Vitamin D: 100 years of discoveries, yet controversy continues.

Over the past 100 years, many major breakthroughs and discoveries have occurred in relation to vitamin D research. These developments include the cure of rickets in 1919, the discovery of vitamin D compounds, advances in vitamin D molecular biology, and improvements in our understanding of endocrine control of vitamin D metabolism. Furthermore, recommended daily allowances for vitamin D have been established and large clinical trials of vitamin D, aimed at clarifying the effect of Vitamin D in the prevention of multiple diseases, have been completed. However, disappointingly, these clinical trials have not fulfilled the expectations many had 10 years ago. In almost every trial, various doses and routes of administration did not show efficacy of vitamin D in preventing fractures, falls, cancer, cardiovascular diseases, type 2 diabetes, asthma, and respiratory infections. Although concerns about side-effects of long-term high-dose treatments, such as hypercalcaemia and nephrocalcinosis, have been around for four decades, some trials from the past 5 years have had new and unexpected adverse events. These adverse events include increased fractures, falls, and hospitalisations in older people (aged >65 years). Several of these clinical trials were powered appropriately for a primary outcome but did not include dose response studies and were underpowered for secondary analyses. Furthermore, more attention should be paid to the safety of high doses of vitamin D supplementation, particularly in older people. In addition, despite universal recommendations by osteoporosis societies for combining calcium supplements with vitamin D there remains insufficient data about their efficacy and effect on fracture risk in the highest risk groups. More trials are needed for people with severe vitamin D deficiency (ie, serum 25-hydroxyvitamin D <25nmol/L [10ng/mL]). In this Personal View, we summarise and discuss some of the major discoveries and controversies in the field of vitamin D.

Individual participant data (IPD)-level meta-analysis of randomised controlled trials to estimate the vitamin D dietary requirements in dark-skinned individuals resident at high latitude.

CONTEXT AND PURPOSE: There is an urgent need to develop vitamin D dietary recommendations for dark-skinned populations resident at high latitude. Using data from randomised controlled trials (RCTs) with vitamin D3-supplements/fortified foods, we undertook an individual participant data-level meta-regression (IPD) analysis of the response of wintertime serum 25-hydroxyvitamin (25(OH)D) to total vitamin D intake among dark-skinned children and adults residing at ≥ 40° N and derived dietary requirement values for vitamin D. METHODS: IPD analysis using data from 677 dark-skinned participants (of Black or South Asian descent; ages 5-86 years) in 10 RCTs with vitamin D supplements/fortified foods identified via a systematic review and predefined eligibility criteria. Outcome measures were vitamin D intake estimates across a range of 25(OH)D thresholds. RESULTS: To maintain serum 25(OH)D concentrations ≥ 25 and 30 nmol/L in 97.5% of individuals, 23.9 and 27.3 µg/day of vitamin D, respectively, were required among South Asian and 24.1 and 33.2 µg/day, respectively, among Black participants. Overall, our age-stratified intake estimates did not exceed age-specific Tolerable Upper Intake Levels for vitamin D. The vitamin D intake required by dark-skinned individuals to maintain 97.5% of winter 25(OH)D concentrations ≥ 50 nmol/L was 66.8 µg/day. This intake predicted that the upper 2.5% of individuals could potentially achieve serum 25(OH)D concentrations ≥ 158 nmol/L, which has been linked to potential adverse effects in older adults in supplementation studies. CONCLUSIONS: Our IPD-derived vitamin D intakes required to maintain 97.5% of winter 25(OH)D concentrations ≥ 25, 30 and 50 nmol/L are substantially higher than the equivalent estimates for White individuals. These requirement estimates are also higher than those currently recommended internationally by several agencies, which are based predominantly on data from Whites and derived from standard meta-regression based on aggregate data. Much more work is needed in dark-skinned populations both in the dose-response relationship and risk characterisation for health outcomes. TRAIL REGISTRATION: PROSPERO International Prospective Register of Systematic Reviews (Registration Number: CRD42018097260).

Steroidogenesis in patients with adrenal incidentalomas: Extended steroid profile measured by liquid chromatography-mass spectrometry after ACTH stimulation and dexamethasone suppression.

OBJECTIVE: Describe the secretion and profile of adrenal steroids in patients with adrenal incidentalomas compared to control subjects. DESIGN, SETTING AND PARTICIPANTS: A prospective study, 73 patients with adrenal incidentalomas, 21 bilateral and 52 unilateral and 34 matched controls in University Hospital. METHODS: Collect fasting blood sample before and 60 min after ACTH test (250 µg IV). One week later, perform overnight 1 mg dexamethasone test. The following steroids were measured by liquid chromatography-mass spectrometry (LC-MS): pregnenolone, 17-OH pregnenolone, 17-OH progesterone, 11-deoxycorticosterone, 11-deoxyortisol, 21-deoxycortisol, corticosterone, cortisol, androstenedione and aldosterone. RESULTS: Mean baseline serum cortisol was higher in incidentalomas, bilateral 361 ± 124, (range 143-665) nmol/L,(p < .0001), unilateral 268 ± 89 3.2 (range 98-507) nmol/L (p < .019) compared to controls 207 ± 100 (range 72-502) nmol/L. ACTH stimulation showed significantly higher levels in bilateral and unilateral cases compared to controls. After dexamethasone, mean serum cortisol levels suppressed in bilaterals 89 ± 69 (range 30-3) nmol/L (p < .0001), 58 ± 52 (range 16-323) nmol/L in unilateral (p < .01) compared to 26 ± 9 (range 7-46) nmol/L in controls. Mean baseline serum corticosterone was higher in bilateral 9.3 ± 4.8 (range 2.4-18.4) nmol/L (p < .005) and unilateral 7.3 ± 5.7 (range 0.1-30.3) nmol/L (p < .01) compared to controls 4.2 ± 2.4 (range 1.1-10.2) nmol/L, after ACTH stimulation significantly increased to higher levels in bilateral (p < .0002) and unilateral cases (p < .044) compared to controls. After dexamethasone, mean levels were 2.5 ± 2.6 (range 0.5-12.5) nmol/L in bilateral (p < .0006), 1.5 ± 1.6 (range 0.3-9.3) nmol/L in unilateral (p < .09) and 0.75 ± 0.46 (range 0.1-2.1) nmol/L in controls. Mean baseline serum 11-deoxycorticosterone (DOC) was higher in bilaterals 0.32 ± 0.23 (range 0.08-1.1) nmol/L (p < .03) compared to controls 0.15 ± 0.21 (range 0.08-1.1) nmol/L. ACTH stimulation increased levels to 3.27 ± 1.72 (range 0.5-7.4) nmol/L in bilateral cases compared to controls 1.369 ± 1.53 (range 0.1-7.1) nmol/L (p < .0001). Dexamethasone decreased levels to baseline (p ns). There were significant differences in serum 21-deoxycortisol (p < .0002) and serum pregnenolone (p < .004) only after ACTH stimulation. CONCLUSIONS: There is increased activity in several steroid biosynthesis pathways and higher steroid levels in bilateral compared to unilateral cases and evidence of hypercortisolism in 30% unilateral and 62% of bilateral incidentalomas.

Correlation Between Size and Function of Unilateral and Bilateral Adrenocortical Nodules: An Observational Study.

OBJECTIVE. Adrenal incidentalomas occur in 5% of adults and can produce autonomous cortisol secretion that increases the risk of metabolic syndrome and cardiovascular disease. The objective of our study was to evaluate the relationship between adrenal nodule size measured on CT and autonomous cortisol secretion. SUBJECTS AND METHODS. In a prospective study of 73 patients 22-87 years old with incidentalomas, unilateral in 52 patients and bilateral in 21 patients, we measured maximum nodule diameter on CT and serum cortisol levels at 8:00 am, 60 minutes after the adrenocorticotropic hormone stimulation test, and after the dexamethasone suppression test. We also studied 34 age-, sex-, and body mass index-matched control subjects. Statistics used were Spearman correlation coefficients, t tests, ANOVA test, and multivariate analysis. RESULTS. The mean maximum diameter of unilateral nodules measured on CT was larger on the right (2.47 ± 0.98 [SD] cm) than on the left (2.04 ± 0.86 cm) (p = 0.01). In the bilateral cases, the mean diameter of the right nodules was 2.69 ± 0.93 cm compared with 2.13 ± 0.89 cm on the left (p = 0.06). Mean baseline serum cortisol level was significantly higher in the patients with incidentalomas (bilateral, 13.1 ± 4.5 mcg/dL [p < 0.001]; unilateral, 9.7 ± 3.2 mcg/dL [p = 0.019]) than in the control subjects (7.5 ± 3.6 mcg/dL). After dexamethasone suppression test, serum cortisol levels were suppressed to less than 1.8 mcg/dL in 100% of control subjects, 33% of patients with bilateral incidentalomas, and 62% of patients with unilateral incidentalomas (p < 0.001). There were significant correlations between maximum nodule diameter on CT and serum cortisol levels after the dexamethasone suppression test (ρ = 0.500; p < 0.001) and at baseline (ρ = 0.373; p = 0.003). CONCLUSION. Increasing size of adrenal nodules is associated with more severe hyper-cortisolism and less dexamethasone suppression; these cases need further evaluation and possibly surgery because of increased risks of metabolic syndrome and cardiovascular mortality.

Advances in osteoporosis from 1970 to 2018.

In 1970, there were no drugs under study for osteoporosis. Estrogen was used, but little was known about the correct dose for preventing bone loss. At that time, fractures were not even recognized as a disease, but regarded as part of normal aging. From 1970 to this year (2018), there have been extensive advances in the osteoporosis field ranging from fracture epidemiology to the remarkable invention of bone density measurements. There have been major advances in therapeutic options available for patients for prevention and treatment of osteoporosis. In parallel, the advances in the laboratory helped elucidate the process of bone remodeling, not only at the macroscopic level but also at the cellular level. This led to rapid advances in translational research from cellular biology to new therapies exemplified by the development of monoclonal antibodies for osteoporosis. Further understanding of the signaling pathways in bone cells will lead to new small molecules made for osteoporosis treatment, perhaps causing less adverse events. University-based research throughout the world has been a leader in most of these advances, and Pharma support for phase 1 to 4 studies helped bring these discoveries to patients. In the osteoporosis field alone, one sees the tremendous value of grant support for university research by National funding agencies such as the National Institute of Health in this country and similar agencies in other countries. There are clinical challenges that have to be solved with long-term compliance with osteoporosis medication if we want to reduce fracture incidence in the long term.

Determination of Free 25(OH)D Concentrations and Their Relationships to Total 25(OH)D in Multiple Clinical Populations.

Context: The optimal measure of vitamin D status is unknown. Objective: To directly measure circulating free 25-hydroxyvitamin D [25(OH)D] concentrations and relationships to total 25(OH)D in a clinically diverse sample of humans. Design: Cross-sectional analysis. Setting: Seven academic sites. Patients: A total of 1661 adults: healthy (n = 279), prediabetic (n = 479), outpatients (n = 714), cirrhotic (n = 90), pregnant (n = 20), nursing home resident (n = 79). Interventions: Merge research data on circulating free 25(OH)D (directly-measured immunoassay), total 25(OH)D (liquid chromatography/tandem mass spectrometry), D-binding protein [DBP; by radial (polyclonal) immunodiffusion assay], albumin, creatinine, intact parathyroid hormone, and DBP haplotype. Main outcome measures: Distribution of free 25(OH)D (ANOVA with Bonferroni correction for post hoc comparisons) and relationships between free and total 25(OH)D (mixed-effects modeling incorporating clinical condition, DBP haplotype with sex, race, estimated glomerular filtration rate (eGFR), body mass index (BMI), and other covariates). Results: Free 25(OH)D was 4.7 ± 1.8 pg/mL (mean ± SD) in healthy persons and 4.3 ± 1.9 pg/mL in outpatients, with levels of 0.5 to 8.1 pg/mL and 0.9 to 8.1 pg/mL encompassing 95% of healthy persons and outpatients, respectively. Free 25(OH)D was higher in patients with cirrhosis (7.1 ± 3.0 pg/mL; P < 0.0033) and nursing home residents (7.9 ± 2.1 pg/mL; P < 0.0033) than in other groups and differed between whites and blacks (P < 0.0033) and between DBP haplotypes (P < 0.0001). Mixed-effects modeling of relationships between free and total 25(OH)D identified clinical conditions (patients with cirrhosis > nursing home residents > patients with prediabetes > outpatients > pregnant women) and BMI (lesser effect) as covariates affecting relationships but not eGFR, sex, race, or DBP haplotype. Conclusions: Total 25(OH)D, health condition, race, and DBP haplotype affected free 25(OH)D, but only health conditions and BMI affected relationships between total and free 25(OH)D. Clinical importance of free 25(OH)D needs to be established in studies assessing outcomes.

Dose ranging effects of vitamin D3 on the geriatric depression score: A clinical trial.

Depression is a common problem affecting millions, usually treated with selective serotonin uptake inhibitors. Interest in vitamin D as a co-therapy was stimulated by some association studies that correlated depression with low serum 25OHD levels. There are few longitudinal studies of vitamin D and depression and most are single doses of vitamin D. In this study we examined the effect of one-year treatment with several doses of vitamin D on the Geriatric depression score (GDS) in older Caucasian and African American women. The clinical trial was a study of seven daily oral doses of vitamin D (400-4800IU/d) in Black and White older women. The trial was a double blind, randomized and placebo controlled lasting 12-months. The main inclusion criterion was serum 25 hydroxyvitamin D (25OHD)≤20ng/mL (50nmol/L). Calcium supplements were given to maintain calcium intake 1000mg/day in young people and 1200-1400mg/day in older women. Data on Geriatric depression (GDS) was collected using the validated long form at baseline and 12-months. The change in serum 25OHD was the primary outcome and GDS was one of the secondary outcomes. Adjustments were made for relevant covariates. Analysis of vitamin D effect was by dose low, medium and high compared to placebo or by quintiles. Serum 25OHD increased as a quadratic curve function to a mean of 46ng/mL (115nmol/L) in white women and 49ng/mL (122.5nmol/L) in black women on the highest dose of 4800 IU. In older women mean GDS scores changed from 3.8 (SD±4.2) at baseline to 3.6 (SD±4.1) at 12 months in whites and from 3.0 (SD±3.7) to 3.02 (SD±4.2) in Blacks. (p=0.790 in whites; p=0.958 in blacks). After 12-months there was no effect of dose on change in GDS score in women treated with different doses of vitamin D (p=0.507 in whites and p=0.340 in blacks). When both Caucasians and African Americans were divided into 3 dose groups, low (400-800 IU), medium (1600-3200 IU) and high (4000-4800 IU) doses, the change in score was 0.8 on low dose, -0.30 on medium dose and -0.31 on high dose compared to 0.11 on placebo (p=0.546). In summary, there was no improvement in GDS scores in Caucasians or African Americans on either increasing doses of vitamin D or quintiles of achieved response in serum 25OHD. The changes were small and not significant perhaps because of the relatively lower numbers of depressed women in the groups. Further studies should recruit larger numbers, 3 dose groups covering a serum25OHD range of 20-60ng/mL and more subjects with clinical depression in order to fully address the question of vitamin D effects on depression.

Dietary Vitamin D Intake for the Elderly Population: Update on the Recommended Dietary Allowance for Vitamin D.

Vitamin D insufficiency and deficiency can be diagnosed with measurements of serum 25-hydroxyvitamin D (25OHD). Most vitamin D is derived from sunlight (80%), so serum 25OHD levels are lowest in late winter and early spring. Dietary vitamin D in North America is small, about 100 to 200 IU daily. A recent review of the literature shows many association studies relating vitamin D deficiency and insufficiency to several diseases. Large randomized trials of vitamin D are underway and soon there may be answers as to whether vitamin D is clinically effective and what level of serum 25OHD is necessary.

Medium doses of daily vitamin D decrease falls and higher doses of daily vitamin D3 increase falls: A randomized clinical trial.

Falls are a serious health problem in the aging population. Because low levels of vitamin D have been associated with increased fall rates, many trials have been performed with vitamin D; two meta-analyses showed either a small effect or no effect of vitamin D on falls. We conducted a study of the effect of vitamin D on serum 25 hydroxyvitamin D (25OHD) and data on falls was collected as a secondary outcome. In a 12-month double blind randomized placebo trial, elderly women, mean age 66 years, were randomized to one of seven daily oral doses of vitamin D or placebo. The main inclusion criterion for study was a baseline serum 25OHD<20ng/ml (50nmol/L). A history of falls was collected at baseline and fall events were collected every 3 months. Results showed that the effect of vitamin D on falls followed a U-shaped curve whether analyzed by dose or serum 25OHD levels. There was no decrease in falls on low vitamin D doses 400, 800 IU, a significant decrease on medium doses 1600, 2400,3200 IU (p=0.020) and no decrease on high doses 4000, 4800 IU compared to placebo (p=0.55). When compared to 12-month serum 25OHD quintiles, the faller rate was 60% in the lowest quintile <25ng/ml (<50nmol/L), 21% in the low middle quintile 32-38ng/ml (80-95nmo/L), 72% in the high middle quintile 38-46ng/ml (95-115nmo/L) and 45% in the highest quintile 46-66ng/ml (115-165nmol/L). In the subgroup with a fall history, fall rates were 68% on low dose, 27% on medium doses and 100% on higher doses. Fall rates on high doses were increased compared to medium doses (Odds Ratio 5.6.95% CI: 2.1-14.8). In summary, the maximum decrease in falls corresponds to a 12- month serum 25OHD of 32-38ng/ml (80-95nmol/L) and faller rates increase as serum 25OHD exceed 40-45ng/ml (100-112.5nmol/L). The Tolerable upper limit (TUL) recently increased in 2010 from 2000 to 4000 IU/day may need to be reduced in elderly women especially in those with a fall history.

Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)2 D3 in Affected Patients.

CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D3 :24,25-(OH)2 D3 ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D3 by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH)2 D3 remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH)2 D3 peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH)2 D3 as a contaminant of the 24,25-(OH)2 D3 peak. In normals, the concentration of 24,25-(OH)2 D3 greatly exceeds 25,26-(OH)2 D3 ; however, 25,26-(OH)2 D3 becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH)2 D3 levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH)2 D3 levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH)2 D3 from 25,26-(OH)2 D3 produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH)2 D3 in IIH patients appears to be multifactorial. © 2017 American Society for Bone and Mineral Research.

Vitamin D and falls - the dosage conundrum.

Falls are a major health problem in elderly individuals. Although intensive physical therapy and management of hazards in the home can reduce falls by 25%, long-term practicality limits their use. Interest in vitamin D as a medical therapy has led to many trials; however, results using daily oral doses of vitamin D have been inconsistent. In the past 5 years, studies on the effect of bolus doses of vitamin D have produced surprising results. Bolus doses of vitamin D, given annually (at a dose of 300,000 IU or 500,000 IU) or monthly (at a dose of 24,000 IU or 60,000 IU) - equivalent to approximate daily doses of 800 IU, 1400 IU and 2,000 IU - result in a significant increase in the number of falls and fractures associated with serum levels of 25-hydroxyvitamin D greater than 40-45 ng/ml (equivalent to 100-112 nmol/l). These unexpected results show increased falls and fractures are adverse events related to vitamin D administration. Until further safety data is available, bolus dosing or daily doses should not exceed 3,000 IU and serum levels of 25-hydroxyvitamin D should not exceed 40-45 ng/ml (equivalent to 100-112 nmol/l) in elderly individuals.

Effect of conjugated estrogens/bazedoxifene on postmenopausal bone loss: pooled analysis of two randomized trials.

OBJECTIVE: Conjugated estrogens/bazedoxifene reduces vasomotor symptoms and prevents postmenopausal bone loss without stimulating the breast and endometrium. We analyzed changes in bone mineral density (BMD) and bone markers using pooled data from two phase-3 trials. METHODS: Selective Estrogens, Menopause, and Response to Therapy (SMART)-1 and SMART-5 were randomized, double-blind, placebo- and active-controlled studies conducted in postmenopausal nonhysterectomized women. BMD and turnover marker data were pooled for women given conjugated estrogens (0.45 or 0.625 mg) plus bazedoxifene 20 mg or placebo over 12 months. Sensitivity analyses were conducted using baseline Fracture Risk Assessment Tool score, age, years since menopause, body mass index, race, and geographic region. RESULTS: There were 1,172 women, mean age 54.9 years, mean 6.21 years since menopause, mean lumbar spine, and total hip T scores -1.05 and -0.58; 58.8% had a Fracture Risk Assessment Tool score less than 5% indicating low fracture risk. At 12 months, adjusted differences (vs placebo) in BMD change in the groups taking conjugated estrogens 0.45 or 0.625 mg plus bazedoxifene 20 mg were 2.3% and 2.4% for lumbar spine, 1.4% and 1.5% for total hip, and 1.1% and 1.5% for femoral neck (all P < 0.001 vs placebo). These increases were unrelated to baseline Fracture Risk Assessment Tool score, age, years since menopause, body mass index, or geographic region. Both doses reduced bone turnover markers (P < 0.001). CONCLUSIONS: Conjugated estrogens/bazedoxifene significantly improved BMD and turnover in a large population of younger postmenopausal women at low fracture risk and is a promising therapy for preventing postmenopausal bone loss.

Biological agents in management of osteoporosis.

Osteoporosis is a skeletal disease associated with an imbalance between formation and resorption, leading to net loss of bone mass, loss of bone microarchitecture, and development of fractures. Bone resorption is primarily due to an activation of osteoclastogenesis and an increase in receptor activator of nuclear factor kappa-B ligand (RANKL) expression, a cytokine involved in the final pathway of the osteoclast cycle.Recent studies of genetic diseases led to the discovery of the wingless-type (Wnt) signaling pathway that plays a major role in bone formation. Further work showed that sclerostin produced by osteocytes and the Dickkopf (DKK1) protein secreted in bone were negative regulators of the Wnt signaling bone formation pathway that act directly by binding to the co-receptors LRP5 and LRP6 of WnT and thereby inhibiting the anabolic Wnt pathway. This understanding of the bone remodeling led to the discovery of new biological drugs that target these pathways and have been evaluated in clinical trials.The current article discusses the role of these newer "biological" agents in management of osteoporosis. Denosumab, a human monoclonal antibody that specifically binds RANKL, blocks the binding of RANK to its ligand markedly reducing bone resorption, increases bone density, and reduces fractures and is approved for osteoporosis. Parathyroid hormone PTH 1-34 (teriparatide) stimulates bone formation through inhibition of sclerostin, DKK1, and frizzled protein; increases BMD; improves microarchitecture; and decreases fractures and is approved for osteoporosis. The anti-sclerostin antibodies (romosozumab, blosozumab) increase bone mass by neutralizing the negative effects of sclerostin on the Wnt signaling pathway. These biologics are being evaluated now in a clinical trial and early data looks promising. Cathepsin K is a proteolytic enzyme that degrades bone matrix and inhibitors such as odanacatib show increasing bone density and perhaps decreased fractures. The potential power of combining these newer antiresorptives with the newer anabolic agents could theoretically increase bone mass rapidly to normal within 1 year and reduce fractures. These newer treatments are revolutionizing the management of osteoporosis.

Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD.

CONTEXT: The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)2D]. OBJECTIVE: We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)2D using 100 µL of serum with a 5-minute run time. DESIGN: The assay uses a newly synthesized internal standard d6-24,25-(OH)2D3 enabling the quantitation of 24,25-(OH)2D3 as well as the determination of the ratio of 25-OH-D3 to 24,25-(OH)2D3, a physiologically useful parameter. SETTING: We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models. RESULTS: The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2% to -5% bias) and exhibited low inter- and intraassay coefficients of variation (4%-7%) and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2) = 0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3 to 24,25-(OH)2D3, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia. CONCLUSIONS: The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

Vitamin D supplementation in young White and African American women.

There is limited information on the effects of vitamin D on serum 25 hydroxyvitamin D (25OHD) in young people and none on African Americans. The main objective of this trial was to measure the effect of different doses of vitamin D3 on serum 25OHD and serum parathyroid hormone (PTH) in young women with vitamin D insufficiency (serum 25OHD ≤ 20 ng/mL (50 nmol/L). A randomized double-blind placebo-controlled trial of vitamin D3 was conducted in young white and African American women, age 25 to 45 years. A total of 198 healthy white (60%) and African American (40%) women were randomly assigned to placebo, or to 400, 800, 1600, or 2400 IU of vitamin D3 daily. Calcium supplements were added to maintain a total calcium intake of 1000 to 1200 mg daily. The primary outcomes of the study were the final serum 25OHD and PTH levels at 12 months. The absolute increase in serum 25OHD with 400, 800, 1600, and 2400 IU of vitamin D daily was slightly greater in African American women than in white women. On the highest dose of 2400 IU/d, the mixed model predicted that mean 25OHD increased from baseline 12.4 ng/mL (95% confidence interval [CI], 9.2-15.7) to 43.2 ng/mL (95% CI, 38.2-48.1) in African American women and from 15.0 ng/mL (95% CI, 12.3-17.6) to 39.1 ng/mL (95% CI, 36.2-42.0) in white women. There was no significant effect of vitamin D dose on serum PTH in either race but there was a significant inverse relationship between final serum PTH and serum 25OHD. Serum 25OHD exceeded 20 ng/mL in 97.5% of whites on the 400 IU/d dose and between 800 and 1600 IU/d for African Americans. The recommended dietary allowance (RDA) suggested by the Institute of Medicine for young people is 600 IU daily. The increase in serum 25OHD after vitamin D supplementation was similar in young and old, and in white and African American women.

Prevention and treatment of postmenopausal osteoporosis.

In the beginning, that is from the 1960's, when a link between menopause and osteoporosis was first identified; estrogen treatment was the standard for preventing bone loss, however there was no fracture data, even though it was thought to be effective. This continued until the Women's Health Initiative (WHI) study in 2001 that published data on 6 years of treatment with hormone therapy that showed an increase in heart attacks and breast cancer. Even though the risks were small, 1 per 1500 users annually, patients were worried and there was a large drop off in estrogen use. In later analyses the WHI study showed that estrogen reduced fractures and actually prevented heart attacks in the 50-60 year age group. Estrogen alone appeared to be safer to use than estrogen+the progestin medroxyprogesterone acetate and actually reduced breast cancer. At the same time other drugs were being developed for bone that belong to the bisphosphonate group and the first generation of compounds showed moderate potency on bone resorption. The second and third generation compounds were much more potent and in a series of large trials were shown to reduce fractures. For the last 15 years the treatment of osteoporosis belonged to the bisphosphonate compounds, most of which reduce fracture rates by 50 percent. With the exception of gastrointestinal irritation the drugs are well tolerated and highly effective. The sophistication of the delivery systems now allow treatment that can be given daily, weekly, monthly and annually either orally or intravenously. Bone remodeling is a dynamic process that repairs microfractures and replaces old bone with new bone. In the last 10 years there has been a remarkable understanding of bone biology so that new therapies can be specifically designed on a biological basis. The realization that RANKL was the final cytokine involved in the resorption process and that marrow cells produced a natural antagonist called Osteoprotegerin (OPG) quickly led to two lines of therapy. First OPG was used as a therapy to block RANKL was initially successful but later antibodies against OPG developed and this line of treatment had to be discontinued. The next step was to develop a monoclonal antibody against RANKL and this proved to be highly effective in blocking bone resorption. It led to development of a drug Denosumab that successfully reduces fractures and is now one of the therapeutic options for osteoporosis treatment. On the anabolic side bone biology research showed that osteocytes produces sclerostin an inhibitor of the anabolic WNT signaling pathway. Recent development of a monoclonal antibody against sclerostin has shown remarkable anabolic activity in bone showing large increases in bone density and fracture trials are now underway. The newer treatments for osteoporosis are likely to be based on our understanding of bone biology and the design of new highly specific compounds with fewer side effects. This review summarizes the diagnosis of postmenopausal osteoporosis and various available non-pharmacological and pharmacological therapies available for its management. This article is part of a Special Issue entitled 'Menopause'.

Vitamin D does not increase calcium absorption in young women: a randomized clinical trial.

It is commonly said that vitamin D should be used to increase calcium absorption. We tested this statement in a dose-response study of vitamin D on calcium absorption. A total of 198 white and African American women, aged 25 to 45 years, with vitamin D insufficiency, serum 25-hydroxyvitamin D (25OHD) <20 ng/mL, were randomized in a double-blind study to vitamin D3 400, 800, 1600, 2400 IU, or placebo. A calcium supplement was given to increase mean calcium intake at baseline from 706 mg/d to 1031 mg/d. Calcium absorption was measured at baseline and after 12 months using a single isotope method with radiocalcium45 and 100 mg of calcium. Mean baseline serum 25OHD was 13.4 ng/mL (33.5 nmol/L) and increased to 40 ng/mL (100 nmol/L) on the highest dose of 2400 IU. Using a multivariate regression analysis with significant predictors, baseline absorption, calcium intake, and weight, there was no increase in 12-month calcium absorption compared with baseline on any dose of vitamin D in either whites or African Americans. There was no significant relationship between 12-month calcium absorption and final serum 25OHD. In an analysis of calcium absorption and serum 25OHD at baseline, serum 25OHD levels were divided into groups: 0 to 5, 6 to 10, 11 to 15, or 16 to 20 ng/mL. There was no evidence of a threshold decrease in calcium absorption or serum 1,25 dihydroxyvitamin D (1,25(OH)2 D) amongst the lowest groups. Vitamin D doses up to 2400 IU daily did not increase calcium absorption. No threshold level of serum 25OHD for calcium absorption was found at baseline or in the longitudinal study, suggesting that active transport of calcium is saturated at very low serum 25OHD levels <5 ng/mL. There is no need to recommend vitamin D for increasing calcium absorption in normal subjects. Very efficient calcium absorption at very low levels of serum 25OHD explains why people do not develop osteomalacia provided that dietary intakes of calcium and phosphorus are adequate.