Influence of carboxylation on osteocalcin detection by mass spectrometry.

RATIONALE: Osteocalcin is a small, abundant bone protein that is difficult to detect using high-throughput tandem mass spectrometry (MS/MS) proteomic approaches from bone protein extracts, and is predominantly detected by non-MS immunological methods. Here, we analyze bovine osteocalcin and its post-translational modifications to determine why a protein of this size goes undetected. METHODS: Osteocalcin was purified from cow bone using well-established methods. Intact osteocalcin or trypsin-digested osteocalcin were separated using an Agilent 1200 series high-performance liquid chromatography (HPLC) system and analyzed using a ThermoScientific LTQ-Orbitrap XL after fragmentation with higher-energy collision dissociation. Data were analyzed using Mascot or Prosight Lite. RESULTS: Our results support previous findings that the cow osteocalcin has up to three carboxylations using both intact osteocalcin and digested forms. Using Mascot, we were able to detect osteocalcin peptides, but no fragments that localized the carboxylations. Full annotation using Prosight Lite of the intact (three carboxylations), N-terminal peptide (one carboxylation), and middle peptide (two carboxylations) showed complete fragmentation was present, but complete neutral loss was observed. CONCLUSIONS: Osteocalcin carboxylation, and its associated neutral losses, makes high-throughput detection of this protein challenging; however, alternative fragmentation or limited purification can overcome these challenges. Copyright © 2016 John Wiley & Sons, Ltd.

Osteocalcin Is Not Associated with the Risk of Type 2 Diabetes: Findings from the EPIC-NL Study.

OBJECTIVE: To investigate whether total osteocalcin (tOC), uncarboxylated osteocalcin (ucOC) and percentage of uncarboxylated osteocalcin (%ucOC) are associated with the risk of type 2 diabetes. METHODS: This nested case control study included 1,635 participants, 833 incident diabetes cases and 802 non-diabetic control participants, aged 21-70 years from the EPIC-NL cohort. Baseline concentrations of tOC, ucOC and %ucOC were assessed. During 10 years of follow-up, diabetes cases were self-reported and verified against information from general practitioners or pharmacists. The association between the different forms of osteocalcin and diabetes risk was assessed with logistic regression adjusted for diabetes risk factors (waist circumference, age, sex, cohort, smoking status, family history of diabetes, hypertension, alcohol intake, physical activity and education) and dietary factors (total energy intake and energy adjusted intake of fat, fiber, protein and calcium). RESULTS: TOC concentration was not associated with diabetes risk, with an odds ratio (OR) of 0.97 (0.91-1.03) for each ng/ml increment after adjustment for diabetes risk factors and dietary factors. No association between ucOC and %ucOC and the risk of diabetes was observed either. In sex stratified analyses (P interaction = 0.07), higher %ucOC tended to be associated with an increased risk of type 2 diabetes in a multivariable model in women (OR 1.05 for each increment of 5% ucOC (1.00-1.11), Ptrend = 0.08), but not in men (OR 0.96 for each increment of 5% ucOC (0.88-1.04)). When waist circumference was replaced by body mass index, none of the osteocalcin forms were associated with the risk of type 2 diabetes in the final model among both women and men. CONCLUSIONS: Available evidence suggests that tOC, ucOC and %ucOC are each not associated with the risk of type 2 diabetes. However, more large-scale cohort studies are needed to clarify the presence of any association between the different forms of osteocalcin and the risk of type 2 diabetes.

Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry.

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0-3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.

A comparison of parathyroid hormone-related protein (1-36) and parathyroid hormone (1-34) on markers of bone turnover and bone density in postmenopausal women: the PrOP study.

Parathyroid hormone-related protein (PTHrP)(1-36) increases lumbar spine (LS) bone mineral density (BMD), acting as an anabolic agent when injected intermittently, but it has not been directly compared with parathyroid hormone (PTH)(1-34). We performed a 3-month randomized, prospective study in 105 postmenopausal women with low bone density or osteoporosis, comparing daily subcutaneous injections of PTHrP(1-36) to PTH(1-34). Thirty-five women were randomized to each of three groups: PTHrP(1-36) 400 µg/day; PTHrP(1-36) 600 µg/day; and PTH(1-34) 20 µg/day. The primary outcome measures were changes in amino-terminal telopeptides of procollagen 1 (PINP) and carboxy-terminal telopeptides of collagen 1 (CTX). Secondary measures included safety parameters, 1,25(OH)2 vitamin D, and BMD. The increase in bone resorption (CTX) by PTH(1-34) (92%) (p < 0.005) was greater than for PTHrP(1-36) (30%) (p < 0.05). PTH(1-34) also increased bone formation (PINP) (171%) (p < 0.0005) more than either dose of PTHrP(1-36) (46% and 87%). The increase in PINP was earlier (day 15) and greater than the increase in CTX for all three groups. LS BMD increased equivalently in each group (p < 0.05 for all). Total hip (TH) and femoral neck (FN) BMD increased equivalently in each group but were only significant for the two doses of PTHrP(1-36) (p < 0.05) at the TH and for PTHrP(1-36) 400 (p < 0.05) at the FN. PTHrP(1-36) 400 induced mild, transient (day 15) hypercalcemia. PTHrP(1-36) 600 required a dose reduction for hypercalcemia in three subjects. PTH(1-34) was not associated with hypercalcemia. Each peptide induced a marked biphasic increase in 1,25(OH)2 D. Adverse events (AE) were similar among the three groups. This study demonstrates that PTHrP(1-36) and PTH(1-34) cause similar increases in LS BMD. PTHrP(1-36) also increased hip BMD. PTH(1-34) induced greater changes in bone turnover than PTHrP(1-36). PTHrP(1-36) was associated with mild transient hypercalcemia. Longer-term studies using lower doses of PTHrP(1-36) are needed to define both the optimal dose and full clinical benefits of PTHrP. © 2013 American Society for Bone and Mineral Research.

Investigating the mechanism for maintaining eucalcemia despite immobility and anuria in the hibernating American black bear (Ursus americanus).

Ursine hibernation uniquely combines prolonged skeletal unloading, anuria, pregnancy, lactation, protein recycling, and lipolysis. This study presents a radiographic and biochemical picture of bone metabolism in free-ranging, female American black bears (Ursus americanus) that were active (spring bears and autumn bears) or hibernating (hibernating bears). Hibernating bears included lactating and non-lactating individuals. We measured serum calcium, albumin, inorganic phosphate, creatinine, bone specific alkaline phosphatase (BSALP), CTX, parathyroid hormone, insulin-like growth factor-I (IGF-l), leptin, 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and sclerostin from 35 to 50 tranquilized hibernating bears and 14 to 35 tranquilized spring bears. We compared metacarpal cortical indices (MCI), measured by digital X-ray radiogrammetry, from 60 hunter-killed autumn bears and 79 tranquilized, hibernating bears. MCI was greater in autumn than winter in younger bears, but showed no seasonal difference in older bears. During hibernation eucalcemia was maintained, BSALP was suppressed, and CTX was in the range expected for anuria. During hibernation 1,25(OH)(2)D was produced despite anuria. 1,25(OH)(2)D and IGF-I were less in hibernating than spring bears. In a quarter of hibernating bears, sclerostin was elevated. Leptin was greater in hibernating than spring bears. In hibernating bears, leptin correlated positively with BSALP in non-lactating bears and with CTX in lactating bears. Taken together the biochemical and radiographic findings indicate that during hibernation, bone turnover was persistent, balanced, and suppressed; bone resorption was lower than expected for an unloaded skeleton; and there was no unloading-induced bone loss. The skeleton appears to perceive that it was loaded when it was actually unloaded during hibernation. However, at the level of sclerostin, the skeleton recognized that it was unloaded. During hibernation leptin appeared anabolic in non-lactating bears and catabolic in lactating bears. We hypothesize that ursine hibernation may represent a natural model in which suppression of the sympathetic nervous system prevents unloading-induced bone loss by influencing leptin's skeletal effects and preventing transmission of loading information.

A 7-day continuous infusion of PTH or PTHrP suppresses bone formation and uncouples bone turnover.

Human in vivo models of primary hyperparathyroidism (HPT), humoral hypercalcemia of malignancy (HHM), or lactational bone mobilization for more than 48 hours have not been described previously. We therefore developed 7-day continuous-infusion models using human parathyroid hormone(1-34) [hPTH(1-34)] and human parathyroid hormone-related protein(1-36) [hPTHrP(1-36)] in healthy human adult volunteers. Study subjects developed sustained mild increases in serum calcium (10.0 mg/dL), with marked suppression of endogenous PTH(1-84). The maximal tolerated infused doses over a 7-day period (2 and 4 pmol/kg/h for PTH and PTHrP, respectively) were far lower than in prior, briefer human studies (8 to 28 pmol/kg/h). In contrast to prior reports using higher PTH and PTHrP doses, both 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] and tubular maximum for phosphorus (TmP/GFR) remained unaltered with these low doses despite achievement of hypercalcemia and hypercalciuria. As expected, bone resorption increased rapidly and reversed promptly with cessation of the infusion. However, in contrast to events in primary HPT, bone formation was suppressed by 30% to 40% for the 7 days of the infusions. With cessation of PTH and PTHrP infusion, bone-formation markers abruptly rebounded upward, confirming that bone formation is suppressed by continuous PTH or PTHrP infusion. These studies demonstrate that continuous exposure of the human skeleton to PTH or PTHrP in vivo recruits and activates the bone-resorption program but causes sustained arrest in the osteoblast maturation program. These events would most closely mimic and model events in HHM. Although not a perfect model for lactation, the increase in resorption and the rebound increase in formation with cessation of the infusions are reminiscent of the maternal skeletal calcium mobilization and reversal that occur following lactation. The findings also highlight similarities and differences between the model and HPT.

Lactation and bone turnover: a conundrum of marked bone loss in the setting of coupled bone turnover.

CONTEXT: Mothers who exclusively breastfeed lose up to 10% of their bone mass. This is primarily mediated by PTHrP, in combination with low estrogen levels. The mechanisms underlying this marked bone loss are unknown. Uncoupling of bone turnover, which is seen in other prototypical states of bone loss, would seem the likely explanation. However, the most current markers of bone turnover have not been studied in human lactation. OBJECTIVES: The purpose of this study was to assess bone formation in lactating humans using the most current bone turnover markers. DESIGN AND PARTICIPANTS: We conducted a prospective cohort study with repeated measures of bone metabolism in a volunteer sample of 49 women, recruited into three study groups: lactating, bottle feeding, and healthy controls. The postpartum women were studied at 6-8 and 12-14 wk postpartum, whereas the controls were studied at the follicular phase of their menstrual cycles. OUTCOME MEASURES: Biochemical markers of bone turnover were assessed. RESULTS: Mean serum C-telopeptide of type I collagen, a sensitive marker of bone resorption, was approximately 2-fold higher in lactating women as compared with bottle-feeding and healthy controls (P = 0.037 and P < 0.001, respectively). Surprisingly, amino-terminal telopeptides of procollagen 1, the most current marker of bone formation, bone-specific alkaline phosphatase, and osteocalcin were all significantly higher in the lactating group as compared with controls (P < 0.001, P = 0.002, and P < 0.001, respectively). CONCLUSIONS: In contrast to prototypical states of rapid bone loss (myeloma, cancer, and immobilization) in which markers of bone turnover display marked uncoupling, lactational bone loss, as assessed in this small exploratory study, is distinct, showing comparably rapid bone loss in the face of apparent osteoclast-osteoblast coupling.

Parathyroid hormone-related protein for the treatment of postmenopausal osteoporosis: defining the maximal tolerable dose.

CONTEXT: PTH is the only approved skeletal anabolic agent for the treatment of human osteoporosis. Unlike PTH, which is a mixed anabolic and catabolic agent, PTHrP displays features suggesting that it may be a pure anabolic agent when intermittently administered. The full dose range of PTHrP is unknown. OBJECTIVES: The primary objective of the study was to define the complete therapeutic window and dose-limiting toxicities of PTHrP. The secondary objective was to determine whether PTHrP retains a pure anabolic profile at the highest usable doses. DESIGN: This was a single-blinded, two-part, dose-escalating clinical trial. SETTING: The study was conducted in a university academic setting. PATIENTS OR OTHER PARTICIPANTS: Participants included 41 healthy postmenopausal women between the ages of 45 and 75 yr. INTERVENTION: INTERVENTIONs included PTHrP(1-36) or placebo in a dose-escalating design for 3 wk. MAIN OUTCOME MEASURES: Safety measures (hypercalcemia, nausea, vomiting, hemodynamics, flushing, miscellaneous) and bone turnover markers were measured. RESULTS: Intermittent PTHrP was administered safely and without serious adverse events in subjects receiving 500 and 625 microg/d for 3 wk. Subjects receiving 750 microg/d developed mild hypercalcemia. Bone turnover markers suggested that even at the highest doses, daily sc PTHrP may not activate bone resorption, i.e. may be purely anabolic. Interestingly, when hypercalcemia occurred, it may have resulted not from bone resorption but from activation of intestinal calcium absorption by 1,25 dihydroxyvitamin D. CONCLUSIONS: In doses as high as 750 microg/d, in contrast to PTH, intermittently administered PTHrP appears to act as a pure skeletal anabolic agent. Surprisingly, PTHrP in the high doses studied activates 1,25 dihydroxyvitamin D production. Dosing information obtained herein can be used to design a longer term head-to-head comparative efficacy trial of PTHrP vs. PTH.

Vitamin K, circulating cytokines, and bone mineral density in older men and women.

BACKGROUND: Vitamin K modulates cytokines involved in bone turnover, including interleukin-6 (IL-6) and osteoprotegerin in vitro. OBJECTIVE: The objective of this study was to assess 1) associations between measures of vitamin K status [plasma phylloquinone and serum percentage of undercarboxylated osteocalcin (%ucOC)] and IL-6, osteoprotegerin, and C-reactive protein (CRP) concentrations and 2) the effect of daily 500 mug phylloquinone supplementation for 3 y on cytokine concentrations. DESIGN: Concentrations of IL-6, osteoprotegerin, and CRP and bone mineral density (BMD) were measured at baseline and after 3 y of follow-up in 379 healthy men and women (60-81 y; 58.5% women) participating in a randomized trial that studied the effect of vitamin K supplementation on bone loss. RESULTS: Cross-sectionally, plasma phylloquinone was inversely associated with IL-6 and CRP, whereas serum %ucOC was inversely associated with IL-6. Osteoprotegerin was associated positively with plasma phylloquinone and inversely with %ucOC. No differences were observed in the 3-y change in IL-6, osteoprotegerin, and CRP concentrations between participants who received phylloquinone supplementation and those who did not. Overall, no association was observed between the 3-y changes in circulating cytokines and BMD. CONCLUSIONS: Poor vitamin K status was associated with high concentrations of cytokines involved in bone turnover, but vitamin K supplementation did not confer a decrease in cytokine concentrations. The healthy status of this cohort may explain a lack of effect of vitamin K supplementation on cytokine concentrations. This trial was registered with www.clinicaltrials.gov as NCT00183001.

Expression of an estrogen receptor agonist in differentiating osteoblast cultures.

Osteoblasts respond in direct and indirect ways to estrogens, and age-dependent changes in hormone levels and bone health can be limited by focused hormone replacement therapy. In this study, we report the release and isolation of an estrogen receptor agonist from osteoblast cultures. This entity reprises many aspects of estradiol activity in isolated osteoblasts, but differs from authentic estradiol by several biochemical and physical criteria. At levels that occur in conditioned medium from differentiating osteoblast cultures, the agonist directly drives gene expression through estrogen-sensitive response elements, activates the obligate osteoblast transcription factor Runx2, and potently enhances Smad-dependent gene expression in response to TGF-beta, but exhibits relatively lesser suppressive effects on gene expression through C/EBP and AP-1-binding protein transcription factors. Estrogen receptor agonist activity is resistant to heating at 100 degrees C and separable from the bulk of the remaining alcohol- and hexane-soluble molecules by C18 chromatography. MS and molecular fragmentation analyses predict a M(r) of 415.2 to 437.2. Therefore, in addition to earlier studies showing that osteoblasts readily respond to and metabolize various sex steroid-like substrates, we find that they also generate a potent estrogen receptor agonist during differentiation in vitro. Changes in the availability of a molecule like this within bone may relate to differences in skeletal integrity with aging or metabolic disease.

The presence of both an energy deficiency and estrogen deficiency exacerbate alterations of bone metabolism in exercising women.

BACKGROUND: Bone loss in amenorrheic athletes has been attributed to energy deficiency-related suppression of bone formation, but not increased resorption despite hypoestrogenism. OBJECTIVE: To assess the independent and combined effects of energy deficiency and estrogen deficiency on bone turnover markers in exercising women. DESIGN: PINP, osteocalcin, U-CTX-I, TT3, leptin, and ghrelin were measured repeatedly, and bone mineral density (BMD) was measured once in 44 exercising women. Resting energy expenditure (REE) was used to determine energy status (deficient or replete) and was corroborated with measures of metabolic hormones. Daily levels of urinary estrone and pregnanediol glucuronides (E1G, PdG), were assessed to determine menstrual and estrogen status. Volunteers were then retrospectively categorized into 4 groups: 1) Energy Replete+Estrogen Replete (EnR+E2R), (n=22), 2) Energy Replete+Estrogen Deficient (EnR+E2D), (n=7), 3) Energy Deficient+Estrogen Replete (EnD+E2R), (n=7), and 4) Energy Deficient+Estrogen Deficient (EnD+E2D), (n=8). RESULTS: The groups were similar (p>0.05) with respect to age (24.05+/-1.75 yrs), weight (57.7+/-2.2 kg), and BMI (21.05+/-0.7 kg/m2). By design, REE/FFM (p=0.028) and REE:pREE (p<0.001) were lower in the EnD vs. EnR group, and the E2D group had a lower REE:pREE (p=0.005) compared to the E2R group. The EnD+E2D group had suppressed PINP (p=0.034), and elevated U-CTX-I (p=0.052) and ghrelin (p=0.028) levels compared to the other groups. These same women also had convincing evidence of energy conservation, including TT3 levels that were 29% lower (p=0.057) and ghrelin levels that were 44% higher (p=0.028) than that observed in the other groups. Energy deficiency was associated with suppressed osteocalcin, and TT3 (p<0.05), whereas estrogen deficiency was associated with decreased E1G (p<0.02), and lower L2-L4 BMD (p=0.033). Leptin was significant in predicting markers of bone formation, but not markers of bone resorption. CONCLUSIONS: When the energy status of exercising women was adequate (replete), there were no apparent perturbations of bone formation or resorption, regardless of estrogen status. Estrogen deficiency in exercising women, in the presence of an energy deficiency, was associated with bone loss and involved suppressed bone formation and increased bone resorption. These findings underscore the importance of avoiding energy deficiency, which is associated with hypoestrogenism, to avoid bone health problems.

A reciprocal regulatory interaction between megakaryocytes, bone cells, and hematopoietic stem cells.

A growing body of evidence suggests that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. MK have been shown to express and/or secrete several bone-related proteins including osteocalcin, osteonectin, bone sialoprotein, osteopontin, bone morphogenetic proteins, and osteoprotegerin. In addition, at least 3 mouse models have been described in which MK number was significantly elevated with an accompanying marked increase in bone mineral density. Mice overexpressing thrombopoietin, the major MK growth factor, have an osteosclerotic bone phenotype. Mice deficient in transcription factors GATA-1 and NF-E2, which are required for the differentiation of MK, exhibited a strikingly increased bone mass. Importantly, recent studies have demonstrated that MK can stimulate osteoblast (OB) proliferation and differentiation in vitro and that they can also inhibit osteoclast (OC) formation in vitro. These findings suggest that MK play a dual role in skeletal homeostasis by stimulating formation while simultaneously inhibiting resorption. Conversely, cells of the osteoblast lineage support hematopoiesis, including megakaryopoiesis. Postnatal hematopoiesis occurs almost solely in the bone marrow (BM), close to or on endosteal surfaces. This finding, in conjunction with the observed contact of OB with hematopoietic cells, has lead investigators to explore the molecular and cellular interactions between hematopoietic cells and cells of the OB lineage. Importantly, it has been shown that many of the cytokines that are critical for normal hematopoiesis and megakaryopoiesis are produced by OB. Indeed, culturing osteoblasts with CD34+ BM cells significantly enhances hematopoietic cell number by both enhancing the proliferation of long-term culture initiating cells and the proliferation and differentiation of MK. These data are consistent with cells in the OB lineage playing a critical role in the hematopoietic niche. Overall, these observations demonstrate the importance of MK-bone cell interactions in both skeletal homeostasis and hematopoiesis.

Repetitive exposure to TGF-beta suppresses TGF-beta type I receptor expression by differentiated osteoblasts.

Transforming growth factor-beta (TGF-beta) has potent, cell phenotype restricted effects. In bone, it controls multiple activities by osteoblasts through three predominant receptors. Of these, the relative amounts of TGF-beta receptor I (TbetaRI) vary directly with TGF-beta sensitivity. The rat TbetaRI gene promoter includes cis-acting elements for transcription factor Runx2. Here we show conservation and selective partitioning of TbetaRI and retention of TGF-beta activity with osteoblast differentiation, Runx2 binding to the TbetaRI gene promoter on osteoblast chromatin, and decreased promoter activity by Runx2 binding site mutation. Furthermore, in contrast to the stimulatory effects induced by single or limited exposure to TGF-beta, we found that osteoblasts became resistant to TGF-beta after high dose and repetitive treatment. TbetaRI protein, mRNA, and gene promoter activity all decreased after three daily TGF-beta treatments, in parallel with a reduction in Runx2 protein and Runx dependent gene expression. In this way, sustained TGF-beta exposure can limit its own effectiveness by suppressing the expression of its primary signaling receptor. This tightly controlled system may constitute a feedback loop to protect against TGF-beta excess, and impose important limitations that are required for the progression of events during skeletal growth, remodeling and repair.

Megakaryocyte-mediated inhibition of osteoclast development.

A growing body of evidence indicates that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. We previously identified a novel regulatory pathway that controls bone formation, which is mediated by MK. In vivo megakaryocytosis resulted in massive bone formation. The co-culture of MK with osteoblasts (OB) resulted in increased OB proliferation in vitro, by a mechanism that required direct cell-to-cell contact. Here, we examined a second MK-mediated pathway that regulates osteoclast (OC) development. We have begun examining the unique inhibitory effect of MK on OC development. Spleen or bone marrow (BM) cells from C57BL/6 mice, as a source of OC precursors, were cultured with M-CSF and RANKL to induce OC development. MK were prepared by culturing fetal liver cells with thrombopoietin and separating cells into MK and non-MK populations. MK were titrated into spleen cell cultures and OC were identified as tartrate-resistant acid phosphatase-positive giant cells with >3 nuclei. There was a significant, P < 0.001, up to 10-fold reduction in OC formed when MK were added to the spleen cell cultures. We determined that 30% (vol:vol) MK conditioned media (CM) were able to completely block OC development from precursors, whereas 3% MK CM resulted in up to a 10-fold reduction in OC development, P < 0.001. These data indicate that a soluble factor(s) was responsible, at least in part, for the inhibition. We examined MK CM for known inhibitors of OC formation, using ELISAs. IL-4 was undetectable in MK CM, whereas IL-10 and IFN-gamma levels were similar in MK and non-MK CM. TGFbeta-1 levels were increased 2-fold in MK CM compared to control CM but were not responsible for the inhibition in OC development. Although, we found a significant increase in the levels of osteoprotegerin (OPG) in MK CM, antibody neutralization studies, MK derived from OPG-deficient mice, and tandem mass spectrophotometry, all confirm that OPG was not responsible for the MK-mediated inhibition of OC development. Overall, these data suggest that an unidentified factor(s) is present in MK CM that inhibits OC development. These studies indicate that MK can play a dual role in skeletal homeostasis by stimulating OB proliferation and simultaneously inhibiting OC development.

Positive linear growth and bone responses to growth hormone treatment in children with types III and IV osteogenesis imperfecta: high predictive value of the carboxyterminal propeptide of type I procollagen.

Extreme short stature is a cardinal feature of severe osteogenesis imperfecta (OI), types III and IV. We conducted a treatment trial of growth hormone in children with OI and followed linear growth velocity, bone metabolism markers, histomorphometrics, and vertebral bone density. Twenty-six children with types III and IV OI, ages 4.5-12 years, were treated with recombinant growth hormone (rGH), 0.1-0.2 IU/kg per day for 6 days/week, for at least 1 year. Length, insulin-like growth factor (IGF-I), insulin-like growth factor binding protein (IGFBP-3), bone metabolic markers, and vertebral bone density by DXA were evaluated at 6-month intervals. An iliac crest biopsy was obtained at baseline and 12 months. Approximately one-half of the treated OI children sustained a 50% or more increase in linear growth over their baseline growth rate. Most responders (10 of 14) had moderate type IV OI. All participants had positive IGF-I, IGFBP-3, osteocalcin, and bone-specific alkaline phosphatase responses. Only the linear growth responders had a significant increase in vertebral DXA z-score and a significant decrease in long bone fractures. After 1 year of treatment, responders' iliac crest biopsy showed significant increases in cancellous bone volume, trabecular number, and bone formation rate. Responders were distinguished from nonresponders by higher baseline carboxyterminal propeptide (PICP) values (p < 0.05), suggesting they have an intrinsically higher capacity for collagen production. The results show that growth hormone can cause a sustained increase in the linear growth rate of children with OI, despite the abnormal collagen in their bone matrix. In the first year of treatment, growth responders achieve increased bone formation rate and density, and decreased fracture rates. The baseline plasma concentration of PICP was an excellent predictor of positive response.

Effects of oral dehydroepiandrosterone on bone density in young women with anorexia nervosa: a randomized trial.

Young women with anorexia nervosa (AN) have subnormal levels of dehydroepiandrosterone (DHEA) and estrogen that may be mechanistically linked to the bone loss seen in this disease. The purpose of this study was to compare the effects of a 1-yr course of oral DHEA treatment vs. conventional hormonal replacement therapy (HRT) in young women with AN. Sixty-one young women were randomly assigned to receive oral DHEA (50 mg/d) or HRT (20 micro g ethinyl estradiol/0.1 mg levonorgestrel). Anthropometric, nutrition, and exercise data were acquired every 3 months, and bone mineral density (BMD) and body composition were measured by dual energy x-ray absorptiometry (DXA) every 6 months over 1 yr. Serum samples were obtained for measurements of hormones, proresorptive cytokines, and bone formation markers, and urine was collected for determinations of bone resorption markers at each visit. In initial analyses, total hip BMD increased significantly and similarly (+1.7%) in both groups. Hip BMD increases were positively correlated with increases in IGF-I (r = 0.44; P = 0.030) and the bone formation marker, bone-specific alkaline phosphatase increased significantly only in the DHEA treatment group (P = 0.003). However, both groups gained significant amounts of weight over the year of therapy, and after controlling for weight gain, no treatment effect was detectable. There was no significant change in lumbar BMD in either group. Both bone formation markers, bone-specific alkaline phosphatase and osteocalcin, increased transiently at 6-9 months in those subjects receiving DHEA compared with the estrogen-treated group (P < 0.05). Both DHEA and HRT significantly reduced levels of the bone resorption markers, urinary N-telopeptides (P < 0.05). There was a positive correlation between changes in IGF-I and changes in weight, body fat determined by DXA, and estradiol for both groups. In addition, patients receiving DHEA exhibited improvement on three validated psychological instruments (Eating Attitudes Test, Anorexia Nervosa Subtest, and Spielberger Anxiety Inventory). Both DHEA and HRT had similar effects on hip and spinal BMD. Over the year of treatment, maintenance of both hip and spinal BMD was seen, but there was no significant increase after accounting for weight gain. Compared with HRT, DHEA appeared to have anabolic effects, evidenced by the positive correlation between increases in hip DXA measurements and IGF-I and significant increases in bone formation markers. Both therapies significantly decreased bone resorption. Replicating results from studies of the elderly, DHEA resulted in improvements in specific psychological parameters in these young women.

Dietary and nondietary determinants of vitamin K biochemical measures in men and women.

Few epidemiological studies that rely on the food frequency questionnaire (FFQ) for dietary assessment have measured biomarkers of vitamin K intake to independently confirm associations between self-reported dietary vitamin K intake and disease risk. Associations were examined between two sensitive biomarkers of vitamin K status, plasma phylloquinone and serum percent undercarboxylated osteocalcin (%ucOC), and self-reported usual phylloquinone intake as estimated from a FFQ. The influence of other dietary and nondietary factors on plasma phylloquinone concentrations was also examined. Dietary phylloquinone intake was estimated using a FFQ in 369 men and 468 women of the Framingham Offspring Study. The prevalence of high %ucOC concentrations (>/= 20%), suggestive of a low vitamin K status, was 44% in men and 54% in women, respectively. After multivariate adjustment, the odds of a high %ucOC was 2.5 greater for women (odds ratio: 2.5; 95% confidence interval [CI]: 1.2-5.1) and almost three times greater for men (odds ratio: 2.8; 95% CI: 1.3-5.9) in the lowest dietary phylloquinone intake quintile category compared to the highest quintile category. Fasting triglyceride concentrations, smoking status and season were associated with plasma phylloquinone concentrations, independent of dietary phylloquinone intake. Phylloquinone and green vegetable intake was linearly associated with plasma phylloquinone, after adjustment for potential confounding factors. There were limitations in the use of the FFQ to predict plasma phylloquinone, evident in an observed plateau effect and required nondietary adjustment factors. Despite these caveats, these findings support the use of a FFQ for a relative assessment of vitamin K status in population-based studies.