Bone and mineral最新文献

X-Linked hypophosphatemic rickets (XLH) is an X-linked dominant disorder that is secondary to renal phosphate wasting. Affected individuals frequently present the following characteristics: short stature, lower-extremity deformity, bone pain, dental abscesses, en-thesopathy, rickets, and osteomalacia. Since the disorder is characterized by evident phenotypic abnormalities, we hypothesized that there would be a high degree of knowledge about the disease in affected kindreds. Thus, we constructed a six-page, self-administered questionnaire to determine whether family members are, in fact, aware of their disease and properly diagnosed and treated. We also designed the survey to determine rates of symptoms thought to be associated with rickets/osteomalacia in a population with a lower referral bias than is usually seen in tertiary care centers. We administered the questionnaire to 234 study subjects (57 affected) who were members of one of three large kindreds. Although 62% of affected individuals knew they had some problem with their bones, only 22.6% were told by a physician that they had rickets or osteomalacia. This apparent lack of awareness occurred in spite of 61.1% of affected subjects complaining of bone or joint problems to their personal physician. Indeed, of those patients who had persistent complaints, only 34.5% were told they had rickets or osteomalacia. Only one patient was taking phosphate and vitamin D. The spectrum of symptoms evident in affected subjects compared with normals included: dental abscesses (54.5% vs. 13.0%, P < 0.001), bone pain (45.5% vs. 28.2%, P = 0.027), back pain (51.8% vs. 35.1%, P = 0.036), joint stiffness (48.2% vs. 16.8%, P < 0.001), joint pain (55.4% vs. 31.1%, P = 0.003), weakness (25.0% vs. 10.7%, P = 0.023), and hearing loss (28.6% vs. 9.8%, P = 0.002). Surprisingly, although affected individuals complained of many symptoms due to XLH, they fractured bones less frequently than controls (20% vs. 38.1%., P = 0.018). Our data demonstrate that, despite the presence of disease in family members, few affected subjects knew that they had XLH. Although the presence of symptoms did increase knowledge of disease status, only one-third of symptomatic individuals knew of their diagnosis.

Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at −70°C/37°C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.

Thirty-six 3-month-old female Wistar rats were labelled with a single intraperitoneal tetracycline injection. Twenty-four animals were subsequently ovariectomized, while the control group of 12 animals underwent sham operations. All animals received the basal Ewos R3 diet and half of the ovariectomized animals (n=12) were given an additional 5% dietary xylitol supplementation. Three months later, following the collection of blood and urine, the animals were killed by decapitation. The tibiae were detached and prepared for chemical and other studies. The weight and density of the tibiae were measured. The right tibiae were dried and pulverized for chemical analysis of calcium, phosphorus and citric acid. The total inorganic fraction was determined by ashing the powdered bone. The left tibiae were cross-sectioned at the tibio-fibular junctions for the measurement of the width of periosteally formed bone, which was identified by tetracycline fluorescence. The examination of mineral content of bone was performed by scanning electron microscopy, using an electron probe microanalytic technique. The results indicate that the supplementation of the diet with 5% xylitol had a protective effect against the loss of bone mineral after ovariectomy in the rat. This was clearly seen in tibial density and in the inorganic fraction of the bone, and in the concentrations of bone Ca and phosphorus. Ovariectomy caused a doubling in periosteal bone formation relative to the controls, whereas the growth of the periosteally formed bone was somewhat reduced following xylitol supplementation as compared with ovariectomy alone. The results support the idea that dietary xylitol after ovariectomy seems to protect against mineral loss and may support a favourable effect of dietary xylitol on the prevention of osteoporosis.

A modified and improved radiographic absorptiometry of the distal radius which enables on-site analysis, called computer assisted X-ray densitometry (CXD), was evaluated from the viewpoint of quality assessment. Its precision and the correlation with dual energy X-ray absorptiometry (DXA) was evaluated in 12 volunteers (mean age 44.7 years). The profile of CXD-measured radial bone mineral density (RBMD) from 142 subjects (75 premenopausal and 67 postmenopausal women, mean ages 44.9 and 50.6 years, respectively) were compared with previous data by other methodologies of bone mineral analysis. The intra-assay coefficient of variation (CV) was 0.617%, the inter-assay CV was 2.064%, and the inter-observer CV was 0.673%o. The correlation between CXD-measured RBMD and DXA-measured RBMD was of statistical significance (r² = 0.733, P < 0.01). The correlation of CXD-measured RBMD with age, height or weight corresponded well with previous reports. CXD-measured RBMD and DXA-measured vertebral bone mineral density (VBMD) also had a significant positive correlation, but their correlation was not so close (r² = 0.149, P < 0.01). The discriminative ability of osteoporosis by CXD was of acceptable level (odd's ratio = 5.72, P < 0.05), when assessed by comparison with bone dystrophy score (BDS) on the plain vertebral radiogram. Although some problems remain in technical standardization, CXD could be an easy, inexpensive, and widely applicable alternative of non-weight bearing cancellous bone densitometry.

Ascites sarcoma 180 (S180A) is a transplantable tumor which causes hypercalcemia in tumor-bearing mice, and stimulates bone resorption without parathyroid hormone-like activity. In the present study, parathyroid hormone-related protein (PTHrP) mRNA could not be detected in total RNA from S180A cells. Bone-resorbing activity (BRA) derived from serum-free conditioned medium of S180A cells (S180A-CM) was coeluted with either transforming growth factor α (TGFα) activity (peak A, approximate M_r 29 kDa) or lymphocyte-activating factor (LAF) activity (peak B, M_r, 20.1–24 kDa) in Bio-Gel P-100 column chromatography. Fractions in peak A and B contained IL-6 but not tumor necrosis factor α (TNFα). Subsequent separation of peak A by reverse-phase high performance liquid chromatography produced a single fraction which contained both BRA and TGFα activity. Recombinant human TGFα-induced bone resorption was completely inhibited by indomethacin. The BRA in peak A was partially inhibited by indomethacin and almost completely inhibited by simultaneous treatment of indomethacin and anti-IL-6 antibody. The BRA in peak B was partially inhibited by neutralization with anti-IL-1α antibody and was completely inhibited by simultaneous treatment with anti-IL-1α and anti-IL-6 antibody in the absence of indomethacin. Bone resorption induced by S180A-CM was associated with an increased production of prostaglandin E₂ (PGE₂) by cal varia. The BRA in S180A-CM, however, was not completely abolished by the simultaneous addition of indomethacin and anti-IL-1α, anti-IL-1β and anti-IL-6 antibodies. Our findings indicate that (1) BRA derived from S180A cells includes TGFα, IL-1α, IL-6 and some other unknown factor(s), distinct from PTHrP, IL-1β and TNFα, and (2) these unknown factors resorb bone in part via a PGE₂-independent pathway.

We reported previously that the variability in bone loss rates among postmenopausal women decreases dramatically during the first few years of followup. In this paper, we have examined the distributions of bone loss rates measured at the calcaneus, distal radius and proximal radius. The incidence of physical impairment was five times greater among women with bone loss rates faster than 2 S.D. below the mean. Because the rate of change in bone density was skewed at the lower end of the distribution (representing rapid bone loss), the influence of values at the extreme ends of the distribution were statistically removed in order to estimate the normal distribution of bone loss rates. For the convenience of clinicians, the upper and lower limits of the 90 and 70% normal ranges are presented. Because average bone loss rates vary with age, normal ranges are provided separately by age group. The width of each normal range decreased by at least half after 3 or 4 years of followup, compared to less than 1 year. Consequently, measured loss rates which were well within the normal range at 1 year were sometimes far outside the normal range for longer followup times. We conclude that followup duration has a profound effect on estimates of the normal range, and must be considered when interpreting the clinical significance of measured loss rates.

Bisphosphonates are now widely used in the treatment of bone diseases, particularly where there is uncontrolled bone resorption, as they are known to be potent inhibitors of osteoclasis. It is still unclear whether the bisphosphonates act by inhibiting osteoclast maturation or by blocking the mechanism of bone resorption, and little is known of their effects on osteoblasts. Recent studies with 3-amino-1, hydroxypropylidene-1,1-bisphosphonic acid (APD) in the treatment of osteolytic metastases in breast cancer have suggested that APD may affect osteoblasts directly. We have now investigated the effects of two novel bisphosphonates, CGP 47072 and CGP 42446A on osteoclastogenesis in fetal rat calvariae cultured on collagen gels and on human osteoblasts (hOB) cultured as expiants from bone taken from patients at surgery. We also compared the action of these new bisphosphonates with that of APD, which at concentrations of 2.5 × 10⁻⁶ M to 2.5 × 10⁻¹⁰ M inhibited osteoclast recruitment, even when this was stimulated by conditioned medium from MCF7 breast cancer cells. This bisphosphonate was particularly potent if cultured with calvaria taken at 19 days gestation, when more immature osteoclast precursors are present. If calvariae from 20 days gestation were used, which contain more mature cells, it produced less inhibition. In contrast, CGP 42446A (2.5 × 10⁻⁶ M to 2.5 × 10⁻⁸ M) was more effective in inhibiting osteoclast maturation in calvariae from 20 days gestation than in those from 19 days. CGP 47072 had a similar pattern of effects but was less potent than either of the other two compounds. APD or CGP 42446A at 10⁻⁵ M significantly inhibited hOB numbers and DNA synthesis, but lower concentrations had little effect. CGP 47072 did not inhibit human osteoblast replication. It is unlikely that these effects are due to calcium chelation, as none of these compounds mimicked results obtained with EDTA, which was effective only at 2.5 × 10⁻⁶ M in reducing osteoclast size and 10⁻⁴ M in human osteoblast cultures. These results demonstrate that all three bisphosphonates are able to inhibit osteoclast formation at low concentrations. APD may be able to influence less mature osteoclast precursors and CGP 42446A and CGP47072 may exert their effects on the fusion of more mature precursor cells on the bone surface. At these concentrations, however, there is little or no effect on osteoblasts.

The effects of medium pH were tested on calvariae, tibiae, and osteoblast-like cells from chick embryos. Bones and isolated cells were incubated for 5 h or 2 days in Hepes-buffered medium at pH values ranging from 6.8 to 8.2. Osteoblast function was evaluated by lactate production, oxygen consumption, alkaline phosphatase activity (AlPase), Ca and inorganic phosphate (P_i) flux, proline hydroxylation, DNA content, and thymidine incorporation. As medium pH was increased, glycolysis, collagen synthesis, and AlPase increased, while Ca efflux decreased. No effect of pH was seen on mitochondrial activity, P_i efflux, or cell number or proliferation. The importance of glycolysis as an endogenous pH regulator was demonstrated by inhibition with iodoacetic acid or glucose restriction and by adding lactate to the medium. The results suggest that the pH of bone interstitial fluid may be regulated by glycolysis and that changes in pH of this compartment may have marked effects on osteoblast function.