Reference intervals for bone turnover markers in Spanish premenopausal women.

BACKGROUND: The aims of this study were to establish robust reference intervals and to investigate the factors influencing bone turnover markers (BTMs) in healthy premenopausal Spanish women. METHODS: A total of 184 women (35-45 years) from 13 centers in Catalonia were analyzed. Blood and second void urine samples were collected between 8 a.m. and 10 a.m. after an overnight fast. Serum procollagen type I amino-terminal propeptide (PINP) and serum cross-linked C-terminal telopeptide of type I collagen (CTX-I) were measured by two automated assays (Roche and IDS), bone alkaline phosphatase (bone ALP) by ELISA, osteocalcin (OC) by IRMA and urinary NTX-I by ELISA. PTH and 25-hydroxyvitamin D (25OHD) levels were measured. All participants completed a questionnaire on lifestyle factors. RESULTS: Reference intervals were: PINP: 22.7-63.1 and 21.8-65.5 µg/L, bone ALP: 6.0-13.6 µg/L, OC: 8.0-23.0 µg/L, CTX-I: 137-484 and 109-544 ng/L and NTX-I: 19.6-68.9 nM/mM. Oral contraceptive pills (OCPs) influenced PINP (p=0.007), and low body mass index (BMI) was associated with higher BTMs except for bone ALP. Women under 40 had higher median values of most BTMs. CTX-I was influenced by calcium intake (p=0.010) and PTH (p=0.007). 25OHD levels did not influence BTMs. Concordance between the two automated assays for PINP and particularly CTX-I was poor. CONCLUSIONS: Robust reference intervals for BTMs in a Southern European country are provided. The effects of OCPs and BMI on their levels are significant, whilst serum 25OHD levels did not influence BTMs. Age, calcium intake, BMI and PTH influenced CTX-I. The two automated assays for measuring PINP and CTX-I are not interchangeable.

Reference interval for osteocalcin in Chinese Han ethnic males from the Fangchenggang Area Male Health and Examination Survey.

BACKGROUND: The aim of this study was to set a reference interval (RI) for osteocalcin (OC) in a healthy Han male population from the Fangchenggang Area Male Health and Examination Survey (FAMHES) project and study the effects of age, BMI, smoking, and alcohol consumption. METHODS: We used data from 2018 Han ethnic males who participated in FAMHES project from September 2009 to December 2009. Serum OC values were measured by electrochemiluminescence immunoassay on COBAS 6000 system E601 (Elecsys module) immunoassay analyzers. RESULTS: OC data does not have a normal distribution or Gaussian pattern (Z = 3.791, p < 0.001). After log-transformation, data took on a normal, Gaussian distribution (Z = 0.968, p = 0.306). The RI of serum OC was 12.49 - 43.94 ng/mL. No difference in OC concentration was noted either between non-smoker or smoker groups (p = 0.629) or non-drinker and drinker groups (p = 0.748). OC levels varied with age (r = -0.371, p < 0.001) and BMI (r = -0.331, p < 0.001), and the age-dependent and BMI-dependent RIs were calculated. CONCLUSIONS: The RIs for serum OC exhibit slight differences compared to previously reported reference ranges. Age-dependent and BMI-dependent RIs for serum OC should be implemented in clinical laboratories.

Validation of a new automated immunoassay for measurement of intact osteocalcin.

Bone turnover is assessed indirectly by measurement of biochemical markers of bone turnover. Osteocalcin, a 49-amino-acid protein is a major noncollagenous protein of bone matrix, synthesized by osteoblasts and odontoblasts. Various assays exist for assessment of osteocalcin and concentrations in the same serum or plasma sample may vary enormously. The used antibodies may recognize intact osteocalcin and/or circulating fragments of osteocalcin. We here describe and validate a new automated immunoassay system for measurement of intact osteocalcin (DPC IMMULITE assay) using monoclonal antibodies (mouse) against the C-terminus of osteocalcin (AA 44-49). For detection polyclonal antibodies (goat) directed against the N-terminus (AA 1-17) conjugated with alkaline phosphatase are used. While different laboratory assays show marked clinical discordance, we evaluated our results comparatively to an established IRMA method (Nichols). We observed a highly significant correlation between both assays (r = 0.9352, p < 0.0001, n = 286) for healthy persons and also for patient samples (osteoporosis, diabetes type 1, rheumatoid arthritis). Very low inter- and intraassay covariance as well as highly significant linearity (analytical recovery near 100%) tested by serial dilutions were demonstrated for the DPC IMMULITE intact osteocalcin assay. We conclude that the IMMULITE assay is a useful method for assessment of intact osteocalcin giving valuable results in comparison to an established non-automated assay.

Comparison of monoclonal and polyclonal antibody-based immunoassays for osteocalcin: a study of sources of variation in assay results.

We have compared two assays for osteocalcin (BGP, bone gla protein), one based on a monoclonal antibody, the other based on a polyclonal serum, in an effort to better understand the factors that contribute to the wide variation reported for osteocalcin normal ranges. The two assays compared well using serum samples, with a correlation coefficient of 0.9071 (n = 48). However, the monoclonal antibody assay returns values increased over the polyclonal assay by approximately 1.5-fold. Gel permeation studies indicate that these larger values are due primarily to increased reactivity in the monoclonal assay with high- and low-molecular-weight forms of osteocalcin in plasma; both assays give similar reactions to intact osteocalcin. Analysis of samples from individuals with increased bone resorption due to parathyroid hormone administration reveals that the decrease seen in osteocalcin values after hormone infusion occurs primarily in the fraction that corresponds to intact osteocalcin. During the course of these studies, we re-evaluated the extinction coefficient for osteocalcin, arriving at the value E = 1.33 mg/ml-1, cm-1. We also observed a significant negative interference in both assays (and a commercial assay) caused by hemolysis. This interference is due to proteolysis of osteocalcin by enzymatic activity released from the lysed red cells.