Effect of 12-months testosterone replacement therapy on bone mineral density and markers of bone turnover in testicular cancer survivors - results from a randomized double-blind trial.

BACKGROUND: Testicular cancer survivors (TCS) are at risk of Leydig cell insufficiency, which is a condition characterized by elevated luteinising hormone (LH) in combination with low levels of testosterone. It has been suggested that this condition is associated with impaired metabolic profile and low bone mineral density (BMD). The primary aim of the randomized double-blind trial NCT02991209 was to evaluate metabolic profile after 12-months testosterone replacement therapy (TRT) in TCS with mild Leydig cell insufficiency. Here we present the secondary outcomes of changes in BMD and markers of bone turnover. METHODOLOGY: In total, 69 TCS with mild Leydig cell insufficiency were randomized 1:1 to 12 months TRT (n = 35) (Tostran, gel, 2%, applied transdermally, with a maximum daily dose of 40 mg) or placebo (n = 34). BMD and markers of bone turnover were evaluated at baseline, after 6- and 12-months TRT, and 3-months post-treatment. Linear mixed effects models were used to analyse changes in BMD, N-terminal propeptide of type 1 procollagen (P1NP) and C-terminal telopeptide of type I collagen (CTX). RESULTS: After 12 months treatment, TRT was not associated with a statistically significant difference in BMD compared to placebo; total body BMD: 0.01 g/cm2 (95% confidence interval (CI): -0.01 - 0.02), BMD of the lumbar spine: 0.01 g/cm2, (95% CI: -0.01-0.03), BMD of the left femoral neck: 0.00, (95% CI: -0.01-0.02). TRT was associated with a small but statistically significant increase in P1NP: 11.65 µg/L (95% CI: 3.96, 19.35), while there was no difference in CTX. CONCLUSION: 12 months of TRT did not change BMD, while there was as small and clinically irrelevant increase in P1NP compared to placebo in TCS with mild Leydig cell insufficiency. The findings need validation in a larger cohort.

Do bone turnover markers reflect changes in bone microarchitecture during treatment of patients with thyroid dysfunction?

PURPOSE: This study aimed to compare changes in the bone turnover markers (BTMs)-C-terminal telopeptide of type I collagen (CTX-I) and procollagen I N-terminal peptide (PINP)-with changes in the bone microarchitecture, assessed by high-resolution peripheral quantitative computed tomography (HR-pQCT), during treatment of patients with thyroid dysfunction. METHODS: In women with newly diagnosed hypo- or hyperthyroidism, HR-pQCT variables, obtained from the tibia and the radius, were compared with BTMs. Data were collected at diagnosis and after at least 12 months of euthyroidism. RESULTS: 73 women completed the study (hypothyroidism, n = 27; hyperthyroidism, n = 46). Among hyperthyroid patients, correlations were found between changes in BTMs and HR-pQCT variables, primarily for cortical variables in the tibia, i.e. cortical thickness (CTX-I, p < 0.001; PINP, p < 0.001), and volumetric bone mass density (vBMD) (CTX-I, p < 0.001; PINP, p < 0.001). Moreover, correlations between BTMs and estimated bone strength were found. In the hypothyroid subgroup, no significant findings existed after adjustment. Following treatment, less decrease in tibial vBMD was seen among patients with increasing CTX-I compared to those with a decreasing CTX-I level (p = 0.009). Opposite findings applied to PINP, as patients with decreasing PINP showed an increase in tibial vBMD, in contrast to a decline in this parameter among patients with increasing PINP (p < 0.001). CONCLUSION: Changes in CTX-I and PINP correlated with HR-pQCT variables during the treatment of women with thyroid dysfunction. To some extent, these BTMs reflected the restoration of bone microarchitecture. CTX-I seems to be the most sensitive BTM in treatment-naïve thyroid diseases, while PINP is more useful for monitoring during treatment. TRIAL REGISTRATION NUMBER: NCT02005250. Date: December 9, 2013.

A Multicenter Study to Evaluate Harmonization of Assays for C-Terminal Telopeptides of Type I Collagen (ß-CTX): A Report from the IFCC-IOF Committee for Bone Metabolism (C-BM).

BACKGROUND: Biochemical bone turnover markers are useful tools to assess bone remodeling. C-terminal telopeptide of type I collagen (ß-CTX) has been recommended as a reference marker for bone resorption in research studies. METHODS: We describe the results of a multicenter study for routine clinical laboratory assays for ß-CTX in serum and plasma. Four centers (Athens GR, Copenhagen DK, Liege BE and Sheffield UK) collected serum and plasma (EDTA) samples from 796 patients presenting to osteoporosis clinics. Specimens were analyzed in duplicate with each of the available routine clinical laboratory methods according to the manufacturers' instructions. Passing-Bablok regressions, Bland-Altman plots, V-shape evaluation method, and Concordance correlation coefficient for ß-CTX values between serum and plasma specimens and between methods were used to determine the agreement between results. A generalized linear model was employed to identify possible variables that affected the relationship between the methods. Two pools of serum were finally prepared and sent to the four centers to be measured in 5-plicates on 5 consecutive days with the different methods. RESULTS: We identified significant variations between methods and between centers although comparison results were generally more consistent in plasma compared to serum. We developed univariate linear regression equations to predict Roche Elecsys®, IDS-iSYS, or IDS ELISA ß-CTX results from any other assay and a multivariable model including the site of analysis, the age, and weight of the patient. The coefficients of determination (R2) increased from approximately 0.80 in the univariate model to approximately 0.90 in the multivariable one, with the site of analysis being the major contributing factor. Results observed on the pools also suggest that long-term storage could explain the difference observed with the different methods on serum. CONCLUSION: Our results show large within- and between-assay variation for ß-CTX measurement, particularly in serum. Stability of the analyte could be one of the explanations. More studies should be undertaken to overcome this problem. Until harmonization is achieved, we recommend measuring ß-CTX by the same assay on EDTA plasma, especially for research purposes in large pharmacological trials where samples can be stored for long periods before they are assayed.

Harmonization of commercial assays for PINP; the way forward.

International Federation of Clinical Chemistry and Laboratory Medicine and The International Osteoporosis Foundation Joint Committee on Bone Metabolism believes that the harmonization of PINP assays is an achievable and practical goal. INTRODUCTION: In order to examine the agreement between current commercial assays, a multi-center study was performed for PINP in serum and plasma. METHODS: The automated methods for PINP (Roche Cobas and IDS iSYS) gave similar results. A significant proportional bias was observed between the two automated assays and the Orion radioimmunoassay (RIA) for PINP. RESULTS: Results from other published studies comparing PINP values among these three assays broadly support our findings. Taken together, these results confirm that harmonized PINP measurements exist between the two automated assays (Roche Cobas and IDS iSYS) when the eGFR is > 30 mL/min/1.73m2, but a significant bias exists between the Orion RIA and the two automated assays. CONCLUSION: Therefore, in subjects with normal renal function, PINP results reported by the Roche Cobas and IDS iSYS assays are similar and may be used interchangeably, and similar reference intervals and treatment targets could be applied for the two automated assays. Harmonization between the automated assays and the RIA is potentially possible with the use of common calibrators and the development of a reference method for PINP. This should also help ensure that any new commercial assay developed in the future will attain similar results. IOF and IFCC are committed to working together towards this goal with the cooperation of the reagent manufacturing industry.

Fracture-induced changes in biomarkers CTX, PINP, OC, and BAP-a systematic review.

To assess the time from fracture until bone turnover markers (BTM), which are biochemical markers reflecting in vivo bone formation and resorptive activity, have returned to a stable level since BTM have been shown to be at least as good as bone mineral density in monitoring the effect of anti-resorptive treatment in osteoporosis. This study searched for articles in PUBMED, CINAHL, Medline, EM-BASE, and Cochrane, and identified 3486 unique articles. These articles were screened based on predefined inclusion and exclusion criteria. Seven articles addressing time to normalization of either CTX, PINP, osteocalcin, or bone-specific alkaline phosphatase after a recent fracture were identified and these were analyzed qualitatively. CTX appeared to return to baseline within 6 months. PINP appeared to return to baseline within 6 months and interestingly dip below baseline after a year. Osteocalcin was elevated throughout the first year after a fracture, with most changes in the first 6 months. Bone-specific alkaline phosphatase (BAP) was increased for up to a year, however with a discrepancy between used assays. Seven studies were identified, showing CTX and PINP to return to baseline within 6 months. OC was elevated for 12 months. BAP was increased for up to a year. However, none of these studies had fasting patients and a long follow-up period with regular measurements. The studies could indicate that the BTM CTX and PINP have returned to baseline within 6 months; however, further studies are needed assessing pre-analytical factors while having a long follow-up. Bone turnover markers appear as good as or better than bone mineral density in monitoring the effect of anti-resorptive medication in osteoporosis. This study tries to identify the time from fracture until BTM are back at baseline. Most studies did not however take pre-analytical variation into consideration. Further research is therefore needed.

Bone turnover markers are differentially affected by pre-analytical handling.

Given that bone turnover markers are often shipped to central laboratories, it is essential to be aware of factors that will affect stability. We have evaluated how sample type, time before separation of blood samples, and time between separation and analysis affect the stability of four bone turnover markers. INTRODUCTION: Bone turnover markers are often shipped to central laboratories for analysis, which require knowledge of the stability of the markers of interest in different sample materials. The aim of the current study was to evaluate how time before separation of blood samples and time between separation and analysis affect the stability of four bone turnover markers in serum and plasma samples. METHODS: Serum, EDTA, and Lithium heparin (LiHep) plasma samples from seven osteoporosis patients and three healthy controls were collected and stored at room temperature for up to 72 h before separation and analysis. After separation, samples were stored at room temperature for up to 72 h and re-analyzed. The bone turnover markers N-terminal pro-collagen type 1 extension pro-peptide (P1NP), bone-specific alkaline phosphatase (BAP), C-terminal teleopeptide cross links of collagen type 1 (CTX), and osteocalcin (OC) were analyzed using the automated iSYS IDS platform. RESULTS: P1NP and BAP were stable in both plasma and serum for 72 h before centrifugation. CTX levels were higher in EDTA plasma at all time points compared to LiHep plasma and serum. The use of EDTA plasma prolonged the stability of CTX as compared to LiHep plasma and serum. Osteocalcin showed high tendency to degrade in all sample types and concentrations were significantly lower after 24 h of storage. CONCLUSIONS: For the bone turnover markers P1NP and BAP, the use of both plasma and serum is recommended. Samples for CTX analysis should be taken as EDTA plasma. Samples for osteocalcin analysis can be taken in either type of plasma or serum, but should be analyzed within 3 h or preserved at - 18 °C.

Comparison of bone turnover markers in peripheral blood and bone marrow aspirate.

BACKGROUND: Bone remodeling takes place in the bone marrow environment. We investigated if levels of bone turnover markers (BTMs) differ between bone marrow and peripheral blood, if the bone marrow is an independent compartment, and how well the measurements in bone marrow correlate with bone mineral density. METHODS: Sixty-six men participated in a placebo controlled study designed to evaluate the effect of 16 weeks supplementation with resveratrol on bone mineral density and BTM. Bone marrow aspirates and blood samples were drawn at baseline and at week 16. Procollagen I N-terminal propeptide (PINP), C-terminal telopeptide of type I collagen (CTx), osteocalcin, bone specific alkaline phosphatase (BAP), and osteoprogeterin (OPG) were analyzed. Bland-Altman analysis was used to compare measurements across compartment to detect possible systematic or proportional differences. Paired t-test was performed if no proportional difference was revealed at the difference vs concentration plot. RESULTS: Measurements of PINP, CTx, and BAP differed proportionally between compartments depending on concentration; at low concentrations absolute values were only slightly different, while at higher average concentrations the levels were much higher in bone marrow than blood. Osteocalcin measures in bone marrow were systematically and significantly lower than in blood (mean ±â€¯SD; 14.4 µg/L ±â€¯5.3 µg/L versus 21.7 µg/L ±â€¯6.0 µg/L respectively, p < 0.001). OPG measures were comparable between compartments (p = 0.69). CTx and OPG measured in blood were negatively associated with lumbar spine BMD (ß = -0.22, p = 0.05 and ß = -0.02, p = 0.02, respectively), whereas both markers measured in bone marrow were not (p = 0.60 and p = 0.50 respectively). None of the BTMs, bone marrow or blood, were associated with total hip BMD. DISCUSSION: The levels of most BTMs differed significantly between bone marrow and peripheral blood, while OPG was comparable. Levels of PINP, CTx, and BAP differed between compartments depending on concentration, suggesting bone marrow to represent a compartment separate from the general circulation. Unexpectedly, osteocalcin was lower in the marrow, a gradient that was independent of concentration. BTMs measured in bone marrow did not show any association with bone mineral density. Although further studies are needed to investigate potential explanatory causes of the differences, BTMs in bone marrow do not seem to contribute further to fracture risk assessment.

Comparison of two automated assays of BTM (CTX and P1NP) and reference intervals in a Danish population.

Bone turnover markers are used for monitoring osteoporosis treatment. Therefore, we evaluated the agreement between different assays for CTX and PINP and established reference intervals in a cohort of 2300 individuals. We found poor agreement between assays and different reference intervals. This highlights the importance of harmonization of the assays. INTRODUCTION: Two reference markers for bone turnover have been proposed: CTX bone resorption and P1NP for bone formation. The purpose of the current study was to establish reference intervals for the two markers in a Danish cohort and to determine the agreement on the two platforms. METHODS: Fasting sera from 2308 individuals (1250 males and 1058 females, age range 24-76 years) participating in the Health2006 study were analyzed for CTX and P1NP using the automated IDS-iSYS analyzer and the automated Cobas e411 analyzer. Participants in anti-osteoporotic treatment were excluded, while subjects on hormonal contraceptives were included. RESULTS: There was significant disagreement between both the two P1NP assays with a mean difference of -3 µg/L (LoA -19 to 14) (p < 0.001) and the two CTX assays with a mean difference of 13 ng/L (LoA-187 to 214) (p < 0.001). For CTX, there was a systematic bias: at low values, Cobas measured a higher value than iSYS and at higher concentrations, iSYS measured increasingly higher values than Cobas. Based on the results, we propose three reference intervals for each sex: 25-29, 30-39, and 40-80 years for men, and 25-29, >30 (pre-menopausal), and >30 years (post-menopausal) for women. CONCLUSIONS: There is significant disagreement between the IDS-iSYS and Roche Cobas assays for both reference markers. Consequently, the reference intervals for an adult, healthy population are different depending on the analysis method used. Therefore, repeated measurements of patient samples used for monitoring of treatment should be done on the same assay. Moreover, assay-specific reference intervals should be used. Harmonization of assays for BTM is highly warranted.

Efficacy of recreational football on bone health, body composition, and physical functioning in men with prostate cancer undergoing androgen deprivation therapy: 32-week follow-up of the FC prostate randomised controlled trial.

UNLABELLED: Androgen deprivation therapy (ADT) for prostate cancer (PCa) impairs musculoskeletal health. We evaluated the efficacy of 32-week football training on bone mineral density (BMD) and physical functioning in men undergoing ADT for PCa. Football training improved the femoral shaft and total hip BMD and physical functioning parameters compared to control. INTRODUCTION: ADT is a mainstay in PCa management. Side effects include decreased bone and muscle strength and increased fracture rates. The purpose of the present study was to evaluate the effects of 32 weeks of football training on BMD, bone turnover markers (BTMs), body composition, and physical functioning in men with PCa undergoing ADT. METHODS: Men receiving ADT >6 months (n = 57) were randomly allocated to a football training group (FTG) (n = 29) practising 2-3 times per week for 45-60 min or to a standard care control group (CON) (n = 28) for 32 weeks. Outcomes were total hip, femoral shaft, femoral neck and lumbar spine (L2-L4) BMD and systemic BTMs (procollagen type 1 amino-terminal propeptide, osteocalcin, C-terminal telopeptide of type 1 collagen). Additionally, physical functioning (postural balance, jump height, repeated chair rise, stair climbing) was evaluated. RESULTS: Thirty-two-week follow-up measures were obtained for FTG (n = 21) and for CON (n = 20), respectively. Analysis of mean changes from baseline to 32 weeks showed significant differences between FTG and CON in right (0.015 g/cm(2)) and left (0.017 g/cm(2)) total hip and in right (0.018 g/cm(2)) and left (0.024 g/cm(2)) femoral shaft BMD, jump height (1.7 cm) and stair climbing (-0.21 s) all in favour of FTG (p < 0.05). No other significant between-group differences were observed. CONCLUSIONS: Compared to standard care, 32 weeks of football training improved BMD at clinically important femoral sites and parameters of physical functioning in men undergoing ADT for PCa.

Soluble urokinase plasminogen activator receptor (suPAR) is a novel, independent predictive marker of myocardial infarction in HIV-1-infected patients: a nested case-control study.

OBJECTIVES: Patients infected with HIV are at increased risk of myocardial infarction (MI). Increased plasma levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) have been associated with increased risk of cardiovascular diseases (CVD), including MI in the general population. We tested suPAR as a predictive biomarker of MI in HIV-1-infected individuals. METHODS: suPAR levels were investigated in a nested case-control study of 55 HIV-1-infected cases with verified first-time MI and 182 HIV-1-infected controls with no known CVD. Controls were matched for age, gender, duration of antiretroviral therapy (ART), smoking and no known CVD. suPAR was measured in the four plasma samples available for each patient at different time-points; 1, Before initiation of ART; 2, 3 months after initiation of ART; 3, 1 year before the case's MI; and 4, The last sample available before the case's MI. RESULTS: In unadjusted conditional regression analysis, higher levels of suPAR were associated with a significant increase in risk of MI at all time-points. Patients in the third and fourth suPAR quartiles had a three- to 10-fold higher risk of MI compared to patients in the lowest suPAR quartile at all time-points. suPAR remained a strong significant predictor of MI, when adjusting for HIV-1 RNA, total cholesterol, triglycerides and high-density lipoprotein. CONCLUSION: Elevated suPAR levels were associated with increased risk of MI in HIV-infected patients, suggesting that suPAR could be a useful biomarker for prediction of first-time MI in this patient group, even years before the event.

Epidemiology of forearm fractures in adults in Denmark: national age- and gender-specific incidence rates, ratio of forearm to hip fractures, and extent of surgical fracture repair in inpatients and outpatients.

UNLABELLED: National epidemiological studies of forearm fractures are scarce. We examined in- and outpatient rates in Denmark, including anatomical location, surgery, hospitalization ratio, recurrent fractures, and ratio of forearm to hip fractures. This may be useful for triangulation in countries with less detailed information. Rates were higher than previously estimated. INTRODUCTION: Despite a significant contribution to the overall burden of osteoporotic, nonvertebral fractures, relatively little information is available about age- and gender-specific incidence rates for many countries including Denmark. METHODS: We used national individual patient data on inpatient and outpatient treatment to calculate rates of forearm fractures, taking readmissions into account, with subtables for distal and proximal fractures. We also calculated ratios of forearm to hip fractures that may be useful when imputing forearm fracture rates from other administrative sources. In addition, we report the rates of hospital admission and the rates of surgical treatment, allowing readers to extrapolate from the number of admissions or surgical procedures to incidence rates, should their data sources be less comprehensive. RESULTS: Forearm fracture rates were 278 per 100,000 patient years in men aged 50+ and 1,110 per 100,000 in women aged 50+. The female to male incidence rate ratio was 4.0 for the age group 50+ but close to unity in persons aged 40 or under. Two thirds of patients were treated on an outpatient basis with little difference across age and gender strata. Four out of five fractures were treated conservatively. The rate of forearm fractures in Denmark was somewhat higher in both genders than recently imputed from hip fracture rates and were close to the rates previously reported in studies from Norway and Sweden. CONCLUSION: The rates of forearm fracture in Denmark are higher than previously estimated and very similar to the high risk reported from studies in Norway and Sweden.

The role of P2X receptors in bone biology.

Bone is a highly dynamic organ, being constantly modeled and remodeled in order to adapt to the changing need throughout life. Bone turnover involves the coordinated actions of bone formation and bone degradation. Over the past decade great effort has been put into the examination of how P2X receptors regulate bone metabolism and especially for the P2X7 receptor an impressive amount of evidence has now documented its expression in osteoblasts, osteoclasts, and osteocytes as well as important functional roles in proliferation, differentiation, and function of the cells of bone. Key evidence has come from studies on murine knockout models and from pharmacologic studies on cells and animals. More recently, the role of P2X receptors in human bone diseases has been documented. Loss-of-functions polymorphisms in the P2X7 receptorare associated with bone loss and increased fracture risk. Very recently a report from a genetic study in multiple myeloma demonstrated that decreased P2X7 receptor function was associated with increased risk of developing multiple myeloma. In contrast, the risk of developing myeloma bone disease and subsequent vertebral fractures was increased in subjects carrying P2X7 receptor gain-of-function alleles as compared to subjects only carrying loss-of-function or normal functioning alleles. It is evident that P2X receptors are important in regulating bone turnover and maintaining bone mass, and thereby holding great potential as novel drug targets for treatment of bone diseases. However, further research is needed before we fully understand the roles and effects of P2X receptors in bone.

Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts.

In vitro models of bone cells are important for the study of bone biology, including the regulation of bone formation and resorption. In this study, we have validated an in vitro model of human osteoblastic cells obtained from bone marrow biopsies from healthy, young volunteers, aged 20-31 years. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro.

Comparison of quantitative ultrasound and dual X-ray absorptiometry in estrogen-treated early postmenopausal women.

Identifying individuals at risk of developing osteoporosis is important in order to initiate early treatment. Many new techniques have been proposed as alternatives for DXA-scanning. Some of these alternatives certainly have advantages, but none have so far been demonstrated to predict fractures better, or even to identify individuals at risk of osteoporosis as well as with the standard method. In this study, comprising a group of women from the Danish Osteoporosis Prevention Study, we wished to investigate whether a technique based on quantitative ultrasound (QUS) could identify individuals with low BMC/BMD as measured by dual X-ray absorptiometry (DXA). Furthermore, we wished to test whether the method could detect differences between untreated individuals and those treated with hormone replacement therapy. We found that QUS could detect differences between the treated and untreated groups, but it was unable to identify women with low BMD, although it might be able to identify persons not at risk of osteoporosis. Low QUS values should be followed by a regular DXA measurement to confirm the presence of osteoporosis.

Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms.

Effective bone remodeling requires the coordination of bone matrix deposition by osteoblastic cells, which may occur via soluble mediators or via direct intercellular communication. We have previously identified two mechanisms by which rat osteoblastic cell lines coordinate calcium signaling among cells: autocrine activation of P2 (purinergic) receptors leading to release of intracellular calcium stores, and gap junction-mediated communication resulting in influx of extracellular calcium. In the current work we asked whether human osteoblastic cells (HOB) were capable of mechanically induced intercellular calcium signaling, and if so, by which mechanisms. Upon mechanical stimulation, human osteoblasts propagated fast intercellular calcium waves, which required activation of P2 receptors and release of intracellular calcium stores but did not require calcium influx or gap junctional communication. After the fast intercellular calcium waves were blocked, we observed slower calcium waves that were dependent on gap junctional communication and influx of extracellular calcium. These results show that human osteoblastic cells can propagate calcium signals from cell to cell by two markedly different mechanisms and suggest that these two pathways may serve different purposes in coordinating osteoblast functions.

The influence of smoking on vitamin D status and calcium metabolism.

OBJECTIVE: To assess the influence of smoking on serum parathyroid hormone (PTH), serum vitamin D metabolites, serum ionized calcium, serum phosphate, and biochemical markers of bone turnover in a cohort of 510 healthy Danish perimenopausal women. DESIGN: A cross-sectional study. SETTING: Copenhagen, Denmark. SUBJECTS: Five-hundred-and-ten healthy women aged 45-58 y, included 3-24 months after last menstrual bleeding. None were using hormone replacement therapy. METHODS: The women were grouped according to their current smoking status. The two groups were compared with regard to serum levels of 25-hydroxyvitamin D (25OHD) and 1, 25-dihydroxyvitamin D (1,25-(OH)2D), intact PTH, ionized calcium and phosphate, osteocalcin, as well as urine pyridinolines. Bone mineral density (BMD) was measured with DEXA-scans. Multiple regression analyses were performed to detect the effect of potentially confounding lifestyle factors, such as calcium and vitamin D intakes, alcohol and coffee consumption, sunbathing, and physical exercise. RESULTS: Fifty percent were current smokers. Smokers had significantly reduced levels of serum 25OHD (P=0.02), 1,25(OH)2D (P=0.001), and PTH (P<0.001). There was no difference in serum ionized calcium between smokers and non-smokers. We found a negative effect of smoking on serum osteocalcin (P=0.01), while urinary pyridinolines were similar in the two groups. The small differences in lifestyle between the two groups could not explain these findings. Smokers had small but significant reductions in bone mineral density. CONCLUSIONS: Smoking has a significant effect on calcium and vitamin D metabolism, which is not likely to be explained by other confounding lifestyle factors. The depression of the vitamin D-PTH system seen among smokers may represent another potential mechanism for the deleterious effects of smoking on the skeleton, and may contribute to the reported risk of osteoporosis among smokers. SPONSORSHIP: Grants from the Karen Elise Jensens Foundation.

Processing-independent analysis in the diagnosis of gastrinomas.

BACKGROUND: This study evaluates whether a new analytic principle, processing-independent analysis (PIA), offers better specificity and sensitivity than the conventional gastrin radioimmunoassay in the diagnosis of gastrinomas. METHODS: Plasma concentrations of alpha-amidated gastrins and the total progastrin product were measured with radioimmunoassay and with PIA, respectively, in 512 samples taken for gastrin measurement and in a selected group of gastrinoma patients (n=10). RESULTS: Among the 512 patients were 9 with gastrinomas. In plasma from these patients the median degree of amidation (ratio of alpha-amidated gastrins to total progastrin product) was 75% (range, 25-98%), whereas in the other groups the medians varied from 41% to 86%. In the second group of gastrinoma patients all had a degree of amidation of less than 50%. CONCLUSIONS: In screening for gastrinomas PIA offered no diagnostic advantages in comparison with conventional gastrin radioimmunoassay. However, in selected patients who in spite of normal or slightly increased concentrations of amidated gastrins were still suspected of having gastrinoma, additional measurement of the total progastrin product showed incomplete processing of progastrin and thus proved helpful in establishing the diagnosis.

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells.

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.

[Cytokines and osteoporosis]. / Cytokiner og osteoporose.

During the last few years, progress has been made towards the understanding of local regulation of bone remodelling especially in relation to osteoporosis. Cytokines have shown to be powerful regulators of bone resorption and formation, though under superior control from oestrogen/testosterone, parathyroidhormone and 1,25(OH)2D3. Some of the cytokines primarily enhance osteoclastic bone resorption e.g. IL-1 (Interleukin-1), TNF (Tumor Necrosis Factor) and IL-6 (Interleukin-6), while others primarily stimulate bone formation e.g. TGF-beta (Transforming Growth Factor), IGF (Insulin-like Growth Factor) and PDGF (Platelet Derived Growth Factor). Another category has complex functions with stimulation of bone formation in vitro but stimulation of bone resorption in vivo; IFN-gamma (Interferon-gamma) belongs to this category. The bone remodelling cycle is delicately regulated, and even a slight disturbance in this regulation can cause a pathological state in the bone such as osteoporosis. This paper will try to give a survey of some of the processes that regulate bone metabolism and hopefully contribute to understanding the changes in the remodelling related to osteoporosis.