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

STATURE AND LOAD AFFECT THE WALK TO RUN TRANSITION SPEED

Military personnel are often required to march “in-step” while carrying heavy loads. For example, the two speeds required to complete the role fitness test for the British Army are close to the preferred walking speed and preferred walk-to-run transition speed (PTS) for healthy adults when unloaded. PTS depends on anthropometry, including stature. Walking at speeds markedly different to PTS has been associated with increased metabolic cost and increased joint loading. There is also limited research into how this PTS is affected by load carriage. To minimise the risk of injury, there is a need to understand how load carriage affects PTS. This study found PTS for male and female personnel decreased with increased load carried, and that female personnel tended to transition from walking to running earlier than male personnel. The relationship between PTS and stature became more positive as load increased, irrespective of sex. Due to the association between deviating from preferred walking gait and increases in joint loading, these findings may have implications for the risk of injury in military personnel who are required to march “in-step”

Smoking and Biochemical, Performance, and Muscle Adaptation to Military Training

PURPOSE: To determine whether physical performance adaptation is impaired in smokers during early stages of military training, and to examine some of the putative mechanistic candidates that could explain any impairment.METHODS: We examined measures of oxidative stress (malondialdehyde (MDA), lipid hydroperoxides), inflammation (C-reactive protein (CRP), interleukin-6), antioxidants (Vitamins A, E and carotenes) and hormones (cortisol, testosterone, insulin-like growth factor-1) in 65 male British Army Infantry recruits (mean ± SD age: 21 ± 3 yr; mass: 75.5 ± 8.4 kg; height: 1.78 ± 0.07 m) at week 1, week 5 and week 10 of basic training. Physical performance (static lift, grip strength, jump height, 2.4 km run time and two-minute press up and sit up scores) was examined and lower-leg muscle and adipose cross-sectional area (CSA) and density measured by peripheral Quantitative Computed Tomography.RESULTS: Basic Military training, irrespective of smoking status, elicited improvement in all physical performance parameters (main time effect; P &lt; 0.05) except grip strength and jump height, and resulted in increased muscle area and decreased fat area in the lower leg (P &lt; 0.05). MDA was higher in smokers at baseline, and both MDA and CRP were greater in smokers during training (main group effect; P &lt; 0.05), than non-smokers. Absolute performance measures, muscle characteristics of the lower leg and other oxidative stress, antioxidant, endocrine and inflammatory markers were similar in the two groups.CONCLUSIONS: Oxidative stress and inflammation were elevated in habitual smokers during basic military training, but there was no clear evidence that this was detrimental to physical adaptation in this population over the timescale studied.</p

Smoking and Biochemical, Performance, and Muscle Adaptation to Military Training

Purpose To determine whether physical performance adaptation is impaired in smokers during early stages of military training, and to examine some of the putative mechanistic candidates that could explain any impairment. Methods We examined measures of oxidative stress (malondialdehyde (MDA), lipid hydroperoxides), inflammation (C-reactive protein (CRP), interleukin-6), antioxidants (Vitamins A, E and carotenes) and hormones (cortisol, testosterone, insulin-like growth factor-1) in 65 male British Army Infantry recruits (mean ± SD age: 21 ± 3 yr; mass: 75.5 ± 8.4 kg; height: 1.78 ± 0.07 m) at week 1, week 5 and week 10 of basic training. Physical performance (static lift, grip strength, jump height, 2.4 km run time and two-minute press up and sit up scores) was examined and lower-leg muscle and adipose cross-sectional area (CSA) and density measured by peripheral Quantitative Computed Tomography. Results Basic Military training, irrespective of smoking status, elicited improvement in all physical performance parameters (main time effect; P < 0.05) except grip strength and jump height, and resulted in increased muscle area and decreased fat area in the lower leg (P < 0.05). MDA was higher in smokers at baseline, and both MDA and CRP were greater in smokers during training (main group effect; P < 0.05), than non-smokers. Absolute performance measures, muscle characteristics of the lower leg and other oxidative stress, antioxidant, endocrine and inflammatory markers were similar in the two groups. Conclusions Oxidative stress and inflammation were elevated in habitual smokers during basic military training, but there was no clear evidence that this was detrimental to physical adaptation in this population over the timescale studied

Effect of carbohydrate feeding on the bone metabolic response to running

Bone resorption is increased after running, with no change in bone formation. Feeding during exercise might attenuate this increase, preventing associated problems for bone. This study investigated the immediate and short-term bone metabolic responses to carbohydrate (CHO) feeding during treadmill running. Ten men completed two 7-day trials, once being fed CHO (8% glucose immediately before, every 20 min during, and immediately after exercise at a rate of 0.7 g CHO·kg body mass-1·h-1) and once being fed placebo (PBO). On day 4 of each trial, participants completed a 120-min treadmill run at 70% of maximal oxygen consumption (VO2 max). Blood was taken at baseline (BASE), immediately after exercise (EE), after 60 (R1) and 120 (R2) min of recovery, and on three follow-up days (FU1-FU3). Markers of bone resorption [COOH-terminal telopeptide region of collagen type 1 (β-CTX)] and formation [NH2-terminal propeptides of procollagen type 1 (P1NP)] were measured, along with osteocalcin (OC), parathyroid hormone (PTH), albumin-adjusted calcium (ACa), phosphate, glucagon-like peptide-2 (GLP-2), interleukin-6 (IL-6), insulin, cortisol, leptin, and osteoprotogerin (OPG). Area under the curve was calculated in terms of the immediate (BASE, EE, R1, and R2) and short-term (BASE, FU1, FU2, and FU3) responses to exercise. β-CTX, P1NP, and IL-6 responses to exercise were significantly lower in the immediate postexercise period with CHO feeding compared with PBO (β-CTX: P=0.028; P1NP: P=0.021; IL-6: P=0.036), although there was no difference in the short-term response (β-CTX: P=0.856; P1NP: P=0.721; IL-6: P=0.327). No other variable was significantly affected by CHO feeding during exercise. We conclude that CHO feeding during exercise attenuated the β-CTX and P1NP responses in the hours but not days following exercise, indicating an acute effect of CHO feeding on bone turnover

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

Effectiveness of parathyroid hormone (PTH) analogues on fracture healing: a meta-analysis

Purpose: This meta-analysis evaluated the evidence of Parathyroid Hormone (PTH) analogues in fracture healing. The use of PTH analogues to prevent osteoporotic fractures is well investigated and studies are emerging on extended indications. One such indication receiving increasing attention is the effect of PTH in fracture healing; however, the overall degree of efficacy remains inconclusive. Methods: A systematic electronic database search of MEDLINE, EMBASE and the Cochrane Library was conducted for relevant articles in August 2019 with no date restrictions. Randomised controlled trials of adults with acute fractures treated with a PTH analogue were included. PTH was compared with a comparator intervention, placebo, or no treatment. Results: PTH analogue treatment improved functional outcomes in a range of fracture types but did not affect the fracture healing rate or reduce pain. Most trials included in this review were in elderly patients with osteoporosis. There was no evidence that PTH treatment caused harm or impeded fracture healing. Conclusions: Meta-analysis of published data supports the use of PTH analogues to improve functional outcomes but not fracture healing rate or pain for different fracture types. The evidence for PTH analogue use in fracture healing is less clear in younger, nonosteoporotic patient populations. Trial design was heterogeneous and of limited quality justifying further original trials

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