FGF23 metabolism, a new paradigm for chronic kidney disease

Introduction:  Fibroblast growth factor-23 (FGF23) is a major regulator of phosphate metabolism often elevated in genetic hypophosphataemic disorders and in chronic kidney disease. Recent studies have identified relationships between FGF23 and various markers of iron status including ferritin. New assays measuring the intact form of FGF23 have been released.  Objective:  To determine the relationship between ferritin and C-terminal and intact FGF23 concentrations in blood.  Method:  FGF23 concentrations were measured using the 2nd generation, two-site enzyme-linked immunosorbent assay for either C-terminal or intact FGF23 (Immutopics Inc., Ca, USA). Ferritin was measured on a COBAS 6000 (Roche Diagnostics). Assay accuracy and precision were monitored using kit controls supplied by the manufacturers.  Results:  We observe a weak negative correlation between measurements of C-terminal and intact FGF23 (Pearson’s rho=0.85 p<0.0001). We observed no statistically significant correlation of ferritin concentrations with either FGF23 C-terminal or intact. However high concentrations of ferritin were observed in samples showing low concentrations of C-terminal FGF23 (<140RU/mL) and intact FGF23 (<122pg/mL).  Conclusion:  Although not statistically significant, we observe a negative relationship between concentrations of ferritin and FGF23. High level of C-terminal FGF23 is found in patients with chronic kidney disease, especially in patients with end-stage renal disease usually regarded as a compensatory response to hyperphosphatemia or phosphate overload. We observed a cluster of patients with retention of both C-terminal and intact FGF23 associated with low levels of ferritin suggesting that metabolism and/or excretion of FGF23 in CDK patients might be an iron dependent mechanism

How accurate is your sclerostin measurement? Comparison between two commercially available sclerostin ELISA kits.

Introduction: Sclerostin (SOST), osteocyte-secreted soluble antagonist of the Wnt/β-catenin signaling pathway, is a potent inhibitor of osteoblastogenesis. Circulating SOST levels have been measured in a plethora of disorders such as ankylosing spondylitis, chronic kidney disease, diabetes, fractures, hypercortisolism, multiple myeloma and spinal cord injury. SOST is a crucial regulator of the skeletal anabolic action of PTH and as so, anti-sclerostin antibodies are being investigated as potential therapeutic molecules for metabolic bone diseases. Accurate measurement of SOST is therefore of utmost importance for the diagnosis of bone disorders and therapy effectiveness. However, reports so far suggests further study is needed before SOST measurements are introduced into routine clinical practice. Objective: To compare two commercially available assays for measurement of circulating SOST. Method: EDTA-plasma samples from 36 anonymised healthy individuals were analyzed using ELISA kit for circulating SOST from Biomedica (Vienna, Austria) and TecoMedical (TECO, Sissach, Switzerland). Both assays are based on immuno-capture using two antibodies which have been raised against human recombinant SOST and are highly specific for this molecule. Results: Circulating SOST levels in EDTA plasma samples were found to be significantly different between TECO and Biomedica assays (36.9 ± 2 and 21.3 ± 1pmol/L, respectively, p<0.001) with discrepancies of up to 32pmol/L. The TECO assay demonstrated less variability between duplicates (2.6±2.4 % and 7.4±6.3 % respectively) and dilution study showed that the biomedical kit over-recovered diluted samples by up to 60%. When samples containing various concentrations of endogenous sclerostin were spiked with a known amount of SOST, recovery was 88.5% and 104% respectively. Conclusion: The variability in values generated from Biomedica and TECO assays has raised questions regarding the specificity of antibodies used by the two manufactures, and whether there is possible interference affecting one of the assays remains unclear. Cross-reactivity experiments are being conducted to determine the source of variation between the two kits. Until such issues are resolved, measurement of sclerostin remains invaluable for understanding the mechanism by which osteocytes regulate bone turnover but should be used in discretion and interpretation should be carried out with guided clinical evidence

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

Circulating α-Klotho Levels Are Inversely Correlated to FGF-23 in Tumour Induced Osteomalacia

Tumour induced osteomalacia is characterised by high circulating levels of fibroblast growth factor 23 (FGF23) due to ectopic secretion from mesenchymal tumors. An abundance of FGF23 drives the hypophosphatemia and low 1,25-dihydroxy vitamin D levels characteristic of this condition. The single-pass trans-membrane protein α-klotho is integral for FGF23-mediated receptor activation and its downstream effects. In addition, α-klotho may have a phosphaturic effect independent of FGF-23. The regulation of α-klotho in the face of high circulating FGF23 is currently unknown. We investigated the relationship between circulating FGF23 and α-klotho in patients with TIO. We identified 15 consecutive plasma FGF-23 requests in subjects with confirmed TIO for testing. FGF-23 was measured using Immutopics C-term ELISA kit and soluble α-klotho was measured using the IBL ELISA kit. The group consisted of 7 males and 8 females with an age of 53 ± 20 years (mean ± SD). Only one subject was not on treatment for TIO at the time of sampling. The average circulating levels of FGF23 and α-klotho were 286 ± 244 RU/ml and 644 ± 309 pg/ml respectively. There was an inverse correlation between FGF-23 and α-klotho (Pearson coefficient - 0.28). This inverse correlation may suggest that increasing levels of FGF23 in TIO down-regulate α-klotho expression in the kidney and parathyroid glands leading to reduced circulating levels of α-klotho. Given α-klotho’s role in mediating FGF23 signalling and that α-klotho may be a phosphaturic factor in its own right; such an adaptive mechanism would help partially reduce the renal phosphate wasting seen in TIO. In summary there is an inverse correlation seen between circulating levels of FGF23 and α-klotho in TIO. This may suggest there is a down-regulation of α-klotho expression to limit the phosphate wasting and resultant hypophosphatemia that is seen in TIO

Assessment of C3-Epi-25-Hydroxyvitamin D concentration in adult serum: LC-MS/MS determination using [2H3] 3-epi-25OHD3 internal standard and NIST traceable commercial 3-epi-25OHD calibrators.

Background: The C-3 Epimer of 25 Hydroxyvitamin D3 (3-Epi-25OHD3) is produced in the liver by the epimerisation pathway of 25-hydroxy vitamin D3. It differs from 25OHD3 in configuration of the hydroxyl group at the third carbon (C-3) position. Despite the fact that little is known regarding its clinical significance, concerns have been raised that isobaric interference may result in over-estimation of total 25OHD when measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Objective: The aim of the study was to assess the occurrence of 3-Epi-25OHD3 in adult serum samples. A LC-MS/MS technique was developed to resolve and quantify 3-Epi-25OHD3 from 25OHD3. The newly available NIST (SRM972a) traceable 3-Epi-25OHD commercial standards were used to ensure assay accuracy. Method: Serum was precipitated with zinc sulphate and acetonitrile containing [2H3]-3-epi-25OHD3 as internal standard. The extract was chromatographed using a 2.6µm 100 x 2.1mm I.D. solid core particle column. Mass detection and quantification were performed by positive electrospray ionization with MS/MS in multiple reaction monitoring mode. Results: The method was able to fully resolved 3-Epi-25OHD3 from 25OHD3. The intraassay CVs for the epimer were 6.3% and 4.1% at 25.4 and 62.1 nmol/L respectively; and interassay CVs were 8.3% and 6.5% at 27.6 and 63.2 nmol/L, respectively. In our sample cohort with 25OHD3 ranged between 3.4 – 165 nmol/L, 3-Epi-25OHD3 was detected in 91.9% of samples (mean = 3.8 nmol/L). No detectable 3-Epi-25OHD2 was found in our sample study. One patient sample had total 25OHD3 of 187 nmol/L that was shown to contain 141 nmol/L of 25OHD3 and 44 nmol/L of 3-Epi-25OHD3. This patient was receiving a high dose of vitamin D supplementation. Conclusion: Using [2H3]-3-epi-25OHD3 as internal standard and NIST aligned calibrators enabled us to obtain an accurate assessment of 3-epi-25OHD concentration in adult serum. Although the concentration of serum 3-epi-25OHD3 was found to be low the presence was observed in the majority of our samples. The findings in this study showed that 3-epi-25OHD3 contributed to the overestimation of 25OHD3 that could potentially resulted in misinterpretation of total vitamin D status

Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma

Background: Teriparatide [recombinant human PTH (1-34)] is an osteoanabolic agent for treatment of osteoporosis. The effect on bone decreases the risk of vertebral and non-vertebral fractures and increases bone mineral density (BMD) in post-menopausal women with osteoporosis. Measurement of PTH (1-34) is valuable in assessing treatment response and concordance with therapy.Aim: To develop and validate a method for quantification of PTH (1-34) using Liquid chromatography tandem mass spectrometry (LC-MS/MS) and to perform comparison with a commercial immunoassay.  Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis® HLB µElution solid phase extraction. Quantification m/z transition 589>656 was used on Waters/Micromass® Quattro Ultima™ Pt mass spectrometer to measure PTH (1-34) in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. PTH (1-34) results obtained from human subjects given Teriparatide (Fortsteo, Eli Lilly, IN, USA) (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK).  Results and Discussion: LC-MS/MS produced a linear calibration curve from 10 to 2000 pg/mL (r2 >0.990). The LLoQ and LLoD for PTH (1-34) were 10 pg/mL and 2.1 pg/mL respectively. The inter- /intra-assay precision (CV%) of the method were 98.3% for four QCs (20, 100, 200, and 800 pg/mL). The mean recovery of PTH (1-34) was 107.2%. Method comparison between the LC-MS/MS and immunoassay using human EDTA plasma samples showed a high correlation (r2 = 0.950). A concentration-dependent, negative bias of 35.5% was observed across the range of 0 – 800 pg/mL. The immunoassay showed a 7% cross reactivity to human PTH (1-84) and 44% to rat PTH (1-34), no interference was observed in the LC-MS/MS method. Matrix effect and cross reactivity to human PTH (1-84) in the immunoassay were the likely contributing factors to the bias between the methods. The oxidised form of PTH (1-34) does not interfere with our LC-MS/MS method.  Conclusion: Our LC-MS/MS method demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The method was successfully used for measurements of PTH (1-34) in rat and human plasma

Measurement of autoantibodies against osteoprotegerin in adult human serum: development of a novel ELISA assay

Introduction: In 2009, neutralizing autoantibodies against OPG (α-OPGAb) blocking the inhibitory effect of OPG on RANK signaling pathway were identified in a man with celiac disease associated with severe osteoporosis. Although this finding was not reproduced in thirty patients presenting coeliac disease and low bone mineral density, Hauser et al (2013) recently detected the presence of α-OPGAb in patients presenting Rheumatoid Arthritis, Systemic Lupus Erythematosus, Spondyloarthritis and Osteoporosis. There is a growing focus on OPG autoantibodies as primary cause of high bone turnover in disorders with unknown etiology. Objective: To develop an enzyme linked immunosorbent assay (ELISA) for detection and quantification of α-OPGAb in patient serum samples. Method: A full-length human recombinant OPG is immobilized on a plate to allow capture of the antibodies from the sera. In a two-step reaction, the αOPGAb is detected using a biotinylated antibody and a horseradish peroxidase-labelled streptavidin. Substrate is incubated in a timed reaction and color development measured in a spectrophotometric microtiter plate reader. The concentration of human α-OPGAb in the samples is determined directly from a 4PL-fit standard curve. Results: Intra-assay imprecision was <5% at 274.4 ± 18.8 and 98.5 ± 2.9 ng/mL. Inter-assay imprecision was <20% at 324.2 ± 53.3 and 166.8 ± 30.6 ng/mL. Linear range was 0-500ng/mL. Lower and upper limit of quantification were 3.9 and 500 ng/mL. Cross reactivity was assessed against human sera containing raised thyroid antibody and RANKL to ensure assay specificity. Using the method presented, we established that the adult population would be considered positive with a titer above the cut-off limit (95%) of 68ng/mL. Our preliminary data suggested that 14% of our sample population (n=136) presented elevated α-OPGAb. Conclusion: We presented a novel ELISA assay for the detection and measurement of anti-OPG autoantibodies in human serum. The validated method showed excellent assay characteristics and is suitable for use in research and clinical hospital laboratories. In patients with severe form of osteoporosis, measurement of OPG autoantibodies could help clinicians identify appropriate treatment options for this particular subgroup of patients

Assessment of vitamin D status using MitraTM volumetric absorptive microsampling (VAMS) device

Introduction: The use of dried blood spot (DBS) sampling for general wellness assessment and in clinical diagnostics has gained popularity as a convenient and less invasive alternative to venous sampling. Collection of blood samples from a finger/heel prick using conventional filter paper suffers from variability in sample volume and spot sizes which undermine the quality of results. We describe the use of a volumetric absorptive microsampler (VAMS), called MitraTM (Torrance, CA, USA) for measurement of 25OHD3 and interpretation of vitamin D status according to current international guidelines.  Method: A liquid-chromatography mass spectrometry (LC-MS/MS) method was used for measurement of 25OHD3 (Tang et al. ASBMR 2015, LB-MO0026). We compared results from patient samples (n=97) collected by VAMS and Whatman® 903 cards extracted as whole spot (wDBS) and sub-punches (spDBS) against plasma 25OHD3 concentration. We investigated the volume displacement effects of haematocrit (Hct) on DBS 25OHD3 measurements and described the use of DBS-to-plasma equivalence value (PEV) to allow accurate interpretation of vitamin D status.  Results: VAMS showed the best assay precision CV (<8.2%) compared to wDBS (<16.6%) and spDBS (<15.1%) across the assay range of 0.1-125 nmol/L, the least variability in recovery and lowest LLoQ (Figure 1). We observed a decrease in DBS 25OHD3 concentration in proportion to the reduction in plasma volume and increase in packed cell volume. The displacement effect of Hct resulted in a strong but negatively biased correlation (r2=0.893, -39.3%) between raw DBS values and plasma concentrations, that was dependent upon the level of Hct present in sample. We demonstrated the use of simple linear regression model to transform raw DBS values into PEVs. In a subsequent cohort of patient samples (n=70), PEVVAMS produced the most accurate interpretation of vitamin D status compared to PEVwDBS and PEVspDBS.  Discussion: We present data supporting the use of VAMS for measurement of 25(OH)D3, particularly in circumstances where venesection may be impossible or difficult and where sample volume may be limited. Although the recovery of analyte remains Hct-dependent, the use of DBS-to-plasma equivalence values improves the clinical applicability and broadens the utility of DBS as a sampling technique

Non-osteoporotic post-menopausal women do not have elevated concentrations of autoantibodies against osteoprotegerin

Introduction: Osteoprotegerin (OPG) plays a protective role in bone remodelling as it provides a ‘decoy’ binding site for RANKL, preventing the stimulation of osteoclasts. Autoantibodies to OPG allow a sustained reaction between RANKL and RANK which in turn increase bone degradation. Autoantibodies against Osteoprotegerin (-OPGAb) first isolated in patients with autoimmune conditions associated with high bone turnover have been shown to be present in 14% of a healthy young adult population. Bone degradation is more prominent in the oldest population, particularly in women. Objective: To define a reference range for OPG autoantibodies in non-osteoporotic post-menopausal women. Method: Using a previously developed sandwich ELISA assay we were able to detect OPG autoantibody in serum samples taken from non-osteoporotic post-menopausal women (ANSAVID study - 60-65yrs). Briefly, -OPGAb are captured by the use of an immobilized full-length human recombinant OPG and detected by the sequential addition of a biotinylated antibody and a horseradish-peroxidase-labelled streptavidin. The concentration of human α-OPGAb in the samples is determined directly from a 4PL-fit standard curve. Results: We established that the population of post-menopausal women who do not present osteoporosis do not have elevated concentration of -OPGAb as compared to a younger healthy population (17-32yrs). This suggest that -OPGAb is not normally occurring with age suggesting that the production of -OPGAb is solely related to pathologic conditions in which the bone is heavily degraded. Conclusion: Comparison of osteoporotic patient samples to the non-osteoporotic post-menopausal women would be interesting to determine whether -OPGAb can be used to identify appropriate treatment options for this particular subgroup of patients

Reference intervals for serum 24,25-Dihydroxyvitamin D and the ratio with 25-Hydroxyvitamin 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 (p4.2 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