Young's modulus and hardness of human trabecular bone with bisphosphonate treatment durations up to 20 years.

Bone modulus from patients with osteoporosis treated with bisphosphonates for 1 to 20 years was analyzed. Modulus increases during the first 6 years of treatment and remains unchanged thereafter. INTRODUCTION: Bisphosphonates are widely used for treating osteoporosis, but the relationship between treatment duration and bone quality is unclear. Since material properties partially determine bone quality, the present study quantified the relationship between human bone modulus and hardness with bisphosphonate treatment duration. METHODS: Iliac crest bone samples from a consecutive case series of 86 osteoporotic Caucasian women continuously treated with oral bisphosphonates for 1.1-20 years were histologically evaluated to assess bone turnover and then tested using nanoindentation. Young's modulus and hardness were measured and related to bisphosphonate treatment duration by statistical modeling. RESULTS: All bone samples had low bone turnover. Statistical models showed that with increasing bisphosphonate treatment duration, modulus and hardness increased, peaked, and plateaued. These models used quadratic terms to model modulus increases from 1 to 6 years of bisphosphonate treatment and linear terms to model modulus plateaus from 6 to 20 years of treatment. The treatment duration at which the quadratic-linear transition (join point) occurred also depended upon trabecular location. Hardness increased and peaked at 12.4 years of treatment; it remained constant for the next 7.6 years of treatment and was insensitive to trabecular location. CONCLUSIONS: Bone modulus increases with bisphosphonate treatment durations up to 6 years, no additional modulus increases occurred after 6 years of treatment. Although hardness increased, peaked at 12.4 years and remained constant for the next 7.6 years of BP treatment, the clinical relevance of hardness remains unclear.

Two-year cortical and trabecular bone loss in CKD-5D: biochemical and clinical predictors.

This prospective two-year study of patients on chronic dialysis measured changes in bone mineral density (BMD). Patients with higher baseline BMD and shorter dialysis vintage lost more bone. Treatment with anti-hypertensives acting on the central nervous system was protective against bone loss. Baseline serum levels of sclerostin and bone-specific alkaline phosphatase predicted bone loss. INTRODUCTION: This prospective 2-year study of chronic kidney disease on dialysis (CKD-5D) patients assessed trabecular and cortical bone loss at the hip and spine and examined potential demographic, clinical, and serum biochemical predictors of bone loss. METHODS: Eighty-nine CKD-5D patients had baseline, year 1, and year 2 bone mineral density (BMD) measurements using dual X-ray absorptiometry (DXA) and quantitative computed tomography (QCT); concurrent blood samples were drawn and clinical variables recorded. No study treatments occurred. RESULTS: The 2-year total hip BMD change was - 5.9% by QCT and - 3.1% by DXA (p < 0.001). Spinal BMD was unchanged. QCT total hip cortical mass and volume decreased (- 7.3 and - 10.0%); trabecular volume increased by 5.9% (ps < 0.001). BMD changes did not vary with age, BMI, race, diabetes, smoking, or exercise. Patients with higher baseline BMD and shorter dialysis vintage lost more bone (p < 0.05). Vitamin D analogs and phosphate binders were not protective against bone loss; cinacalcet was protective by univariate but not by multivariable analysis. CNS-affecting antihypertensives were protective against loss of BMD, cortical mass, cortical volume (ps < 0.05) and trabecular mass (p = 0.007). These effects remained after adjustment. BSAP correlated with changes in BMD, cortical mass, and volume (p < 0.01) as did sclerostin (inversely). CONCLUSIONS: There was severe cortical bone loss at the hip best recognized by QCT. Patients with shorter dialysis vintage and less pre-existing bone loss lost more bone, while treatment with CNS-acting antihypertensives was protective. BSAP and sclerostin were useful markers of bone loss.

Stiffness and strength of bone in osteoporotic patients treated with varying durations of oral bisphosphonates.

UNLABELLED: Apparent modulus and failure stress of trabecular bone structure from 45 women with osteoporosis treated with bisphosphonates for varying durations were studied using finite element analyses and statistical modeling. Following adjustments for patient age and bone volume, increasing bisphosphonate treatment duration for up to 7.3 years was associated with treatment-time-dependent increases in bone apparent modulus and failure stress. Treatment durations exceeding 7.3 years were associated with time-dependent decreases in apparent modulus and failure stress from the peak values observed. INTRODUCTION: The purpose of this study was to clarify the relationship between bisphosphonate (BP) treatment duration and human bone quality. This study quantified changes in the apparent modulus and failure stress of trabecular bone biopsied from patients with osteoporosis who were treated with BPs for widely varying durations. METHODS: Forty-five iliac crest bone samples were obtained from women with osteoporosis who were continuously treated with oral BPs for varying periods of up to 16 years. Micro-CT imaging was used to develop three-dimensional virtual models of the trabecular bone from these samples. Apparent modulus and failure stress of these virtual models were determined using finite element analyses (FEA). Polynomial regression and cubic splines, adjusted for relevant (age and BV/TV) covariates, were used to statistically model the data and quantify the relationships between BP treatment duration and apparent modulus or failure stress. RESULTS: Second-order polynomial models were needed to relate apparent modulus or failure stress to BP treatment duration. These models showed that these bone quality parameters (a) increased with increasing BP treatment duration up to approximately 7.3 years, (b) reached a maximum at this (~7.3 years) time, and then (c) declined with BP treatment durations exceeding ~7.3 years. A similar result was obtained by modeling with cubic splines. CONCLUSIONS: Changes in FEA-derived apparent stiffness and failure stress are attributable to changes in trabecular bone structure, which in turn are related to the duration of BP treatment. These relationships are evident even after adjustments are made in the statistical models for changes in age and BV/TV. According to these models, increases in trabecular bone apparent stiffness and failure stress linked to BPs cease and appear to reverse after approximately 7.3 years of treatment. Conclusions regarding optimal BP therapy duration await study of additional bone quality parameters.

The new kidney disease: improving global outcomes (KDIGO) guidelines - expert clinical focus on bone and vascular calcification.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) defines a triad of interrelated abnormalities of serum biochemistry, bone and the vasculature associated with chronic kidney disease (CKD). The new kidney disease: improving global outcomes (KDIGO) guidelines define the quality and depth of evidence supporting therapeutic intervention in CKD-MBD. They also highlight where patient management decisions lack a strong evidence base. Expert interpretation of the guidelines, along with informed opinion, where evidence is weak, may help develop effective clinical practice. The body of evidence linking poor bone health and reservoir function (the ability of bone to buffer calcium and phosphorus) with vascular calcification and cardiovascular outcomes is growing. Treating renal bone disease should be one of the primary aims of therapy for CKD. Evaluation of the biochemical parameters of CKD-MBD (primarily phosphorus, calcium, parathyroid hormone and vitamin D levels) as early as CKD Stage 3, and an assessment of bone status (by the best means available), should be used to guide treatment decisions. The adverse effects of high phosphorus intake relative to renal clearance (including stimulation of hyperparathyroidism) precede hyperphosphatemia, which presents late in CKD. Early reduction of phosphorus load may ameliorate these adverse effects. Evidence that calcium load may influence progression of vascular calcification with effects on mortality should also be considered when choosing the type and dose of phosphate binder to be used. The risks, benefits, and strength of evidence for various treatment options for the abnormalities of CKD-MBD are considered.

Femoral bone mineral density reflects histologically determined cortical bone volume in hemodialysis patients.

UNLABELLED: We evaluated the associations between dual energy X-ray absorptiometry (DXA) and histologically determined cancellous and cortical bone volume by controlling for vascular calcifications and demographic variables in hemodialysis (HD) patients. Femoral bone mineral density (f-BMD) was associated with cortical porosity. INTRODUCTION: Assessment of bone mass in chronic kidney disease patients is of clinical importance because of the association between low bone volume, fractures, and vascular calcifications. DXA is used for noninvasive assessment of bone mass whereby vertebral results reflect mainly cancellous bone and femoral results reflect mainly cortical bone. Bone histology allows direct measurements of cancellous and cortical bone volume. The present study evaluates the association between DXA and histologically determined cancellous and cortical bone volumes in HD patients. METHODS: In 38 HD patients, DXA was performed for assessment of bone mass, anterior iliac crest bone biopsies for bone volume, and multislice computed tomography for vascular calcifications. RESULTS: While lumbar bone mineral density (l-BMD) by DXA was not associated with histologically measured cancellous bone volume, coronary Agatson score showed a borderline statistically significant association (P = 0.055). When controlled for age and dialysis duration, f-BMD by DXA was associated with cortical porosity determined by histology (P = 0.005). CONCLUSIONS: The usefulness of l-BMD for predicting bone volume is limited most probably because of interference by soft tissue calcifications. In contrast, f-BMD shows significant association with cortical porosity.

The five most commonly used intact parathyroid hormone assays are useful for screening but not for diagnosing bone turnover abnormalities in CKD-5 patients.

BACKGROUND/AIMS: Assessment of bone turnover for management of renal osteodystrophy is part of routine care in chronic kidney disease Stage 5 (CKD-5) patients. Measurement of intact parathyroid hormone (iPTH) is the most commonly used surrogate marker for bone turnover in these patients. The current study was conducted to evaluate the predictive value of the five most commonly used iPTH assays for bone turnover. METHODS: In a cross-sectional study, 84 CKD-5 patients underwent bone biopsy and blood drawings for determination of iPTH and total serum alkaline phosphatase (AP). RESULTS: Histologically, patients presented with a broad range of bone turnover abnormalities as determined by activation frequency and bone formation rate/bone surface. Results of the five iPTH assays in each patient correlated but were significantly different. There were also significant differences between iPTH measurements at the same bone turnover level. Using Kidney Disease Outcome Quality Initiative recommended iPTH ranges, all assays showed comparably poor diagnostic performance. At 80% specificity, cut-off values of the 5 iPTH assays for low bone turnover varied from 165 to 550 pg/ml and for high bone turnover from 404 to 1,003 pg/ml. Sensitivities at these cutoffs remained below acceptable standards. Addition of AP measurements to iPTH did not improve diagnostic accuracy. CONCLUSIONS: Precise assessment of bone turnover will require utilization of established and novel bone markers reflecting effects of bone turnover rather than measuring only iPTH or other effectors.

Absence of adynamic bone disease in African-Americans with CKD stage 5 after 3 years of vitamin D therapy guided by iPTH and the PTH-(1-84)/N-terminally truncated PTH fragments ratio.

BACKGROUND: Secondary hyperparathyroidism is a frequent complication of chronic kidney disease (CKD). The goal of treatment is to achieve circulating levels of parathyroid hormone (PTH) associated without oversuppression of bone turnover. This is commonly achieved by treatment with vitamin D analogs. Doses of vitamin D compounds are usually monitored by measurement of circulating levels of PTH. STUDY DESIGN: To prospectively assess the effects on bone histology of two different protocols for dosing vitamin D. SETTING AND PARTICIPANTS: African-American patients from the same geographic area, managed by the same team of physicians in three dialysis clinics were studied. Patients were treated with vitamin D for 3 years and underwent bone biopsies for assessment of bone turnover. Dosing of vitamin D during the 3 years prior to the biopsy was done following two different guidelines. One group was treated following K/DOQI guidelines adapted to the bio-intact PTH assay (Protocol A), the other group was managed (Protocol B) following K/DOQI guidelines for intact PTH and/or the ratio of PTH-(1-84)/N-terminally truncated fragments (PTH ratio). PREDICTOR: Levels of circulating PTH and/or PTH ratio. OUTCOME: Prevalence of low bone turnover. MEASUREMENTS: Qualitative and quantitative assessment of bone histology after tetracycline labeling. RESULTS: 7 out of 22 patients managed following Protocol A were found to have low bone turnover (32%) by bone histology. None of the 21 patients managed by Protocol B for guidance of vitamin D therapy, had low bone turnover. LIMITATIONS: Lack of bone biopsy at the beginning of study. CONCLUSIONS: This report indicates that the additional information provided by the PTH ratio represents a distinct advantage in avoiding low bone turnover over the use of a single PTH assay to guide vitamin D dosing in African-American patients with CKD Stage 5 on dialysis.

Improvements in renal osteodystrophy in patients treated with lanthanum carbonate for two years.

AIMS: To investigate the evolution of renal osteodystrophy in patients on maintenance dialysis, treated with lanthanum carbonate (LC) vs. standard phosphate-binder therapy (Stx). MATERIALS AND METHODS: This was a 2-year, randomized, prospective, open-label study during which patients on dialysis received LC titrated to a maximum of 3,000 mg/day or their previous phosphate binder treatment with the aim to achieve target phosphorus levels of < or = 5.9 mg/dl. Paired bone biopsy samples for histomorphometric analysis were available at baseline and 1 year (LC 32, Stx 33), and at baseline and 2 years (LC 32, Stx 24). RESULTS: With similar phosphorus control, Stx was associated with numerically higher serum calcium levels at most visits. Results of osteocalcin and bone-specific alkaline phosphatase in LC patients were higher throughout the study and correlated with parameters of bone formation; however, the differences were not significant. Histological changes in bone turnover and volume were analyzed with respect to normal ranges. There was an improvement in bone turnover in the LC group, which was significant in the 1-year group, and an improvement in bone volume which was significant in the 2-year group. No significant changes in bone turnover or bone volume were observed in the Stx groups. In the 2-year LC group, 1 patient had osteomalacia at baseline and end of therapy, and a mineralization defect developed in 2 other patients. Several possible factors for a mineralization defect were present in these patients, but no single cause could be clearly identified. Histomorphometric parameters of bone, including formation and mineralization, did not correlate with bone lanthanum. No mineralization defect was observed in the Stx groups. CONCLUSION: These findings show that similar phosphorus control with Stx and LC results in higher bone turnover after 1 year and higher bone volume after 2 years with LC.

An assessment of cinacalcet HCl effects on bone histology in dialysis patients with secondary hyperparathyroidism.

AIMS: Cinacalcet lowers plasma parathyroid hormone (PTH) levels in patients with secondary hyperparathyroidism (sHPT), but the bone histologic response has not been described. This prospective, double-blind, placebo-controlled trial assessed the effects of cinacalcet on bone histology and serum markers of bone metabolism in dialysis patients with sHPT. METHODS: Patients with intact PTH (iPTH) > or = 300 pg/ml were randomly assigned 2:1 to receive cinacalcet or placebo with concurrent vitamin D and/or phosphate binder therapy. Cinacalcet (30 - 180 mg/day) was used to achieve iPTH levels < or = 200 pg/ml. Bone biopsies were performed before and after one year of treatment. RESULTS: Baseline and end-of-study data were available from 32 patients (19 cinacalcet, 13 placebo). Baseline bone turnover was elevated in 27, reduced in 3 and normal in 2 patients. Serum bone-specific alkaline phosphatase (BSAP) and N-telopeptide (NTx) were elevated. Cinacalcet treatment decreased PTH and diminished activation frequency, bone formation rate/bone surface, and fibrosis surface/bone surface. Adynamic bone was observed in three patients receiving cinacalcet; in two of these, PTH levels were persistently low (< 100 pg/ml). The histomorphometric parameter changes in bone corresponded to PTH, BSAP and NTx reductions. Bone mineralization parameters remained normal. CONCLUSIONS: Treatment with cinacalcet lowered PTH and reduced bone turnover and tissue fibrosis among most dialysis patients with biochemical evidence of sHPT.

Influence of the parathyroid glands on bone metabolism.

Bone is a classic target tissue for parathyroid hormone (PTH), whose calciotropic effect is mediated largely via catabolic actions on this tissue. Paradoxically, PTH also exerts anabolic actions, with intermittent injections of PTH or its amino-terminal fragments causing an increase in bone formation and bone mass, actions that form the basis for the use of PTH in the treatment of osteoporosis. Besides vitamin D, PTH is the only other known bone anabolic agent. High-affinity PTH receptors (PTH-1R) have been detected on osteoblasts and osteoclasts (albeit in lower numbers). Bone turnover, which includes activation of osteoclasts and osteoblasts, appears to be best reflected not by absolute concentrations of PTH (which can vary based on the assay and antibody used) but by a balance of circulating full-length PTH-(1-84) and amino-terminally truncated C-PTH fragments. When PTH-(1-84) is predominant, bone turnover is promoted. Among PTH fragments, PTH-(7-84) appears to be the most potent antagonist of PTH-(1-84). The mechanisms involved in these effects are unclear although mediation via unique C-terminal receptors has been suggested. We propose that, within the range of total PTH (100-1000 pg mL(-1)), the ratio of PTH-(1-84)/C-PTH fragment is a valuable tool for diagnosis of bone turnover. Data indicate that at PTH levels < 100-150 pg mL(-1) and > 1000 pg mL(-1), the ratio looses its predictive power. Assay type, patient characteristics (race, underlying renal disease) and treatment attributes (vitamin D, corticosteroids, phosphate binders) have an impact on the PTH ratio, and care should be used in interpreting assay results and making subsequent treatment decisions.

Changes in total parathyroid hormone (PTH), PTH-(1-84) and large C-PTH fragments in different stages of chronic kidney disease.

INTRODUCTION: Loss of renal function is accompanied by progressive increase in serum levels of intact parathyroid hormone (iPTH) in patients with end-stage renal disease (ESRD). There is a paucity of data regarding levels of PTH-(1-84) and its large carboxyl-terminal fragments (large C-PTH fragments) and progressive loss of kidney function in patients with chronic kidney disease (CKD). The current study was undertaken to describe the glomerular filtration rate (GFR)-dependent plasma concentrations of PTH-(1-84) and related large C-PTH fragments in adult patients with CKD by using different commercially available PTH assays. METHODS: We studied 80 Caucasian patients with CKD stages 1-5 without renal replacement therapy. Creatinine clearance was calculated by the Modification of Diet in Renal Disease (MDRD) formula. Levels of iPTH were determined by second-generation assays (iPTH Elecsys system, Roche Diagnostics; DUO total iPTH, Scantibodies Laboratory, Inc.; iPTH, Nichols Institute Diagnostics). Third-generation assays were used to measure PTH-(1-84) (CAP (cyclase activating PTH), Scantibodies; Bio-Intact PTH, Nichols). Levels of large C-PTH fragments and ratios of PTH-(1-84)/large C-PTH fragments were calculated and statistical analyses performed. RESULTS: Levels of iPTH and PTH-(1-84) showed CKD stage-dependent increases. Variations among the assays increased with progressive loss of kidney function. The assay from Scantibodies showed a GFR-dependent decrease of the ratio 1-84 PTH / large C-PTH fragment that was not observed with the Nichols assay. CONCLUSION: Increasing variations among the assays with progression of CKD emphasize the fact that the interpretation of measurements must take into consideration the specific assay. We found evidence for a possible preferential increase of the level of large C-PTH fragments over 1-84 PTH in a CKD stage-dependent manner (Scantibodies). The clinical implications of this finding have to be further evaluated by bone biopsy studies.

Renal osteodystrophy: what's in a name? Presentation of a clinically useful new model to interpret bone histologic findings.

Renal osteodystrophy begins early in the course of chronic kidney disease and occurs almost without exception in all patients with Stage 5 disease (CKD-5). Bone biopsies and evaluation of mineralized bone sections after double tetracycline-labeling are currently considered the gold standard for diagnosis and classification of renal osteodystrophy. Nevertheless, bone biopsies are rarely employed. This is, at least in part, related to the paucity of nephrologists trained in performance of the procedure and the fact that reports of the histologic results are not easily translatable to clinical practice. Results are usually given qualitatively, using non-uniform classifications or by histomorphometric evaluations which are esoteric to most nephrologists. We suggest here that histomorphometric evaluation can be reserved for research and special situations. Also, the customarily used qualitative classification should be replaced by a clinically useful nomenclature, provided the interpretation is done by an individual with sufficient experience in bone pathology. We present a new interactive nomenclature for renal osteodystrophy that addresses abnormalities of turnover, abnormalities of bone balance, and abnormalities of mineralization. The new nomenclature, thus, includes disorders of high- and low-turnover with consideration of the interrelation with positive or negative bone balance with or without mineralization defect. In this schema, changes in bone status are described as deviations from a norm, and treatment is geared toward normalizing values rather than creating any absolute change in one direction or another. It is hoped that such a classification will be easily usable, clinically more relevant, and more amenable to individualized treatment guidance.

Utilization of bone histomorphometry in renal osteodystrophy: demonstration of a new approach using data from a prospective study of lanthanum carbonate.

AIM: The aim of this study was to develop a novel approach to the analysis of bone histomorphometric data and outcomes presentation that would simplify the characterization of renal osteodystrophy and facilitate clinical decision-making. METHODS AND MATERIALS: Data were derived from a randomized trial of dialysis patients treated for one year with a dose of lanthanum carbonate or calcium carbonate (up to 3750 mg/day and 9000 mg/day, respectively). Histomorphometric analyses of baseline and end-of-study bicortical transiliac bone biopsies were performed. Activation frequency, bone formation rate/bone surface, osteoclast surface/ bone surface, osteoblast surface/bone surface, mineralization lag time, and osteoid thickness were determined to provide a measure of overall bone cell activity (bone formation, bone resorption, bone turnover) and risk of developing osteopenia (bone balance). A novel approach of qualitatively grouping these numerical data as "improved", "unchanged", or "worsened" based on deviation from normal was used to facilitate interpretation of clinical relevance. RESULTS: Using our method, lanthanum carbonate was shown to improve histomorphometric parameters measured. These improvements were superior to those produced by calcium carbonate. These data add valuable clinical relevance to the previously published qualitative data from the same cohort [D'Haese et al. 2003]. Lanthanum carbonate moderated extreme forms of renal osteodystrophy, whereas calcium carbonate treatment increased the incidence of adynamic and predominant hyperparathyroid bone disease. CONCLUSIONS: This study provides an approach to the prospective evaluation of bone disease progression with therapy, and its application supports the safety and greater efficacy of one-year lanthanum carbonate versus calcium carbonate therapy as a means to normalize bone turnover in dialysis patients.

Contrasting mammalian PTH promoters: identification of transcription factors controlling PTH gene expression.

Parathyroid hormone (PTH) is a key component in the maintenance of calcium and phosphate homeostasis. The steady-state expression of the PTH gene can be modeled as a balance between transcriptional activators and repressors. During renal failure, the gradual loss of kidney function is often accompanied by increased circulating concentrations of PTH and decreased synthesis of 1,25-di-hydroxyvitamin D3 (1,25(OH)2D3). The latter finding results in impaired calcium absorption and the removal of a known repressor of PTH gene transcription. Current regimens for treating secondary hyperparathyroidism associated with renal insufficiency are focused on boosting activities that repress PTH gene transcription or secretion of the hormone, and involve the use of vitamin D and its analogues or calcimimetic agents. However, in recent years, concerns have arisen over the use of the steroid hormone and alternative treatments are being sought. Here, we present new information regarding transcription factors controlling PTH gene expression, which include the specificity proteins (Sp) and the nuclear factor Y (NF-Y) complex. A highly conserved DNA response element for the Sp proteins has been identified in mammalian promoters, while an NF-Y binding site is uniquely positioned in the human promoter. Both of these factors are expressed in the parathyroid gland and their DNA elements appear to be functioning as activators of PTH gene expression. Further elucidation of such pathways may offer novel approaches for treating hyperparathyroidism associated with renal failure via suppression of transcriptional activators. That work is currently in progress.

Parathyroid hormone assays--evolution and revolutions in the care of dialysis patients.

Renal osteodystrophy may present with low, normal, or high bone turnover. An ideal parathyroid hormone (PTH) assay should discriminate between the bioactive whole PTH-(1-84) molecule and PTH fragments, including the PTH-(7-84) fragment. Most dialysis patients have "intact" PTH (iPTH) levels between 65 and 450 pg/ml, which are poorly predictive of bone turnover state, making the iPTH test of limited value for bone turnover prediction. iPTH levels higher than 500 pg/ml can be observed in some dialysis patients with low bone turnover, while iPTH levels as low as 100 pg/ml have been found in patients with bone turnover above normal, indicating the standard second generation iPTH assay is not a reliable sole indicator of bone turnover. The whole PTH immunoradiometric assay, a third generation assay, uses a detection antibody that recognizes antigenic determinants at the extreme amino-terminal (1-4) end of the PTH molecule, making the assay specific for biologically active whole PTH-(1-84). Comparing results using the whole PTH and iPTH assays, the PTH-(7-84) level is indirectly determined and the PTH-(1-84)/PTH-(7-84) ratio can be calculated. It was shown that PTH-(7-84) inhibits the calcemic effect of PTH-(1-84) and its stimulatory effect on bone turnover. In the interpretation of results using the PTH-(1-84)/PTH-(7-84) ratio, it must be taken into consideration that second generation "intact" PTH assays have different cross-reactivity with PTH-(7-84). Therefore, when comparing or analyzing PTH-(1-84)/PTH-(7-84) ratios, the employed PTH assays must be identical. The whole PTH assay and the PTH-(1-84)/PTH-(7-84) ratio allow more meaningful interpretation of PTH trends, and offer a non-invasive means to more accurately diagnose bone disease in this population.

Improved assessment of bone turnover by the PTH-(1-84)/large C-PTH fragments ratio in ESRD patients.

BACKGROUND: The "intact" parathyroid hormone (PTH) assay recognizes PTH-(1-84) as well as amino terminally truncated PTH fragments, that is, large carboxyterminal PTH fragments (C-PTH fragments). The present study investigated whether the use of the plasma PTH-(1-84)/C-PTH fragment ratio enhances the noninvasive assessment of bone turnover in patients on dialysis. METHODS: Bone biopsies and blood samples for determinations of routine indices of bone turnover and PTH peptides were obtained in 51 adult patients on dialysis not treated with drugs affecting bone such as vitamin D or corticosteroids. Blood levels of large C-PTH fragments were calculated by subtracting PTH-(1-84) from "intact" PTH. Patients were classified according to their levels of bone turnover based on histomorphometrically obtained results of activation frequency. Prediction of bone turnover by the various blood indices was done by using proper statistical methods. In addition, hypercalcemia was induced by calcium gluconate infusion in a subset of patients, and levels of PTH-(1-84), "intact" PTH, and PTH-(1-84)/C-PTH fragment ratio were determined. RESULTS: The PTH-(1-84)/C-PTH fragment ratio was the best predictor of bone turnover. A ratio> 1 predicted high or normal bone turnover (sensitivity 100%), whereas a ratio <1 indicated a high probability (sensitivity 87.5%) of low bone turnover. Calcium infusion resulted in decrease in PTH-(1-84)/C-PTH fragment ratio. CONCLUSIONS: The PTH-(1-84)/C-PTH fragment ratio predicts bone turnover with acceptable precision for biological measurements. Moreover, a change in serum calcium levels is one of the regulators of the relative amount of circulating PTH-(1-84) and its large C-PTH fragments.

Hypercalcemia and human nature.

Patients on hemodialysis may develop severe and symptomatic hypercalcemia if skeletal buffering is ineffective. We report a case of persistent hypercalcemia with apparent extrarenal vitamin D synthesis. Associated aluminium intoxication was suggested on desferrioxamine challenge and adynamic uremic osteodystrophy confirmed on bone biopsy. Plasma calcitriol did not suppress with corticosteroids but did with ketoconazole. No other evidence for underlying granulomatous disease was found. We discuss our approach to less usual causes of hypercalcemia, and emphasise the pitfalls associated with factitious disorders.

Isolation of genomic DNA sequences that bind vitamin D receptor complexes.

Vitamin D signaling is believed to be transduced by a heterodimeric receptor complex that binds to specific sequences of DNA termed vitamin D response elements (VDREs) in the promoter regions of target genes. However, recent studies have suggested that considerable flexibility exists in the types of binding sites the vitamin D receptor (VDR) is capable of recognizing, including some that bind VDR homodimers. In this report, a screening method involving immunoselection and PCR amplification was utilized to examine genomic binding sites for the receptor. Four individual fragments ranging in size from ca. 250-320 bp were nominally isolated from the amplified pool of captured fragments for further analysis. Each of the four sequences was capable of forming specific, unique VDR complexes using recombinant human VDR (rhVDR) alone or rhVDR heteromers formed in conjunction with the addition of recombinant human retinoid X receptor alpha (rhRXRalpha). Two of these fragments exhibited significant hormone-dependent repression of luciferase activity when linked to a thymidine kinase driven reporter vector. DNaseI footprinting revealed specific binding over DR+3 or related half-site sequences found within both of these DNA fragments. The results from this study demonstrate that specific, functional binding sites for the VDR can be successfully isolated from genomic DNA and should aid in the discovery of genes regulated by the steroid hormone.

Evidence of functional vitamin D receptors in rat hippocampus.

The steroid hormone vitamin D has important biological roles in calcium transport, cell growth, and cell differentiation. Its cellular activities are mediated by high affinity interaction with the vitamin D receptor. In brain, autoradiographic, immunohistologic, and messenger RNA expression studies implicate a number of neuronal systems, including the hippocampus, as potential targets of vitamin D. However, cellular distribution and protein expression, and binding of the receptor to vitamin D response elements have yet to be established in hippocampus. This investigation was undertaken to characterize the vitamin D receptor in rat hippocampus with western blot, immunocytochemistry, and gel shift analyses. The presence of the receptor protein in hippocampus extracts was revealed with western blotting using an anti-rat vitamin D receptor antiserum. In vivo and in vitro immunocytochemical results confirmed the presence of vitamin D receptor in neuronal and glial cells. In the hippocampus, the receptor was localized in pyramidal and granule cell layers, CA1, CA2, and CA3 subfields and in the dentate gyrus. Double labeling for the vitamin D receptor and glial fibrillary acidic protein revealed that glia also expressed the receptor protein. Gel shift analyses evaluated with the murine osteopontin vitamin D response element indicated a specific, bound receptor-containing complex from hippocampal extracts. Altogether, these findings clearly document the localization of vitamin D receptor in rat hippocampus and that hippocampus contains vitamin D receptors capable of specifically binding to DNA. In combination with reports of a neuroprotective role for vitamin D in hippocampal cell survival, these data suggest that the endogenous vitamin D receptor may mitigate processes related to cellular homeostasis, perhaps through a calcium buffering mechanism.