A Liquid Chromatography/Tandem Mass Spectrometry Method for Determination of 25-Hydroxy Vitamin D2 and 25-Hydroxy Vitamin D3 in Dried Blood Spots: A Potential Adjunct to Diabetes and Cardiometabolic Risk Screening

Background

Now emerging as an important risk factor for type 1 diabetes, vitamin D deficiency is also associated with obesity, metabolic syndrome, and type 2 diabetes and has been identified as a potential cardiometabolic risk factor. A simple, accurate screening test for 25-hydroxy vitamin D [25(OH)D] deficiency is needed. We developed a liquid chromatography/tandem mass spectrometry assay for 25-hydroxy vitamin D₂ [25(OH)D₂] and 25-hydroxy vitamin D₃ [25(OH)D₃] in dried blood spots.

Method

Blood spots were collected by finger stick simultaneously with serum samples obtained by venipuncture from healthy volunteers. Disks punched from the dried blood spots were sonicated with an internal standard solution of deuterated 25(OH)D₃ (26,26,26,27,27,27-d₆). Methanol was added to precipitate proteins prior to extraction with hexane. The extracted samples were dried and reconstituted in 50:50 methanol:H₂O before injection into a Varian 320-MS TQ mass spectrometer.

Results

Blood spot assay precision was good over the reportable range: interassay coefficients of variation were 13, 13, and 11% at concentrations of 14, 26, and 81 ng/ml, respectively, for 25-hydroxy vitamin D₃ and 12% at 23 ng/ml for 25(OH)D₂. The 25(OH)D₃ assay was linear from 3.5 to 75 ng/ml (R > 0.99). Blood spot and serum values showed excellent correlation for 25(OH)D₂ (R = 0.90, n = 54) and 25(OH)D₃ (R = 0.91, n = 83).

Conclusions

This blood spot assay for 25(OH)D₂ and 25(OH)D₃ provides a convenient and cost-effective alternative to serum assays and can be automated. This may be valuable in large-scale screening for risk of type 1 diabetes, for cardiometabolic risk screening, and for monitoring vitamin D supplementation     

Calcium and Phosphorus Regulatory Hormones and Risk of Incident Symptomatic Kidney Stones

Background and objectives

Calcium and phosphorus regulatory hormones may contribute to the pathogenesis of calcium nephrolithiasis. However, there has been no prospective study to date of plasma hormone levels and risk of kidney stones. This study aimed to examine independent associations between plasma levels of 1,25-dihydroxyvitamin D (1,25[OH]₂D), 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, fibroblast growth factor 23 (FGF23), parathyroid hormone, calcium, phosphate, and creatinine and the subsequent risk of incident kidney stones.

Design, setting, participants, & measurements

This study was a prospective, nested case-control study of men in the Health Professionals Follow-Up Study who were free of diagnosed nephrolithiasis at blood draw. During 12 years of follow-up, 356 men developed an incident symptomatic kidney stone. Using risk set sampling, controls were selected in a 2:1 ratio (n=712 controls) and matched for age, race, and year, month, and time of day of blood collection.

Results

Baseline plasma levels of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, parathyroid hormone, calcium, phosphate, and creatinine were similar in cases and controls. Mean 1,25(OH)₂D and median FGF23 levels were higher in cases than controls but differences were small and statistically nonsignificant (45.7 versus 44.2 pg/ml, P=0.07 for 1,25[OH]₂D; 47.6 versus 45.1 pg/ml, P=0.08 for FGF23). However, after adjusting for body mass index, diet, plasma factors, and other covariates, the odds ratios of incident symptomatic kidney stones in the highest compared with lowest quartiles were 1.73 (95% confidence interval, 1.11 to 2.71; P for trend 0.01) for 1,25(OH)₂D and 1.45 (95% confidence interval, 0.96 to 2.19; P for trend 0.03) for FGF23. There were no significant associations between other plasma factors and kidney stone risk.

Conclusions

Higher plasma 1,25(OH)₂D, even in ranges considered normal, is independently associated with higher risk of symptomatic kidney stones. Although of borderline statistical significance, these findings also suggest that higher FGF23 may be associated with risk.     

Plasma Vitamin D Level and Change in Albuminuria and eGFR According to Sodium Intake

Background and objectives

Low circulating 25-hydroxyvitamin D [25(OH)D] and high sodium intake are both associated with progressive albuminuria and renal function loss in CKD. Both vitamin D and sodium intake interact with the renin-angiotensin-aldosterone system. We investigated whether plasma 25(OH)D or 1,25-dihydroxyvitamin D [1,25(OH)₂D] is associated with developing increased albuminuria or reduced renal function and whether these associations depend on sodium intake.

Design, setting, participants, & measurements

Baseline plasma 25(OH)D and 1,25(OH)₂D were measured by liquid chromatography tandem mass spectrometry, and sodium intake was assessed by 24-hour urine collections in the general population–based Prevention of Renal and Vascular End-Stage Disease cohort (n=5051). Two primary outcomes were development of urinary albumin excretion >30 mg/24 h and eGFR (creatinine/cystatin C–based CKD Epidemiology Collaboration) <60 ml/min per 1.73 m². Participants with CKD at baseline were excluded. In Cox regression analyses, we assessed associations of vitamin D with developing increased albuminuria or reduced eGFR and potential interaction with sodium intake.

Results

During a median follow-up of 10.4 (6.2–11.4) years, 641 (13%) participants developed increased albuminuria, and 268 (5%) participants developed reduced eGFR. Plasma 25(OH)D was inversely associated with increased albuminuria (fully adjusted hazard ratio [HR] per SD higher, 0.86; 95% confidence interval [95% CI], 0.78 to 0.95; P=0.003) but not reduced eGFR (HR, 0.99; 95% CI, 0.87 to 1.12; P=0.85). There was interaction between 25(OH)D and sodium intake for risk of developing increased albuminuria (P interaction =0.03). In participants with high sodium intake, risk of developing increased albuminuria was inversely associated with 25(OH)D (lowest versus highest quartile: adjusted HR, 1.81; 95% CI, 1.20 to 2.73, P<0.01), whereas this association was nonsignificant in participants with low sodium intake (HR, 1.29; 95% CI, 0.94 to 1.77; P=0.12). Plasma 1,25(OH)₂D was not significantly associated with increased albuminuria or reduced eGFR.

Conclusions

Low plasma 25(OH)D is associated with higher risk of developing increased albuminuria, particularly in individuals with high sodium intake, but not of developing reduced eGFR. Plasma 1,25(OH)2D is not associated with risk of developing increased albuminuria or reduced eGFR.     

In vitro metabolism of 25-hydroxyvitamin D3 by isolated rat kidney cells

Cells were dispersed from rat kidney after enzymatic digestion of the extracellular matrix. When the cells were suspended in a serum-free medium and incubated with ³H-labeled 25-hydroxyvitamin D₃ (25-OH-D₃) several polar metabolites, including 1,25-(OH)₂[³H]D₃ and 24,25-(OH)₂[³H]D₃ were produced. The specific activities of the 25-OH-D₃:1- and 24-hydroxylases in isolated rat kidney cells were 10-100 times greater than in avian kidney homogenates. The rates of production of 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ were linear over a wide range in cell densities (0.65-5.0 × 10⁶ cells per ml) and substrate concentrations (3.5-70 nM). The rate of production of 24,25-(OH)₂[³H]D₃ from 25-OH-[³H]D₃ by cells isolated from rats fed control diet was linear with time for up to 30 min, while the synthesis of 1,25-(OH)₂[³H]D₃ was linear for over 90 min. The specific activity of the 25-OH-D₃:1-hydroxylase was increased in kidney cells from vitamin D-deficient rats (11.5 fmol/min per 10⁶ cells) as well as calcium-deficient rats (8.1 fmol/min per 10⁶ cells) when compared to cells from rats fed the control diet (2.0 fmol/min per 10⁶ cells). Also, the specific activity of the 25-OH-D₃:24-hydroxylase was reduced in cells from the vitamin D-deficient rats (<0.2 fmol/min per 10⁶ cells) and calcium-deficient rats (5.1 fmol/min per 10⁶ cells) compared to the controls (15.2 fmol/min per 10⁶ cells). On the basis of these results, as well as previous in vivo studies, we conclude that the metabolism of 25-OH-D₃ by freshly isolated rat kidney cells reflects the in vivo activities of the renal vitamin D-metabolizing enzymes and may prove useful as an assay.     

Serum 25-Hydroxyvitamin D and Change in Estimated Glomerular Filtration Rate

Summary

Background and objectives

Mounting evidence suggests that 1,25-dihydroxyvitamin D prevents the progression of chronic kidney disease (CKD). It is not clear whether “nutritional” forms of vitamin D affect GFR.

Design, setting, participants, & measurements

We tested whether serum 25-hydroxyvitamin D concentration (25(OH)D), a measure of total vitamin D intake from cutaneous synthesis and dietary consumption, is associated with loss of estimated GFR among 1705 older adults with predominantly normal baseline kidney function participating in the Cardiovascular Health Study. Baseline 25(OH)D was measured by HPLC-tandem mass spectrometry. GFR was estimated at baseline and 4 years later using the CKD-EPI formula, with rapid GFR loss defined as 12 ml/min per 1.73 m² or more over 4 years.

Results

Rapid GFR loss was observed for 207 participants (12%). Each 10 ng/ml lower 25(OH)D was associated with a 25% greater risk of rapid GFR loss (95% confidence interval [CI] 5%, 49%, P = 0.01), adjusting for potential confounding characteristics. Compared with 25(OH)D ≥30 ng/ml, 25(OH)D concentrations 15 to 29 ng/ml and <15 ng/ml were associated with 29% (95% CI −13%, 91%) and 68% (95% CI 1%, 177%) greater adjusted risks of rapid GFR loss, respectively. Magnitudes of association were largest among participants with diabetes. Results were similar evaluating a composite outcome of rapid GFR loss, end stage renal disease, and death.

Conclusions

Insufficient 25(OH)D may be a modifiable risk factor for early GFR loss. We recommend clinical trials to determine whether vitamin D supplementation prevents the development and progression of CKD.     

Nutritional Vitamin D Supplementation in Dialysis: A Randomized Trial

Background and objectives

Vitamin D (25-hydroxyvitamin D; 25[OH]D) deficiency is common in patients initiating long-term hemodialysis, but the safety and efficacy of nutritional vitamin D supplementation in this population remain uncertain.

Design, setting, participants, & measurements

This randomized, placebo-controlled, parallel-group multicenter trial compared two doses of ergocalciferol with placebo between October 2009 and March 2013. Hemodialysis patients (n=105) with 25(OH)D levels ≤32 ng/ml from 32 centers in the Northeast United States were randomly assigned to oral ergocalciferol, 50,000 IU weekly (n=36) or monthly (n=33), or placebo (n=36) for a 12-week treatment period. The primary endpoint was the achievement of vitamin D sufficiency (25[OH]D >32 ng/ml) at the end of the 12-week treatment period. Survival was assessed through 1 year.

Results

Baseline characteristics were similar across all arms, with overall mean±SD 25(OH)D levels of 21.9±6.9 ng/ml. At 12 weeks, vitamin D sufficiency (25[OH]D >32 ng/ml) was achieved in 91% (weekly), 66% (monthly), and 35% (placebo) (P<0.001). Mean 25(OH)D was significantly higher in both the weekly (49.8±2.3 ng/ml; P<0.001) and monthly (38.3±2.4 ng/ml; P=0.001) arms compared with placebo (27.4±2.3 ng/ml). Calcium, phosphate, parathyroid hormone levels, and active vitamin D treatment did not differ between groups. All-cause and cause-specific hospitalizations and adverse events were similar between groups during the intervention period. Lower all-cause mortality among ergocalciferol-treated participants was not statistically significant (hazard ratio, 0.28; 95% confidence interval, 0.07 to 1.19).

Conclusions

Oral ergocalciferol can increase 25(OH)D levels in incident hemodialysis patients without significant alterations in blood calcium, phosphate, or parathyroid hormone during a 12-week period.     

Temporal Trajectory of B-Type Natriuretic Peptide in Patients with CKD Stages 3 and 4, Dialysis,and Kidney Transplant

Background and objectives

B-type natriuretic peptide (BNP) concentration predicts outcome in patients undergoing dialysis. Because survival and cardiovascular risk change across the CKD continuum, serial changes in BNP were compared in patients at different CKD stages and after kidney transplantation.

Design, setting, participants, & measurements

Patients with CKD stages 3 and 4 (CKD 3–4), dialysis patients, and kidney transplant recipients (KTRs) from one center had two measurements of BNP taken a median of 161 days apart in 2003–2004 and were followed until July 2012. Both BNP-32 (Triage BNP; Biosite Diagnostics) and NT-BNP-76 (proBNP; Roche Diagnostics) were assayed. The interaction between change in log-transformed BNP concentration over time and patient group was tested by fitting regression models on panel data with random effects. Survival after the second measurement was compared by tertile of change in BNP.

Results

Patients with CKD 3–4 (n=48), dialysis patients (n=102), and KTRs (n=73) were followed for a median of 5.7, 4.8, and 5.9 years, respectively. The interaction between patient group and BNP measurements over time was significant for NT-BNP-76 (P<0.001) and BNP-32 (P<0.01). Median NT-BNP-76 increased in dialysis patients and those with CKD 3–4 from 3850 pg/ml (interquartile range [IQR], 1776–12,323 pg/ml) to 18,830 pg/ml (IQR, 6114–61,009 pg/ml; P<0.001) and from 698 pg/ml (IQR, 283–2922 pg/ml) to 2529 pg/ml (IQR, 347–9277 pg/ml; P=0.002), respectively. Change was not significant for KTRs or comparisons made with BNP-32. Survival rate was significantly lower for patients with the highest tertile of change in NT-BNP-76 among patients with CKD 3–4 (P=0.02), but not in the dialysis or KTR groups. In 11 patients who received a kidney transplant during the study, median NT-BNP-76 decreased from 9607 pg/ml (IQR, 2292–31,282 pg/ml) to 457 pg/ml (IQR, 203–863 pg/ml) after transplant (P<0.01).

Conclusions

The temporal trajectory of BNP differs between dialysis patients and those with CKD 3–4 and KTRs. This has important implications for the development of BNP-guided management strategies in CKD.     

Fibroblast growth factor 23, left ventricular mass,and left ventricular hypertrophy in community-dwelling older adults

Objectives

In chronic kidney disease (CKD), high FGF23 concentrations are associated with left ventricular hypertrophy (LVH), cardiovascular events, and death. The associations of FGF23 with left ventricular mass (LVM) and LVH in the general population and the influence of CKD remains uncertain.

Methods

C-terminal plasma FGF23 concentrations were measured, and LVM and LVH evaluated by echocardiogram among 2255 individuals ≥65 years in the Cardiovascular Health Study. Linear regression analysis adjusting for demographics, cardiovascular, and kidney related risk factors examined the associations of FGF23 concentrations with LVM. Analyses were stratified by CKD status and adjusted linear and logistic regression analysis explored the associations of FGF23 with LVM and LVH.

Results

Among the entire cohort, higher FGF23 concentrations were associated with greater LVM in adjusted analyses (β = 6.71 [95% CI 4.35–9.01] g per doubling of FGF23). 32% (n = 624) had CKD (eGFR <60 mL/min/1.73 m² and/or urine albumin-to-creatinine ratio >30 mg/g). Associations were stronger among participants with CKD (p interaction = 0.006): LVM β = 9.71 [95% CI 5.86–13.56] g per doubling of FGF23 compared to those without CKD (β = 3.44 [95% CI 0.77, 6.11] g per doubling of FGF23). While there was no significant interaction between FGF23 and CKD for LVH (p interaction = 0.25), the OR (1.46 95% CI [1.20–1.77]) in the CKD group was statistically significant and of larger magnitude than the OR for in the no CKD group (1.12 [95% CI 0.97–1.48]).

Conclusion

In a large cohort of older community-dwelling adults, higher FGF23 concentrations were associated with greater LVM and LVH with stronger relationships in participants with CKD.     

Fibroblast Growth Factor 23 and Cardiovascular Mortality after Kidney Transplantation

Background and objectives

Circulating fibroblast growth factor 23 (FGF23) is associated with adverse cardiovascular outcomes in CKD. Whether FGF23 predicts cardiovascular mortality after kidney transplantation, independent of measures of mineral metabolism and cardiovascular risk factors, is unknown.

Design, setting, participants, & measurements

The association between plasma C-terminal FGF23 and cardiovascular mortality was analyzed in a single-center prospective cohort of 593 stable kidney transplant recipients (mean age ± SD, 52±12 years; 54% male; estimated GFR, 47±16 ml/min per 1.73 m²), at a median of 6.1 (interquartile range, 2.7–11.7) years after transplantation. Multivariate Cox regression models were built, adjusting for measures of renal function and mineral metabolism; Framingham risk factors; the left ventricular wall strain markers midregional fragment of pro–A-type natriuretic peptide (MR-proANP) and N-terminal-pro brain natriuretic peptide (NT-proBNP); and copeptin, the stable C-terminal portion of the precursor of vasopressin.

Results

In multivariate linear regression analysis, MR-proANP (β=0.20, P<0.001), NT-proBNP (β=0.18, P<0.001), and copeptin (β=0.26, P<0.001) were independently associated with FGF23. During follow-up for 7.0 (interquartile range, 6.2–7.5) years, 128 patients (22%) died, of whom 66 (11%) died due to cardiovascular disease; 54 (9%) had graft failure. FGF23 was associated with an higher risk of cardiovascular mortality in a fully adjusted multivariate Cox regression model (hazard ratio [HR], 1.88 [95% confidence interval (CI), 1.11 to 3.19]; P=0.02). FGF23 was also independently associated with all-cause mortality (full model HR, 1.86 [95% CI, 1.27 to 2.73]; P=0.001). Net reclassification improved for both cardiovascular mortality (HR, 0.07 [95% CI, 0.01 to 0.14]; P<0.05) and all-cause mortality (HR, 0.11 [95% CI, 0.05 to 0.18]; P<0.001).

Conclusions

Plasma FGF23 is independently associated with cardiovascular and all-cause mortality after kidney transplantation. The association remained significant after adjustment for measures of mineral metabolism and cardiovascular risk factors.     

Associations of FGF-23 and sKlotho with Cardiovascular Outcomes among Patients with CKD Stages 2–4

Background and objectives

CKD–mineral and bone disorders (CKD-MBD) measures contribute to cardiovascular morbidity in patients with CKD. Among these, fibroblast growth factor (FGF)-23 and its coreceptor Klotho may exert direct effects on vascular and myocardial tissues. Klotho exists in a membrane-bound and a soluble form (sKlotho). Recent experimental evidence suggests sKlotho has vasculoprotective functions.

Design, settings, participants, & measurements

Traditional and novel CKD-MBD variables were measured among 444 patients with CKD stages 2–4 recruited between September 2008 and November 2012 into the ongoing CARE FOR HOMe study. Across tertiles of baseline sKlotho and FGF-23, the incidence of two distinct combined end points was analyzed: (1) the first occurrence of an atherosclerotic event or death from any cause and (2) the time until hospital admission for decompensated heart failure or death from any cause.

Results

Patients were followed for 2.6 (interquartile range, 1.4–3.6) years. sKlotho tertiles predicted neither atherosclerotic events/death (fully adjusted Cox regression analysis: hazard ratio [HR] for third versus first sKlotho tertile, 0.75 [95% confidence interval (95% CI), 0.43–1.30]; P=0.30) nor the occurrence of decompensated heart failure/death (HR for third versus first sKlotho tertile, 0.81 [95% CI, 0.39–1.66]; P=0.56). In contrast, patients in the highest FGF-23 tertile had higher risk for both end points in univariate analysis. Adjustment for kidney function attenuated the association between FGF-23 and atherosclerotic events/death (HR for third versus first FGF-23 tertile, 1.23 [95% CI, 0.58–2.61]; P=0.59), whereas the association between FGF-23 and decompensated heart failure/death remained significant after adjustment for confounders (HR for third versus first FGF-23 tertile, 4.51 [95% CI, 1.33–15.21]; P=0.02).

Conclusions

In this prospective observational study of limited sample size, sKlotho was not significantly related to cardiovascular outcomes. FGF-23 was significantly associated with future decompensated heart failure but not incident atherosclerotic events.     

Changes in vitamin D levels in patients with systemic lupus erythematosus: Effects on fatigue,disease activity,and damage

Objective

To analyze whether changes in serum 25‐hydroxyvitamin D (25[OH]D) levels affect activity, irreversible organ damage, and fatigue in systemic lupus erythematosus (SLE).

Methods

We performed an observational study of 80 patients with SLE included in a previous cross‐sectional study of 25(OH)D, reassessed 2 years later. Oral vitamin D₃ was recommended in those with low baseline 25(OH)D levels. The relationship between changes in 25(OH)D levels from baseline and changes in fatigue (measured by a 0–10 visual analog scale [VAS]), SLE activity (measured by the Systemic Lupus Erythematosus Disease Activity Index [SLEDAI]), and irreversible organ damage (measured by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index [SDI]) were analyzed.

Results

Sixty patients took vitamin D₃. Mean 25(OH)D levels increased among all treated patients (P = 0.044), in those with baseline vitamin D levels <30 ng/ml (P < 0.001), and in those with baseline vitamin D levels <10 ng/ml (P = 0.005). Fifty‐seven patients (71%) still had 25(OH)D levels <30 ng/ml and 5 (6%) had 25(OH)D levels <10 ng/ml. Inverse significant correlations between 25(OH)D levels and the VAS (P = 0.001) and between changes in 25(OH)D levels and changes in the VAS in patients with baseline 25(OH)D levels <30 ng/ml (P = 0.017) were found. No significant correlations were seen between the variation of the SLEDAI or SDI values and the variation in 25(OH)D levels (P = 0.87 and P = 0.63, respectively).

Conclusion

Increasing 25(OH)D levels may have a beneficial effect on fatigue. Our results do not support any effects of increasing 25(OH)D levels on SLE severity, although they are limited by the insufficient 25(OH)D response to the recommended regimen of oral vitamin D₃ replacement.     

Plasma vitamin D metabolite levels in phosphorus deficient rats during the development of vitamin D deficient rickets

Plasma levels of the vitamin D metabolites were related to changes in bone morphology during the development of rickets in rats deprived of phosphorus and vitamin D. Weanling rats were studied at 1, 3, and 5 wk after onset of diets deficient in phosphorus or in both phosphorus and vitamin D. Bone histology and morphometry were carried out and measurements were made of ⁴⁵Ca and ³²P absorption, serum Ca and P, and plasma 25(OH)D₃, 24,25 (OH)₂D₃ and 1,25 (OH)₂D₃. After 1 wk of vitamin D restriction, the plasma levels of 25(OH)D₃ and 24,25(OH)₂D₃ were non-detectable (<0.5 and <0.8 ng/ml). The plasma 1,25(OH)₂D₃ level was elevated at 1 wk (105.5 pg/ml) and fell to 19 pg/ml by 5 wk. At 1 wk mild rachitic lesions in epiphyseal cartilage were observed despite the elevated 1,25(OH)D₃ level. Serum Ca and P levels and values for ⁴⁵Ca and ³²P absorption decreased and the severity of the rickets increased with the fall in plasma levels of 1,25(OH)₂D₃. In vitamin D replete, phosphate deficient rats the epiphyseal cartilage was normal throughout the 5 wk study peroid. Our results provide further evidence that physiological levels of 1,25 (OH)₂D₃ will not prevent rickets without adequate plasma concentrations of either 25(OH)D₃ or 24,25(OH)₂D₃.     

Independent association of severe vitamin D deficiency as a risk of acute myocardial infarction in Indians

Background

Association of vitamin D deficiency with coronary heart disease (CHD) has been widely reported. Emerging data has shown high prevalence of vitamin D deficiency among Indians. However, this association has not been studied in Indians.

Methods

A case-control study with 120 consecutive cases of first incident acute myocardial infarction (MI) and 120 age and gender matched healthy controls was conducted at All India Institute of Medical Sciences, New Delhi. The standard clinical and biochemical risk factors for MI were assessed for both cases and controls. Serum 25 (OH) vitamin D assay was performed from stored samples for cases and controls using radioimmunoassay.

Results

Vitamin D deficiency [25(OH) D < 30 ng/ml] was highly prevalent in cases and controls (98.3% and 95.8% respectively) with median levels lower in cases (6 ng/ml and 11.1 ng/ml respectively; p < 0.001). The cases were more likely to have diabetes, hypertension and consume tobacco and alcohol. They had higher waist hip ratio, total and LDL cholesterol. Multivariate logistic regression analysis revealed severe vitamin D deficiency [25(OH) vitamin D < 10 ng/ml] was associated with a risk of MI with an odds ratio of 4.5 (95% CI 2.2–9.2).

Conclusions

This study reveals high prevalence of vitamin D deficiency among cases of acute MI and controls from India, with levels of 25 (OH)D being significantly lower among cases. Despite rampant hypovitaminosis, severe vitamin D deficiency was associated with acute MI after adjusting for conventional risk factors. This association needs to be tested in larger studies in different regions of the country.     

The effect of cholecalciferol supplementation on vitamin D levels and insulin sensitivity is dose related in vitamin D-deficient HIV-1-infected patients

Objective

The aim of this study was to explore the effects of cholecalciferol supplementation on vitamin D levels, bone mineral density (BMD), body fat distribution and insulin sensitivity in vitamin D‐deficient HIV‐1‐infected patients.

Methods

Twenty vitamin D‐deficient HIV‐1‐infected patients were prospectively treated with 2000 IU cholecalciferol/day for 14 weeks, whereafter treatment was continued with half this dosage until 48 weeks. BMD, body fat distribution, 1,25‐dihydroxy vitamin D₃ (1,25(OH)₂D₃), fasting glucose, insulin, adiponectin, leptin, interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α were measured at baseline, and at 24 and 48 weeks. Parathyroid hormone (PTH), 25‐hydroxy vitamin D₃ [25(OH)D₃], cholesterol and triglycerides were measured at baseline, and at 12, 24 and 48 weeks.

Results

After 24 weeks, cholecalciferol supplementation significantly increased 25(OH)D₃ and 1,25(OH)₂D₃ levels and decreased PTH and insulin sensitivity. After 48 weeks, however, only 25(OH)D₃ levels remained significantly different from baseline, while the other parameter levels returned to baseline, suggesting a dose–response effect. Cholecalciferol had no effect on BMD, adipokines and triglycerides.

Conclusions

The effect of cholecalciferol treatment in this cohort appears to be dose dependent. Cholecalciferol dosages of ≥2000 IU are necessary to achieve 1,25(OH)₂D₃ levels that significantly decrease PTH, but also negatively affect insulin sensitivity. The results of this hypothesis‐driven explorative study need to be confirmed in larger clinical trials.     

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3

To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10⁻¹⁰–10⁻⁸ M 1,25-(OH)₂D₃ or 10⁻⁹–10⁻⁷ M 24,25-(OH₂)D₃, and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [³⁵S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)₂D₃ caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)₂D₃ responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)₂D₃; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)₂D₃. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification.     

Heterogeneity in Serum 25‐Hydroxy‐Vitamin D Response to Cholecalciferol in Elderly Women with Secondary Hyperparathyroidism and Vitamin D Deficiency

OBJECTIVES: To compare the effects on parathyroid hormone (PTH) and 25‐hydroxy‐vitamin D (25(OH)D) of two dosing regimens of cholecalciferol in women with secondary hyperparathyroidism (sHPTH) and hypovitaminosis D and to investigate variables affecting 25(OH)D response to cholecalciferol. DESIGN: Randomized‐controlled trial with 6‐month follow‐up. SETTING: Two osteoporosis centers in northern Italy. PARTICIPANTS: Sixty community‐dwelling women aged 65 and older with sHPTH and hypovitaminosis D, creatinine clearance greater than 65 mL/min and without diseases or drugs known to influence bone and vitamin D metabolism. INTERVENTION: Cholecalciferol 300,000 IU every 3 months, once at baseline and once at 3 months (intermittent D₃ group) or cholecalciferol 1,000 IU/day (daily D₃ group). MEASUREMENTS: Serum PTH, 25(OH)D, calcium, bone‐specific alkaline phosphatase, β‐C‐terminal telopeptide of type I collagen, phosphate, 24‐hour urinary calcium excretion. RESULTS: The two groups had similar baseline characteristics. All participants had vitamin D deficiency [25(OH)D<20 ng/mL)], and 36 subjects (60%) had severe deficiency (<10 ng/mL), with no difference between the groups (severe deficiency: intermittent D₃ group, n=18; daily D₃ group, n=18). After 3 and 6 months, both groups had a significant increase in 25(OH)D and a reduction in PTH. Mean absolute increase±standard deviation of 25(OH)D at 6 months was higher in the intermittent D₃ group (22.7±11.8 ng/mL) than in the daily D₃ group (13.7±6.7 ng/mL, P<.001), with a higher proportion of participants in the intermittent D₃ group reaching desirable serum concentration of 25(OH)D ≥ 30 ng/mL (55% in the intermittent D₃ group vs 20% in the daily D₃ group, P<.001). Mean percentage decrease of PTH in the two groups was comparable, and at 6 months, a similar proportion of participants reached normal PTH values. 25(OH)D response to cholecalciferol showed a wide variability. In a logistic regression analysis, body mass index and type of treatment appeared to be significantly associated with normalization of 25(OH)D values. CONCLUSION: Cholecalciferol 300,000 IU every 3 months was more effective than 1,000 IU daily in correcting vitamin D deficiency, although the two groups achieved similar effects on PTH at 6 months. Only 55% of the higher‐dose intermittent group reached desirable concentrations of 25(OH)D, suggesting that yet‐higher doses will be required for adequate vitamin D repletion.     

Serum 25-hydroxyvitamin D level is negatively associated with serum phosphorus level among stage 3a-5 chronic kidney disease patients

Background

Serum 25-hydroxyvitamin D (25(OH)D) negatively correlates with serum phosphorus level of stage 3a-5 chronic kidney disease (CKD) patients. So far, no explanation has been provided for this negative association.

Follow-Up Renal Assessment of Injury Long-Term After Acute Kidney Injury (FRAIL-AKI)

Background and objectives

Novel urinary kidney damage biomarkers detect AKI after cardiac surgery using cardiopulmonary bypass (CPB-AKI). Although there is growing focus on whether AKI leads to CKD, no studies have assessed whether novel urinary biomarkers remain elevated long term after CPB-AKI. We assessed whether there was clinical or biomarker evidence of long-term kidney injury in patients with CPB-AKI.

Design, setting, participants, & measurements

We performed a cross-sectional evaluation for signs of chronic kidney injury using both traditional measures and novel urinary biomarkers in a population of 372 potentially eligible children (119 AKI positive and 253 AKI negative) who underwent surgery using cardiopulmonary bypass for congenital heart disease at Cincinnati Children’s Hospital Medical Center between 2004 and 2007. A total of 51 patients (33 AKI positive and 18 AKI negative) agreed to long-term assessment. We also compared the urinary biomarker levels in these 51 patients with those in healthy controls of similar age.

Results

At long-term follow-up (mean duration±SD, 7±0.98 years), AKI-positive and AKI-negative patients had similarly normal assessments of kidney function by eGFR, proteinuria, and BP measurement. However, AKI-positive patients had higher urine concentrations of IL-18 (48.5 pg/ml versus 20.3 pg/ml [P=0.01] and 20.5 pg/ml [P<0.001]) and liver-type fatty acid–binding protein (L-FABP) (5.9 ng/ml versus 3.9 ng/ml [P=0.001] and 3.2 ng/ml [P<0.001]) than did AKI-negative patients and healthy controls.

Conclusions

Novel urinary biomarkers remain elevated 7 years after an episode of CPB-AKI in children. However, there is no conventional evidence of CKD in these children. These biomarkers may be a more sensitive marker of chronic kidney injury after CPB-AKI. Future studies are needed to understand the clinical relevance of persistent elevations in IL-18, kidney injury molecule-1, and L-FABP in assessments for potential long-term kidney consequences of CPB-AKI.     

Relations of vitamin D status,gender and type 2 diabetes in middle-aged Caucasians

Vitamin D (Vit D) deficiency may be linked to the development of obesity-associated complications such as insulin resistance and type 2 diabetes. We therefore evaluated the relationship of Vit D serum concentrations with metabolic parameters and type 2 diabetes in middle-aged Caucasian men and women. One thousand six hundred and thirty-one Caucasians (832 males, 58.8 ± 9.7 years; 799 females, 59.7 ± 10.7 years) were evaluated in a cross-sectional study. Vit D status was assessed by measuring the serum concentration of 25-hydroxyvitamin D₃ [25(OH)D₃]. Type 2 diabetes prevalence was ascertained by medical history, fasting plasma glucose concentrations, oral glucose tolerance testing and/or glycosylated hemoglobin. Men displayed higher crude or seasonally adjusted 25(OH)D₃ serum concentrations than women (24.64 ± 10.98 vs. 22.88 ± 11.6 ng/ml; P < 0.001). Strong associations between body mass index (BMI) and 25(OH)D₃ were observed in both genders (P < 0.001). Seasonally adjusted levels of 25(OH)D₃ revealed stronger associations with type 2 diabetes in women than men (P < 0.001). However, adjustment for BMI and other confounding variables revealed an independent inverse association of 25(OH)D₃ with diabetes only in women (P < 0.001), whereas the association was abrogated in men. Using a 15 ng/ml 25(OH)D₃ cutoff for binary comparison, adjusted odds ratios for having newly diagnosed or known type 2 diabetes more than doubled (2.95 [95 % CI 1.37–4.89] and 3.26 [1.59–6.68], respectively), in women below the cutoff. We conclude that in women, but not in men, low 25(OH)D₃ serum levels are independently associated with type 2 diabetes. These findings suggest sex-specific effects of Vit D in the pathogenesis of type 2 diabetes.     

VITAMIN D METABOLISM IN HYPERTHYROIDISM

The serum concentrations of 25-hydroxycholecalciferol (25 OH D₃), 24,25-dihydroxycholecalciferol [24,25(OH)₂D₃] and 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] were measured in twenty-one patients with untreated hyperthyroidism. Compared with control subjects, 25 OH D₃ concentrations were not altered, 24,25(OH)₂D₃ concentrations were increased, although not significantly and 1,25(OH)₂D₃ concentrations were decreased (P= 0.01). Following oral carbimazole therapy, 24,25(OH)₂D₃ concentrations fell (P<0.01), 1,25(OH)₂D₃ concentrations increased (P<0.01) and 25 OH D₃ concentrations were unchanged. The altered 1,25(OH)₂D₃ and 24,25(OH)₂D₃ concentrations found in hyperthyroidism are probably due to the effects of thyroid hormone on bone and mineral metabolism. Increased serum calcium and phosphate concentrations with secondary hypoparathyroidism result in stimulation of the renal 24-hydroxylase and suppression of the 1-hydroxylase enzymes. In addition, serum 24,25(OH)₂D₃ concentrations were significantly correlated with serum triiodothyronine levels (T3) (r= 0.66, P<0.002) before treatment. This may indicate a direct stimulatory effect of T3 on 24-hydroxylase activity. No relationship was found between serum 1,25(OH)₂D₃ concentrations before therapy and serum T3.