Sex hormone status may modulate rate of expansion of proximal femur diameter in older women alongside other skeletal regulators.

CONTEXT: Little is known of associations between hip geometry and skeletal regulators. This is important because geometry is a determinant of both hip function and resistance to fracture. OBJECTIVE: We aimed to determine the effects of sex hormone status and other candidate regulators on hip geometry and strength. SUBJECTS AND METHODS: A random sample of 351 women aged 67-79 had two to four hip dual-energy x-ray absorptiometry scans performed over 8 yr of follow-up. Hip structural analysis software was used to measure subperiosteal diameter (PD) and the distance from the center of mass to the lateral cortical margin (d-lat) on three 5-mm-thick cross-sectional regions: narrow neck, intertrochanter, and shaft. Section modulus (Z), bone mineral density (grams per centimeter squared), and an index of bone mineral content (cross-sectional area) were calculated as estimators of bone strength. Serum analytes measured at baseline included SHBG, estradiol, PTH, creatinine, albumin, vitamin D metabolites, and glutamate- and gamma-carboxyglutamate-osteocalcin (OC). A linear mixed model was used to model associations with predictor variables, including testing whether the predictors significantly modified the effect of aging. RESULTS: Aging was associated with increasing PD and d-lat, and higher baseline SHBG significantly modified this effect, in the case of PD, increasing the rates of change at the narrow neck region by 19% for SHBG level 2 sd higher than population mean (P = 0.026). Higher baseline creatinine was independently associated with faster increases in PD and d-lat with aging (P < 0.041). Z declined faster with aging if baseline PTH was higher, and higher albumin had a contrary effect. Z was positively associated with free estradiol and inversely associated with SHBG and glutamate-OC. CONCLUSION: These results show large effects of SHBG on the regulation of proximal femur expansion and bending resistance, probably acting as a surrogate for low bioavailable estrogen. Potentially important effects for fracture resistance in old age were also revealed for PTH, markers related to renal function and the nutritional markers albumin and undercarboxylated OC.

A prospective study of change in bone mineral density over one year in adults with cystic fibrosis.

BACKGROUND: Low bone mineral density (BMD) is prevalent in adults with cystic fibrosis. To identify appropriate therapeutic strategies and the optimal time for intervention, it is necessary to document the natural history of cystic fibrosis related low BMD. METHODS: 114 adults with cystic fibrosis underwent bone densitometry a median (25-75% interquartile range) of 12 (12-13) months after initial assessment of bone density. BMD was measured in the lumbar spine, femoral neck, total hip, and distal forearm on recruitment to the trial and at follow up. RESULTS: In patients or=25 years of age (n=59, mean (SD) age 30.3 (5.4) years) in whom no annual change in BMD would normally be expected, BMD decreased by 1.9% (95% CI -2.9 to -0.8) per year in the femoral neck (p<0.001), by 1.5% (95% CI -2.4 to -0.6) per year in the total hip (p=0.001), and by 0.8% (95% CI -1.5 to -0.1) per year in the distal forearm (p=0.026). There was no significant annual change in lumbar spine BMD in either patient cohort. CONCLUSIONS: Reduced rates of bone accretion and accelerated rates of bone loss explain the high prevalence of low BMD in adults with cystic fibrosis.

The impact of atmospheric pollution on vitamin D status of infants and toddlers in Delhi, India.

AIMS: To compare the vitamin D status of 34 children, 9-24 months old, living in an area of Delhi renowned for high levels of atmospheric pollution (Mori Gate), with a comparable age matched group of children from a less polluted (Gurgaon) area of the city. METHODS: Serum concentrations of calcium, alkaline phosphatase (ALP), parathyroid hormone (PTH), 25-hydroxyvitamin D (25(OH)D), and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured. Haze scores, regarded as a surrogate marker of solar UVB radiation reaching ground level, were measured in both areas. RESULTS: Mean 25(OH)D of children in the Mori Gate area was 12.4 (7) ng/ml, compared with 27.1 (7) ng/ml in children living in the Gurgaon area (p < 0.001). The median ALP (p < 0.05) and mean PTH (p < 0.001) concentrations were higher in children living in the Mori Gate area than in the Gurgaon area. The mean haze score in the Mori Gate area (2.1 (0.5)) was significantly lower (p < 0.05) than in the Gurgaon area (2.7 (0.4)), indicating less solar UVB reaching the ground in Mori Gate. CONCLUSION: We suggest that children living in areas of high atmospheric pollution are at risk of developing vitamin D deficiency rickets and should be offered vitamin D supplements.

Vitamin D nutrition and bone disease in adults.

The consequences of vitamin D deficiency upon the skeleton are well known and management in the absence of renal failure is relatively straightforward. Vitamin D, either by mouth or parenterally will correct the deficiency and heal the osteomalacia. The mechanisms underlying the causation of vitamin D deficiency are now better understood and indicate the importance of underlying calcium malabsorption and secondary hyperparathyroidism leading to 1,25(OH)2D-induced catabolism of 25(OH)D and possibly also of vitamin D itself. In such situations, e.g., gastrointestinal and pancreaticobiliary disease, calcium supplementation in addition to vitamin D is indicated. The reasons behind nutritional vitamin D deficiency and the possible role of meat in protecting from osteomalacia await further elucidation, but from epidemiological studies, calcium deficiency, per se, is not implicated in the etiopathogenesis. The concept of vitamin D insufficiency is poorly understood, and difficult to define since a single value or close range of serum 25(OH)D values is unlikely to predict the needs of all subjects. Oral calcium intake and renal function are also likely to be relevant to the level of 25(OH)D which is found to be sufficient or insufficient for any given individual to maintain a normal serum calcium level without secondary hyperparathyroidism. There is increasing evidence that vitamin D insufficiency, by leading to sustained hyperparathyroidism, is prejudicial to the skeleton, particularly cortical bone. Since it is without symptoms until fractures occur, it should be actively sought in those clinical situations now recognized as contributing to risk. It can only be identified by the periodic measurement of serum 25(OH)D and the calcitropic hormones PTH and 1,25(OH)2D. In addition, BMD should be measured in a predominantly cortical site such as the proximal forearm, as well as the more conventional sites of spine and hip. The implications of these recommendations are an increase in the use of assays for PTH and vitamin D metabolites in the groups of subjects discussed in this review. Patients with chronic malabsorption states might reasonably be expected to have measurements performed twice-yearly. When vitamin D insufficiency is found, treatment with either vitamin D, calcium or both will be necessary, depending on the etiology of the insufficiency state in the inividual. In some malabsorptive states, calcium malabsorption is the cause of hyperparathyroidism and oral calcium alone can be used to reverse excess PTH activity in those with an adequate state of vitamin D nutrition. However, even in those vitamin D replete individuals, vitamin D catabolism will be enhanced and a small additional oral dose of vitamin D can do no harm. Regular monitoring of PTH and vitamin D metabolites will remain a necessity to ensure continued efficacy of treatment. Current recommendations for dietary supplements of vitamin D are clearly inadequate [61]. There is compelling evidence for supplements of 800 IU per day in the elderly and other high risk populations. Such a dose is safe and without side effects. The available evidence suggests that this should be combined with calcium supplements of 1200 mg/day [19] and that the current UK recommendations for a daily calcium intake of 700 mg contrast with those from the USA at 1,200 mg for people over 50 years old. Physicians need to be aware of both the small but important problem of vitamin D depletion and osteomalacia with its sometimes ambiguous presentation, and the more common but covert vitamin D (and calcium) insufficiency with its widespread and varied clinical associations.

Bisphosphonates in the treatment of Charcot neuroarthropathy: a double-blind randomised controlled trial.

AIMS/HYPOTHESIS: The management of charcot neuroarthropathy, a severe disabling condition in diabetic patients with peripheral neuropathy, is currently inadequate with no specific pharmacological treatment available. We undertook a double-blind randomised controlled trial to study the effect of pamidronate, a bisphosphonate, in the management of acute diabetic Charcot neuroarthropathy. METHODS: Altogether 39 diabetic patients with active Charcot neuroarthropathy from four centres in England were randomised in a double-blind placebo-controlled trial. Patients received a single infusion of 90 mg of pamidronate or placebo (saline). Foot temperatures, symptoms and markers of bone turnover (bone specific alkaline phosphatase and deoxypyridinoline crosslinks) were measured over the 12 months, in 10 visits. All patients also had standard treatment of the Charcot foot. RESULTS: Mean age of the study group (59 % Type II (non-insulin-dependent) diabetes mellitus) was 56.3 +/- 10.2 years. The mean temperature difference between active and control groups was 3.6 +/- 1.7 degrees C and 3.3 +/- 1.4 degrees C, respectively. There was a fall in temperature of the affected foot in both groups after 2 weeks with a further reduction in temperature in the active group at 4 weeks (active and placebo vs baseline; p = 0.001; p = 0.01, respectively), but no difference was seen between groups. An improvement in symptoms was seen in the active group compared with the placebo group (p < 0.001). Reduction in bone turnover (means +/- SEM) was greater in the active than in the control group. Urinary deoxypyridinoline in the pamidronate treated group fell to 4.4 +/- 0.4 nmol/mmol creatinine at 4 weeks compared with 7.1 +/- 1.0 in the placebo group (p = 0.01) and bone-specific alkaline phosphatase fell to 14.1 +/- 1.2 u/l compared with 18.6 +/- 1.6 u/l after 4 weeks, respectively (p = 0.03). CONCLUSION/INTERPRETATION: The bisphosphonate, pamidronate, given as a single dose leads to a reduction in bone turnover, symptoms and disease activity in diabetic patients with active Charcot neuroarthropathy.

Effect of intravenous pamidronate on bone mineral density in adults with cystic fibrosis.

BACKGROUND: Low bone mineral density (BMD) is prevalent in adults with cystic fibrosis. The aim of this study was to assess the effect of intravenous pamidronate on BMD in these subjects. METHODS: Patients were invited to participate if they had a BMD Z score of -2 or less in the lumbar spine, proximal femur, or distal forearm. Patients were randomised to receive either 30 mg intravenous pamidronate every 3 months + 1 g calcium daily (pamidronate group) or 1 g calcium daily (control group). All pancreatic insufficient patients were prescribed oral vitamin D supplements. RESULTS: After 6 months of treatment the pamidronate group (n=13) showed a significant increase in absolute BMD compared with the control group (n=15) in the lumbar spine (mean difference 5.8% (CI 2.7% to 8.9%)) and total hip (mean difference 3.0% (CI 0.3% to 5.6%)). However, the pamidronate group showed a reduction in BMD compared with the control group in the distal forearm (mean difference -1.7% (CI -3.7% to 0.3%)). The use of pamidronate was associated with a high incidence of bone pain in non-corticosteroid treated individuals. CONCLUSION: Intravenous pamidronate increases axial BMD in adults with cystic fibrosis, but the high incidence of bone pain associated with this treatment might limit its use.

Down-regulation of human osteoblast PTH/PTHrP receptor mRNA in end-stage renal failure.

BACKGROUND: Resistance to the action of parathyroid hormone (PTH) has been demonstrated in end-stage renal failure and is considered to be important in the pathogenesis of secondary hyperparathyroidism. The mechanism of resistance is unknown. However, altered regulation of cellular PTH/PTH-related protein (PTH/PTHrP) receptor (PTH1R) has been assumed to be important. METHODS: We have used in situ hybridization to examine PTH1R mRNA expression by osteoblasts in human bone and have compared the expression in high- and low-turnover renal bone disease, high-turnover nonrenal bone disease (healing fracture callus and Pagetic bone), and normal bone. Bone biopsies were formalin fixed, ethylenediaminetetraacetic acid decalcified, and paraffin wax embedded. A 1.8 kb PTH1R cDNA probe, labeled with 35S, was used, and the hybridization signal was revealed by autoradiography. The density of signal over osteoblasts was quantitated using a semiautomated Leica image analysis software package. RESULTS: The mean density of PTH1R mRNA signal over osteoblasts in renal high-turnover bone was only 36% of that found in nonrenal high-turnover bone (P < 0.05) and 51% of that found in normal bone (P < 0.05). Osteoblast PTH1R mRNA signal in adynamic bone from individuals with diabetes mellitus was 28% of normal bone (P < 0.05) and 54% of that found in renal high-turnover bone (P < 0.05). CONCLUSIONS: These results demonstrate a down-regulation of osteoblast PTH1R mRNA in end-stage renal failure in comparison to normal and high-turnover bone from otherwise healthy individuals, and provide an insight into the mechanisms of "skeletal resistance" to the actions of PTH.

1 alpha,25-dihydroxyvitamin D(3) inhibits angiogenesis in vitro and in vivo.

Modulation of angiogenesis is now a recognized strategy for the prevention and treatment of pathologies categorized by their reliance on a vascular supply. The purpose of this study was to evaluate the effect of 1 alpha,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)], the active metabolite of vitamin D(3), on angiogenesis by using well-characterized in vitro and in vivo model systems. 1,25(OH)(2)D(3) (1 x 10(-9) to 1 x 10(-7) mol/L) significantly inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting and elongation in vitro in a dose-dependent manner and had a small, but significant, inhibitory effect on VEGF-induced endothelial cell proliferation. 1, 25(OH)(2)D(3) also inhibited the formation of networks of elongated endothelial cells within 3D collagen gels. The addition of 1, 25(OH)(2)D(3) to endothelial cell cultures containing sprouting elongated cells induced the regression of these cells, in the absence of any effect on cells present in the cobblestone monolayer. Analysis of nuclear morphology, DNA integrity, and enzymatic in situ labeling of apoptosis-induced strand breaks demonstrated that this regression was due to the induction of apoptosis specifically within the sprouting cell population. The effect of 1,25(OH)(2)D(3) on angiogenesis in vivo was investigated by using a model in which MCF-7 breast carcinoma cells, which had been induced to overexpress VEGF, were xenografted subcutaneously together with MDA-435S breast carcinoma cells into nude mice. Treatment with 1,25(OH)(2)D(3) (12.5 pmol/d for 8 weeks) produced tumors that were less well vascularized than tumors formed in mice treated with vehicle alone. These results highlight the potential use of 1,25(OH)(2)D(3) in both the prevention and regression of conditions characterized by pathological angiogenesis.

The role of insulin-like growth factor I in age-related changes in calcium homeostasis in men.

The aim of this study was to evaluate hormonal influences on age-related changes in calcium homeostasis in men. We recruited 178 healthy men, ages 20-79 (about 30 per decade). We measured serum calcium, phosphate, urinary calcium, and creatinine clearance. Dietary calcium intake and use of fish oils were determined by questionnaire. Fractional calcium absorption was estimated using the stable strontium technique in a subgroup of 60 men. PTH, 1, 25-dihydroxyvitamin D [1,25(OH)(2)D], 25-hydroxyvitamin D (25OHD), serum insulin-like growth factor I (IGF-I), and testosterone were measured in all men. There was no change in serum calcium with age. There were decreases in serum phosphate, urinary calcium, and creatinine clearance with age (P: < 0.02). Dietary calcium was unchanged. Strontium absorption decreased (P: < 0.01), and PTH increased (P: < 0.001) with age. The data for 1,25OH(2)D were biphasic, reaching a peak at age 55 yr (P: = 0.003). There was a linear increase in 25OHD with age (P: = 0.009) that persisted after correcting for seasonal variation and was positively associated with fish oil use, therefore, the age-related changes in 25OHD were masked by self medication. There were log-linear decreases in IGF-I and testosterone with age (P: < 0.0001). Strontium absorption was not related to 25OHD or 1,25(OH)(2)D, but was positively correlated with IGF-I. 1,25(OH)(2)D correlated negatively with serum phosphate and calcium, but not PTH or creatinine clearance. IGF-I was positively associated with creatinine clearance, serum calcium, and phosphate and negatively associated with PTH (P: < 0.001). In this cross-sectional study of otherwise healthy, normally aging men, age-related decreases in IGF-I seem to have a greater impact on mineral absorption than does vitamin D status.

Altered calbindin mRNA expression and calcium regulating hormones in rat diabetic pregnancy.

Offspring of rats with diabetes mellitus are at risk of reduced calcium and bone mineral content. Altered expression of the maternal calcium binding proteins, calbindin-D(9K) and calbindin-D(28K), which are involved in renal and placental calcium transport, may underlie these problems.We have investigated the effect of diabetes on circulating concentrations of regulatory hormones with respect to calbindin-D mRNA concentrations. Three rat groups were studied; control (CP), streptozotocin-induced diabetic (DP), and insulin-treated diabetic (DPI) pregnant rats. Calbindin-D(9K) and calbindin-D(28K) mRNA abundance in placenta and maternal kidney were measured at days 7, 15, 18 and 21 of gestation, together with serum or plasma concentrations of 1,25 dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)), parathyroid hormone (PTH), PTH-related protein (PTHrP), calcitonin, oestradiol and IGF-I. An increase in placental calbindin-D(9K) mRNA abundance between days 18 and 21 in CP and DPI rats was severely blunted in the DP rats. In contrast, renal calbindin-D(28K) mRNA abundance was greater at days 7, 15 and 18 in DP compared with CP rats, as was calbindin-D(9K) at day 18. Calcitonin concentrations showed no differences between the groups, and both PTH and IGF-I were reduced over the first half of gestation, unlike the calbindins. In contrast, the concentrations of PTHrP and 1,25(OH)(2)D(3) were reduced at term in the DP group compared with the other two groups. Plasma oestradiol concentrations were lower in DP than in CP rats at days 7, 15 and 18, and most striking was the absence in DP rats of the peak of oestradiol seen at day 18 in CP rats. Despite the similarity between changes in placental calbindin mRNA and 1,25(OH)(2)D(3), previous work has shown placental calbindin-D(9K) regulation to be vitamin-D-independent. These studies produce suggestive evidence, therefore, that PTHrP and oestradiol may be involved in the altered calbindin-D expression by kidney and placenta in rat diabetic pregnancy.

Hip fracture and bone histomorphometry in a young adult with cystic fibrosis.

A 25-yr-old male with cystic fibrosis sustained a fragility fracture of the left femoral neck, which required surgical correction. He had several risk factors for the development of low bone density and despite treatment with an oral bisphosphonate, his bone mineral density reduced further. The patient died 2 yrs after sustaining the fracture. Bone specimens obtained at post mortem demonstrated severe cortical and trabecular osteopenia, but the histological features were not typical of osteoporosis or osteomalacia. Osteoporosis is thought to be a common complication of cystic fibrosis. The novel histomorphometric appearances reported here suggest that the bone disease of cystic fibrosis may be more complex and possibly unique. Labelled bone biopsies are required to clarify the bone defect leading to low bone density in cystic fibrosis patients so that appropriate therapeutic strategies can be developed.

Low bone mineral density in adults with cystic fibrosis.

BACKGROUND: Patients with cystic fibrosis have several risk factors for the development of low bone mineral density (BMD). To identify the prevalence and clinical correlates of low BMD in adult patients with cystic fibrosis, densitometry was performed in 151 patients (83 men) aged 15-52 years. METHODS: BMD was measured in the lumbar spine (L1-4) using dual energy x ray absorptiometry (DXA) and quantitative computed tomography (QCT). It was also measured in the proximal femur (total hip and femoral neck) using DXA, and in the distal and ultra distal forearm using single energy x ray absorptiometry (SXA). Biochemical markers of bone turnover, vitamin D levels, parathyroid hormone levels, and a variety of anthropometric variables were also assessed. RESULTS: The mean (SD) BMD Z score was -0.73 (0.85) in the distal forearm, -0.31 (0.92) in the ultra distal forearm, -1.21 (1. 18) in the lumbar spine using DXA, -0.56 (1.36) in the lumbar spine using QCT, -1.25 (1.30) in the femoral neck, and -1.01 (1.14) in the total hip. 34% of patients had a BMD Z score of -2 or less at one or more skeletal sites. Body mass index (0.527, p = 0.01), percentage predicted forced expiratory volume in one second (0.388, p = 0.01), and physical activity (0.249, p = 0.05) were positively related to the mean BMD Z score. Levels of C reactive protein (-0.328, p = 0. 01), parathyroid hormone (-0.311, p = 0.01) and biochemical markers of bone turnover (osteocalcin -0.261 and bone specific alkaline phosphatase -0.249, p = 0.05) were negatively related to the mean BMD Z score. Vitamin D insufficiency (25-hydroxyvitamin D <15 ng/ml) was prevalent (53/139 patients, 38%) despite supplementation with 900 IU vitamin D per day. CONCLUSIONS: Low bone density is prevalent in adult patients with cystic fibrosis. Current levels of vitamin D supplementation appear to be inadequate.

Prostaglandin E2 regulates vitamin D receptor expression, vitamin D-24-hydroxylase activity and cell proliferation in an adherent human myeloid leukemia cell line (Ad-HL60).

The effects of prostaglandin E2, forskolin, and phorbol 12-myristate 13-acetate on cell proliferation, cell surface antigen expression, vitamin D-24-hydroxylase activity and vitamin D receptor (VDR) expression have been studied in an adherent variant (Ad-HL60) of the human HL60 promyelomonocytic leukemia cell line. Ad-HL60 cells have a more differentiated phenotype than the nonadherent HL60 cells from which they were derived and, unlike the parent cell line, constitutively express vitamin D-24-hydroxylase activity. Treatment of Ad-HL60 cells with 1 microM PGE2 resulted in a decrease in the rate of cell proliferation (cell numbers were approximately 23% of control values after 72 h treatment), a change in expression of leukocyte surface antigens (decreased CD13 and CD14, increased CD11b and CD49d expression), an increase in the synthesis of 24,25-dihydroxyvitamin D3 from substrate 25-hydroxyvitamin D3 (control 5.76 +/- 0.17, 72 h PGE2-treated cells 12.10 +/- 1.90 pmol/h/10(6) cells), and an increase in receptors for the active metabolite of vitamin D, 1 alpha,25-dihydroxyvitamin D3, from 3910 to 11285 receptors per cell in control and 7-day treated cells, respectively. Prostaglandin E2 may be acting via a mechanism involving cyclic AMP in these cells, as we have also demonstrated that 10 microM forskolin, an adenylate cyclase activator, has similar effects. Phorbol 12-myristate 13-acetate had little effect on any of the parameters measured in this cell line.

Vitamin D receptors in the rheumatoid lesion: expression by chondrocytes, macrophages, and synoviocytes.

OBJECTIVES: The active form of vitamin D3, 1 alpha,25 dihydroxyvitamin D3 (1,25D3), through its interaction with vitamin D receptors (VDR), is reported to effect a variety of anabolic and catabolic events, especially in bone and cartilage tissues. As cartilage degradation and tissue remodelling are characteristic features of the rheumatoid lesion, the distribution and expression of VDR at sites of cartilage erosion was examined. METHODS: Immunolocalisation techniques using a rat monoclonal antibody to VDR and an alkaline phosphatase conjugated avidin/biotin detection system were used to examine VDR in 18 specimens of cartilage-pannus junction, 10 specimens of rheumatoid synovium or cartilage tissue, and four primary cultures of adherent rheumatoid synovial cells (RSC). For comparison, VDR expression was examined in 10 specimens of normal, healthy age matched articular cartilage. RESULTS: VDR was demonstrated in 15 of 18 cartilage-pannus junctions either at the interface (8 of 18), within the pannus tissue (12 of 18), and by chondrocytes often close to the erosive lesion (10 of 18). All the rheumatoid synovial tissue and 5 of 10 cartilage specimens showed cells with positive staining, but the extent of this was variable. Negligible VDR staining was observed for normal cartilage. Primary cultures of RSC also showed variability in both the numbers and proportions of macrophages or synovial fibroblasts stained for VDR (range 10-50%), this being more common in cultures with a high proportion of macrophages. CONCLUSIONS: VDR expression has been demonstrated by most specimens of cartilage-pannus junction; was associated with various cell types, including chondrocytes, but not exclusively with CD68+ macrophages. The focal nature of VDR expression within the rheumatoid lesion suggests a contributory role for 1 alpha,25D3 in the pathophysiological processes of rheumatoid arthritis.

Autocrine control of vitamin D metabolism in synovial cells from arthritic patients.

OBJECTIVE: This study was designed to investigate whether 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3), produced by activated synovial fluid macrophages, promotes its own catabolism by upregulating vitamin D-24-hydroxylase (24-OHase) in synovial fibroblasts through a vitamin D receptor (VDR) mediated mechanism. METHODS: Synovial macrophages and fibroblasts were derived from patients with rheumatoid arthritis. Expression of VDR and 24-OHase mRNAs was determined using in situ hybridisation. Vitamin D hydroxylase activity was determined by incubating cells with [3H]-25-(OH)D3, or [3H]-1,25-(OH)2D3, and metabolite synthesis quantified using high performance liquid chromatography. RESULTS: 1, 25-(OH)2D3 increased expression of mRNA for both VDR and 24-OHase in fibroblasts by approximately threefold over 24 hours. 1,25-(OH)2D3 increased fibroblast 24-OHase activity, yielding 24-hydroxylated, and more polar, metabolites. In co-culture, fibroblasts were able to catabolise macrophage derived 1,25-(OH)2D3. CONCLUSIONS: 1, 25-(OH)2D3 is produced by macrophages in vitro at biologically relevant concentrations and can increase its own catabolism by synovial fibroblasts; this effect is probably mediated via upregulation of both synovial fibroblast VDR and 24-OHase.

Novel mutations in the 1alpha-hydroxylase (P450c1) gene in three families with pseudovitamin D-deficiency rickets resulting in loss of functional enzyme activity in blood-derived macrophages.

Pseudovitamin D-defiency rickets (PDDR) is an autosomal recessive disorder characterized by hypocalcemia, rickets (which are resistant to treatment with vitamin D), and low or undetectable serum levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). The symptoms are corrected with 1,25(OH)2D treatment, and the disease is now believed to result from a defect in the cytochrome P450 component (P450c1; CYP27B1) of the renal 25-hydroxyvitamin D-1alpha-hydroxylase (1-OHase). We have studied genomic DNA from three families with PDDR and have identified the same homozygous mutation in the P450c1 gene in two of the index cases, causing a frameshift in exon 8, resulting in a premature stop codon in the heme-binding domain. The two cases in the third kindred were compound heterozygotes with missense mutations in exons 6 and 9. We have also identified a C/T polymorphism in intron 6 of the P450c1 genomic DNA. Interferon gamma-inducible 1-OHase activity in blood-derived macrophages was shown by 1,25(OH)2D synthesis in all control cells tested (37-184 fmol/h/106 cells) and those from the PDDR family parents (34-116 fmol/h/106 cells) but was totally absent from the patients' cells, indicating a defect in their macrophage 1-OHase, similar to the presumed renal defect. The assumption of similarity between the renal and macrophage P450c1 was supported by our ability to clone a 514 bp sequence, including the heme-binding region of the macrophage P450c1 cDNA from controls, which was identical to that published for both the renal and keratinocyte P450c1 cDNAs.

Bone loss in celiac disease is related to secondary hyperparathyroidism.

Celiac disease is a major cause of intestinal malabsorption. Previous studies have demonstrated that celiac disease is associated with significant osteoporotic bone loss. These studies have suggested that successful treatment of the malabsorption is associated with amelioration of the bone loss. Such studies have failed to examine bone mass at peripheral skeletal sites which is more likely to be responsive to changes in parathyroid hormone (PTH) in response to calcium malabsorption. We have examined bone density in the lumbar spine, femoral neck, and distal forearm in 35 patients with celiac disease who had been established on gluten-free diet. In addition, the concentrations of PTH and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured. Bone density was below that expected for the subject's age and gender at all sites. This was most marked in the distal forearm where the bone density was 1.40 SD below expected (p < 0.0001). In the forearm, there was a negative relationship between bone density and PTH concentration (r = -0.49, p = 0.009). In the forearm and lumbar spine, there was a negative relationship between 1,25(OH)2D concentration and bone density. Bone mass was not related to the concentration of 25-hydroxyvitamin D at any of the skeletal sites measured. Bone density is reduced in the peripheral skeleton in celiac disease and this deficit persists despite treatment with apparent normalization at axial skeletal sites. This reduction in bone mass is related to the presence of secondary hyperparathyroidism which should be sought in all patients with treated celiac disease.

Expression of genes involved in calcium absorption in human duodenum.

BACKGROUND: The role of vitamin D metabolites in the regulation of expression of genes involved in dietary calcium absorption by the human intestine remains poorly understood despite much work in animals. MATERIALS AND METHODS: To investigate this, we measured the expression of transcripts for two of these genes, calbindin-D9k and the basolateral membrane calcium pumping ATPase, PMCA1, in duodenal endoscopic biopsies from 40 subjects. Northern blots were hybridized with previously characterized probes, and the signal was quantified by phosphor imaging. These transcript levels were related to circulating vitamin D metabolites and also analysed according to the vitamin D receptor gene TaqI polymorphism determined in DNA from blood. RESULTS: Plasma 1,25-dihydroxycholecalciferol correlated significantly with calbindin-D9k RNA (r = 0.4, P < 0.02) but not with PMCA1. Plasma 25-OH-cholecalciferol was not correlated with transcripts for either gene and, furthermore, the mean levels of these transcripts did not differ significantly when grouped by vitamin D receptor genotype. CONCLUSION: In normal humans, 1, 25-dihydroxycholecalciferol has a small but significant relationship to duodenal expression of calbindin-D9k, but not to PMCA1 expression.