Plasma Osteocalcin Research Articles

Influence of N- and/or P-restriction on bone metabolism in young goats.

Ruminants can recycle nitrogen (N) and phosphorus (P), which are essential for vital body processes. Reduced N- and P-intake in ruminants is desirable for economic and ecologic reasons. Simultaneous modulation of mineral homoeostasis and bone metabolism occurs in young goats. This study aimed to investigate potential effects of dietary N- and/or P-restriction on molecular changes in bone metabolism. The twenty-eight young male goats were fed a control diet, an N-reduced diet, a P-reduced diet or a combined N- and P-reduced diet for 6-8 weeks. The N-restricted goats had lower plasma Ca concentration and higher plasma osteocalcin (OC) and CrossLaps concentrations. The P-restricted goats had reduced plasma inorganic phosphate (Pi) concentrations and increased plasma Ca concentrations. Due to the initiation of a signalling pathway that inhibits the fibroblast growth factor 23 (FGF23) expression, this was lower with P-restriction. Consequently, lower Pi concentrations were the main factor influencing the reduction in FGF23. The changes in mineral homoeostasis associated with P-restriction led to a reduction in OC, bone mineral content and mineral density. Simultaneously, bone resorption potentially increased with P-restriction as indicated by an increased receptor activator of NF-κB ligand/osteoprotegerin (OPG) ratio and an increase in OPG mRNA expression. Additionally, the increased mRNA expression of the calcitonin receptor during P-restriction points to a higher number of osteoclasts. This study demonstrates an impairment of bone remodelling processes in young goats by N- or P-restriction. With P-restriction, bone mineralisation rate was potentially reduced and bone quality impaired, while with N-restriction, bone remodelling increased.

Association between uncarboxylated osteocalcin, blood glucose, and BMI among Saudi diabetic patients: an evaluation study.

Uncarboxylated osteocalcin is an important osteocalcin enzyme found in the bloodstream and is a crucial protein for maintaining calcium binding in bones, controlling blood sugar levels, and balancing body minerals. Due to the lack of data, the current study intends to investigate the relationship between uncarboxylated osteocalcin levels and DM-II in Saudi patients. For 138 patients, case-control research was conducted in 2021-2023, with 69 type II diabetes mellitus patients and 69 matching healthy control participants. An enzyme immunoassay kit was used to quantify the levels of uncarboxylated osteocalcin in fasting blood samples, and an automated analyzer evaluated Hb1Ac, fasting blood glucose, enzymes, electrolytes, lipid, and kidney profiles. Data processing and analysis were carried out using GraphPad Prism statistical software. According to our study, patients with type II diabetes mellitus had considerably lower levels of uncarboxylated osteocalcin than healthy controls. According to the correlation analysis, uncarboxylated osteocalcin and fasting blood sugar had a negative relationship. In the overweight BMI group, uncarboxylated osteocalcin was considerably higher in control subjects. We concluded that, in Saudi type II diabetes mellitus patients, the compromised glucose level is associated with diminished serum uncarboxylated osteocalcin. This study has limitations, such as a small sample size and only measuring the uncarboxylated form of plasma osteocalcin. Future research is needed to understand how anti-diabetic drugs affect undercarboxylated osteocalcin's effect on metabolic control and provide more efficient techniques and resources in diabetes and osteoporosis prevention and care.

Impact of Long-Term Swimming Exercise on Rat Femur Bone Quality.

Considering the conflicting evidence regarding the potential long-term detrimental effect of swimming during growth on femur quality and fracture risk, our aim was to investigate the effect of eight months of swimming on femur quality. Twenty male eight-week-old Wistar rats were assigned into a swimming (SW; n = 10; 2 h/day, 5 days/week) or active control group (CG; n = 10, housed with running wheel) for eight months. Plasma osteocalcin and C-terminal telopeptide of type I collagen concentrations (ELISA) were assessed at baseline, four, and eight months of protocol. Femur structure (micro-computed tomography), biomechanical properties (three-point bending), and cellular density (histology) were determined after the protocol. SW displayed a lower uncoupling index, suggesting higher bone resorption, lower empty lacunae density, cortical and trabecular femur mass, femur length and cortical thickness, and higher cortical porosity than CG (p < 0.05). Although both biomarkers' concentrations decreased in both groups throughout the experiment (p < 0.001), there were no significant differences between groups (p > 0.05). No differences were also found regarding biomechanical properties, bone marrow adiposity, and osteocyte and osteoclast densities (p > 0.05). Long-term swimming was associated with unbalanced bone turnover and compromised femur growth, lower femur mass, and deteriorated cortical bone microarchitecture. However, femur trabecular microarchitecture and biomechanical properties were not affected by swimming.

Plasma undercarboxylated osteocalcin dynamics with glycemic stress reflects insulin sensitivity and beta-cell function in humans with and without T2DM

This study aimed to better understand the relationship between bone-related biomarkers and nutrient stress in the context of metabolic health. We investigated plasma osteocalcin (OC) during an oral glucose challenge and experimental hyperinsulinemia in Type 2 diabetes (T2DM) and lean healthy controls (LHC). Older individuals with obesity and T2DM (n = 9) and young LHCs (n = 9) underwent a 75g oral glucose tolerance test (OGTT) and a 40 mU/m2/min hyperinsulinemic-euglycemic clamp. Plasma undercarboxylated OC (ucOC) and total OC were measured at baseline, 60mins, and 120mins of the OGTT and clamp via ELISA. In addition, plasma alkaline phosphatase (ALP), leptin, adiponectin, Vitamin D and insulin were measured and indices of insulin sensitivity and β-cell function were derived. The T2DM group had lower (p<0.05) ucOC and ucOC:total OC ratio than LHC during both the OGTT and clamp. Further, baseline ucOC was positively correlated to indices of β-cell function and negatively correlated to indices of insulin resistance when both groups were combined (all p<0.05). Suppression of OC observed in T2DM may be related to glucose intolerance and insulin resistance. Similarly, our data suggest that the observed phenotypic differences between groups are likely a product of long-term glucose dysregulation rather than acute flux in glucose or insulin.

Mouse skeletal muscle adaptations to different durations of treadmill exercise after the cessation of FOLFOX chemotherapy.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is a treatment for colorectal cancer that can induce persistent fatigue and metabolic dysfunction. Regular exercise after chemotherapy cessation is widely recommended for cancer patients and has been shown to improve fatigue resistance in mice. However, gaps remain in understanding whether the early systemic and skeletal muscle adaptations to regular exercise are altered by prior FOLFOX chemotherapy treatment. Furthermore, the effects of exercise duration on early metabolic and skeletal muscle transcriptional adaptations are not fully established. Purpose: Investigate the effects of prior FOLFOX chemotherapy treatment on the early adaptations to repeated short- or long-duration treadmill exercise, including the fasting regulation of circulating metabolic regulators, skeletal muscle COXIV activity and myokine/exerkine gene expression in male mice. Methods: Male C57BL6/J mice completed 4 cycles of FOLFOX or PBS and were allowed to recover for 4-weeks. Subsets of mice performed 14 sessions (6d/wk, 18m/min, 5% grade) of short- (10min/d) or long-duration (55min/d) treadmill exercise. Blood plasma and muscle tissues were collected 48-72h after the last exercise bout for biochemical analyses. Results: Long-duration exercise increased fasting plasma osteocalcin, LIF, and IL-6 in healthy PBS mice, and these changes were ablated by prior FOLFOX treatment. Slow-oxidative soleus muscle COXIV activity increased in response to long-duration exercise in PBS mice, which was blocked by prior FOLFOX treatment. Fast-glycolytic plantaris muscle COXIV activity increased with short-duration exercise independent of FOLFOX administration. There was a main effect for long-duration exercise to increase fasting muscle IL-6 and COXIV mRNA expression independent of FOLFOX. FOLFOX administration reduced muscle IL-6, LIF, and BDNF mRNA expression irrespective of long-duration exercise. Interestingly, short-duration exercise suppressed the FOLXOX induction of muscle myostatin mRNA expression. Conclusion: FOLFOX attenuated early exercise adaptations related to fasting circulating osteocalcin, LIF, and IL-6. However, prior FOLFOX treatment did not alter the exercise adaptations of plantaris muscle COXIV activity and plasma adiponectin. An improved understanding of mechanisms underlying exercise adaptations after chemotherapy will provide the basis for successfully treating fatigue and metabolic dysfunction in cancer survivors.

Osteocalcin associates with bone mineral density and VDR gene polymorphisms in type 1 and type 2 diabetes.

Bone metabolism is impaired in diabetes mellitus (DM). Our objective is to evaluate the association of bone turnover markers (BTM) and vitamin D receptor (VDR) gene polymorphisms with bone mineral density (BMD) in DM type 1 (T1D) and DM type 2 (T2D). A total of 165 patients (53 T1D and 112 T2D) were enrolled. BMD was measured by dual-energy X-ray absorptiometry (DEXA). Plasma osteocalcin (OC), beta-CrossLaps (β-CTX) and N-amino terminal propeptide of type I collagen (P1NP) and VDR gene polymorphisms were evaluated. Participants were 53 T1D (41 years [31-48]) and 112 T2D (60 years [51-66]). BMD were not statistically different between the groups. OC (p<0.001) and P1NP levels (p<0.001) were higher in patients with T1D. The areas under the curve for the prediction of bone pathology were 0.732 (p=0.038) for OC in T1D and 0.697 (p=0.007) in T2D. A significant association was found between lower lumbar BMD and the A allele of BsmI (p=0.03), the A allele of ApaI (p=0.04) and the allele C of the Taql (p=0.046). Also, a significant correlation was found with higher OC levels and the G allele of BsmI (p=0.044), C allele of ApaI (p=0.011), T allele of Taql (p=0.006) and with C allele of FokI (p=0.004). The high negative predictive value of the cut-off point for OC suggests that could be useful in excluding the risk suffering bone loss, allowing offering a personalized clinical approach to prevent this pathology.

Thyroid Hormones Are Not Associated with Plasma Osteocalcin Levels in Adult Population with Normal Thyroid Function.

Thyroid hormones (THs) play an indispensable role in skeletal development and bone remodeling. Some studies have reported associations of THs with serum osteocalcin (OC) levels, but the results are quite inconsistent and the molecular mechanism of their simultaneous or interdependent activity on bone is almost unknown. Therefore, the aim of this study was to determine the possible associations of plasma THs with plasma OC levels and the possible mediating effect of OC on the relationship between THs and bone mineral density (BMD). For this purpose, out of the initial 1981 participants, we selected healthy euthyroid participants controlled for available confounding factors that can affect thyroid function and bone metabolism (N = 694). Given our results, we could not confirm any associations of THs with plasma OC levels nor the mediating effect of OC on the relationship between THs and BMD in euthyroid population. In the group of women controlled for menopause status (N = 396), we found a significant negative association of body mass index (BMI) with OC levels (β = −0.14, p = 0.03). We also found a negative association of free triiodothyronine (fT3) (β = −0.01, p = 0.02) and age (β = −0.003, p < 0.001) with BMD, and a positive association of BMI (β = 0.004, p < 0.001) and male gender (β = 0.1, p < 0.001) with BMD. In addition, we found significantly higher plasma OC levels and lower values of BMD in postmenopausal euthyroid women compared with premenopausal euthyroid women. In our opinion, the results of previous studies suggesting an association between circulating THs and serum OC levels may be influenced by an inconsistent selection of participants and the influence of confounding factors.

Investigating the Effects of Diet-Induced Pre-Diabetes on the Functioning of Calcium-Regulating Organs in Male Sprague Dawley Rats: Effects on Selected Markers.

Derangements to the functioning of calcium-regulating organs have been associated with type 2 diabetes mellitus (T2DM), a condition preceded by pre-diabetes. Type 2 diabetes has shown to promote renal calcium wastage, intestinal calcium malabsorption and increased bone resorption. However, the changes to the functioning of calcium-regulating organs in pre-diabetes are not known. Subsequently, the effects of diet-induced pre-diabetes on the functioning of calcium-regulating organs in a rat model for pre-diabetes was investigated in this study. Male Sprague Dawley rats were separated into two groups (n=6, each group): non-pre-diabetic (NPD) group and a diet-induced pre-diabetic (DIPD) group for 20 weeks. After the experimental period, postprandial glucose and HOMA-IR were analysed in addition to plasma and urinary calcium concentrations. Gene expressions of intestinal vitamin D (VDR), intestinal calbindin-D9k, renal 1-alpha hydroxylase and renal transient receptor potential vanilloid 5 (TRPV5) expressions in addition to plasma osteocalcin and urinary deoxypyridinoline concentrations were analysed at week 20. The results demonstrated significantly increased concentrations of postprandial glucose, HOMA-IR and urinary calcium in addition to unchanged plasma calcium levels in the DIPD group by comparison to NPD. Renal TRPV5, renal 1-alpha hydroxylase, intestinal VDR and intestinal calbindin-D9k expressions were increased in the DIPD group by comparison to NPD. Furthermore, plasma osteocalcin levels were increased and urine deoxypyridinoline levels were decreased in the DIPD group by comparison to NPD. These observations may suggest that calcium-regulating organs compensate for the changes to calcium homeostasis by inducing increased renal calcium reabsorption, increased intestinal calcium absorption and decreased bone resorption followed by increased bone formation.

Impact of hydrocortisone replacement on bone mineral density and bone turnover markers in patients with primary adrenal insufficiency.

A cross-sectional study was conducted in 37 PAI patients treated with HC. BMD and selected bone turnover markers (β-crosslaps and osteocalcin) were measured. A stepwise binary logistic regression model was applied to determine the independent variables associated with low BMD. Osteoporosis was noted in 14.3% and osteopenia in 34.3% of cases. These patients were older (p=0.01) and received higher daily HC dose compared to patients with normal BMD (p=0.01). BMD values in the lumbar spine and the femoral neck were negatively correlated with daily HC dose (r=-0.36, p=0.03 and r=-0.34, p=0.05, respectively). Plasma osteocalcin was negatively correlated with disease duration (r=-0.38, p=0.02) and cumulative HC dose (r=-0.43, p<0.01). In multivariate analysis, a daily HC dose ≥12 mg/m2/day was independently associated with a higher risk of osteopenia/osteoporosis [OR (95% CI), 9.0 (1.1-74.6); p=0.04]. Impaired bone mineralization in patients with PAI is correlated with HC dose. A daily HC dose ≥12 mg/m2/day was associated with an increased risk of osteopenia and osteoporosis in these patients.

Dose-response effects of alcohol on biochemical markers of bone turnover in non-human primates: Effects of species, sex and age of onset of drinking

PurposeAlcohol consumption suppressed bone turnover in male non-human primates; however, it is unclear the extent to which this effect depends upon biological variables. Using archived plasma samples, we investigated whether sex, age of onset of alcohol intake, and species influence the effects of graded increases in alcohol consumption on bone turnover markers. Methods91 male and female macaques (rhesus and cynomolgus), ranging in age from 4 years (adolescent) to 10 years (adult) were required to increase their consumption of ethanol in 30-day increments: 0 g/kg/day, followed by 0.5 g/kg/day, 1.0 g/kg/day, and, finally, 1.5 g/kg/day. Plasma osteocalcin (formation), plasma CTX (resorption) and osteocalcin to CTX ratio (turnover balance) were measured during these intervals to assess the dose-response effects of alcohol. ResultsWe detected no relationship between dose and osteocalcin when all monkeys were combined, but there was a significant effect of sex (lower levels in females) and interactions between alcohol dose and sex (osteocalcin levels increased with dose in rhesus females). In contrast, we detected a negative linear dose-response relationship for ethanol and CTX. We did not detect a relationship between dose and osteocalcin to CTX ratio overall, but there was a significant positive relationship detected in females (no change in males). Increased age predicted lower biomarker levels for both osteocalcin and CTX. Species was a significant predictor for osteocalcin and the osteocalcin to CTX ratio in these models. ConclusionThese findings indicate that age, sex, and species influence bone turnover and support the concept that factors beyond quantity of alcohol affect skeletal response to alcohol consumption.

Effects of vitamin D and high dairy protein intake on bone mineralization and linear growth in 6- to 8-year-old children: the D-pro randomized trial

Vitamin D and dairy protein may stimulate bone mineralization and linear growth in children, but previous studies show inconsistent results and have not examined their combined effects. To investigate combined and separate effects of vitamin D supplementation and high-protein (HP) compared with normal-protein (NP) yogurt intake on children's bone mineralization and linear growth. In a 2×2-factorial trial, 200 healthy, 6- to 8-year-old, Danish, children with light skin (55°N) were randomized to 20µg/d vitamin D3 or placebo and to substitute 260g/d dairy with HP (10g protein/100g) or NP (3.5g protein/100g) yogurt for 24 weeks during an extended winter. Outcomes were total body less head (TBLH) and lumbar spine bone mineral density (BMD), bone mineral content (BMC), and bone area (BA) by dual-energy X-ray absorptiometry, height, and biomarkers of bone turnover and growth. The primary outcome was TBLH BMD. In total, 184 children (92%) completed the study. The baseline serum 25-hydroxyvitamin D was 80.8±17.2nmol/L, which increased by 7.2±14.1nmol/L and decreased by 32.3±17.5nmol/L with vitamin D and placebo, respectively. The baseline protein intake was 15.4±2.4 energy percentage (E%), which increased to 18.3±3.4 E% with HP. There were no vitamin D-yogurt interactions and no main effects of either intervention on TBLH BMD. However, vitamin D supplementation increased lumbar spine BMD and TBLH BMC compared to placebo, whereas HP groups showed lower increments in lumbar spine BMD, TBLH BMC and BA, and plasma osteocalcin compared to NP groups. Height, growth factors, and parathyroid hormone levels were unaffected. Although there were no effects on whole-body BMD, vitamin D increased bone mass and spinal BMD, whereas high compared with normal dairy protein intake had smaller incremental effects on these outcomes. This supports a recommended vitamin D intake of around 20µg/d during winter but not use of HP dairy products for improved bone mineralization among healthy, well-nourished children. This trial was registered at clinicaltrials.gov as NCT03956732.

Growth Hormone As Antiaging Factor in Old Bones.

Aging induces changes in bone. Growth hormone (GH) is reduced by aging, and age-related changes observed in old bones might be due to a decrease in the GH/insulin-like growth factor-I (IGF-I) axis. GH administration on aged individuals is controversial. This study aimed to assess the effect of systemic GH treatment on bone properties, bone metabolism, and bone mineral density (BMD) in long bone of old rats. Aged Wistar rats were treated with GH at a dose of 2 mg/kg/day during 10 weeks. Plasma osteocalcin, IGF-I, and carboxy-terminal telopeptide of type I collagen levels were measured. Cross-sectional bone areas and BMD were measured by morphometric and densitometric analysis, respectively. Femora were analyzed by three point-bending testing. t-Test was used for statistical evaluation. p < 0.05 was considered to be significant. Significantly enhanced bone area, at the expense of the cortical area, was found in treated rats. The densitometric analysis showed 11% higher BMD in the experimental group. Significantly higher bone flexural modulus, stiffness, and ultimate load were observed in the treated rats. Plasma osteocalcin and IGF-I levels were significantly increased in the treated group, while the resorption marker concentration remained unchanged. Within the limitations of this experimental study, systemic GH administration has shown to enhance biomechanical properties, BMD, cortical mass, and plasma IGF-I and osteocalcin in old treated rats, compared to the control group; consequently, GH could be considered as an alternative therapy against age-related changes in the bone.

Abstract P060: Plasma Osteocalcin And Incident Diabetes Mellitus: The Atherosclerosis Risk In Communities (ARIC) Study

Introduction: Diabetes is a major risk factor for cardiovascular disease. Osteocalcin is a vitamin K-dependent, bone-derived hormone that functions as an endocrine regulator of energy metabolism, male fertility, and cognition. Early studies of endocrine effects of osteocalcin have shown that genomic deletion of osteocalcin in mice resulted in a diabetic phenotype (i.e. glucose intolerance, and insulin resistance). However, results from clinical studies have shown mixed associations between blood levels of osteocalcin and risk of incident type 2 diabetes mellitus. Hypothesis: Lower values of plasma osteocalcin would be associated with an increased risk of diabetes. Methods: A total of 11,557 ARIC participants without diabetes at baseline were followed from ARIC visit 3 (1993-1995) through 2018. Diabetes cases were identified through self-report on annual and semi-annual follow-up phone calls. Plasma osteocalcin data was measured using an aptamer-based proteomic profiling platform (SomaLogic). We used Cox regression to evaluate the association of quintiles of plasma osteocalcin and incident diabetes. The primary model adjusted for age, sex, and race-center. Results: Participants were age 60 ± 5.6 years at visit 3, 56% identified as female, 21% identified as Black. There were 3,031 incident diabetes cases over a median follow-up of 17.9 years. Mean ± SD was 10.053 ± 0.775. When comparing the highest quintile of plasma osteocalcin (values 10.42 to 14.66) to the lowest quintile (values 9.03 to 9.52), there was no association with incident diabetes (HRs [95% CIs]: 0.92 [0.81, 1.02]). There was also no significant trend across the quintiles (p = 0.19). Results were similar when adjusting for additional potential confounders, and when limiting the follow-up time to 10 years. Conclusions: These data do not support the hypothesis that total plasma osteocalcin, as measured by Somalogic proteomic panel, is a biomarker associated with diabetes risk. It is possible that total plasma or serum osteocalcin and/or other isoforms of osteocalcin protein (i.e. gamma carboxylated or uncarboxylated osteocalcin) measured via other validated methodologies may be linked to diabetes.

Effects of boric acid supplementation on bone health in crossbred calves under tropical condition

IntroductionBoron (B) is thought to play key role in proper bone growth and development as well as have some role in regulation of minerals such as calcium (Ca), phosphorus (P) and magnesium (Mg) which act synergistically with vitamin D. ObjectivePresent study was planned in two phases to assess the effect of optimum and supranutritional levels of (B) in the form of boric acid (BA) supplementation on bone health of growing cross bred calves. MethodDuring Phase-1, twenty four male crossbred calves were blocked into four groups (n = 6) on the basis of their body weight (154.83 ± 8.5 kg), age (7–9 months) and were supplemented with 0 (C), 2.6 (T-1), 5.4 (T-2) and 10.7 (T-3) g BA for appropriate B (0.175 adjustment factor to calculate B form BA) consumption i.e. 0, 100, 200 and 400 ppm in each group respectively, for 90 days. During phase 2, twenty-one male crossbred calves were divided into 3 groups (n = 7) on the basis of their body weight (103.76 ± 4.34 kg) and age (5–8 months). All the groups were on similar dietary regimen with additional supplementation of boric acid as 0 g (control); 3.6 g (200 ppm B; T-1) and 10.8 g (600 ppm B; T-2), respectively for a period of 120 d. ResultsFrom the first experiment it is reported that plasma levels of bovine alkaline phosphatase (BALP), type I collagen cross-linked N-telopeptide (NTx) and Ca were significantly (P < 0.05) affected in T-2 and T-3 groups as compared to T-1 and control groups. Whereas, plasma osteocalcin (OCN) concentration was found to be higher in T-2 and T-3 groups as compared to control group. However, plasma concentrations (ng/mL) of tartrate resistant acid phosphatase (TRAP) remained unaltered due to dietary treatments. Based on the results, another experiment was conducted to validate the above findings and further to determine the effect of still higher i.e supranutritional levels of BA supplementation on bone health of calves. Results revealed that supplementation of BA in T-2 group had no beneficial effect on bone health as the plasma concentration of BALP, OCN, NTx, 25 (OH) vitamin D and Ca as compared to T-1 group in phase 2. Other possible attributes of bone health i.e. plasma concentration of Mg, P, parathyroid hormone (PTH), and calcitonin were not affected by BA supplementation at any levels. ConclusionOverall from present study it can be concluded that supplementation of boric acid 3.6 g/d (equivalent to 200 ppm B) in the diet of growing animals has positive effect on bone health related biomarkers (OCN, NTx and BALP) and supplementation of supranutritional level of BA i.e. 10.8 g (equivalent to 600 ppm B) level had neither additional beneficial nor harmful effect on bone health of calves.

Relationship Between Chronic Kidney Disease, Glucose Homeostasis, and Plasma Osteocalcin Carboxylation and Fragmentation

Chronic kidney disease (CKD) is associated with reduced insulin sensitivity, through mechanisms that are not well understood. Low vitamin K intake and incomplete carboxylation of the vitamin K-dependent protein osteocalcin may promote insulin resistance. We assessed relationships of osteocalcin concentration, carboxylation, and fragmentation with CKD and glucose homeostasis in a cross-sectional study. We included 87 participants without diabetes: 50 (27 female) with moderate to severe CKD (estimated glomerular filtration rate <60mL/min/1.73m2 not treated with dialysis) and 37 (17 female) healthy controls. Total osteocalcin was measured by immunoassay, and osteocalcin carboxylation and fragmentation status by liquid chromatography-electrospray ionization-based mass spectrometric immunoassay. Endpoints included glucose tolerance (based on 2-hour oral glucose tolerance test), insulin sensitivity (hyperinsulinemic-euglycemic clamp), and pancreatic beta-cell function (intravenous glucose tolerance test). The total plasma osteocalcin concentration was higher in the CKD group (mean [standard deviation] 102.9 [147.5]) than that in the control group (53.6 [51.1] ng/mL, P=.03), and more osteocalcin was circulating as fragments. The extent of osteocalcin carbocylation did not differ between individuals with and without CKD. Osteocalcin concentration, carboxylation, and fragmentation were not associated with any measure of glucose homeostasis in multivariable-adjusted analyses. In CKD, circulating osteocalcin concentrations are elevated, in part due to larger proportions of fragmented forms. However, osteocalcin carboxylation status is not significantly different between individuals with and without CKD. Our data also do not provide support for the hypothesis that differences in osteocalcin carboxylation may explain reduced insulin sensitivity in individuals with CKD.

Validation of an enzyme-linked immunoassay assay for osteocalcin, a marker of bone formation, in dried blood spots.

Investigating factors that contribute to bone loss and accretion across populations in remote settings is challenging, particularly where diagnostic tools are scarce. To mitigate this challenge, we describe validation of a commercial ELISA assay to measure osteocalcin, a biomarker of bone formation, from dried blood spots (DBS). We validated the Osteocalcin Human SimpleStep ELISA kit from Abcam (ab1951214) using 158 matched plasma and DBS samples. Passing-Bablok regression analysis assessed the relationships between plasma and DBS osteocalcin concentrations. Dilutional linearity and spike and recovery experiments determined if the DBS matrix interfered with osteocalcin measurement, and intra- and inter-assay coefficients of variation (CVs) were calculated. Limit of detection, analyte stability, and specific forms of osteocalcin measured by the kit were also investigated. Mean plasma osteocalcin value was 218.2 ng/mL (range 64.6-618.1 ng/mL). Linear relationships existed between plasma and DBS concentrations of osteocalcin, with no apparent bias in plasma vs DBS concentrations. There was no apparent interference of the DBS matrix with measurement of osteocalcin in DBS. Intra-assay CV for DBS was ~8%, while average inter-assay CV was 14.8%. Limit of detection was 0.34 ng/mL. Osteocalcin concentrations were stable in DBS stored at -28°C and room temperature, but not those stored at 37°C. This ELISA kit detects total osteocalcin. Osteocalcin, a bone formation biomarker, can be measured from DBS. Combined with a previously validated DBS assay for TRACP-5b, a bone resorption biomarker, these assays have the potential to help researchers disentangle the many factors contributing to bone strength.

Plasma Osteocalcin Levels, Status of Oral Disease and Alteration in Mandibular Bone Density in Postmenopausal Women

The present study was undertaken for assessing plasma osteocalcin levels, status of oral disease, and alteration in mandibular bone density in postmenopausal women (PMW). In all, 80 premenopausal women and 80 PMW were enrolled. For analyzing the oral dryness, clinical score of oral dryness (CSOD) on a scale of up to 10 was used. Complete dental profiling of all the patients was done. Digital panoramic radiographs were taken for all the patients. Bone mineral density (BMD) was evaluated by measuring the following parameters: mandibular cortical index (MCI), panoramic mandibular index (PMI), mandibular cortical width (MCW), and fractal dimension (FD). Osteocalcin levels were evaluated with enzyme-linked immunosorbent assay technique. All the results were recorded and analyzed. Mean osteocalcin levels of PMW (453.12 ng/mL) were significantly higher in comparison to the premenopausal women (249.28 ng/mL). Postmenopausal women had significantly higher CSOD and number of peri-apical radiolucencies in comparison to premenopausal women. Bone mineral density as assessed by MCI was found to be negatively and significantly correlated with oral disease status and osteocalcin levels. Significant difference was obtained while comparing the MCI inbetween the two study groups. There is significantly higher prevalence of oral lesions along with oral dryness in postmenopausal women. Also, thinning of mandibular cortex is significantly higher in postmenopausal women. Higher plasma osteocalcin levels help in predicting osteopenia/osteoporosis at an early stage in such patients. In PMW, special considerations should be made while planning for dental implant therapy.

Increased Plasma Osteocalcin, Oral Disease, and Altered Mandibular Bone Density in Postmenopausal Women.

An association between oral diseases and postmenopausal status has been recognized. However, the relationship between all oral disease, mandibular bone density, health status, and osteocalcin (OCN) bone markers in postmenopausal dental patients has not been reported. This study was therefore to verify the differences in plasma OCN levels, dental, periodontal, and oral mucosal disease, and mandibular bone density alterations from panoramic radiograph and systemic parameters in postmenopausal women, compared to premenopausal women. Oral, radiographic, and blood examination were performed in 92 females. Dental, periodontal, and oral mucosal statuses were recorded. Health profile parameters were collected from medical charts. Plasma OCN was evaluated by enzyme-linked immunosorbent assay. Forty-two (45.7%) participants were postmenopausal with a higher median age (55 (51, 62) years) than the premenopausal group (43 (38, 45) years). Overweight or obesity, hypercholesterolemia, and impaired fasting blood sugar were more prevalent in postmenopause. The average postmenopausal OCN level (425.62 ng/mL) was significantly higher than the premenopausal group (234.77 ng/mL, p < 0.001). The average number of missing teeth, mean attachment loss, alveolar bone loss, periapical lesion count, and clinical oral dryness score were also significantly higher in postmenopause (p=0.008, < 0.001, 0.031, 0.006, and 0.005, respectively). However, mandibular bone density determined by mandibular cortical index was lower in postmenopause (p < 0.001). The panoramic mandibular index, mandibular cortical width, fractal dimension, and other oral mucosal disease did not differ between the groups. Postmenopause was associated with elevated plasma OCN (β = 0.504, p < 0.001) when related covariates were adjusted. Elevated plasma OCN, oral mucosal dryness, high number of periapical radiolucencies and missing teeth, and lower mandibular bone density from panoramic radiograph were prevalent in postmenopausal women. Dentists should suspect an increased risk of low bone mineral density in postmenopausal patients who display these clinical and radiographic findings, and they should be referred for further examination. Plasma OCN may interconnect a relationship between postmenopausal status and the low mandibular bone density.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n=13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22h/d, 7 d/wk, for a total of 12months. Control animals (n=9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. Monkeys in the EtOH group consumed an average of 2.8±0.2 (mean±SE) g/kg/d of EtOH (30±2% of total calories), resulting in an average blood EtOH concentration of 88.3±8.8mg/dl 7hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

Circulating sclerostin levels in relation to nutritional status, sex hormones and selected bone turnover biochemical markers levels in peri- and postmenopausal women

Hormonal changes during the peri- and postmenopausal age, especially decreasing estradiol levels as the result of the expired ovarian function, are an established link of the pathogenesis of postmenopausal osteoporosis. The objective of the study was to examine the association between the circulating sclerostin levels and nutritional status, sex hormones and selected bone markers turnover levels in peri- and postmenopausal women. The study enrolled 84 stable-body mass women (31 perimenopausal and 54 postmenopausal). Anthropometric measurements and serum estrone, testosterone, androstenedione, DHEA-S, osteocalcin, β-CTx, 25-OHVitamin D and sclerostin levels were obtained. There were not any differences between body mass, BMI, body fat and waist circumference between the study groups. The serum androstenedione and DHEA-S levels were similar in both study groups. However, estrone and total testosterone levels were observed to be notably higher in the perimenopausal group, unlike in the postmenopausal group (124.1 pg/mL vs. 98.3 pg/mL, p < 0.01 and 0.3 pg/mL vs. 0.22 pg/mL, p < 0.01, respectively). Higher plasma osteocalcin and β-CTx levels were shown in the postmenopausal rather than in the perimenopausal group (19.8 ng/mL vs. 16.8 ng/mL, p < 0.001 and 0.35 ng/mL vs. 0.29 ng/mL, p < 0.05, respectively). Plasma sclerostin and 25-OH-Vitamin D levels were similar. There was not any correlation between plasma sclerostin levels and the other studied parameters. In the multivariate regression analyses, sclerostin levels were proportional to the androstenedione ones (b = 0.06; p < 0.05) but inversely related to the log10(testosterone) levels (b = -0.18; p < 0.05). Circulating sclerostin levels are similar in peri- and postmenopausal women and are related to the androstenedione and testosterone levels regardless of the nutritional status.