Paul Glendenning

Current assays overestimate 25-hydroxyvitamin D3 and underestimate 25-hydroxyvitamin D2 compared with HPLC: need for assay-specific decision limits and metabolite-specific assays

Background: Clinical demand for quick, cheap, precise and accurate 25-hydroxyvitamin D (25(OH)D) results has led to the development of a variety of assay methods. Lack of standardization of these methods has resulted in intermethod disagreement and challenged whether current assays recognize 25(OH)D2 and 25(OH)D3 equally. Methods: We studied 172 patient samples from hip fracture cases using DiaSorin (DS) and IDS radioimmunoassays and the Nichols Advantage-automated protein binding assay (NA-CLPBA) in comparison to high-performance liquid chromatography (HPLC). 52 patient samples were analysed before and after three months treatment with 1000 IU of daily ergocalciferol (vitamin D2). Results: Linear regression analysis in pre-treatment samples demonstrated a positive Y-intercept for each immunoassay compared with HPLC, and a slope that varied from 0.64 (IDS) to 0.97 (DS, NA-CLPBA). Bland Altman analysis demonstrated that all the three assays had a proportional positive bias relative to HPLC at values from 20 to 50 nmol/L. Regression analysis of post-treatment samples demonstrated a slope that was not significantly different from zero for the IDS and NA-CLPBA and 0.2 for the DS method, with a positive intercept for all assays of between 8 and 22, indicating less than 50% of 25(OH)D2 measured by HPLC was detected. Conclusions: These results demonstrate the need for assay-specific decision limits for 25(OH)D3 in order to define appropriate thresholds for treatment institution. Treatment with vitamin D2 may not be accurately monitored with any of the three commercial assays studied. Clinicians and biochemists who continue to use 25(OH)D assays need to be urgently informed of these issues.

Effects of three-monthly oral 150,000 IU cholecalciferol supplementation on falls, mobility, and muscle strength in older postmenopausal women: A randomized controlled trial

Daily vitamin D in addition to calcium supplementation reduces falls and fractures in older women. However, poor adherence to therapy is a common clinical problem. To examine the effects of supervised oral 3‐monthly vitamin D therapy on falls, muscle strength, and mobility, we conducted a 9‐month randomized, double‐blind, placebo‐controlled trial in 686 community‐dwelling ambulant women aged over 70 years. Participants received either oral cholecalciferol 150,000 IU every 3 months (n = 353) or an identical placebo (n = 333). All participants were advised to increase dietary calcium intake. Falls data were collected 3‐monthly. At baseline, 3, 6, and 9 months, muscle strength was measured by a handheld dynamometer and mobility by the Timed Up and Go (TUG) test. Serum 25 hydroxyvitamin D (25OHD) was measured in a subgroup of 40 subjects. Mean age at baseline was 76.7 ± 4.1 years. The average serum 25OHD value at baseline was 65.8 ± 22.7 nmol/L. By 3, 6, and 9 months after supplementation, 25OHD levels of the vitamin D group were approximately 15 nmol/L higher than the placebo group. Calcium intake did not change significantly between baseline (864 ± 412 mg/day) and 9 months (855 ± 357 mg/day). Faller rates in the two groups did not differ: vitamin D group, 102 of 353 (29%); placebo group, 89 of 333 (27%). At 9 months, compared to placebo or baseline, muscle strength, and TUG were not altered by vitamin D. In conclusion, oral cholecalciferol 150,000 IU therapy administered 3‐monthly had neither beneficial nor adverse effects on falls or physical function. These data together with previous findings confirm that intermittent large doses of vitamin D are ineffective or have a deleterious effect on falls. Thus despite adherence issues with daily vitamin D replacement, an intermittent, high‐dose vitamin D regimen cannot be supported as a strategy to reduce falls and fractures.

Serum 25-hydroxyvitamin D levels in vitamin D-insufficient hip fracture patients after supplementation with ergocalciferol and cholecalciferol☆

Vitamin D insufficiency is commonly associated with hip fracture. However, the equipotency of ergocalciferol and cholecalciferol supplementation in this patient group has not been studied in a randomized trial using high-performance liquid chromatography (HPLC) measurement of serum 25-hydroxyvitamin D (25OHD). The objective of this study was to determine if ergocalciferol and cholecalciferol are equipotent therapies in vitamin D-insufficient hip fracture patients. Ninety five hip fracture inpatients with vitamin D insufficiency (25OHD<50 nmol/L) were randomized, double-blind, to treatment with ergocalciferol 1000 IU/day (n=48) or cholecalciferol 1000 IU/day (n=47) for three months. All participants were also given a placebo matching the alternative treatment to maintain blinding of treatment allocation. The primary endpoint was total serum 25OHD measured by HPLC. Secondary endpoints included 25OHD measured by radioimmunoassay (RIA), intact parathyroid hormone (iPTH), and bioactive (1-84) whole PTH (wPTH). Seventy patients (74%) completed the study with paired samples for analysis. Cholecalciferol supplementation resulted in a 31% greater increase in total HPLC-measured 25OHD (p=0.010) and 52% greater rise in RIA-measured 25OHD (p<0.001) than supplementation with an equivalent dose of ergocalciferol. Changes in iPTH and wPTH were not significantly different between calciferol treatments (p>0.05). In vitamin D-insufficient hip fracture patients, supplementation with cholecalciferol 1000 IU/day for three months was more effective in increasing serum 25OHD than an equivalent dose of ergocalciferol. However, the lack of difference in PTH lowering between calciferol treatments raises questions about the biological importance of this observation.

Issues of methodology, standardization and metabolite recognition for 25-hydroxyvitamin D when comparing the DiaSorin radioimmunoassay and the Nichols Advantage automated chemiluminescence protein-binding assay in hip fracture cases

Background: Deficiency of vitamin D is commonly associated with hip fracture and treatment with vitamin D reduces hip fracture rates. Consequently, the demand for assays to measure 25-hydroxyvitamin D (25-OHD) has increased. The Nichols Advantage chemiluminescence protein-binding assay (CLPBA) for 25-OHD is a first-generation automated immunoassay with decreased turnaround time, reduced manual handling and non-radioactive label. Methods: We compared the CLPBA to the DiaSorin radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) for the measurement of 25-OHD using 161 samples from hip fracture patients and samples before and after institution of ergocalciferol (vitamin D2) therapy. Results: A negative bias for the CLPBA at concentrations below 30 nmol/L and a positive bias at 25-OHD values above 30 nmol/L compared with the RIA resulted in diagnostic discordance for one in three samples when using 30 and 50 nmol/L as decision limits. HPLC analysis confirmed the presence of a negative bias for the CLPBA at low values. Both immunoassays under-estimate 25-hydroxyvitamin D2. Conclusions: The discordance between 25-OHD values may be due to differences in standardization of each assay relative to HPLC. Our results emphasize the need for assay-specific clinical decision limits.

Efficacy and safety of pharmacological agents in managing osteoporosis in the old old: Review of the evidence

INTRODUCTION Osteoporosis and fracture risk increase exponentially in postmenopausal females. This places a significant burden in terms of morbidity, mortality and costs that are likely to increase with an ageing population. Despite this there is very limited data on pharmacological management of osteoporosis in this high risk group. OBJECTIVES OF THIS REVIEW: To review the published literature on the clinical efficacy and safety of specific anti osteoporosis treatments in the reduction in fracture risk in females >or=75 years of age. The following major endpoints were used in this review: SEARCH METHODS FOR IDENTIFICATION OF STUDIES: We performed an electronic search of Medline (1970 to June 2007) and the Cochrane Library (1996 to June 2007). Our search strategy included MeSH terms for osteoporosis and treatments. We reviewed the reference list of identified articles for additional relevant published trials. RESULTS Two hundred and fifty-two potentially relevant abstracts were identified. Only six publications were deemed to meet full eligibility criteria and one met most criteria. There is evidence for significant vertebral fracture relative risk reduction(RR) at 1 year for Risedronate (RR 81%; p<0.001), Teriparatide (RR 65%; p<0.05) and Strontium Ranelate (RR 59%; p=0.002) and 3 years for Risedronate (RR 44%; p=0.003), Alendronate (RR 38%; p<0.05) and Strontium Ranelate (RR 32%; p=0.013). There is evidence for significant non-vertebral fracture relative risk reduction at 1 year for Strontium Ranelate (RR 41%; p=0.027) but not Teriparatide (p=0.66) and 3 years for Strontium Ranelate (RR 31%; p=0.011) but not Risedronate (p=0.66). The only study to report a reduction in hip fracture at 3 years is the TROPOS study with Strontium Ranelate (RR 36%; p=0.046). DISCUSSION This review reinforces the irony that the least evidence is available for fragility fracture reduction in the group at greatest risk; the old old and those with non vertebral and hip fracture. Although there is good evidence for the benefit of the bisphosphonates (Alendronate and Risedronate), Teriparatide and Strontium Ranelate in vertebral fracture reduction, there are very limited data for non vertebral and hip fracture reduction. Strontium Ranelate is the only agent to date that has demonstrated a reduction in non vertebral and hip fracture events in this high risk elderly female population. Perhaps we need to adopt different strategies in managing older patients with osteoporosis as their fracture risks and treatment strategies may be quite different from younger populations.

High prevalence of osteoporosis in cardiac transplant recipients and discordance between biochemical turnover markers and bone histomorphometry

All patients attending the cardiac trans‐plantation clinic at the Royal Perth Hospital were investigated to determine the prevalence of osteoporosis and to assess changes in bone metabolism and histomorphometry in a cohort of cardiac transplant recipients.

Estrogen and androgen regulation of plasma membrane calcium pump activity in immortalized distal tubule kidney cells.

The ATP dependent plasma membrane calcium pump (PMCA) is a regulator of renal calcium reabsorption. The effect of estrogen and dihydrotestosterone to increase the activity of the PMCA in membrane vesicle preparations from a distal tubule cell line was investigated. 17beta Estradiol (10(-10)M) increased PMCA activity (1.5 +/- 0.2-fold increase compared to control) with 24 h, but not 1 or 5 h, of exposure, an effect that was blocked by the addition of the estrogen antagonist ICI 164384. alpha Estradiol did not increase PMCA activity. Dihydrotestosterone (10(-11)M ) resulted in a dose dependent increase in PMCA activity (1.5+/-0.1-fold increase compared to control) with 24h, but not 1 or 5h, of exposure, an effect that was blocked by the androgen receptor agonist flutamide. Testosterone (10(-5)M) also increased PMCA activity (1.9+/-0.3-fold increase compared to control). Neither estrogen nor dihydrotestosterone increased PMCA protein expression in MDBK cells, indicating that these hormones increase PMCA activity by regulating PMCA activity rather than PMCA expression. These results demonstrate receptor dependent stimulatory effects of both estrogen and dihydrotestosterone to increase PMCA activity. and have significance for our understanding of estrogen and androgen deficient states on calcium transport.

Association between 25-hydroxyvitamin D, somatic muscle weakness and falls risk in end-stage renal failure

Objective  Suboptimal levels of 25‐hydroxyvitamin D (25OHD) are common in haemodialysis patients (Chronic Kidney disease‐5D: CKD‐5D) and may be associated with reduced muscle strength and increased falls risk. We tested the hypothesis that 25OHD levels may be independently associated with falls risk in CKD‐5D.

Endotoxin induced TNF and IL-10 mRNA production is higher in male than female donors: correlation with elevated expression of TLR4.

Modification of cytokine production by gender hormones has been postulated to affect disease susceptibility and outcome. Here we investigate the effect of gender and the menstrual cycle on production of cytokines. Mononuclear cells were isolated every week for 10 consecutive weeks from healthy pre-menopausal women and men. TNF and IL-10 mRNA and protein levels were measured as well as membrane CD14 and intracellular TLR4 protein. Endotoxin stimulation of mononuclear cells from men produced more TNF and IL10 mRNA than cells from women. TLR4 expression was also significantly higher in cells from men. These gender differences in the immune response may help to elucidate the sexual dimorphism observed in infectious diseases.

Diagnosis of primary hyperparathyroidism: controversies, practical issues and the need for Australian guidelines

Abstract