BACKGROUND:Postmenopausal women with higher visceral adipose tissue (VAT) present with suppressed bone resorption (lower C-terminal crosslinking telopeptide of type I collagen; CTX-1) and turnover (lower osteocalcin) but whether this blunts the effect of calcium is unknown. OBJECTIVES:The primary outcome of this study was the effect of VAT on changes in CTX-1 after intake of 2 forms of calcium. Secondary outcomes included changes in parathyroid hormone (PTH), serum calcium, phosphorus, and alkaline phosphatase (ALP). METHODS:Randomized open three period crossover trial conducted between 2017 and 2019 at the University of South Australia among 77 lean and overweight postmenopausal women (53-79 y) with BMI <25 kg/m2 and >27 kg/m2, respectively. Participants received a single dose of milk (1000 mg calcium), calcium carbonate tablet (1000 mg calcium), and fruit juice (no calcium) in random order with a 7-d washout period. Blood samples were collected at baseline and hourly for 5 h. Data was analyzed by repeated measures ANOVA of log-transformed data. RESULTS:At baseline, women with higher VAT had significantly lower CTX-1 and higher PTH (44% lower and 30% higher, respectively, between Q4 and Q1, P < 0.0001). VAT had no influence on the acute changes in CTX-1 or PTH with calcium or juice. A suppression of 44% in CTX-1 was seen with calcium carbonate and milk and a suppression of 18% with juice. PTH was suppressed more with calcium carbonate (47%) compared to milk (22%). Milk calcium reduced PTH and CTX-1 at 2 h, whereas calcium carbonate reduced PTH in 1 h. The suppression in CTX-1 was slower with lowest concentrations at 4-5 h. CONCLUSIONS:Intake of 1000 mg calcium from milk or from calcium carbonate is effective in acutely suppressing bone resorption in postmenopausal women irrespective of visceral fat. This trial is registered at http://www.ANZCTR.org.au/ACTRN12617000779370.aspx as ACTRN 12617000779370).

A correction to this paper has been published: https://doi.org/10.1007/s00223-021-00839-y

Adiposity increases estrogen receptor (ER)‐positive postmenopausal breast cancer risk. While mechanisms underlying this relationship are uncertain, dysregulated sex‐steroid hormone production and insulin signaling are likely pathways. Our aim was to quantify mediating effects of fasting insulin and free estradiol in the adiposity and ER‐positive postmenopausal breast cancer association. We used data from a case–cohort study of sex hormones and insulin signaling nested within the Melbourne Collaborative Cohort Study. Eligible women, at baseline, were not diagnosed with cancer, were postmenopausal, did not use hormone therapy and had no history of diabetes or diabetes medication use. Women with ER‐negative disease or breast cancer diagnosis within the first follow‐up year were excluded. We analyzed the study as a cumulative sampling case–control study with 149 cases and 1,029 controls. Missing values for insulin and free estradiol were multiply imputed with chained equations. Interventional direct (IDE) and indirect (IIE) effects were estimated using regression‐based multiple‐mediator approach. For women with body mass index (BMI) >30 kg/m2 compared to women with BMI 18.5–25 kg/m2, the risk ratio (RR) of breast cancer was 1.75 (95% confidence interval [CI] 1.05–2.91). The estimated IDE (RR) not through the mediators was 1.03 (95% CI 0.43–2.48). Percentage mediated effect through free estradiol was 72% (IIE‐RR 1.56; 95% CI 1.11–2.19). There was no evidence for an indirect effect through insulin (IIE‐RR 1.12; 95% CI 0.68–1.84; 28% mediated). Our results suggest that circulating free estradiol plays an important mediating role in the adiposity–breast cancer relationship but does not explain all of the association.

The protective effect of obesity on bone health has been challenged by studies that link visceral adiposity to poor bone microarchitecture in young obese men and women. In postmenopausal women, the role of visceral adipose tissue (VAT) on bone turnover markers (BTMs) has not been investigated. The aim was to investigate the impact of VAT on BTMs, total bone mineral density (BMD), vitamin D metabolites and parathyroid levels (1-84 PTH) levels in postmenopausal women. A total of 76 lean and overweight women (without osteoporosis) underwent VAT measurements by dual-energy X-ray absorptiometry (iDXA). Blood samples were analyzed for serum C-terminal telopeptide of type 1 collagen (CTX-1), osteocalcin, bone-specific alkaline phosphatase (bone ALP), 1–84 PTH and vitamin D (25 hydroxyvitamin D, 25(OH)D) levels. VAT volumes ranged from 91 to 3392 cm3 and body mass index (BMI) ranged from 18.3 to 53.9 kg/m2. Women in the highest VAT quartile had significantly lower CTX-1, 25(OH)D, osteocalcin and the highest BMD (p < 0.05, for all). While VAT positively associated with BMD, after controlling for BMI, VAT was a negative predictor of BMD (β = 0.368, p < 0.05). VAT was an independent negative predictor of CTX-1 (β = −0.263, p < 0.05) and osteocalcin levels (β = −0.277, p < 0.05). Among all measures of adiposity, VAT was the strongest independent determinant of BMD and BTMs. In clinical settings, VAT, and not BMI, may be a sensitive predictor of bone health in obese women.

BACKGROUND Obesity has been regarded to be protective against fracture in spite of its association with low levels of vitamin D. Vitamin D is the key regulator of bone metabolism and its deficiency contributes to higher level of parathyroid hormone (PTH), leading to the activation of bone turnover. METHODS We studied 161 subjects of which 65 were young healthy subjects and 96 were elderly subjects. We measured creatinine, 25(OH)D, 1,25(OH)2D, PTH, albumin, and calcium plasma levels, we evaluated physical activity, and we calculated BMI. A sub-cohort of elderly subjects also underwent DXA scans. RESULTS Overweight and obese subjects, as well as underweight ones, had lower levels of vitamin D but normal serum concentrations of 1,25(OH)2D and PTH was higher in underweight and obese subjects. Moreover, we found a nonlinear relationship between body mass index (BMI) and PTH with a significant U-shaped exponential regression. Regardless of BMI, 25(OH)D mean levels were higher in subjects who practice physical activity. CONCLUSIONS These findings suggest that physical activity and BMI had a significant effect on the metabolism of bone and vitamin D, but the effect of BMI was different in underweight, normal weight or obese subjects. In obesity the real vitamin D deficiency could be estimate by serum 1,25(OH)2D concentrations whose lower levels contribute to the higher PTH production and consequently to bone loss and to a greater fracture risk.

This review advances the discussion about the future of laboratory medicine in the 2020s. In five major topic areas: 1. the "big picture" of healthcare; 2. pre-analytical factors; 3. Analytical factors; 4. post-analytical factors; and 5. relationships, which explores a next decade perspective on laboratory medicine and the likely impact of the predicted changes by means of a number of carefully focused questions that draw upon predictions made since 2013. The "big picture" of healthcare explores the effects of changing patient populations, the brain-to-brain loop, direct access testing, robots and total laboratory automation, and green technologies and sustainability. The pre-analytical section considers the role of different sample types, drones, and biobanks. The analytical section examines advances in point-of-care testing, mass spectrometry, genomics, gene and immunotherapy, 3D-printing, and total laboratory quality. The post-analytical section discusses the value of laboratory medicine, the emerging role of artificial intelligence, the management and interpretation of omics data, and common reference intervals and decision limits. Finally, the relationships section explores the role of laboratory medicine scientific societies, the educational needs of laboratory professionals, communication, the relationship between laboratory professionals and clinicians, laboratory medicine financing, and the anticipated economic opportunities and outcomes in the 2020's.

BACKGROUND:Serum total 25-hydroxyvitamin D is a measure of the total circulating 25-hydroxyvitamin D concentration and is the primary measurement for estimating vitamin D status. A number of automated immunoassays are commercially available, and in an attempt to standardize the assays the Vitamin D Standardization Program (VDSP) was established in 2010. Therefore, the aim of the current project is to evaluate the status of the standardization of routinely used 25-hydroxyvitamin D assays. METHODS:200 patient serum samples were measured in Spring 2017 on seven different assays for 25-hydroxyvitamin D. Samples were measured in duplicate for the evaluation of precision. A certified standard reference material (SRM972a) from The National Institute of Standardization and Technology (NIST) was measured to evaluate the accuracy of the assays. Finally, the agreement of the assays of clinically categorizing patients into vitamin D deficiency, inadequacy or adequacy was evaluated. RESULTS:All seven assays achieved precision below the VDSP requirement of CV < 10%. However, only two of the assays achieved an accuracy bias <5% when measuring the SRM972a. When comparing methods using Deming regression, substantial proportional and/or systematic bias was found between many of the assays. Finally, when evaluating the ability of the assays to categorize patients into "vitamin D deficiency" (25-hydroxyvitamin D concentration < 30 nmol/L (<12 ng/mL)), "vitamin D inadequacy" (30-50 nmol/L (12-20 ng/mL)), "vitamin D adequacy" (50-250 nmol/L (20-100 ng/mL)) and "risk of toxicity" (>250 nmol/L (>100 ng/mL)), clinically relevant differences between assays were detected. Especially in the deficiency group, major discrepancies were found as the percentage of patients ranged from 1.5%-14.3% between the assays.. CONCLUSIONS:In conclusion, some of the commercially available assays have been standardized with performance as required by the VDSP. However, several of the assays do still not comply with the VDSP requirements even eight years after the program was started. This may have clinical consequences for patients, and manufacturers are therefore encouraged to continue their work on standardizing serum 25-hydroxyvitamin D assays.

Objectives: Vitamin D is involved in various physiologic and pathologic processes in the human body. The aim of this study was to determine the prevalence of vitamin D deficiency and the association of adiposity indicators with 25-hydroxyvitamin D (25[OH]D) in Chinese children and adolescents. Methods: This was a population-based, nationwide, multicenter cross-sectional study involving 10 696 participants (51.2% boys) 6 to 18 y of age. Total body fat mass was assessed by dual-energy x-ray absorptiometry, and measures of body mass index (BMI), fat mass index (FMI), fat mass percentage (FMP), and plasma 25(OH) D concentrations were obtained. Results: The adjusted mean of 25(OH)D was 39.3 nmol/L for all participants, 40.7 nmol/L for boys, and 37.9 nmol/L for girls. Of the children, 30% had vitamin D deficiency (25[OH]D <30 nmol/L) and 80% had vitamin D insufficiency (25[OH]D <50 nmol/L). The prevalence rates of vitamin D deficiency and insufficiency were higher in girls (31%, 83.4%, respectively) than in boys (22.8%, 78.7%, respectively). An L-shape relationship between age and 25(OH)D was observed in all children, with a threshold age of 14 y. Also, there was an inverted U-shaped association of BMI with 25(OH)D, and multivariable linear models shown FMI and FMP were inversely associated with 25(OH)D concentrations, particularly in boys (beta = 0.86 and 0.83, respectively, all P < 0.05). Conclusions: Vitamin D deficiency was widespread and its sex-specific association with an excess of body fat in Chinese children and adolescents. The findings indicate that targeted screening and treatment guidelines may be useful. (C) 2019 Elsevier Inc. All rights reserved.

Insulin‐like growth factors (IGFs) and insulin‐like growth factor binding proteins (IGFBPs) have been implicated in the aetiology of several cancers. To better understand whether anthropometric, behavioural and sociodemographic factors may play a role in cancer risk via IGF signalling, we examined the cross‐sectional associations of these exposures with circulating concentrations of IGFs (IGF‐I and IGF‐II) and IGFBPs (IGFBP‐1, IGFBP‐2 and IGFBP‐3). The Endogenous Hormones, Nutritional Biomarkers and Prostate Cancer Collaborative Group dataset includes individual participant data from 16,024 male controls (i.e. without prostate cancer) aged 22–89 years from 22 prospective studies. Geometric means of protein concentrations were estimated using analysis of variance, adjusted for relevant covariates. Older age was associated with higher concentrations of IGFBP‐1 and IGFBP‐2 and lower …

The process of osteoblast switching to alternative mesenchymal phenotypes is incompletely understood. In this study, we tested the ability of the osteoblast/preosteocyte osteogenic cell line, MLO-A5, to also differentiate into either adipocytes or chondrocytes. MLO-A5 cells expressed a subset of skeletal stem cell markers, including Sca-1, CD44, CD73, CD146, and CD166. Confluent cultures of cells underwent differentiation within 3 days upon the addition of osteogenic medium. The same cultures were capable of undergoing adipogenic and chondrogenic differentiation under lineage-appropriate culture conditions, evidenced by lineage-specific gene expression analysis by real-time reverse-transcription-PCR, and by Oil Red O and alcian blue (pH2.5) staining, respectively. Subcutaneous implantation of MLO-A5 cells in a gel foam into NOD SCID mice resulted in a woven bone-like structure containing embedded osteocytes and regions of cartilage-like tissue, which stained positive with both alcian blue (pH2.5) and safranin O. Together, our findings show that MLO-A5 cells, despite being a strongly osteogenic cell line, exhibit characteristics of skeletal stem cells and display mesenchymal lineage plasticity in vitro and in vivo. These unique characteristics suggest that this cell line is a useful model with which to study aging and disease-related changes to the mesenchymal lineage composition of bone.

Background Biochemical bone turnover markers (BTM) are useful tools to assess bone remodeling at the cellular level. N-terminal propeptide of type I procollagen (PINP) has been recommended as a reference marker for bone formation in research studies. Methods We describe the results of a multicenter study for routine clinical laboratory assays for PINP in serum and plasma. Four centers (Athens, Greece [GR], Copenhagen, Denmark [DK], Liege, Belgium [BE] and Sheffield, United Kingdom [UK]) collected serum and plasma (EDTA) samples from 796 patients presenting to osteoporosis clinics. Specimens were analyzed in duplicate with each of the available routine clinical laboratory methods according to the manufacturers' instructions. Passing-Bablok regressions, Bland-Altman plots, V-shape evaluation method and the concordance correlation coefficient for PINP values between serum and plasma specimens and between methods were used to determine the agreement between results. A generalized linear model was employed to identify possible variables that affected the relationship between the methods. Results We showed that both EDTA plasma and serum were suitable for PINP determination. We observed a significant proportional bias between Orion radioimmunoassay and the automated methods for PINP (Roche Cobas and IDS iSYS), which both gave very similar results. The multivariate model did not improve the excellent correlation that was observed between the methods. Conclusions Harmonization of PINP assays is possible by applying a correction factor or correctly assigning the values of the calibrators. This work will benefit from further collaboration between assays manufacturers and clinical laboratory professionals.

OBJECTIVE To identify DBP gene rs4588 and rs7041 polymorphisms and associate with participants' serum 25(OH)D and BMD levels. STUDY DESIGN Cross-sectional descriptive study design. PLACE AND DURATION OF STUDY Section of Chemical Pathology and Molecular Pathology, Department of Pathology and Laboratory Medicine, The Aga Khan University, Karachi, from July 2014 to September 2015. METHODOLOGY Blood samples from 98 young adults, out of 101 samples collected, were genotyped for GC rs4588 and rs7041 polymorphisms by polymerase chain reaction-based restriction fragment length polymorphism assay. Questionnaires were administered to obtain information on demographics and anthropometric characteristics, BMD was assessed with heel ultrasound and 25(OH)D was measured using a Chemiluminescence immunoassay. RESULTS High prevalence of vitamin D deficiency was noted in the study population n=87 (86.1%) having median (IQR) 25(OH)D levels of 14.9 (20.9) ng/ml, in males and 12.1 (51.8) ng/ml in females. The C/C genotype of SNP rs4588 had the highest proportion n=50 (51%), whereas for rs7041 genotype G/T was most frequently observed n=53 (54%) in subjects. Highest 25(OH)D levels were observed within the homozygous genotypes C/C median 25(OH)D 14.0 (49.6) and G/G (median 25(OH)D 14.9 (37.1) ng/ml. Statistically significant relationship was noted between rs7041 genotype G/T and BMD (p 0.037). CONCLUSION Hypovitaminosis D was frequently found in young adults. Furthermore, G/T variant of rs7041 polymorphism was associated with lower 25(OH)D serum levels.

Active vitamin D (1,25(OH)2D) has been shown to regulate numerous cell processes in mammary cells. Degradation of 1,25(OH)2D is initiated by the mitochondrial enzyme, 25-hydroxyvitamin D 24-hydroxylase (CYP24 A1), and provides local control of 1,25(OH)2D bioactivity. Several reports of the association between elevated CYP24 A1 activity and breast cancer incidence, suggest that CYP24 A1 may be a target for therapeutic intervention. Whether CYP24A1 activity within the mammary epithelium regulates 1,25(OH)2D levels and mammary gland development is yet to shown. We have used a conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium to demonstrate reduced terminal end bud number, ductal outgrowth and branching during puberty and alveologenesis at early pregnancy, by inhibiting proliferation but not apoptosis in both basal and luminal MECs. In vitro study showed increased sensitivity of luminal MECs to lower levels of 1,25(OH)2D with the ablation of Cyp24a1 activity. In summary, Cyp24a1 within MECs plays an important role in modulating postnatal and pregnancy-associated mammary gland development which provides support for inhibiting CYP24 A1 as a potential approach to activating the vitamin D pathway in breast cancer prevention and therapy.

We conducted a phenome-wide Mendelian randomization analysis (MR-PheWAS) to survey health effects associated with high normal serum calcium. We found causal evidence for conditions related to renal function, bone and joint health, and cardiovascular risk. These conditions collectively suggest that tissue calcification may be a key mechanism through which serum calcium influences health. Calcium is essential for the normal functioning of the cardiovascular system, muscles, and nerves. In this MR-PheWAS study, we sought to capture the totality of health effects associated with high normal serum calcium. We used data from up to 337,535 UK Biobank participants, and tested for associations between calcium genetic score (calcium-GS) and 925 disease outcomes, with follow-up analyses using complementary MR methods. Calcium-GS was robustly associated with serum calcium concentration (F statistics = 349). After multiple testing correction (P < 1.62E-4), we saw genetic evidence for an association between high serum calcium and urinary calculus (OR per 1 mg/dl 3.5, 95%CI 1.3–9.2), renal colic (9.1, 95%CI 2.5–33.5), and allergy/adverse effect of penicillin (2.2, 95%CI 1.5–3.3). Secondary analyses with independent replication from consortia meta-analyses suggested further effects on myocardial infarction and osteoarthrosis. We found causal evidence for effects of high normal serum calcium with conditions related to renal function, bone and joint health, and cardiovascular risk, which may collectively reflect influences on tissue calcification and immune function.

Vitamin D, along with calcium, is generally considered necessary for bone health and reduction of fractures. However, he effects of improving vitamin D status have not always been observed to improve bone mineral density (BMD). We have investigated whether varying vitamin D status in humans, as measured by serum 25(OH)D levels, relate to micro-structural and histomorphetric measures of bone quality and quantity, rather than density. Intertrochanteric trabecular bone biopsies and serum samples were collected from patients undergoing hip arthroplasty (65 females, 38 males, mean age 84.8 ± 8.3 years) at Royal Adelaide Hospital. Estimated GFR, serum ionized calcium, alkaline phosphatase, albumin, supplement and medication intake prior to surgery were taken from patient case records. Serum 25(OH)D, 1,25(OH)2D, and parathyroid hormone (PTH) levels were measured by immunoassays. Trabecular bone structural indices were determined by high-resolution micro-CT. Mean wall thickness (MWT) was measured on toluidine blue-stained histological sections. Bone mRNA levels for vitamin D metabolising enzymes CYP27B1 and CYP24A1 were measured by qRT-PCR. While serum 25(OH)D levels did not associate with bone volume/tissue volume (BV/TV%), serum 25(OH)D levels were strongly and independently associated with MWT (r = 0.81 p < 0.0001) with values significantly greater in patients with higher serum 25(OH)D levels. Furthermore, serum 25(OH)D levels were negatively associated with Bone Surface/Bone Volume (BS/BV) (r = −0.206, p < 0.05) and together with bone CYP27B1 and CYP24A1 mRNA accounted for 10% of the variability of BS/BV (p = 0.001). These data demonstrate that serum 25(OH)D is an independent positive predictor of micro-structural and bone formation measures and may be dependent, in part, on its metabolism within the bone.

Insulin‐like growth factors (IGFs) and insulin‐like growth factor binding proteins (IGFBPs) have been implicated in the aetiology of several cancers. To better understand whether anthropometric, behavioural and sociodemographic factors may play a role in cancer risk via IGF signalling, we examined the cross‐sectional associations of these exposures with circulating concentrations of IGFs (IGF‐I and IGF‐II) and IGFBPs (IGFBP‐1, IGFBP‐2 and IGFBP‐3). The Endogenous Hormones, Nutritional Biomarkers and Prostate Cancer Collaborative Group dataset includes individual participant data from 16,024 male controls (i.e. without prostate cancer) aged 22–89 years from 22 prospective studies. Geometric means of protein concentrations were estimated using analysis of variance, adjusted for relevant covariates. Older age was associated with higher concentrations of IGFBP‐1 and IGFBP‐2 and lower concentrations of IGF‐I, IGF‐II and IGFBP‐3. Higher body mass index was associated with lower concentrations of IGFBP‐1 and IGFBP‐2. Taller height was associated with higher concentrations of IGF‐I and IGFBP‐3 and lower concentrations of IGFBP‐1. Smokers had higher concentrations of IGFBP‐1 and IGFBP‐2 and lower concentrations of IGFBP‐3 than nonsmokers. Higher alcohol consumption was associated with higher concentrations of IGF‐II and lower concentrations of IGF‐I and IGFBP‐2. African Americans had lower concentrations of IGF‐II, IGFBP‐1, IGFBP‐2 and IGFBP‐3 and Hispanics had lower IGF‐I, IGF‐II and IGFBP‐3 than non‐Hispanic whites. These findings indicate that a range of anthropometric, behavioural and sociodemographic factors are associated with circulating concentrations of IGFs and IGFBPs in men, which will lead to a greater understanding of the mechanisms through which these factors influence cancer risk.

Vitamin D activities in osteocytes regulate important aspects of bone mineral metabolism and contribute to homeostasis of plasma calcium, phosphate, fibroblast growth factor-23 (FGF23), and 1,25(OH)2D3. These activities arise from plasma 1,25(OH)2D3 (endocrine activities) and from bone cell-synthesized 1,25(OH)2D3 (autocrine activities). 1,25(OH)2D3 regulates expression of the important osteocyte-specific genes including FGF23 and SOST. 1,25(OH)2D3 stimulates the maturation of cells throughout the osteoblast lineage as well as enhancing the terminal differentiation of osteoblasts into osteocytes. Although osteocytes are major regulators of osteoclastogenesis, the activity of 1,25(OH)2D3 to induce the osteoclastogenic cytokine, receptor activator of nuclear factor κ B ligand, particularly in vivo, is unclear at this time. Bone formation is also regulated by osteocytes at least partly under the control of 1,25-dihydroxyvitamin D (1,25(OH)2D) to both inhibit and enhance mineralization. 1,25(OH)2D stimulates cells in the osteocyte lineage to produce pyrophosphate (PPi), a strong inhibitor of mineralization. However, when alkaline phosphatase activity is high, then increased PPi enhances mineral deposition through the generation of inorganic phosphate within mineralization vesicles.