Intact Osteocalcin Research Articles

Biological activity of anastrozole in postmenopausal patients with advanced breast cancer: effects on estrogens and bone metabolism

BackgroundTo study the short-term biological effect of anastrozole on serum estrogens, androgens, 17-hydroxyprogesterone (17OH-PGR), gonadotrophins, sex hormone binding globulin (SHBG) and bone metabolism markers. Materials and methodsThirty-four consecutive patients with advanced breast cancer received anastrozole 1 mg/day. Blood samples were taken before commencement of treatment and at 2, 4, 8and 12 weeks during treatment to measure serum levels of estrogens (E1, E2 and E1-S), androgens [androstenedione (Δ4), dihydrotestosterone (DHT), testosterone (TST), free TST, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S)], 17OH-PGR, SHBG and gonadotrophins. As an indicator of bone resorption, we measured serum levels of C-terminal telopeptide of type I collagen (ICTP) and the cross-linked N-telopeptide of type I collagen (NTx), and for osteoblastic activity, intact osteocalcin (BGP) and bone alkaline phosphatase (BAP). ResultsAfter 2 weeks E1 and E1-S levels decreased on average by 56% (range 23.1–88.8%) and 75.8% (range 52.4–87.2%), respectively; E2 decreased on average by 62% (range 31.4–89.6%). No significant changes were detected in levels of androgens or 17OH-PGR. There was a significant increase in gonadotrophins over time (P = 0.0001 for both luteinizing hormone and follicle-stimulating hormone), and a significant decrease in SHBG (P = 0.0001). A progressive significant increase in bone metabolism serum markers was detected in all patients: BAP, P = 0.039; BGP, P = 0.016; ICTP, P = 0.0021; and NTx, P = 0.0013. In particular, patients with bone metastases had a statistically significant increase of bone resorption markers (ICTP, P = 0.0019; NTx, P = 0.025) and borderline for bone formation markers. In patients without bone disease, BAP, BGP and ICTP remained unchanged, whereas serum NTx significantly increased (P = 0.019). ConclusionsAnastrozole is a selective aromatase inhibitor as it does not modify serum levels of androgens and 17OH-PGR. In our experience no relationship was found in the short-term period between serum estrogen suppression and bone metabolism.

Impaired secretion of parathyroid hormone, but not refractoriness of osteoblast, is a major mechanism of low bone turnover in hemodialyzed patients with diabetes mellitus

Diabetic bone disease is characterized by low bone turnover resulting from either impaired secretion of parathyroid hormone (PTH) or refractoriness of osteoblasts to PTH. The present study was performed to elucidate which factor contributes more to the reduction in bone turnover by comparison between 64 hemodialyzed patients with diabetes mellitus and 106 hemodialyzed patients without diabetes mellitus. Only men were enrolled to avoid the influence of the menstrual cycle on bone metabolism. Serum intact PTH (iPTH) levels were significantly lower in hemodialyzed patients with diabetes than those without diabetes, although no significant difference existed in age, duration of hemodialysis therapy, or serum calcium or phosphate levels. Of the biochemical markers measured, serum intact osteocalcin (iOC) and deoxypyridinoline levels were significantly lower in patients with diabetes, although serum bone-specific alkaline phosphatase (BAP) and pyridinoline levels did not differ significantly between the two groups of patients. When patients were restricted to those with serum iPTH levels greater than 180 pg/mL, this parameter correlated significantly in a positive manner with both serum iOC and BAP levels and negatively with bone mineral density at distal radius 1/3. Regression slopes between iPTH levels and these parameters were not significantly different between the two groups of patients, indicating the absence of refractoriness of bone to PTH in patients with diabetes. In conclusion, our findings suggest that impaired PTH secretion, but not refractoriness of osteoblasts to PTH, may be responsible for the low bone turnover in hemodialyzed patients with diabetes. © 2002 by the National Kidney Foundation, Inc.

Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.

For ambulatory pediatric outpatients,reports of abnormalities of bone metabolism associated with anti-epileptic drugs are inconsistent and may be difficult to interpret. The effects of long-term anti-epileptic therapy (mainly valproic acid and/or carbamazepine) on bone mineral status were evaluated in ambulatory epileptic patients(seven males and 11 females) aged 5.5-15.9 years. Bone mineral density (BMD) at the lumbar spine was measured by dual-energy X-ray absorptiometry and markers of bone and mineral metabolism were determined. The mean BMD was decreased by 9% in our patients relative to the control, and five patients (all males)showed osteopenia, defined as BMD SD scores less than - 1.5. Serum levels of minerals, intact parathyroid hormone and 1alpha,25(OH)2 vitamin D were within the normal ranges. In most patients, serum levels of intact osteocalcin, carboxyterminal propeptide of type I procollagen and pyridinoline cross-linked telopeptide of type I collagen were reduced relative to the corresponding mean control values. The BB genotype by BsmI restriction fragment length polymorphism, associated with low BMD, was not found in our patients. The dietary calcium intake in the osteopenic patients was significantly lower than that of the non-osteopenic patients. Our results indicate that long-term anti-epileptic treatment induces a state of decreased bone turnover in children, resulting in osteopenia preferentially in males. The alterations may be due, at least in part, to direct effects of the drugs on bone cells; and that low calcium intake could be an aggravating factor for anti-epileptic-associated osteopenia.

Clinical usefulness serum int-P I NP levels as a bone formation marker in hemodialysis patients

Since intact collagen I amino-terminal propeptide (P I NP) is known to be cleared from blood via liver, its level in blood is hypothesized to be independent of renal function. Serum P I NP, in contrast to serum bone alkaline phosphatase (BAP) and intact osteocalcin (OC), did not show any significant change between before and after a hemodialysis session. Serum P I NP correlated significantly in a positive manner with the other bone formation markers, bone-specific alkaline phosphatase and osteocalcin and also with intact parathyroid hormone in the patients on hemodialysis. In summary, serum P I NP may be very useful for prediction of radius bone loss as well as other serum bone formation markers and evaluating the osteoblastic function in hemodialyzed patients.

Effect of continuous combined therapy with vitamin K 2 and vitamin D 3 on bone mineral density and coagulofibrinolysis function in postmenopausal women

Objectives: To investigate the therapeutic effect of combined use of vitamin K 2 and D 3 on vertebral bone mineral density in postmenopausal women with osteopenia and osteoporosis. Subjects and methods: We enrolled 172 women with vertebral bone mineral density <0.98 g/cm 2 (osteopenia and osteoporosis) as measured by dual-energy X-ray absorptiometry. In this study, we employed the criteria for diagnosis of osteopenia and osteoporosis using dual energy X-ray absorptiometry proposed by the Japan Society of Bone Metabolism in 1996. Subjects were randomized into four groups (each having 43 subjects in vitamin K 2 therapy group, vitamin D 3 therapy group, vitamin K 2 and D 3 combined therapy group, or a control group receiving dietary therapy alone) and treated with respective agents for 2 years, with bone mineral density was measured prior to therapy and after 6, 12, 18, and 24 months of treatment. The bone metabolism markers analyzed were serum type 1 collagen carboxyterminal propeptide (P1CP), serum intact osteocalcin, and urinary pyridinoline. Tests of blood coagulation function consisted of measurement of activated partial thromboplastin time (APTT) and analysis of concentrations of antithrombin III (AT III), fibrinogen, and plasminogen. Results: Combined therapy with vitamin K 2 and D 3 for 24 months markedly increased bone mineral density (4.92±7.89%), while vitamin K 2 alone increased it only 0.135±5.44%. The bone markers measured, revealed stimulation of both bone formation and resorption activity. We observed an increase in coagulation and fibrinolytic activity that was within the normal range, suggesting that balance was maintained in the fibrinolysis–coagulation system. Conclusions: Continuous combination therapy with vitamin K 2 and D 3 may be useful for increasing vertebral bone mass in postmenopausal women. Furthermore, the increase in coagulation function observed during this therapy was within the physiological range, and no adverse reactions were observed.

A Strategy for the Management of Elderly Women with Primary Hyperparathyroidism: A Comparison of Etidronate Therapy with Parathyroidectomy

Objective: To evaluate the efficacy of etidronate (EHDP) on lumbar spine bone mineral density (LSBMD) and total bone mineral density (TBMD) in elderly women with primary hyperparathyroidism (PHPT), we compared changes in LSBMD and TBMD between patients treated by EHDP therapy and parathyroidectomy (PTX). Subjects and Methods: Twenty-two PHPT patients were enrolled and randomized into two groups; 9 received EHDP and 13 underwent PTX. All patients were followed up for 1 year by measuring LSBMD, TBMD, serum calcium, inorganic phosphate, parathyroid hormone, 1,25-dihydroxyvitamin D, serum alkaline phosphatase, intact osteocalcin, urinary pyridinoline (U_pyd) and urinary deoxypyridinoline (U_dpd). The presence of spinal fractures was evaluated by X-ray photography before and after treatment. Results: EHDP treatment produced a significant increase in LSBMD of 10% compared with pretreatment levels after 1 year (p < 0.03, compared to baseline), while PTX produced a significant increase in LSBMD of 20% compared to pretreatment levels (p < 0.01). However, TBMD remained unchanged for 1 year after both EHDP administration and PTX. Among biochemical bone turnover markers, EHDP administration resulted in significant decreases in alkaline phosphatase by 78%, U_pyd by 64% and U_dpd by 37% after 12 months compared with the pretreatment levels (p < 0.05) and intact osteocalcin by 67% after 6 months (p < 0.05). There were no differences in the fracture rate between the EHDP and PTX groups during 1 year. Conclusion: EHDP administration results in a somewhat lower increase in LSBMD than that following PTX and suppresses bone formation and resorption in elderly PHPT patients for 1 year. We conclude that PTX is preferable to EHDP therapy for the management of elderly PHPT patients; however, EHDP administration should also be considered for elderly patients with many complications or who are unfit for surgery.

Time-dependent effects of vitamin K2 (menatetrenone) on bone metabolism in postmenopausal women.

Vitamin K is known to mediate carboxylation of glutamyl residues of osteocalcin. We evaluated the effects of vitamin K2 (menatetrenone) treatment (45 mg/day) for 48 weeks on the markers of bone formation and resorption, bone mineral density (BMD), and the incidence of vertebral fractures in 34 Japanese postmenopausal women (aged 48-82 years). Serum levels of alkaline phosphatase (ALP) increased gradually and became significant at 48 weeks after menatetrenone treatment, while urinary excretion of deoxypyridinoline (DPD) decreased transiently but significantly at 4 weeks. Serum levels of both intact osteocalcin (OC) and carboxylated OC (Gla-OC) increased rapidly and significantly within 4 weeks and sustained their high values up to 48 weeks after the treatment, while those of undercarboxylated OC (Glu-OC) decreased reciprocally. These results can be interpreted to suggest that Glu-OC was converted to Gla-OC in vivo. On the other hand, lumbar BMD values showed no significant change and only one subject with a previous vertebral fracture had one newly occurring vertebral fracture. These results indicate that menatetrenone treatment of postmenopausal women constantly elevates bone formation markers as well as converts Glu-OC to Gla-OC. Thus, vitamin K2 treatment may promote bone formation, at least as measured biochemically in these subjects.

Serum osteocalcin in relation to calcaneal bone mineral density in elderly men and women: a 5-year follow-up

A 5-year follow-up study investigated serum concentrations of total (tOC) and intact (iOC) osteocalcin in relation to calcaneal bone mineral density (BMD). The study comprised two cohorts, 75- and 80-year-olds, both resident in the city of Jyväskylä, Finland. Baseline OC and BMD were obtained for 161 men and 233 women, of whom 83 men and 189 women participated in follow-up bone measurements. The mean concentration of tOC increased from 9.6 +/- 4.3 to 13.2 +/- 8.5 microg/l (P = 0.001) in men and from 11.2 +/- 4.9 to 14.0 +/- 6.1 microg/l (P < 0.001) in women, whereas mean iOC decreased from 6.4 +/- 3.0 to 5.9 +/- 3.0 microg/l (P = 0.273) and from 7.7 +/- 3.7 to 6.9 +/- 3.4 microg/l (P = 0.021) in men and women, respectively. TOC and iOC levels correlated inversely with BMD and change in BMD in both sexes (r ranged from -0.223 to -0.422 and P = 0.048 < or = 0.001). When we divided the baseline tOC and iOC values into four quartiles, the decrease in BMD was significantly greater in the third tOC quartiles in women and in the fourth tOC and iOC quartiles in men when compared with the lower quartiles. During the 5-year period, 19 men and 59 women sustained at least one fracture. These individuals with fractures had significantly higher iOC values and tended to have higher tOC values compared with the nonfracture group at baseline (P = 0.038 and 0.087, respectively). Our results indicate that baseline serum tOC and iOC were associated with bone loss and predicted fracture in the two cohorts of independently living elderly men and women.

Impaired secretion of parathyroid hormone is coherent to diabetic hemodialyzed patients

Diabetic bone disease is characterized by low bone turnover resulting from impaired secretion of parathyroid hormone (PTH). However, it was suggested that the difference in duration of hemodialysis (HD) therapy and age of patients between HD patients with and without diabetes mellitus (DM) may be responsible for a significant reduction in serum intact PTH (iPTH) level in HD patients with DM. The present study showed that although such major factors affecting PTH secretion as age, sex, HD duration, and serum calcium, phosphate, and magnesium levels did not differ significantly between HD patients with and without DM, serum iPTH levels were still significantly lower in HD patients with than without DM. Among biochemical markers for bone metabolism, serum levels of intact osteocalcin (iOC) and deoxypyridinoline (DPD) were significantly lower in HD patients with than without DM, whereas serum bone-specific alkaline phosphatase, pyridinoline, and β-crosslaps did not differ significantly between the two groups of patients. In summary, our findings indicate that PTH secretion may be significantly impaired in HD patients with DM compared with those without DM, and serum iOC and DPD are bone markers sensitive enough to detect low bone turnover in HD patients with DM. © 2001 by the National Kidney Foundation, Inc.

Nitrogen Metabolism and Bone Metabolism Markers in Healthy Adults during 16 Weeks of Bed Rest

The associations between nitrogen metabolism and bone turnover during bed rest are still not completely understood. We measured nitrogen balance (nitrogen intake minus urinary nitrogen excretion) and biochemical metabolic markers of calcium and bone turnover in six males before head-down tilt bed rest (baseline), during 2, 10, and 14 weeks of immobilization, and after reambulation. The changes in nitrogen balance were highest between baseline and week 2 (net change, -5.05 +/- 1.30 g/day; 3.6 +/- 0.6 g/day at baseline vs -1.45 +/- 1.3 g/day at week 2; P<0.05). In parallel, serum intact osteocalcin (a marker of bone formation) was already reduced and renal calcium and phosphorus excretions were increased at week 2 (P <0.05). Fasting serum calcium and phosphorus values and renal excretion of N-telopeptide (a bone resorption marker) were enhanced at weeks 10 and 14 (P <0.05-0.001), whereas serum concentrations of parathyroid hormone, calcitriol, and type I collagen propeptide (a marker of bone collagen formation) were decreased at week 14 (P <0.05-0.01). Significant associations were present between changes of serum intact osteocalcin and 24-h calcium excretion (P <0.001), nitrogen balance and 24-h phosphorus excretion (P <0.001), nitrogen balance and renal N-telopeptide excretion (P <0.05), and between serum osteocalcin and nitrogen balance (P <0.025). Bone formation decreases rapidly during immobilization in parallel with a higher renal excretion of intestinally absorbed calcium. These changes appear in association with the onset of a negative nitrogen balance, but decreased bone collagen synthesis and enhanced collagen breakdown occur after a time lag of several weeks.

Development of low-turnover bone diseases after parathyroidectomy and autotransplantation.

Parathyroidectomy and immediate autotransplantation (PTX-AT) has been shown to decrease bone pain and increase bone mineral density. However, adynamic bone disease (ABD) has been predicted to develop if the serum intact parathyroid hormone (i-PTH) level remains lower than normal for a long period of time. Therefore, we investigated the bone histology of patients whose serum i-PTH levels did not increase over 70 pg/mL for 1 year after PTX-AT. Four chronic hemodialysis patients were investigated. The serum intact osteocalcin (i-OC) level was measured and histomorphometry for cancellous bone was performed 1 year after the operation. Tetracycline hydrochloride was administered in the 12 weeks after PTX-AT. The serum i-PTH levels were 20.5 +/- 15.0 pg/mL and i-OC levels were 19.5 +/- 0.9 ng/mL. Histomorphometric analyses showed the osteoclast surface to be 0.1% in two cases and 0% in the other two cases, the eroded surface was 7.7 +/- 6.1%, and the fibrosis volume and osteoblast surface were 0% in all four cases. Osteoid volume, osteoid surface and osteoid thickness were lower in cases 1-3, but higher in case 4. All tetracycline labelings were in contact with the mineralization front in cases 1 and 3, but some were not in cases 2 and 4. Serum i-PTH and i-OC levels indicated that ABD developed in these four cases. Histomorphometric analyses revealed that ABD developed in case 1, while either ABD or low-turnover osteomalacia developed in cases 2 and 4, and low-turnover osteomalacia was observed in case 3 after PTX-AT. In conclusion, i-PTH should not be maintained at lower levels to avoid low-turnover bone diseases.

Effect of Low Molecular Weight Heparin on Bone Metabolism and Hyperlipidemia in Patients on Maintenance Hemodialysis

The effect of low molecular weight heparin (LMWH) on serum lipid profile in hemodialysis remains controversial and its effect on bone metabolism has not been studied. A crossover study was conducted in 40 patients on stable hemodialysis using unfractionated heparin (UFH) for more than 24 months. These patients were then treated with a LMWH (nadroparin-Ca) for 8 months during hemodialysis and subsequently switched back to UFH for 12 months. Serum lipid profile, biochemical markers for bone metabolism, and bone densitometry (BMD) were monitored at four-month intervals while all medications remained unchanged. Cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), lipoprotein(a) (Lp(a)), apolipoprotein B (Apo B) were raised in 35%, 29%, 12%, 24% and 24% of patients respectively. High-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A1 (Apo A-1) were reduced in 47% and 9% of patients. Bone-specific alkaline phosphatase (BALP) and intact osteocalcin (OSC), both reflecting osteoblastic activity, were raised in 65% and 94% of patients. Tartrate-resistant acid phosphatase (TRACP) reflecting osteoclastic activity and parathyroid hormone (PTH) were elevated in 35% and 88% of patients. Following LMWH treatment, TC, Tg, Lp(a) and Apo B were reduced by 7%, 30%, 21% and 10% respectively (p<0.05 or <0.01) while Apo A-1 were raised by 7% (p<0.01). Simultaneously, TRACP was reduced by 13% (p<0.05). These biochemical changes were detected soon after 4 months of LMWH administration. Although BMD values in our patients were lower than those of age-matched normal subjects, significant changes were not observed with LMWH treatment. After switching back to UFH for hemodialysis, these biochemical indices reverted to previous values during UFH treatment with a significant higher level in TC and Apo B while serum Apo A-1 remained elevated. Our study suggests LMWH may partially alleviate hyperlipidemia and, perhaps, osteoporosis associated with UFH administration in patients on maintenance hemodialysis.

Biochemical Markers of Bone Turnover and Response of Bone Mineral Density to Intervention in Early Postmenopausal Women: An Experience in a Clinical Laboratory

Markers of bone formation and resorption may be useful as early indicators of response to therapy. Our aim in this study was to investigate the use of bone markers for monitoring of intervention for bone loss in early postmenopausal women and to assess the relationships between these markers and changes in bone mineral density (BMD). Subjects were randomly assigned to the following groups: a control group; a group receiving calcium alone; groups receiving calcium plus low or conventional doses of conjugated equine estrogen; and groups receiving calcium plus low or conventional doses of calcitriol. At baseline and at 1 and 3 months after intervention, we measured serum intact osteocalcin, serum N-terminal midfragment osteocalcin, serum C-terminal telopeptide of type I collagen (CTx), urinary deoxypyridinoline cross-links, and urinary CTX: The BMD of the lumbar spine and the femoral neck was measured at baseline and after 1 and 2 years of intervention. No marker changed significantly in the control group except urinary CTx, which increased at 3 months. Serum CTx decreased in all regimens at 1 or 3 months of intervention. In addition, the changes of all markers at 3 months were inversely associated with the change in the BMD of the lumbar spine at 1 or 2 years (r = -0.144 to -0.314), whereas only the changes of bone resorption markers at 3 months were inversely correlated with the changes in femoral BMD at 1 or 2 years (r = -0.143 to -0.366). Biochemical markers of bone turnover appear to be of use in assessing early response to therapy. Bone resorption markers, especially serum CTx, are better indicators than bone formation markers for estimating the response to intervention in early postmenopausal women. However, the early changes in bone markers were weakly related to the later changes in BMD.

DISTURBANCE OF BONE METABOLISM TURNOVER INDUCED BY A SINGLE BOUT ANAEROBIC EXERCISE IN YOUNG MALES

Generally exercise increases and maintains of bone mass. However, long duration exercise and/or high intense exercise disturbs bone metabolism turnover, leading to bone loss. For elucidation of the bone turnover induced by a bout of intense anaerobic exercise, nine healthy young males (aged 23–28 years) completed a bout of intense anaerobic exercise by bicycle ergometer for 3min. Bone resorption (deoxypyridinoline: Dpyr, tartrate resistant acid phosphatase: TrACP and cross-linked carboxyl terminal propeptide of type 1collagen: ICTP) and formation marker (intact osteocalcin: I-OC) were measured. Peripheral blood mononuclear cells (PBMC) were isolated and cultured for 48hrs, and the released cytokines of bone resorption, interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha, were analyzed. After the exercise, urinary calcium excretion increased approximately 3.2 and 3.8 fold as much as the base at 45 and 135 min. (p < 0.01), respectively. Dpyr excretion similarly increased approximately 1.5 fold at 180min. (p < 0.01). TrACP and ICTP increased by 25 and 13% (p < 0.01 and p < 0.05), respectively, at 15min. However, i-OC did not change significantly. Both of IL-6 and TNF-alpha promptly increased approximately 1.5 fold at 15min. (p < 0.01). The exercise promptly induced metabolic acidosis around pH 7.2 with increase of lactate about 10 fold as much as the base. These results suggest that a bout intense anaerobic exercise induced metabolic acidosis, uncoupling of bone metabolism with increased osteoclastic and unchanged osteoblastic activity and hypersecretion of PBMC cytokines. Bone loss by prolonged severe anaerobic exercise might be caused by hypersecretion of bone resorbing cytokines and metabolic uncoupling of bone.

Carboxylation of osteocalcin may be related to bone quality: a possible mechanism of bone fracture prevention by vitamin K.

Vitamin K is essential for the carboxylation of glutamic acid residues, such as osteocalcin. Recent studies have reported that vitamin K reduces vertebral and hip fractures without increasing bone mass in patients with osteoporosis, suggesting that vitamin K could affect bone quality. However, the mechanism is unknown. To investigate the involvement of the carboxylation of osteocalcin in bone quality, the present preliminary study examined serum bone markers and ultrasound velocity, a possible indicator of bone quality, in 14 healthy prepubertal children (eight boys and six girls) aged between 7 and 12 years. Venous blood was collected between 0800 and 0900 h after an overnight fast, and serum levels of intact, carboxylated and undercarboxylated osteocalcin, bone-specific alkaline phosphatase and type I procollagen carboxyl extension peptide were measured. Speed of sound in the right tibia was measured using a SoundScan 2000 Compact (Myriad Ultrasound System, Rehovot, Israel). As a result, there was no significant correlation between the serum bone markers and the Z score for the speed of sound. In contrast, the ratio of serum carboxylated osteocalcin to serum intact osteocalcin was positively correlated with the Z score for the speed of sound (r = 0.621, P = 0.016). These findings suggest, for the first time, that carboxylation of osteocalcin is related to bone quality. Further studies are needed to clarify the role of carboxylation of osteocalcin in bone, and this will provide a new insight into the mechanism of vitamin K treatment in osteoporosis.

Lung metastatic calcification in long-term dialysis patients, detected by single photon emission computed tomography (SPECT)

Background. Metastatic lung calcification (MLC) is an important complication in long-term dialysis patients, causing respiratory disturbance. We used single photon emission computed tomography (SPECT) in lung (L-SPECT) to detect the distribution of MLC. Methods. L-SPECT with 99mtechnetium hydroxymethylenediphosphonate was performed in 59 patients who had been on maintenance dialysis for more than 5 years. The accumulation count ratios were calculated from counts in the upper lung field (UL), lower lung field (LL), basal lung field along the diaphragm (BASE), and the cardiac pool (POOL). Distribution patterns of radionuclide were classified into four groups. Pulmonary function tests and blood examinations were performed. Results. The median values for UL/POOL, LL/POOL, and BASE/POOL were 1.71, 1.85, and 2.21, respectively. On the SPECT images, 28 patients had positive lung accumulation, versus 7 on the planar images. Patterns and frequency were: group I (none or slight accumulation), 32.2%; group II (accumulation only in the basal lung field), 33.8%; group III (accumulation in the whole lower lung or above), 18.6%; and group IV (diffuse accumulation extending to the whole lung field), 15.2%. Intact osteocalcin level was 6.3 ± 5.3 ng/ml in groups III + IV (large group) vs 94.2 ± 184.3 in groups I + II (small group) (P < 0.01). The number of patients with declining diffusion capacity was 20.0% in the large group, vs 5.1% in the small group. All patients in group IV had an intact parathyroid hormone (i-PTH) level of under 65 pg/ml. Conclusions. L-SPECT is very useful for the early detection of MLC, the extension of which may reduce diffusion capacity. Diffuse calcification over the whole lung is considered to be related to a low-turnover bone state concomitant with low PTH level.

Development and application of a synthetic peptide-based osteocalcin assay for the measurement of bone formation in mouse serum.

The mouse is frequently used as an animal model to study skeletal mechanisms relevant to humans. Biochemical markers of bone formation and resorption provide one of the key parameters for assessing skeletal metabolism. One biochemical marker that has proven to be useful in the studies of mouse skeletal metabolism is osteocalcin. Assay for osteocalcin is available in the mouse. The present study describes development of an osteocalcin radioimmunoassay (RIA) using a synthetic peptide. Intact osteocalcin purified from mouse bone extracts shows parallel displacement with synthetic peptide. Sensitivity of the RIA was 19 ng/ml. The average (n = 9) intra- and interassay coefficient of variation for two controls was less than 10%; the averaged recoveries were 106%. The osteocalcin concentration measured by peptide RIA shows a high correlation (r = 0.88, n = 117, P < 0.0001) with an intact osteocalcin assay. In addition, when the intact assay and peptide assays were applied to evaluate skeletal perturbation, similar results were obtained. Accordingly, osteocalcin levels measured by both intact and peptide-based RIA in 8-week C57BL/6J (n = 8) mice treated with PTH 1-34 were twofold higher compared with the vehicle-treated control group. Further studies of the application of the peptide-based RIA for osteocalcin revealed that osteocalcin levels in 4-week postovariectomized (OVX) C57BL/6N mice (n = 10) were 80% higher than the sham-operated (n = 10) mice receiving vehicle. OVX mice receiving weekly injections of estradiol (400 microg/kg body weight) were 38% lower compared with the OVX group treated with vehicle. In conclusion, the peptide-based RIA has analytical and a discriminative power similar to that of the intact osteocalcin assay but has the advantage that the resources for this assay are much easier to accrue.

Effect of soy protein on bone metabolism in postmenopausal Japanese women.

We conducted a cross-sectional study of the effects of soybean protein intake on bone mineral density and biochemical markers in 85 postmenopausal Japanese women. Nutrients in the diet of postmenopausal Japanese women visiting the osteoporosis unit, including subjects with normal lumbar spine bone mineral density (L2-4 BMD), were investigated by questionnaire, and the calculated daily energy, protein, soy protein and calcium intake were obtained. L2-4 BMD was measured with dual-energy X-ray absorptiometry, and assays done of serum alkaline phosphatase (ALP) and serum intact osteocalcin (IOC) as bone formation markers and urinary pyridinoline (UPYR) and urinary deoxypyridinoline (UDPYR) as bone resorption markers. Soy protein intake was significantly associated with the Z-score for L2-4 BMD (r = 0.23,p = 0.038) and UDPYR (r = -0.23, p = 0.034). Stepwise multiple regression analyses showed that soy protein intake is significantly associated with the Z-score for L2-4 BMD (beta = 0.225, p = 0.04) and UDPYR (beta = -0.08, p = 0.03) among four nutritional factors. These results suggest that high soy protein intake is associated with a higher bone mineral density and a lower level of bone resorption, but further studies are needed to confirm the causal dynamic mechanisms.

A new assay method that detects only intact osteocalcin. Two-step non-invasive diagnosis to predict adynamic bone disease in haemodialysed patients.

We studied the usefulness of a new assay method that detects only the intact human osteocalcin molecule in haemodialysed patients. Iliac bone biopsy specimens obtained from 62 haemodialysed patients were analysed. Bone formation rates (BFR/BS) correlated positively with serum intact osteocalcin concentrations (n=62), osteocalcin concentrations assayed by a conventional method (n=31), parathyroid hormone (PTH) concentrations (n=62), and total alkaline phosphatase concentrations (r=0.602, 0. 588, 0.650, and 0.401 respectively). Based on ROC curve and Youden index analysis, the optimal cut-off value to distinguish adynamic bone disease from a mild lesion was 195 pg/ml of serum PTH concentration (Youden index=0.233) or 30 ng/ml of serum intact osteocalcin concentration (Youden index=0.628). The optimal cut-off value to distinguish between hyperparathyroid bone and a mild lesion was 455 pg/ml of serum PTH level (Youden index=0.63) or 50 ng=ml of serum intact osteocalcin concentration (Youden index=0.634). Since both ROC curve and Youden index suggested that the serum PTH concentration was not a good marker to distinguish adynamic bone from a mild lesion or hyperparathyroid bone, we designed a two-step procedure. The first step was the diagnosis of adynamic bone (cut-off: 65 pg/ml) or hyperparathyroid bone (cut-off: 455 pg/ml) according to serum PTH concentration. In a second step, we assessed serum intact osteocalcin concentration in patients with serum PTH concentrations between 65 and 455 pg/ml. The cut-off values for adynamic and hyperparathyroid bone in this diagnostic approach were 30 and 70 ng/ml respectively. As a result, 49 out of 62 patients were diagnosed properly. The Youden index of this two-step diagnosis was 0.527 and 0.661 for adynamic bone and hyperparathyroid bone respectively. Sensitivity markedly improved to 94.4% and 96.2% for adynamic bone and hyperparathyroid bone respectively, without sacrificing specificity (84.0 and 88.8% respectively). Measurement of serum intact osteocalcin concentration is useful for the diagnosis of adynamic bone in haemodialysed patients. A two-step procedure involving also simultaneous measurement of serum PTH concentration further improved the sensitivity of each individual marker while maintaining specificity.

Comparison of the analytical and clinical performance characteristics of an N-MID versus an intact osteocalcin immunoradiometric assay

Osteocalcin is the most abundant non-collagenous protein in bone, reflecting its formation. It was reported that the instability of intact osteocalcin results from the cleavage of the C-terminal sequence of osteocalcin to produce a large N-terminal osteocalcin fragment. A two-site immunoassay for detecting both the N-terminal osteocalcin fragment and the intact osteocalcin was developed that were both independent of an unstable C-terminal sequence. The aim of this study is to investigate the performance of an N-MID osteocalcin immunoradiometric assay and to compare it with an intact osteocalcin assay. Ten serum samples were repeatedly frozen and thawed up to seven times. The variability of the values of N-MID osteocalcin was less than that of the intact osteocalcin. For stability of osteocalcin in serum after storage, the mean value of N-MID was 94.3% of the initial value after 7 days at 4°C, whereas the intact was 73.4%. The reduction of intact values were significantly larger than that of N-MID after 2, 5 and 7 days. At −30°C, the values of N-MID did not change for up to 10 weeks. The concentrations of osteocalcin measured by an N-MID osteocalcin and an intact osteocalcin were investigated in 27 premenopausal subjects, 27 postmenopausal subjects, and 68 osteoporotic patients (23 with vertebral fractures and 45 with hip fractures). The percent mean increase of osteocalcin in postmenopausal subjects over premenopausal subjects was 98% in N-MID versus 42% in the intact assay. The z-scores of N-MID and intact showed similar results in all groups. N-MID osteocalcin significantly correlated with intact osteocalcin ( r=0.755), and other biochemical markers for bone formation, such as bone specific alkaline phosphatase ( r=0.606) and C-terminal propeptide of type I procollagen (PICP) ( r=0.568). An N-MID IRMA had better stability during storage than intact and had the discriminative ability which is similar to the intact assay in postmenopause and osteoporosis. Therefore, an N-MID osteocalcin IRMA could improve the clinical utility and evaluation of osteocalcin.