How Accurate is Your Sclerostin Measurement? Comparison Between Three Commercially Available Sclerostin ELISA Kits.

Sclerostin, bone formation antagonist is in the spotlight as a potential biomarker for diseases presenting with associated bone disorders such as chronic kidney disease (CDK-MBD). Accurate measurement of sclerostin is therefore important. Several immunoassays are available to measure sclerostin in serum and plasma. We compared the performance of three commercial ELISA kits. We measured sclerostin concentrations in serum and EDTA plasma obtained from healthy young (18-26 years) human subjects using kits from Biomedica, TECOmedical and from R&D Systems. The circulating sclerostin concentrations were systematically higher when measured with the Biomedica assay (serum: 35.5 ± 1.1 pmol/L; EDTA: 39.4 ± 2.0 pmol/L; mean ± SD) as compared with TECOmedical (serum: 21.8 ± 0.7 pmol/L; EDTA: 27.2 ± 1.3 pmol/L) and R&D Systems (serum: 7.6 ± 0.3 pmol/L; EDTA: 30.9 ± 1.5 pmol/L). We found a good correlation between the assay for EDTA plasma (r > 0.6; p < 0.001) while in serum, only measurements obtained using TECOmedical and R&D Systems assays correlated significantly (r = 0.78; p < 0.001). There was no correlation between matrices results when using the Biomedica kit (r = 0.20). The variability in values generated from Biomedica, R&D Systems and TECOmedical assays raises questions regarding the accuracy and specificity of the assays. Direct comparison of studies using different kits is not possible and great care should be given to measurement of sclerostin, with traceability of reagents. Standardization with appropriate material is required before different sclerostin assays can be introduced in clinical practice.

Early renal impairment affects hormonal regulators of calcium and bone metabolism and Wnt signalling and the response to vitamin D supplementation in healthy older adults.

Bone and renal metabolism are regulated by common factors and there is extensive cross-talk between these organs (the 'renal-bone-axis'). Ageing is associated with physiological changes including reduced bone mass, renal function and tissue sensitivity to regulatory hormones, impacting the renal-bone axis. We aimed to investigate the influence of estimated Glomerular Filtration Rate (eGFR) on plasma concentrations of vitamin D metabolites, Wnt signalling and bone metabolism in a dose ranging vitamin D3 RCT (12,000 IU, 24,000 IU, 48,000 IU/month for 1 year; n = 379, >70 y) with a baseline eGFR > 30 mL/min/1.73 m2. Participants were categorised on basis of eGFR (≥60 or mL/min/1.73 m2) based on 5 commonly used algorithms for eGFR. Differences between eGFR categories were tested with ANCOVA. Before supplementation commenced, a lower eGFR was associated with significantly higher concentrations of c-terminal and intact Fibroblast Growth Factor-23 (cFGF23; iFGF23), intact Parathyroid Hormone (iPTH) and Sclerostin (SOST) and lower Klotho, 1,25-dihydroxy Vitamin D (1,25(OH)2D) and Dickkopf-related Protein 1 (DKK1) concentrations. Differences between eGFR groups in 25-hydroxy Vitamin D (25(OH)D), 24,25-dihydroxy Vitamin D (24,25(OH)2D) and iPTH were only detected with eGFR based on Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) and Modification in Diet for Renal Disease (MDRD-4) algorithms. Differences in Bone Mineral Density and Content (BMD; BMC) and bone turnover markers were detected only with Cockcroft-Gault (CG). Pre- and post- supplementation comparisons showed differences in the response to supplementation by eGFR group. Plasma 25(OH)D, 24,25(OH)2D, 1,25(OH)2D and DKK1 increased and iPTH and C-terminal telopeptide (CTX) decreased in both groups. Plasma iFGF23, bone specific alkaline phosphatase (BAP) and Procollagen 1 intact N-terminal Propeptide (PINP) increased and phosphate decreased only in the group with eGFR ≥ 60 mL/min/1.73 m2. Findings were largely consistent across all eGFR algorithms. Post-supplementation, cFGF23, iFGF23, iPTH and SOST remained significantly higher in the lower eGFR group. Plasma 1,25(OH)2D and Klotho did no longer differ between eGFR groups. This was found for all eGFR algorithms, with the exception of iPTH and iFGF23, which were not significantly different with eGFR based on CG. Differences in BMD and BMC were detected with CKD-EPI-creatinine and MDRD-4 but not GC. This study showed that even a moderate decline in eGFR is associated with alterations in vitamin D metabolism, Wnt signalling and bone turnover markers. Renal function influenced the response to vitamin D supplementation. Supplementation increased Vitamin D metabolites in the group with moderate renal impairment to concentrations comparable to those found in the group with normal renal function. However, although CTX decreased, an increase in bone formation markers was not found in the group with eGFR 60 mL/min/1.73 m2. In conclusion, vitamin D supplementation had beneficial effects on markers of the renal-bone axis in older people with both normal and impaired renal function.

Vitamin D Supplementation for 12 Months in Older Adults Alters Regulators of Bone Metabolism but Does Not Change Wnt Signaling Pathway Markers.

Vitamin D status and supplementation regulates bone metabolism and may modulate Wnt signaling. We studied the response of hormonal regulators of bone metabolism, markers of Wnt signaling and bone turnover and bone mineral density (BMD) and bone mineral content (BMC) in a randomized vitamin D intervention trial (12,000 IU, 24,000 IU, 48,000 IU/mo for 1 year; men and women aged >70 years; n = 379; ISRCTN35648481). Associations with total and free 25(OH)D concentrations were analyzed by linear regression. Baseline vitamin D status was (mean ± SD) 25(OH)D: 40.0 ± 20.1 nmol/L. Supplementation dose-dependently increased total and free 25(OH)D concentrations and decreased plasma phosphate and parathyroid hormone (PTH) (all p < 0.05). The procollagen 1 intact N-terminal (PINP)/C-terminal telopeptide (CTX) ratio, C-terminal fibroblast growth factor-23 (cFGF23), and intact FGF23 (iFGF23) significantly increased with no between-group differences, whereas Klotho was unchanged. 1,25(OH)2D and PINP significantly increased in the 24 IU and 48,000 IU groups. Sclerostin (SOST), osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), BMD, BMC, and CTX remained unchanged. Subgroup analyses with baseline 25(OH)D <25 nmol/L (n = 94) provided similar results. Baseline total and free 25(OH)D concentrations were positively associated with 1,25(OH)2D, 24,25(OH)2D (p < 0.001), vitamin D binding protein (DBP) (p < 0.05), BMD, and BMC (p < 0.05). Associations with PTH (p <0.001), cFGF23 (p < 0.01), and BAP (p < 0.05) were negative. After supplementation, total and free 25(OH)D concentrations remained positively associated only with 24,25(OH)2D (p < 0.001) and DBP (p < 0.001) and negatively with estimated glomerular filtration rate (eGFR) (p < 0.01). PTH and SOST were significantly associated only with free 25(OH)D. There were no significant relationships with BMD and BMC after supplementation. The decrease in PTH and increase in PINP/CTX ratio suggest a protective effect of supplementation on bone metabolism, although no significant effect on BMD or pronounced changes in regulators of Wnt signaling were found. The increase in FGF23 warrants caution because of its negative association with skeletal and cardiovascular health. Associations of total and free 25(OH)D with biomarkers were similar and known positive associations between vitamin D status and BMD were confirmed. The change in associations after supplementation might suggest a threshold effect. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Reference intervals for serum 24,25-dihydroxyvitamin D and the ratio with 25-hydroxyvitamin D established using a newly developed LC-MS/MS method.

24,25(OH)2D is the product of 25(OH)D catabolism by CYP24A1. The measurement of serum 24,25(OH)2D concentration may serve as an indicator of vitamin D catabolic status and the relative ratio with 25(OH)D can be used to identify patients with inactivating mutations in CYP24A1. We describe a LC-MS/MS method to determine: (1) the relationships between serum 24,25(OH)2D and 25(OH)D; (2) serum reference intervals in healthy individuals; (3) the diagnostic accuracy of 24,25(OH)2D measurement as an indicator for vitamin D status; 4) 24,25(OH)2D cut-off value for clinically significant change between inadequate and sufficient 25(OH)D status. Serum samples of healthy participants (n=1996) from Army recruits and patients (n=294) were analysed. The LC-MS/MS assay satisfied industry standards for method validation. We found a positive, concentration-dependent relationship between serum 24,25(OH)2D and 25(OH)2D concentrations. The 25(OH)D:24,25(OH)2D ratio was significantly higher (P<.001) at 25(OH)D<50 nmol/L. The reference interval for 25(OH)D:24,25(OH)2D ratio in healthy subjects was 7-23. Measurement of serum 24,25(OH)2D can be used as predictor of vitamin D status, a concentration of>4.2 nmol/L was identified as a diagnostic cut-off for 25(OH)D replete status. One patient sample with an elevated 25(OH)D:24,25(OH)2D ratio of 32 and hypercalcaemia who on genetic testing confirmed to have a biallelic mutation of CYP24A1. Our study demonstrated the feasibility of a combined 24,25(OH)2D and 25(OH)D assessment profile. Our established cut-off value for 24,25(OH)2D and ratio reference ranges can be useful to clinicians in the investigation of patients with an impaired calcium/phosphate metabolism and may point towards the existence of CYP24A1 gene abnormalities.