Still, median 1,25(OH)2D values remained lower in patients with 25(OH)D deficiency with 28 (IQR 21–36) ng/l and 25(OH)D insufficiency with 37 (25–48) ng/l as compared with 54 (37–72) ng/l in individuals with 25(OH)D values 75 nmol/l or more (P < 0.001).
Univariable and multivariable analyses of factors influencing 1,25(OH)2D levels revealed a distinct pattern from 25(OH)D (Table 2). While season and ethnicity were not associated with 1,25(OH)2D levels, we observed a negative correlation with hepatitis C seropositivity. On the contrary, previous AIDS and higher CD4 cell counts were associated with low levels and BMI with high 1,25(OH)2D levels. Although use of NNRTI showed no significant correlation in multivariable analysis, use of TDF correlated with higher 1,25(OH)2D. Consistently, the 1-hydroxylation rates in TDF-treated patients were 25% higher (P = 0.002) in the absence of 25(OH)D deficiency, that is at values 30 nmol/l or higher. In vitamin D-deficient patients, however, use of TDF did not stimulate the 1,25(OH)2D-to-25(OH)D ratio any further (P = 0.6).
Calculated glomerular filtration rate and serum alkaline phosphatase
With a median baseline calculated glomerular filtration rate (cGFR) of 109 ml/min (IQR 93–124), all but one patient had cGFR values higher than 30 ml/min and, therefore, no structural reason for a 1-hydroxylation deficiency in the kidney. Neither TDF-containing nor TDF-sparing cART significantly changed cGFR compared with baseline (data not shown).
Use of TDF was associated with a significant increase in sAP from a median of 69 (IQR 55–84) to 92 (70–116) U/l (P = 0.01) as compared with 65 (55–86) to 79 (65–98) U/l (P = 0.5) in patients on a TDF-sparing regimen and 70 (58–93) to 80 (67–98) U/l in patients on protease inhibitors (P = 0.8). Regression analysis provided a correlation coefficient of 6.6 (1.0–12.2; P = 0.02) between higher 1-hydroxylation and increases in sAP between baseline and the 1-year follow-up in patients exposed to TDF.
With 42–52% in spring and 14–18% in fall, vitamin D deficiency was highly prevalent in our HIV-positive patients. This prevalence is higher than the 10.5 or 14% reported from Boston or Sydney [18,20], but compares well with the 29% value described in the Netherlands . In order to reach the target level of 75 nmol/l or higher for maximal vitamin D benefits, as many as 76–95% of the patients would qualify for vitamin D supplementation. Again, such high numbers have been reported earlier [22,23]. From recent assessments of vitamin D levels in western countries, it can be assumed that the prevalence of vitamin D insufficiency and deficiency is high in the general population . A comparison with deficiency rates in the general Swiss population is difficult, as recent data are missing. However, the rate of vitamin D deficiency in our HIV-positive cohort was clearly higher than the 6–15% rate found in the only assessment in the general Swiss population during winter . Consistent with earlier reports , non-white ethnicities with darker skin pigmentation, in particular black patients, were most strongly affected by vitamin D deficiency. The reasons for the negative correlation with active IDU are unknown, both malnutrition and limited sunlight exposure may play a role. The positive correlation with the time since HIV diagnosis may be related to a generally poorer health condition of patients who have been recently diagnosed. The observed trend of lower 25(OH)D levels in individuals with higher BMI is probably caused by vitamin D sequestration in body fat . However, this association must be interpreted with caution, as body weight was normal in almost all patients.
Although cART exposure had no significant impact on vitamin D levels, NNRTI use was associated with significantly lower 25(OH)D levels. This correlation, with a consecutively increased risk of developing hyperparathyroidism, has been documented earlier  and may be caused by an increased catabolism of 25(OH)D through induction of CYP450 by NNRTIs. Consistently, a case report has described severe 25(OH)D deficiency after starting efavirenz .
Neither NNRTI nor protease inhibitor medication showed a significant effect on 1,25(OH)2D levels in our analyses, although protease inhibitor-treated patients had shown lower 1,25(OH)2D values in an earlier study . Many enzymes of the cytochrome P450 cytochrome family, which are known to interact with NNRTIs and/or protease inhibitors, are involved in vitamin D metabolism; for example, 25-hydroxylation in the liver (CYP27A1, CYP2R1 or CYP3A4), 1-hydroxylation in the kidney (CYP27B1) or the catabolism of activated vitamin D (CYP3A4 rather than CYP24) . In vitro, inhibitory effects of ritonavir, indinavir and nelfinavir on CYP450 enzymes resulted in a net reduction of activated vitamin D, suggesting that protease inhibitor use could contribute to bone demineralization .
Further clinical data are needed to evaluate the clinical net effect of NNRTIs and protease inhibitors on bone metabolism in general and on vitamin D in particular.
In clinical routine, vitamin D assessment is based on 25(OH)D measurements, because of its stability with a 2-week half-life in blood circulation . At low 25(OH)D levels, parathyroid hormone (PTH) stimulates the tubular reabsorption of calcium, phosphaturia and the 1-hydroxylation of 25(OH)D. This is consistent with our finding of a logarithmic increase in the 1-hydroxylation ratio with decreasing 25(OH)D levels, particularly with levels below 30 nmol/l . In our patients, this compensatory increase in 1-hydroxylation partially compensated the seasonal differences in 25(OH)D. Our data underline that 1,25(OH)2D should not be used to diagnose vitamin D deficiency, as normal values may hide both 25(OH)D deficiency and secondary hyperparathyroidism.
Surprisingly, a history of AIDS-defining events was associated with lower 1,25(OH)2D, but not with lower 25(OH)D levels, which would have suggested impaired nutritional supply and/or lower sun exposure. AIDS may result in an inflammation-related impairment of 1-hydroxylation. As a matter of fact, earlier studies have reported low 1,25(OH)2D levels in patients with advanced HIV infection and immunologic hyperactivation [31,32]. The significant negative correlation of hepatitis C seropositivity with 1,25(OH)2D, but not with 25(OH)D, found in our analysis might be related to an inflammation-related impairment of 1-hydroxylation due to replicating hepatitis. However, the hypothesis of an inflammation-related impairment of 1-hydroxylation is contradicted by the unexplained negative correlation of 1,25(OH)2D with CD4 cell counts.
TDF-use correlated with both higher serum 1,25(OH)2D and serum alkaline phosphatase levels indicating osteoblast activation . This pattern was not seen in protease inhibitor-treated or zidovudine-treated patients (data not shown). Importantly, higher 1,25(OH)2D levels were not due to higher 25(OH)D, but to an increased rate of 1-hydroxylation. 1-Hydroxylation is not only stimulated by PTH in response to low calcium and 25(OH)D levels but also, possibly mediated through phosphatonins [33,34], by hypophosphatemia . Several publications have documented TDF-related proximal renal tubulopathy with excessive phosphate losses [36,37], but also decreasing bone mineral density . The stimulated 1-hydroxylation observed in our TDF-treated patients may represent an autoregulatory process to compensate for drug-related phosphate losses by the stimulation of intestinal phosphate resorption and/or mobilization of bone phosphate through higher 1,25(OH)2D levels. Stimulating intestinal phosphate resorption may thereby be protective, whereas mobilizing bone phosphate may promote osteopenia and osteoporosis in these individuals.
This study has some limitations. The population studied was homogenous (mostly men and white) and relatively healthy. Its retrospective nature prevented broader analyses, such as the evaluation of calcium and phosphate metabolism, and no conclusions on the potential benefit of supplementation can be drawn. The stringent inclusion criteria including the requirement of availability of samples, CD4 cell measurements at all three time points as well as documentation of successful cART by a HIV viral load less than 50 copies/ml resulted in a relatively small overall sample size. Therefore, documented associations will need confirmation by independent studies. Given the numerous HIV-independent risk factors for vitamin D deficiency in our patients, it is almost impossible to establish a HIV-negative control group to assess the influence of HIV on vitamin D levels. On the contrary, we believe that our study design comparing patients before and after cART initiation is most suitable to elucidate the impact of viral control and antiretroviral drug classes.
On the basis of our prevalence data and the growing recognition of vitamin D-related health benefits, we suggest systematic screening for vitamin D deficiency in all HIV-positive patients. Seasonal changes thereby have to be taken into account. If many benefits of vitamin D supplementation have already been documented in the general population, an adequate supply may be even more important in HIV-infected individuals with their additional risk factors for osteopenia, neoplasias or cardiovascular disease. In the general population, supplementation with a minimum of 700–800 IU of 25(OH)D per day has been shown to reduce the risk for hip and nonvertebral fractures [5,8]. Alternatively, 100 000 IU of oral 25(OH)D every 4 months or 300 000 IU every 12 months might be used . The role of vitamin D supplementation in HIV-positive patients, and specific treatment goals in particular, must be defined by prospective studies with HIV-specific endpoints such as disease progression or change in CD4 cell counts. Also, the impact of NNRTIs on 25(OH)D and TDF on 1,25(OH)2D needs further consideration.
This study was performed within the framework of the Swiss HIV Cohort Study, supported by the Swiss National Science Foundation (grant 33CSCO-108787).
N.J.M., C.A.F., B.L. and H.F. conceived the study. N.J.M. and A.V. were responsible for measurement of vitamin D levels. N.J.M., C.A.F., L.E., P.S., T.D., L.M., and A.C. were responsible for collecting patient data. N.J.M., C.A.F. and H.F. drafted the manuscript. B.L., H.B. and C.A.F. were responsible for statistical analysis. All authors read and gave feedback to the final version of the manuscript.
Swiss HIV Cohort Study
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Keywords:© 2010 Lippincott Williams & Wilkins, Inc.
1,25-dihydroxyvitamin D; 25-hydroxyvitamin D; combined antiretroviral therapy; deficiency; HIV; seasonality; tenofovir