Magnetic solid-phase extraction of high molecular weight peptides using stearic acid-functionalized magnetic hydroxyapatite nanocomposite: determination of some hypothalamic agents in biological samples.

Therapeutic peptides have an important effect on physiological function and human health, so it is momentous to quantify and detect low levels of these biomolecules in biological samples for treatment and diagnostic purposes. In the present study, an efficient magnetic solid-phase extraction (MSPE) method was developed based on stearic acid-functionalized magnetic hydroxyapatite nanocomposite (MHAP/SA) as a novel and cost-effective adsorbent for extraction of five hypothalamic-related peptides (goserelin, octreotide, triptorelin, somatostatin, and cetrorelix) from biological samples. To characterize the morphology and physicochemical properties of MHAP/SA, Fourier transform infrared spectroscopy (FT-IR), energy-dispersive X-ray spectroscopy (EDS), field emission scanning microscopy (FE-SEM), CHNS elemental analysis, Brunauer-Emmett-Teller (BET), and vibrating sample magnetometry (VSM) were applied. Under optimum conditions, the proposed method (MSPE-HPLC-UV) represented favorable linearity with R2 ≥ 0.9987, suitable intra- and inter-day precisions (RSD ≤ 6.9% and RSD ≤ 8.1%, respectively, n = 3), and limits of detection and quantification in the range of 0.75-1.12 ng mL-1 and 2.50-3.75 ng mL-1, respectively. Eventually, the proposed method was used for the extraction and quantification of target therapeutic peptides in plasma and urine samples, and satisfactory relative recoveries were achieved in the range of 90.6-110.3%.

Bone turnover in pregnancy, measured by urinary CTX, is influenced by vitamin D supplementation and is associated with maternal bone health: findings from the Maternal Vitamin D Osteoporosis Study (MAVIDOS) trial.

BACKGROUND: The pattern of change in maternal bone turnover throughout pregnancy is poorly characterized. OBJECTIVES: We investigated changes across pregnancy in a marker of maternal bone resorption, urinary C-terminal telopeptide of type I collagen (CTX), the influence of gestational vitamin D supplementation, and associations between CTX and maternal postnatal bone indices. METHODS: MAVIDOS (the Maternal Vitamin D Osteoporosis Study) is a randomized, double-blind, placebo-controlled trial of 1000 IU cholecalciferol/d compared with placebo from 14 weeks of gestation to birth. Maternal second-void urinary α- and ß-CTX were measured (ELISA) at 14 and 34 weeks of gestation; DXA was performed within 2 wk postpartum. The Mann-Whitney Rank Sum test, Spearman's rank correlation, and linear regression were used to compare median CTX values within and between groups from early to late pregnancy, and associations with maternal bone outcomes. RESULTS: In total, 372 women had CTX and 25-hydroxyvitamin D [25(OH)D] measured in early and late pregnancy. CTX at 14 and 34 weeks of gestation were correlated in both placebo (r = 0.31) and cholecalciferol (r = 0.45) groups (P < 0.0001). Median CTX increased from 14 to 34 weeks of gestation in both groups (n = 372 total) [placebo (n = 188): from 223.6 to 449.7 µg/mmol creatinine; cholecalciferol (n = 184): from 222.3 to 419.3 µg/mmol creatinine; P = 0.03 for placebo compared with cholecalciferol difference in CTX at 34 weeks of gestation]. The conditional mean ± SD increase in CTX [z-score (SD)] from early to late pregnancy was greater in the placebo group (n = 188) than in the cholecalciferol group (n = 184) (placebo: 0.16 ± 0.92; cholecalciferol: -0.16 ± 1.06; P-difference < 0.01). Higher CTX at 34 weeks of gestation was associated, similarly in both groups, with lower maternal total hip and lumbar spine bone mineral content and bone mineral density (BMD) (e.g., lumbar spine BMD: ß = -0.02 g · cm-2 · SD-1 increase in CTX; 95% CI: -0.027, -0.002 g · cm-2 · SD-1; P = 0.02, n = 283). CONCLUSIONS: Maternal urinary CTX, a bone resorption marker, rises through pregnancy, although to a lesser degree with gestational cholecalciferol supplementation, and is inversely associated with maternal bone mass postpartum.This trial was registered at www.isrctn.com as ISRCTN 82927713 and eudract.ema.europa.eu as EudraCT 2007-001716-23.

Changes in levels of cartilage oligomeric proteinase and urinary C-terminal telopeptide of type II collagen in subjects with knee osteoarthritis after dextrose prolotherapy: A randomized controlled trial.

OBJECTIVE: To assess the effects of dextrose prolotherapy in patients with knee osteoarthritis on the levels of serum cartilage oligomeric proteinase and urinary C-terminal telopeptide of type II collagen, and on the Western Ontario McMaster Universities Index and numerical rating scale score for pain. METHODS: A randomized controlled trial, in which participants were randomly allocated into 2 groups, receiving injections of either hyaluronic acid or dextrose prolotherapy. The hyaluronic acid group received 5 injections, 1 each on weeks 1, 2, 3, 4 and 5, and the dextrose prolotherapy group received 3 injections, 1 each on weeks 1, 5 and 9. Serum cartilage oligomeric proteinase, urinary C-terminal telopeptide of type II collagen, Western Ontario McMaster Universities Index score, and numerical rating scale score for pain were measured at baseline and 3 weeks after the last injection. Comparative analysis was conducted using Wilcoxon test within groups and analysis of covariance (ANCOVA) test between groups. RESULTS: A total of 47 participants (21 allocated to hyaluronic acid, 26 allocated to dextrose prolotherapy) completed the protocol. Both interventions resulted in significant improvements in numerical rating scale scores for pain, total Western Ontario McMaster Universities Index scores, and its subscales score. However, the dextrose prolotherapy outperformed hyaluronic acid in numerical rating scale score for pain and level of urinary C-terminal telopeptide of type II collagen, with score changes differences of 0.93 (p = 0.042) and 0.34 (p = 0.048), respectively. No significant changes in level of serum cartilage oligomeric proteinase were found in either group. CONCLUSION: Dextrose prolotherapy is an alternative injection therapy for knee osteoarthritis, which was found to be associated with a significant reduction in urinary C-terminal telopeptide of type II collagen compared with hyaluronic acid injection. Neither injection method resulted in reduced serum cartilage oligomeric proteinase.

Effects of Equol Supplement on Bone and Cardiovascular Parameters in Middle-Aged Japanese Women: A Prospective Observational Study.

OBJECTIVE: To examine changes in the bone and cardiovascular parameters and tolerability in middle-aged Japanese women taking equol supplement for a year. DESIGN: This was a prospective observational study. SUBJECTS AND SETTING: Participants were 74 women receiving outpatient care at Hamasite Medical Clinic, Minato-ku, Tokyo, from 2013 to 2015. INTERVENTIONS: Participants received per oral equol-containing supplement, 10 mg/day. OUTCOME MEASURES: The primary outcome measures were percent changes in bone and cardiovascular parameters after 1 year supplementation with equol. The secondary measures included factors affecting the parameter changes and adverse effects associated with equol use for a year. RESULTS: Reduction in arterial stiffness was observed after 12 months of equol supplement (1402.3 cm/s vs.1367.3 cm/s, p < 0.001). Significant reductions in respective parameters were observed in women with moderate and high risk for arteriosclerosis (median [95% confidence interval]: -3.2% [-5.79 to -0.74]; -12.65% [-18.52 to -4.28]; respectively); hypertriglyceridemia -45.53% [-70.24 to -5.58]; bone resorption risk (-15.15% [-23.71 to 1.56]; and bone fracture risk -26.68% [-76.43 to -5.99]. All 15 women with high baseline parathyroid hormone levels had achieved a median of 50% [-54.11 to -31.69] reduction from their baseline values. These associations were further confirmed in the results of multiple linear regression analysis. There were no reported adverse events or abnormal findings in the blood chemistry, Pap smear, mammography, and transvaginal ultrasound during periodic follow-ups. CONCLUSION: One year equol supplement was tolerable and induced improvement of certain bone and cardiovascular parameters, especially in higher risk groups. Further controlled studies are needed to explore long-term equol use for wellbeing of middle-aged women.

Vitamin D and Calcium Addition during Denosumab Therapy over a Period of Four Years Significantly Improves Lumbar Bone Mineral Density in Japanese Osteoporosis Patients.

This study investigated whether or not vitamin D and calcium supplementation affected bone metabolism and bone mineral density (BMD) over a period of four years of denosumab therapy in patients with primary osteoporosis. Patients were divided into a denosumab monotherapy group (22 cases) or a denosumab plus vitamin D and calcium supplementation group (combination group, 21 cases). We measured serum bone alkaline phosphatase (BAP), tartrate-resistant acid phosphatase (TRACP)-5b, urinary N-terminal telopeptide of type-I collagen (NTX), and BMD of the lumbar 1-4 vertebrae (L-BMD) and bilateral hips (H-BMD) at baseline and at 12, 24, 36, and 48 months of treatment. There were no significant differences in patient background. Serum BAP, TRACP-5b, and urinary NTX were significantly and comparably inhibited in both groups from 12 to 48 months versus baseline values. L-BMD was significantly increased at every time point in both groups, while H-BMD was significantly increased at every time point in the combination group only. There were significant differences between the groups for L-BMD at 24, 36, and 48 months (P < 0.05) and for H-BMD at 12 months (P < 0.05). Compared with denosumab monotherapy, combination therapy of denosumab plus vitamin D and calcium significantly increased H-BMD at 12 months and L-BMD from 24 to 48 months. These findings indicate that continuous vitamin D and calcium supplementation is important, especially for 12 months to improve H-BMD and from 24 to 48 months to improve L-BMD.

Changes in Bone Metabolism in Morbidly Obese Patients After Bariatric Surgery: A Meta-Analysis.

BACKGROUND: The aim is to evaluate via meta-analysis bone metabolism and bone mineral density (BMD) in morbidly obese patients before and after bariatric surgery. METHODS: We searched Medline, EMBASE, and the Cochrane Library for relevant studies published before January 2014. The following outcomes were evaluated: serum calcium, serum parathyroid hormone (PTH), serum 25-hydroxyvitamin D [25(OH)D], serum or urinary N-telopeptide (NTX), bone-specific alkaline phosphatase (BSAP), and bone mineral density (BMD). RESULTS: Ten studies, including 344 patients, met our inclusion criteria. Results showed a significant decrease in serum calcium (MD = -0.10, 95 %CI -0.14 to -0.07, P < 0.00001) and increase in serum PTH (MD = 12.41, 95 %CI 6.51 to 18.31, P < 0.00001) but no significant difference in serum 25(OH)D (MD = 1.35, 95 %CI -1.12 to 3.83, P = 0.28) following bariatric surgery. There were significant increases in serum or urinary NTX (MD = 18.49, 95 %CI 3.33 to 33.66, P = 0.02) and BSAP (MD = 7.47, 95 %CI 0.21 to 14.72, P = 0.04) but a significant decrease in BMD (MD = -0.08, 95 %CI -0.13 to -0.04, P < 0.00001) after bariatric surgery. CONCLUSION: BMD was significantly decreased, while bone turnover was elevated, and bone remodeling was accelerated following bariatric surgery. Basal bone metabolism should be evaluated preoperatively. To prevent secondary hyperparathyroidism and bone loss, calcium and vitamin D should be monitored closely and supplemented accordingly after the surgery.

Comparison of clinical cut-points and treatment targets for urine NTX and plasma ßCTX-I in osteoporosis.

OBJECTIVE: We undertook to identify levels for plasma ß isomerised carboxy-terminal telopeptides of type I collagen (p-ßCTX-I) that are comparable to currently used urine amino-terminal telopeptides of type I collagen (u-NTX) cut-points and treatment targets in osteoporosis. DESIGN AND METHODS: Fasting morning samples were collected from patients attending tertiary hospitals and clinics for investigation of metabolic bone disease. Patients with Paget's disease or <20years of age were excluded. Second void spot urine for NTX and plasma (EDTA) samples were utilised. Urine was analysed routinely and plasma stored at -20C until analysis by enzyme-linked immunosorbent assay (ELISA) (Immunodiagnostic Systems plc), E170 (Roche Diagnostics) and IDS-iSYS (Immunodiagnostic Systems plc) methods. The relationship of u-NTX with each p-ßCTX-I method's results was assessed by Passing and Bablok regression, and p-ßCTX-I levels equivalent to u-NTX cut-points and targets were interpolated. RESULTS: One hundred and forty six patients were included. Spearman correlation coefficients ranged from 0.71 to 0.75 for the three ßCTX-I assays. The equivalent ßCTX-I concentrations for NTX/Cr values of 21 (fracture risk reduction target following risedronate therapy), 27 (healthy pre-menopausal women's mean value), and 38 (threshold for reduction of BMD on calcium alone) nmol BCE/mmol were 230, 312 and 462ng/L for the automated Roche assay and 271, 395 and 624ng/L for the automated IDS i-SYS assay respectively. CONCLUSIONS: The p-ßCTX-I equivalent to the only available fracture outcome based absolute treatment threshold of 21nmol BCE/mmol established for u-NTX, is close to 250ng/L but will vary between p-ßCTX-I assays.

Urinary N telopeptide levels in predicting the anti-nociceptive responses of zoledronic acid and paclitaxel in a rat model of bone metastases.

The present study investigated the hypothesis that urinary levels of N telopeptide (NTx) can be used to predict the anti­nociceptive responses of zoledronic acid and paclitaxel on bone metastases in a rat model. Rats were implanted with intra­femur Walker 256 carcinoma cells or control solution, and were treated with either normal saline, zoledronic acid or paclitaxel on the 10th day following surgery. Mechanical allodynia was recorded and the urine collagen­crosslinked NTx values were measured prior to, and 7, 14 and 21 days following the injections. Bone sections and osteoclasts were stained on the 14th day (4 days post­injection). Furthermore, the mRNA and protein expression levels of c­fos in the spinal cord and acid­sensing ion channel 3 (ASIC3) in the dorsal root ganglion (DRG) were analyzed. The mechanical allodynia of rats was attenuated from day 14 in the zoledronic acid group and from day 21 in the paclitaxel group. A positive correlation was observed between the anti­nociceptive responses of zoledronic acid and paclitaxel, and the urinary levels of NTx (r=0.619; P<0.001). The mRNA levels of c­fos in the spinal cord and ASIC3 in the DRG in the zoledronic acid group were reduced 14 and 21 days after inoculation, and this reduction was observed in the paclitaxel group 21 days after inoculation. Low dose paclitaxel was observed to have a weaker anti­nociceptive effect on bone cancer pain, with a later­onset, compared with zoledronic acid. The results suggested that urinary levels of NTx may predict the anti­nociceptive responses of zoledronic acid and paclitaxel in a rat model of bone metastases.

Potassium Bicarbonate Supplementation Lowers Bone Turnover and Calcium Excretion in Older Men and Women: A Randomized Dose-Finding Trial.

The acid load accompanying modern diets may have adverse effects on bone and muscle metabolism. Treatment with alkaline salts of potassium can neutralize the acid load, but the optimal amount of alkali is not established. Our objective was to determine the effectiveness of two doses of potassium bicarbonate (KHCO3 ) compared with placebo on biochemical markers of bone turnover, and calcium and nitrogen (N) excretion. In this double-blind, randomized, placebo-controlled study, 244 men and women age 50 years and older were randomized to placebo or 1 mmol/kg or 1.5 mmol/kg of KHCO3 daily for 3 months; 233 completed the study. The primary outcomes were changes in 24-hour urinary N-telopeptide (NTX) and N; changes in these measures were compared across the treatment groups. Exploratory outcomes included 24-hour urinary calcium excretion, serum amino-terminal propeptide of type I procollagen (P1NP), and muscle strength and function assessments. The median administered doses in the low-dose and high-dose groups were 81 mmol/day and 122 mmol/day, respectively. When compared with placebo, urinary NTX declined significantly in the low-dose group (p = 0.012, after adjustment for baseline NTX, gender, and change in urine creatinine) and serum P1NP declined significantly in the low-dose group (p = 0.004, adjusted for baseline P1NP and gender). Urinary calcium declined significantly in both KHCO3 groups versus placebo (p < 0.001, adjusted for baseline urinary calcium, gender, and changes in urine creatinine and calcium intake). There was no significant effect of either dose of KHCO3 on urinary N excretion or on the physical strength and function measures. KHCO3 has favorable effects on bone turnover and calcium excretion and the lower dose appears to be the more effective dose. Long-term trials to assess the effect of alkali on bone mass and fracture risk are needed.

Effect of calcium supplementation on bone resorption in pregnancy and the early postpartum: a randomized controlled trial in Mexican women.

BACKGROUND: Calcium needs are physiologically upregulated during pregnancy and lactation to meet demands of the developing fetus and breastfeeding infant. Maternal calcium homeostasis is maintained by hormonal adaptive mechanisms, thus, the role of dietary calcium supplementation in altering maternal responses to fetal-infant demand for calcium is thought to be limited. However, increased calcium absorption is directly related to maternal calcium intake and dietary supplementation has been suggested to prevent transient bone loss associated with childbearing. METHODS: In a double-blind, randomized placebo-controlled trial, we randomly assigned 670 women in their first trimester of pregnancy to 1,200 mg/day calcium (N = 334) or placebo (N = 336). Subjects were followed through 1-month postpartum and the effect on urinary cross-linked N-telopeptides (NTx) of type I collagen, a specific marker of bone resorption, was evaluated using an intent-to-treat analysis. Women with a baseline and at least one follow-up measurement (N = 563; 84%) were included. Subsequent analyses were conducted stratifying subjects by compliance assessed using pill counts. In random subsets of participants, bone-specific alkaline phosphatase (BAP) (N = 100) and quantitative ultrasound (QUS) (N = 290) were also measured. RESULTS: Calcium was associated with an overall reduction of 15.8% in urinary NTx relative to placebo (p < 0.001). Among those who consumed ≥50%, ≥67%, and ≥75% of pills, respectively, the effect was associated with 17.3%, 21.3%, and 22.1% reductions in bone resorption (all p < 0.001). There was no significant effect of calcium on bone formation measured by BAP. However, by 1-month postpartum, those in the calcium group had significantly lower NTx/BAP ratios than those in the placebo group (p = 0.04) indicating a net reduction in bone loss in the supplement group by the end of follow-up. Among subjects who consumed ≥50% and ≥75% of pills, respectively, calcium was also associated with an increase of 26.3 m/s (p = 0.03) and 59.0 m/s (p = 0.009) in radial SOS relative to placebo by 1-month postpartum. CONCLUSIONS: Calcium administered during pregnancy and the early postpartum period, to women with intakes around adequacy, was associated with reduced bone resorption and, thus, may constitute a practical intervention to prevent transient skeletal loss associated with childbearing. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT00558623.

Decrease of a specific biomarker of collagen degradation in osteoarthritis, Coll2-1, by treatment with highly bioavailable curcumin during an exploratory clinical trial.

BACKGROUND: The management of osteoarthritis (OA) remains a challenge. There is a need not only for safe and efficient treatments but also for accurate and reliable biomarkers that would help diagnosis and monitoring both disease activity and treatment efficacy. Curcumin is basically a spice that is known for its anti-inflammatory properties. In vitro studies suggest that curcumin could be beneficial for cartilage in OA. The aim of this exploratory, non-controlled clinical trial was to evaluate the effects of bio-optimized curcumin in knee OA patients on the serum levels of specific biomarkers of OA and on the evaluation of pain. METHODS: Twenty two patients with knee OA were asked to take 2x3 caps/day of bio-optimized curcumin (Flexofytol®) for 3 months. They were monitored after 7, 14, 28 and 84 days of treatment. Pain over the last 24 hours and global assessment of disease activity by the patient were evaluated using a visual analog scale (100 mm). The serum levels of Coll-2-1, Coll-2-1NO2, Fib3-1, Fib3-2, CRP, CTX-II and MPO were determined before and after 14 and 84 days of treatment. RESULTS: The treatment with curcumin was globally well tolerated. It significantly reduced the serum level of Coll2-1 (p<0.002) and tended to decrease CRP. No other significant difference was observed with the other biomarkers. In addition, curcumin significantly reduced the global assessment of disease activity by the patient. CONCLUSION: This study highlighted the potential effect of curcumin in knee OA patient. This effect was reflected by the variation of a cartilage specific biomarker, Coll2-1 that was rapidly affected by the treatment. These results are encouraging for the qualification of Coll2-1 as a biomarker for the evaluation of curcumin in OA treatment. TRIAL REGISTRATION: NCT01909037 at clinicaltrials.gov.

Analysis of the human urine endogenous peptides by nanoparticle extraction and mass spectrometry identification.

Peptides in urine are excreted by kidney from the blood and tissues, which are composed of a large amount of hormones, cytokines, regulatory factors and the metabolized fragments of proteins. The peptide distribution in urine will reflect the physiological and pathophysiological processes in body. In past, limited information was reported about the composition of the peptides in urine. One possible reason is that the peptides in urine are fairly low abundant and there are high concentrations of salts and organic metabolites in the urine. In this report, we extracted the peptides from human urine by highly ordered mesoporous silica particles with the pore size of 2 nm, which will exclude the high molecular weight proteins over 12 kDa. The extracted peptides were then separated into fractions according to their molecular weight by size exclusion chromatography. Each of the fractions was further analyzed by MALDI-TOF MS and µRPLC-MS/MS. Totally, 193 peptides were identified by two-dimensional SEC/µRPLC-MS/MS analysis. By analyzing the progenitor protein of the peptides; we found that two-thirds of the proteins differed from the reported urine proteome database, and the high abundant proteins in urine proteome were less detected in the urine peptidome. The developed extraction and separation methods were efficient for the profiling of the endogenous peptides in human urine. The peptidome in human urine was complementary to the human urinary proteome and may provide an emerging field for biomarker discovery.

The effect of bovine colostrum supplementation in older adults during resistance training.

Bovine colostrum is the first milk secreted by cows after parturition and has high levels of protein, immunoglobulins, and various growth factors. We determined the effects of 8 weeks of bovine colostrum supplementation versus whey protein during resistance training in older adults. Males (N = 15, 59.1 ± 5.4 y) and females (N = 25, 59.0 ± 6.7 y) randomly received (double-blind) 60 g/d of colostrum or whey protein complex (containing 38 g protein) while participating in a resistance training program (12 exercises, 3 sets of 8-12 reps, 3 days/ week). Strength (bench press and leg press 1-RM), body composition (by dual energy x-ray absorptiometry), muscle thickness of the biceps and quadriceps (by ultrasound), cognitive function (by questionnaire), plasma insulin-like growth factor-1 (IGF-1) and C-reactive protein (CRP, as a marker of inflammation), and urinary N-telopeptides (Ntx, a marker of bone resorption) were determined before and after the intervention. Participants on colostrum increased leg press strength (24 ± 29 kg; p < .01) to a greater extent than participants on whey protein (8 ± 16 kg) and had a greater reduction in Ntx compared with participants on whey protein (-15 ± 40% vs. 10 ± 42%; p < .05). Bench press strength, muscle thickness, lean tissue mass, bone mineral content, and cognitive scores increased over time (p < .05) with no difference between groups. There were no changes in IGF-1 or CRP. Colostrum supplementation during resistance training was beneficial for increasing leg press strength and reducing bone resorption in older adults. Both colostrum and whey protein groups improved upper body strength, muscle thickness, lean tissue mass, and cognitive function.

Severe vitamin D-deficiency and increased bone turnover in patients with hepatitis B from northeastern China.

INTRODUCTION: Vitamin D plays a key role in maintaining calcium homeostasis and skeletal health. The liver is critically involved in vitamin D metabolism, as 25-hydroxyvitamin D3 (25(OH)D3) is synthesized in the liver. Therefore liver dysfunction may lead to vitamin D deficiency and bone problems. The aim of this study was to examine vitamin D status and bone turnover markers in hepatitis B patients from northeastern China. METHODS: We recruited 39 patients with hepatitis B (23 noncirrhotic and 16 cirrhotic) and 48 healthy controls in Shenyang, a metropolitan city in northeastern China, and measured serum 25(OH)D3 levels and serum and urinary bone turnover markers in these subjects. RESULTS: Serum 25(OH)D3 levels in the patients with or without cirrhosis were markedly lower compared to the nonhepatitis controls (19.2 ± 1.2 and 18.5 ± 1.3 vs. 31.6 ± 1.3 nmol/L control), whereas serum and urinary bone turnover markers (alkaline phosphatase, C-terminal telopeptide of type I collagen, and pyridinoline) were significantly higher in these patients than in the controls. Moreover, serum levels of osteoprotegerin, a bone mass-regulating protein, were substantially reduced in the patients, with the lowest seen in patients with cirrhosis (2.7 ± 1.1 and 1.4 ± 0.4 vs. 3.4 ± 0.7 pg/mL control). Serum 25(OH)D3 levels below 30 nmol/L were positively correlated with serum osteoprotegerin levels in this cohort. CONCLUSIONS: Severe vitamin D deficiency is very common in hepatitis B patients in northeastern China, which negatively impacts their bone health. These data strongly suggest a need to treat these patients with vitamin D supplementation to protect their bone health.

Effects of RANK-ligand antibody (denosumab) treatment on bone turnover markers in a girl with juvenile Paget's disease.

CONTEXT: Juvenile Paget's disease (JPD) is an extremely rare, yet painful and debilitating bone disease with onset occurring during early childhood. JPD can be caused by loss of function of osteoprotegerin, resulting in subsequent stimulation of osteoclasts via the receptor activator of nuclear factor-κB (RANK) pathway. Increased bone turnover and lack of bone modeling lead to severe deformities, frequent fractures, short stature, and loss of hearing. SETTING: The treatment for JPD is challenging and has previously been based on administration of either calcitonin or bisphosphonates. However, with the development of denosumab, a receptor activator of nuclear factor-κB-ligand (RANKL) antibody, a treatment targeting the pathophysiology of JPD may be available. We report the effects of denosumab treatment on an 8-year-old girl with a severe form of JPD. PATIENT: Before starting the denosumab treatment regimen, the patient had been treated for 3.5 years with iv pamidronate. INTERVENTION AND OUTCOME: The administration of denosumab resulted in improved disease control compared with bisphosphonate, as assessed by monitoring markers of bone turnover. Alkaline phosphatase levels dropped within the normal range and remained at normal levels for 5 months after the final dose of denosumab. Additionally, bone pain was more efficiently controlled with denosumab. However, concomitant with the first injection, severe hypocalcemia developed, for which the patient was hospitalized and iv calcium supplementation was required for 13 days. CONCLUSIONS: Denosumab appears to be significantly effective for osteoclast inhibition for the treatment of JPD. However, in our patient, denosumab administration was associated with severe hypocalcemia, indicating that close monitoring of calcium levels is required during treatment.

Safety and efficacy of polycalcium for improving biomarkers of bone metabolism: a 4-week open-label clinical study.

Polycalcium is a mixture of Polycan and calcium lactate-gluconate 1:9 (w/w) with demonstrated antiosteoporosis activity in vitro and in vivo studies. These studies were a 4-week open-label, single-center trial to evaluate the efficacy of oral Polycalcium on bone metabolism and safety. In total, 30 healthy women (range 40-60 years) were administered 400 mg of Polycalcium for 4 weeks. The primary efficacy parameter was urinary deoxypyridinoline (DPYR) levels, and serum osteocalcin (OSC), bone-specific alkaline phosphatase (BALP), urinary cross-linked C-telopeptide of type-1 collagen (CTx), urinary cross-linked N-telopeptide of type-1 collagen (NTx), calcium (Ca), and phosphorus (P) levels, which were evaluated for comparison before and after administration of Polycalcium. After 4 weeks of Polycalcium administration, 27 subjects completed the test plan. Three subjects withdrew their consent to participate. The values of blood OSC, BALP, serum Ca, and serum P from baseline to 4 weeks of treatment were changed by -28.44%, 14.37%, 6.11%, and 1.42%, respectively. Biomarkers of bone resorption: urinary DPYR, serum CTx, serum NTx, urinary Ca, and urinary P, at baseline after 4 weeks of treatment were changed by -13.40%, 6.67%, -5.13%, -22.43%, and -3.04%, respectively. Additionally, when considering the subjects' adverse effects and the results of the blood and urine tests over the 4-week trial period, the dose of 400 mg Polycalcium showed efficacy for improving bone metabolism and was well tolerated and safe. Polycalcium was apparently safe and efficacious.

Effects of vitamin E on bone turnover markers among US postmenopausal women.

Increased oxidative stress and inflammation resulting from aging and declining estrogen levels can lead to increased bone loss in postmenopausal women. Alpha-tocopherol and gamma-tocopherol, the two predominant isomers of vitamin E, have antioxidant and anti-inflammatory properties, but their effects on bone metabolism have not been well studied in humans. We examined the associations between dietary and total (diet and supplements) alpha-tocopherol intake, serum alpha-tocopherol and gamma-tocopherol levels and their ratio, and bone turnover markers (BTMs) among postmenopausal women aged ≥45 years. We used cross-sectional data from the National Health and Nutrition Examination Survey 1999­2002. Multiple regression models with adjustments for relevant confounders were used to examine the associations between intake and serum levels of tocopherols, and serum bone-specific alkaline phosphatase (BAP), a biomarker of bone formation, and urinary N-telopeptides/creatinine (uNTx/Cr), a biomarker of bone resorption. The study sample included 497 postmenopausal women who were not taking estrogen, steroids, or osteoporosis medications, were free from kidney and liver disease, cancer, and rheumatoid arthritis, and were fasting >9 hours prior to examination. Participants had a mean age of 65.5 ± 0.6 years and over 45% used vitamin E (alpha-tocopherol) supplements in the past month. Vitamin E supplement users had significantly lower serum gamma-tocopherol, higher serum alpha-tocopherol levels, and higher ratio of serum alpha-tocopherol to gamma-tocopherol than nonusers. High serum gamma-tocopherol levels and low ratio of serum alpha-tocopherol to gamma-tocopherol were associated with increased BAP levels (p < 0.01 for both). There were no associations between any of the vitamin E variables and uNTx/Cr. In conclusion, we hypothesize that gamma-tocopherol may uncouple bone turnover, resulting in more bone formation than resorption. Vitamin E supplements in the form of alpha-tocopherol suppress serum gamma-tocopherol levels and may have negative effects on bone formation. Further research is needed to investigate the potential anabolic effect of gamma-tocopherol from food sources on bone.

Salivary bone turnover markers in healthy pre- and postmenopausal women: daily and seasonal rhythm.

No studies had investigated circadian and circannual rhythms of bone biomarkers in whole saliva. We evaluated the salivary daily and seasonal rhythm of carboxy-terminal telopeptide of type I collagen (CTX) and bone alkaline phosphatase (b-ALP). Forty clinical and oral healthy ambulatory pre- and postmenopausal women from two southern Argentine cities: Comodoro Rivadavia (latitude 45º S) and Ushuaia (latitude 54º S) were included in the study. CTX levels were evaluated in serum, urine, and saliva, and b-ALP levels were measured in serum and saliva. In both groups of women, salivary CTX showed a maximum percentage of change early in the morning (80%) and a minimum in the late afternoon (45%), similarly to the pattern observed in urinary samples. No daily rhythm was observed in serum or salivary b-ALP. 25-Hydroxyvitamin D levels decreased in winter vs. summer (p < 0.01) without differences between the two studied groups. Conversely, parathormone reached higher levels in winter (p < 0.05) which induced a slight non-significant increment in salivary CTX and b-ALP levels. The results showed that, as in serum and urinary samples, salivary CTX exhibits daily and a slight seasonal rhythmicity. Whole non-stimulated saliva is a useful tool to detect several oral and systemic diseases because it has important advantages compared to serum and urinary samples. Then, it may also be a promising sample to test changes in bone metabolism contributing to diagnose and to monitor the therapy of several metabolic bone diseases.

Alfacalcidol-supplemented raloxifene therapy has greater bone-sparing effect than raloxifene-alone therapy in postmenopausal Japanese women with osteoporosis or osteopenia.

Vitamin D insufficiency is prevalent in osteopenic and osteoporotic postmenopausal women. The persistent increase in circulating parathyroid hormone (PTH) caused by vitamin D insufficiency reduces bone density response to antiresorptive agents in these postmenopausal women. It is not well known whether administration of raloxifene might increase serum PTH secondary to the suppression of serum calcium in postmenopausal women with osteopenia or osteoporosis. We tried to assess whether raloxifene might affect serum PTH and whether the addition of alfacalcidol to raloxifene therapy could have favorable effects on bone mineral density (BMD) and bone turnover as compared to raloxifene-alone therapy in postmenopausal Japanese women with osteoporosis or osteopenia (≤2.0 SD based on young Japanese women). A total of 169 subjects were randomly assigned to groups receiving 60 mg raloxifene (R), or 1 µg alfacalcidol (D), or a combination of both (R + D) for 2 years. Serum levels of 25-hydroxyvitamin D [25(OH)D] were measured at randomization. The modified 'intention to treat' method was used. We compared the groups using a Tukey-Kramer test for changes in L- and TH-BMD and calcium metabolism when significant differences were found using one-way ANOVA. The parameters in each group during the experimental period were analyzed by means of paired t tests. Baseline 25(OH)D and i-PTH were 23.7 ng/ml and 38.4 pg/ml, respectively. At 6 months, i-PTH demonstrated a significant increase (+21.0%) in the R-group whereas significant decreases in i-PTH were observed in the D-group and combination-group (-15.9 and -8.9%, respectively). There were significant increases in L-BMD in the R + D-group (+4.1% at 1 year and +4.7% at 2 years, P < 0.0001) and in the R-group (+2.9% at 1 year and +2.8% at 2 years, P < 0.001), but the difference between the groups did not reach a significant level. Vitamin D status at randomization did not affect the subsequent BMD response in coadministration of alfacalcidol with raloxifene. Supplementation with alfacalcidol to raloxifene therapy demonstrates a greater bone-sparing effect by suppressing the secondary increment of serum PTH than when raloxifene is used alone.

An effective oral vitamin D replacement therapy in persons with spinal cord injury.

BACKGROUND/OBJECTIVE: Vitamin D deficiency is prevalent in chronic spinal cord injury (SCI). A 3-month course of oral vitamin D(3) to 'normalize' serum vitamin D levels was investigated. DESIGN: Prospective drug-intervention study. SETTING: VA Medical Center; private rehabilitation facility. METHODS: Seven individuals with chronic SCI and vitamin D deficiency completed 3 months of oral vitamin D(3) (i.e. cholecalciferol) supplementation. At screening, baseline, and months 1 and 3, blood was collected for serum calcium, 25 hydroxyvitamin D [25(OH)D], intact parathyroid hormone (iPTH), and N-telopeptide (NTx); 24-hour urine for calcium, creatinine, and NTx was performed. Oral vitamin D(3) (2000 IU daily) and elemental calcium (1.3 g daily) were prescribed for 90 days. The results are expressed as mean ± standard deviation (SD). Analysis of variance with a Fisher's post-hoc analysis was performed to test for differences between study visits. Subjects were classified as deficient (<20 ng/ml), relatively deficient (20-30 ng/ml), or not deficient (>30 ng/ml) in 25(OH)D. RESULTS: Serum 25(OH)D levels were greater at months 1 and 3 than at baseline (26 ± 6 and 48 ± 17 vs. 14 ± 2 ng/ml; P = 0.005). Six of seven subjects were no longer deficient [25(OH)D >30 ng/ml] by month 3. Serum iPTH levels were significantly decreased at month 1 and month 3; serum NTx levels were significantly lower at month 3 than at baseline. Serum and urinary calcium levels remained within the normal range. CONCLUSION: A daily prescription of 2000 IU of oral vitamin D(3) for 3 months safely raised serum 25(OH)D levels into the normal range in persons with chronic SCI on calcium supplementation.