment.]5f2$, 百拇医药
DISCUSSION]5f2$, 百拇医药
The present study, which is the continuation of our earlier work, addressed several issues concerning folate and vitamin B-12 metabolism in HIV-infected patients. In the previous studies, we showed that 20% of HIV-infected patients (before HAART) had a low vitamin B-12 concentration (12). Nevertheless, few of them had a deficiency as determined by the dUST (4). Abnormalities in vitamin B-12 binding proteins may have been the cause of these low vitamin B-12 concentrations (13).
Given the elevated Hcy concentrations in vitamin B-12 or folate deficiency (5–7), we sought to investigate this metabolite rather than to perform the time-consuming dUST in HIV-infected patients. Although some authors previously evaluated plasma Hcy concentrations in HIV-infected patients, few subjects with a low serum vitamin B-12 concentration were included and seldom did the subjects also have a low folate concentration (17, 18). In the present study, we measured serum Hcy concentrations in a large group of HIV-infected patients with a low vitamin B-12 or red blood cell folate concentration..lt@cr, 百拇医药
Not surprisingly, < 30% of the HIV-infected patients with a low serum vitamin B-12 concentration had hyperhomocysteinemia. Moreover, most of the patients with hyperhomocysteinemia and a low vitamin B-12 concentration had a low red blood cell folate concentration. In contrast, 100% and 51.5% of patients with a red blood cell folate concentration below percentiles 2.5 and 10, respectively, had hyperhomocysteinemia.
These results are similar to those obtained by using the dUST (4). Few HIV-infected patients with a low serum vitamin B-12 concentration alone have a real vitamin B-12 deficiency (1, 4). By contrast, serum Hcy concentrations confirmed the presence of a real deficiency in most of the HIV-infected patients who had a low red blood cell folate concentration and a normal vitamin B-12 concentration, as the dUST had shown in our earlier study. Moreover, the dUST showed that the deficiency was due to folate (4).;&ctl1, 百拇医药
In patients with low concentrations of both red blood cell folate and vitamin B-12, Hcy concentrations cannot determine whether a patient has vitamin B-12 deficiency, folate deficiency, or both. The dUST or the serum methylmalonic acid concentration would probably be useful in these few patients (19). If these tests are not available, treatment with both vitamins is recommended. On the basis of our findings and on those of earlier studies (4), the measurement of red blood cell folate concentrations in HIV-infected patients who have a low serum vitamin B-12 concentration is probably as satisfactory as the measurement of serum Hcy concentrations, because among HIV-infected patients with a low vitamin B-12 concentration, those with a low red blood cell folate concentration are more likely to have a real deficiency.
In HIV-infected patients, as in patients with other pathologies (9, 20), a low vitamin B-12 concentration may be related to changes in the vitamin B-12 binding proteins and not to a real deficiency. This decrease in serum vitamin B-12 concentration is correlated with neutrophil counts, the main source of cobalophilin, which is the major vitamin B-12 binding protein in plasma (13).%, 百拇医药
Furthermore, a low vitamin B-12 concentration is an adverse prognostic factor in HIV-infected patients (21). Low vitamin B-12 concentrations may be attributed to low white blood cell counts, a factor associated with more advanced HIV disease (12, 22).%, 百拇医药
The present study showed that patients receiving HAART have a lower prevalence of reduced vitamin B-12 (8.7% compared with 27%) and higher concentrations of hemoglobin, leukocytes, CD4, and CD8. Moreover, as in the non-HAART group, vitamin B-12 concentrations in the HAART group were positively related to neutrophil counts, and this relation was independent of Hcy concentrations. The new and old data correlate well with the hypothesis that vitamin B-12 is a prognostic factor (12, 21, 22) and that leukocytes (12, 22), as the main source of cobalophilin, are related to the vitamin B-12 concentrations found in these patients.
Finally, the response to vitamin B-12 and folate treatment was assessed in a group of HIV-infected patients. As expected, all patients with a low red blood cell folate or vitamin B-12 concentration and hyperhomocysteinemia responded to treatment. However, because the decrease in Hcy concentration occurred in response to treatment with both vitamins, it was not possible to ascertain whether the response was specifically due to one vitamin or to the other or to both. In patients with hyperhomocysteinemia and normal concentrations of these vitamins, a decrease in or a normalization of Hcy concentration was observed. A similar response was observed in patients with hyperhomocysteinemia and vitamin B-12 and folate concentrations within their respective reference ranges when vitamin treatment was administered (23–25).#?nn.4j, 百拇医药
The potential implications of hyperhomocysteinemia for HIV-related cardiovascular disorders warrant further investigation. In this regard, hyperhomocysteinemia may contribute to the cardiovascular morbidity (8) of HIV-infected patients receiving HAART. In summary, our data lend support to the assessment of Hcy and red blood cell folate concentrations in HIV-infected patients with a low vitamin B-12 concentration in order to detect patients with a real vitamin B-12 deficiency.
ACKNOWLEDGMENTSi#8#:(, http://www.100md.com
We are indebted to George von Knorring for reviewing the English version of this manuscript.i#8#:(, http://www.100md.com
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2 Supported by the SANTPAU-CITRAN program. \, http://www.100md.com
ABSTRACT\, http://www.100md.com
Background: Before the advent of highly active antiretroviral therapy (HAART), 20% and 10% of HIV-infected patients had low vitamin B-12 and red blood cell folate (RBCF) concentrations, respectively. However, few patients had real vitamin B-12 deficiency.\, http://www.100md.com
Objective: We evaluated the prevalence of low vitamin B-12 and RBCF concentrations in HIV-infected patients receiving HAART and the usefulness of serum homocysteine (sHcy) for differentiating patients with deficiency from those with harmlessly low vitamin B-12.\, http://www.100md.com
Design: The prevalence of low vitamin B-12 and RBCF was evaluated in 126 HIV-infected patients receiving HAART. Moreover, sHcy concentrations were evaluated in 40 HIV-infected patients with low vitamin B-12 and in 37 HIV-infected patients with low RBCF and were compared with those in 128 HIV-infected patients with normal vitamin B-12 and RBCF. sHcy was used to monitor treatment with vitamin B-12 and folic acid in 28 patients (24 with low vitamin B-12 and RBCF and 4 with hyperhomocysteinemia but normal vitamin B-12 and RBCF).
Results: The prevalence of low vitamin B-12 was significantly lower in patients receiving HAART than in previously studied patients who did not receive HAART (8.7% compared with 27%). Nine of the 40 patients (22.5%) with low vitamin B-12 ( 200 pmol/L) had hyperhomocysteinemia (> 17.5 µmol homocysteine/L). Nineteen (51.4%) of the 37 patients with low RBCF ( 580 nmol/L, percentile 10) had hyperhomocysteinemia. Among the 9 patients with an RBCF concentration 450 nmol/L (percentile 2.5), all had hyperhomocysteinemia. The treatment with vitamin B-12 and folic acid normalized sHcy concentrations.7k]])t0, 百拇医药
Conclusions: The prevalence of low vitamin B-12 decreased after the introduction of HAART. The study of sHcy is useful for detecting HIV-infected patients with low vitamin B-12 and real deficiency.7k]])t0, 百拇医药
Key Words: HIV • vitamin B-12 • folate • homocysteine • highly active antiretroviral therapy • HAART7k]])t0, 百拇医药
INTRODUCTION
Low concentrations of vitamin B-12 and red blood cell folate were found in 20% and 10% of HIV-infected patients, respectively (1). However, during the clinical evaluation of HIV-infected patients who have a low vitamin B-12 concentration, it is necessary to ascertain whether these patients have a real deficiency or merely a harmlessly low vitamin B-12 concentration. This situation resembles that of iron deficiency during pregnancy and in persons with multiple myeloma (2), in which a low vitamin B-12 concentration can be found without a real vitamin B-12 deficiency. Some tests can help to distinguish patients with a real deficiency from those without a deficiency.!{%(f0}, 百拇医药
The deoxyuridine suppression test (dUST) is used to determine megaloblastic alterations by measuring changes in the incorporation of labeled thymidine (3). Our group showed that among HIV-infected patients with low vitamin B-12, most did not have a deficiency, except when a low red blood cell folate concentration was observed. However, the dUST demands a bone marrow sample, which is time consuming to obtain; as a result, few laboratories currently use this test routinely (4).
Some authors have suggested that the study of abnormalities in vitamin B-12 metabolites and in folate metabolism is an alternative to the dUST (5). In this regard, methylmalonic acid and homocysteine (Hcy) were found to have excellent sensitivity for diagnosing vitamin B-12 or folate deficiency (5, 6).y, 百拇医药
Because Hcy concentrations increase in vitamin B-12 and folate deficiencies (6), Hcy measurement may be a good tool for detecting and managing low vitamin B-12 concentrations in HIV-infected patients. Moreover, Hcy evaluation is routinely used in many laboratories and could easily be used in HIV-infected patients with low vitamin B-12 (7–9) and in monitoring the response to treatment.y, 百拇医药
Almost all studies on vitamin B-12 and folate metabolism in HIV-infected patients were carried out before the advent of highly active antiretroviral therapy (HAART), a treatment with 3 antiretroviral drugs including a protease inhibitor (1, 10). Today, in the age of HAART, the prevalence of low serum vitamin B-12 concentrations and low red blood cell folate concentrations should be reappraised in HIV-infected patients undergoing this therapy because many HIV-infected patients develop macrocytosis (11). The present study was undertaken to ascertain whether the prevalence of low concentrations of these vitamins is lower now than it was before the advent of HAART and whether serum Hcy concentrations can be used to identify patients with a real vitamin B-12 deficiency and to monitor responses to treatment.
SUBJECTS AND METHODSu(+4t, 百拇医药
Evaluation of serum vitamin B-12 and red blood cell folate concentrations in HIV-infected patients receiving HAARTu(+4t, 百拇医药
To evaluate the prevalence of abnormalities in these variables, a group of HIV-infected patients receiving HAART (taking 3 antiretroviral drugs) was compared with a group of previously studied HIV-infected patients who did not receive HAART. The HAART group consisted of all the HIV-infected patients who received HAART in the SANTPAU-CITRAN (Centre d’Investigació, Tractament i Rehabilitació Adictes a Narcòtics) program for 11 mo (126 patients). The non-HAART group was made up of 109 patients who either received treatment with one antiretroviral drug or received no treatment with antiretroviral drugs. The subjects in the non-HAART group were recruited from 1989 to 1992. The characteristics of these patients were published elsewhere (12, 13). The study was performed in accord with the Helsinki Declaration of 1975 as revised in 1983, and all subjects provided written informed consent.
Assessment of serum Hcy concentrations in the diagnosis of HIV-infected patients with low serum vitamin B-12 or red blood cell folate concentrations3@, 百拇医药
Serum Hcy concentrations in HIV-infected patients with a low serum vitamin B-12 concentration (" 200 pmol/L; n = 40) or a low red blood cell folate concentration (" 580 nmol/L; n = 37) were compared with those in a group consisting of all the HIV-infected patients with a serum vitamin B-12 concentration > 200 pmol/L and a red blood cell folate concentration > 580 nmol/L who were recruited in the 11-mo period (128 HIV-infected patients who did or did not receive HAART). The patients with low concentrations of serum vitamin B-12 or red blood cell folate were recruited from patients who agreed to participate in the SANTPAU-CITRAN program from 1996 until 2001.3@, 百拇医药
In this study, the patients were considered to have a low concentration of serum vitamin B-12 and red blood cell folate if serum vitamin B-12 and red blood cell folate concentrations were below percentile 10 ( 200 pmol/L and 580 nmol/L, respectively). The rationale for using percentile 10 was based on the fact that a considerable percentage of patients below this cutoff had hyperhomocysteinemia (14). We investigated serum Hcy concentrations in a population of 230 patients with different disorders. Ninety-two percent of patients with a red blood cell folate concentration below percentile 2.5 (60 patients) had hyperhomocysteinemia, as did 47% of patients (n = 54) with a red blood cell folate concentration between percentiles 10 and 2.5 (AF Remacha, unpublished observation, 2000). Moreover, the patients with concentrations below percentile 2.5 of serum vitamin B-12 ( 150 pmol/L) and red blood cell folate ( 450 nmol/L) concentrations were also evaluated. The reference values for red blood cell folate were calculated from 159 healthy persons ( ± SD: 837 ± 235 nmol/L; percentile 2.5: 450 nmol/L; percentile 97.5: 1750 nmol/L).
Evaluation of treatment with vitamin B-12 and folic acid in a group of HIV-infected patients with low vitamin B-12 or red blood cell folate concentrations2'*kv, 百拇医药
Twenty-eight patients were recruited from the same 11-mo period. This group consisted of 22 of 24 patients receiving HAART who had low vitamin concentrations, 2 patients who had low vitamin concentrations but were not receiving HAART, and 4 of the 8 control subjects (receiving HAART or not) who had hyperhomocysteinemia and vitamin B-12 and red blood cell folate concentrations ">=" 200 pmol/L and 580 nmol/L, respectively. Twenty-eight of the 34 possible candidates were treated (2 patients rejected the treatment, one patient died, and 3 candidates were lost before starting the treatment).2'*kv, 百拇医药
One milligram vitamin B-12 (cyanocobalamin; intramuscular) per week and 5 mg folic acid (oral)/d were administered to all patients for 6 wk. Vitamin B-12, red blood cell folate, and serum Hcy concentrations were measured before and after treatment.
Methodsi9i?7, http://www.100md.com
Serum vitamin B-12 and red blood cell folate concentrations were measured by using CEDIA vitamin B-12 and folate in a Hitachi 911 automatic analyzer (Roche Diagnostics, Mannheim, Germany). In the historical group, a radioassay (Amersham, Buckinghamshire, United Kingdom) was used to measure concentrations of vitamin B-12 and folate (reference values: 450–1300 nmol/L) (12, 13).i9i?7, http://www.100md.com
Serum Hcy concentrations were measured by using 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) as the derivative and HPLC with fluorescence detection according to the method of Vester and Rasmussen (15). Homocysteic acid was used as the internal standard. This procedure was used for all serum samples, including those of a group of healthy persons who were used as a reference population.i9i?7, http://www.100md.com
For serum Hcy measurement, blood was collected in sterile, evacuated serum-separator tubes containing a gel and clot activator. The samples were kept at room temperature until centrifugation for 20 min at 2000 x g and room temperature. The interval between the phlebotomy and the centrifugation ranged from 1 to 3 h, after which the sample was maintained at -20 °C until use. The serum sample used in the Hcy measurement was also used for the vitamin B-12 measurement.
Several studies showed a difference in Hcy concentrations between samples maintained at room temperature and those maintained at 4 °C (15, 16). Thus, with the use of the same HPLC procedure, the reference range for serum Hcy was calculated in 71 healthy persons (hemoglobin concentration > 120 g/L, mean corpuscular volume (MCV) < 100 fL, serum vitamin B-12 concentration > 200 pmol/L, red blood cell folate concentration > 600 nmol/L, and serum creatinine concentration < 100 µmol/L; age range: 20–84 y). The mean (± SD) reference value for serum Hcy was 7.5 ± 4.5 µmol/L. Because the Hcy distribution had a long tail on the right side, percentiles were used for calculating the reference range (percentiles 2.5–97.5: 2–17.5 µmol/L), and values for serum Hcy > 17.5 µmol/L were considered to be elevated (hyperhomocysteinemia). This reference population was not related to the reference population used for red blood cell folate.xo;9, http://www.100md.com
Statistical procedures
Calculations were made by using SPSS 10.0 (SPSS Inc, Chicago). Quantitative variables were expressed as the mean ± SD, and qualitative variables were expressed as a percentage of positive cases. For comparisons involving quantitative variables, the paired or unpaired Student’s t test was used; CIs were calculated when the differences were significant. For comparisons involving qualitative variables, the chi-square test was used. A simple or multiple regression analysis was performed to study relations between variables.p|m, 百拇医药
RESULTSp|m, 百拇医药
Evaluation of serum vitamin B-12 and red blood cell folate concentrations in HIV-infected patients receiving HAARTp|m, 百拇医药
Among the 126 patients in the HAART group, a serum vitamin B-12 concentration 150 and 200 pmol/L was detected in 2 (1.6%) and 11 (8.7%) patients, respectively. In contrast, among the 109 patients in the non-HAART group who were receiving treatment with only one or no antiretroviral drugs, 20 (18%) and 30 (27%) patients had a serum vitamin B-12 concentration " 150 and 200 pmol/L, respectively. The 2 groups differed significantly in these percentages (chi-square test, P < 0.0001 for both concentrations of serum vitamin B-12).
A red blood cell folate concentration " 450 or 580 nmol/L was observed in 1 (0.8%) and 13 (10.3%) patients in the HAART group, respectively, and in 19 (17.4%) and 24 (22%) patients in the non-HAART group, respectively. There was a significant difference between the 2 groups in the percentage of subjects who had a red blood cell folate concentration " 450 nmol/L (chi-square test, P < 0.0001).jyk3z1], 百拇医药
The patients in the non-HAART group had significantly lower values for hemoglobin, MCV, leukocytes, CD4, and CD8 (Table 1) than did the patients in the HAART group. As shown previously in the non-HAART group (12, 13), there was a relation in the HAART group between serum vitamin B-12 concentrations and granulocytes (r = 0.33, P < 0.0001); this relation was independent of other variables (hemoglobin, MCV, age, sex, red blood cell folate, and serum homocysteine).jyk3z1], 百拇医药
fig.ommittedjyk3z1], 百拇医药
TABLE 1 Vitamin B-12 and folate metabolism in 2 groups of HIV-infected patients1
Role of serum Hcy in the diagnosis of HIV-infected patients with low serum vitamin B-12 or red blood cell folate concentrationsfj/k, 百拇医药
Evaluation of patients with a low serum vitamin B-12 concentrationfj/k, 百拇医药
Only 9 of 40 patients (22.5%) with a low vitamin B-12 concentration (ranging from 60 to 198 pmol/L) had hyperhomocysteinemia (Table 2). The red blood cell folate concentration was also low in 6 patients with a low serum vitamin B-12 concentration. Five of 6 patients with both a low red blood cell folate concentration (" 580 nmol/L) and a low serum vitamin B-12 concentration had hyperhomocysteinemia (mean serum Hcy: 32.8 ± 16.8 µmol/L; range: 12.5–54 µmol/L). Hcy concentrations were significantly higher (Student’s t test, P = 0.017) in these 6 patients than in the 34 HIV-infected patients with a low serum vitamin B-12 concentration and normal red blood cell folate concentration (mean serum Hcy: 8.7 ± 6.2 µmol/L; range: 1–24 µmol/L). Only 4 of 34 patients (11.8%) with a low vitamin B-12 concentration and a normal red blood cell folate concentration had hyperhomocysteinemia.
fig.ommitted*9z3./, http://www.100md.com
TABLE 2 Characteristics of HIV-infected patients with low serum vitamin B-12 or red blood cell folate (RBCF) concentrations1*9z3./, http://www.100md.com
Serum Hcy concentrations in the group with a low vitamin B-12 concentration without a low red blood cell folate concentration did not differ significantly from those of the control group (Student’s t test, P = 0.74). Four of 15 patients (26.7%) with a serum vitamin B-12 concentration " 150 pmol/L had a high Hcy concentration. However, 3 of 4 patients with an elevated serum Hcy concentration had a low red blood cell folate concentration. HIV-infected patients with a low vitamin B-12 concentration and hyperhomocysteinemia had neutrophil counts similar to those of patients with a low vitamin B-12 concentration and a normal Hcy concentration (Student’s t test, P = 0.152).*9z3./, http://www.100md.com
Evaluation of patients with a low red blood cell folate concentration*9z3./, http://www.100md.com
Nineteen (51.4%) of 37 patients with a low red blood cell folate concentration (ranging from 313 to 580 nmol/L; the latter concentration corresponded to percentile 10 of the reference population) had hyperhomocysteinemia. Among the 9 patients who had a red blood cell folate concentration 450 nmol/L (percentile 2.5 of the reference population), all of them (100%) had an elevated serum Hcy concentration.
In contrast with the patients with a low vitamin B-12 concentration, the 31 patients with a low red blood cell folate concentration without a low vitamin B-12 concentration had significantly higher (Student’s t test, P = 0.002) serum Hcy concentrations than did the patients in the control group (20 ± 19 compared with 8.3 ± 7.8 µmol/L). A negative relation was found between serum Hcy concentrations and red blood cell folate concentrations. Multiple regression analysis that included the variables age, sex, hemoglobin, MCV, red blood cell folate, vitamin B-12, and creatinine showed that red blood cell folate concentrations (r = -0.42) and creatinine concentrations (r = 0.3) were independent variables related to serum Hcy concentrations.4\0sm, 百拇医药
Effect of treatment with vitamin B-12 and folic acid in a group of HIV-infected patients with low vitamin B-12 or red blood cell folate concentrations4\0sm, 百拇医药
Among the 24 patients who had a low serum vitamin B-12 concentration and a low red blood cell folate concentration, serum Hcy concentrations in the 14 patients who had a high Hcy concentration decreased to normal values after treatment with vitamin B-12 and folic acid (Table 3). The treatment decreased Hcy concentrations > 50% in 4 of 10 patients whose starting serum Hcy concentration was normal.
fig.ommitted]5f2$, 百拇医药
TABLE 3 Effect of vitamin B-12 and folate on serum homocysteine (Hcy) concentrations in HIV-infected patients1]5f2$, 百拇医药
A decrease in Hcy concentrations was also observed in the 4 patients who had a high serum Hcy concentration and normal serum vitamin B-12 and red blood cell folate concentrations. The Hcy concentrations were normalized in 3 of the 4 patients. As expected, significant increases in serum vitamin B-12 (before and after treatment: 334 ± 183 and 437 ± 146 pmol/L, respectively; P = 0.0002) and red blood cell folate (before and after treatment: 880 ± 778 and 1881 ± 1115 nmol/L, respectively; P < 0.0001) occurred after treatment.]5f2$, 百拇医药
DISCUSSION]5f2$, 百拇医药
The present study, which is the continuation of our earlier work, addressed several issues concerning folate and vitamin B-12 metabolism in HIV-infected patients. In the previous studies, we showed that 20% of HIV-infected patients (before HAART) had a low vitamin B-12 concentration (12). Nevertheless, few of them had a deficiency as determined by the dUST (4). Abnormalities in vitamin B-12 binding proteins may have been the cause of these low vitamin B-12 concentrations (13).
Given the elevated Hcy concentrations in vitamin B-12 or folate deficiency (5–7), we sought to investigate this metabolite rather than to perform the time-consuming dUST in HIV-infected patients. Although some authors previously evaluated plasma Hcy concentrations in HIV-infected patients, few subjects with a low serum vitamin B-12 concentration were included and seldom did the subjects also have a low folate concentration (17, 18). In the present study, we measured serum Hcy concentrations in a large group of HIV-infected patients with a low vitamin B-12 or red blood cell folate concentration..lt@cr, 百拇医药
Not surprisingly, < 30% of the HIV-infected patients with a low serum vitamin B-12 concentration had hyperhomocysteinemia. Moreover, most of the patients with hyperhomocysteinemia and a low vitamin B-12 concentration had a low red blood cell folate concentration. In contrast, 100% and 51.5% of patients with a red blood cell folate concentration below percentiles 2.5 and 10, respectively, had hyperhomocysteinemia.
These results are similar to those obtained by using the dUST (4). Few HIV-infected patients with a low serum vitamin B-12 concentration alone have a real vitamin B-12 deficiency (1, 4). By contrast, serum Hcy concentrations confirmed the presence of a real deficiency in most of the HIV-infected patients who had a low red blood cell folate concentration and a normal vitamin B-12 concentration, as the dUST had shown in our earlier study. Moreover, the dUST showed that the deficiency was due to folate (4).;&ctl1, 百拇医药
In patients with low concentrations of both red blood cell folate and vitamin B-12, Hcy concentrations cannot determine whether a patient has vitamin B-12 deficiency, folate deficiency, or both. The dUST or the serum methylmalonic acid concentration would probably be useful in these few patients (19). If these tests are not available, treatment with both vitamins is recommended. On the basis of our findings and on those of earlier studies (4), the measurement of red blood cell folate concentrations in HIV-infected patients who have a low serum vitamin B-12 concentration is probably as satisfactory as the measurement of serum Hcy concentrations, because among HIV-infected patients with a low vitamin B-12 concentration, those with a low red blood cell folate concentration are more likely to have a real deficiency.
In HIV-infected patients, as in patients with other pathologies (9, 20), a low vitamin B-12 concentration may be related to changes in the vitamin B-12 binding proteins and not to a real deficiency. This decrease in serum vitamin B-12 concentration is correlated with neutrophil counts, the main source of cobalophilin, which is the major vitamin B-12 binding protein in plasma (13).%, 百拇医药
Furthermore, a low vitamin B-12 concentration is an adverse prognostic factor in HIV-infected patients (21). Low vitamin B-12 concentrations may be attributed to low white blood cell counts, a factor associated with more advanced HIV disease (12, 22).%, 百拇医药
The present study showed that patients receiving HAART have a lower prevalence of reduced vitamin B-12 (8.7% compared with 27%) and higher concentrations of hemoglobin, leukocytes, CD4, and CD8. Moreover, as in the non-HAART group, vitamin B-12 concentrations in the HAART group were positively related to neutrophil counts, and this relation was independent of Hcy concentrations. The new and old data correlate well with the hypothesis that vitamin B-12 is a prognostic factor (12, 21, 22) and that leukocytes (12, 22), as the main source of cobalophilin, are related to the vitamin B-12 concentrations found in these patients.
Finally, the response to vitamin B-12 and folate treatment was assessed in a group of HIV-infected patients. As expected, all patients with a low red blood cell folate or vitamin B-12 concentration and hyperhomocysteinemia responded to treatment. However, because the decrease in Hcy concentration occurred in response to treatment with both vitamins, it was not possible to ascertain whether the response was specifically due to one vitamin or to the other or to both. In patients with hyperhomocysteinemia and normal concentrations of these vitamins, a decrease in or a normalization of Hcy concentration was observed. A similar response was observed in patients with hyperhomocysteinemia and vitamin B-12 and folate concentrations within their respective reference ranges when vitamin treatment was administered (23–25).#?nn.4j, 百拇医药
The potential implications of hyperhomocysteinemia for HIV-related cardiovascular disorders warrant further investigation. In this regard, hyperhomocysteinemia may contribute to the cardiovascular morbidity (8) of HIV-infected patients receiving HAART. In summary, our data lend support to the assessment of Hcy and red blood cell folate concentrations in HIV-infected patients with a low vitamin B-12 concentration in order to detect patients with a real vitamin B-12 deficiency.
ACKNOWLEDGMENTSi#8#:(, http://www.100md.com
We are indebted to George von Knorring for reviewing the English version of this manuscript.i#8#:(, http://www.100md.com
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