Immunohistochemical observations on bone morphogenetic protein in normal and abnormal conditions.

Localization of bone morphogenetic protein (BMP) in human tissues and cells is important for investigating the mechanism of bone induction. A stable cell line secreting monoclonal antibody against bovine BMP (bBMP-McAb) was obtained by the hybridoma technique. The result of immunohistochemical staining (ABC method) showed that BMP is distributed along collagen fibers of normal bone, in periosteal cells, and in mesenchymal cells of marrow stroma. Little BMP can be found in bone cells of lamellar bone or in calcified bone matrix. BMP may be abundant in human tooth anlagen such as predentin, cells of the outer and inner enamel epithelium, and cells of dental sac generating bone. BMP is found in the cytoplasm of tumor cells of osteosarcoma and chondrosarcoma. Immunohistochemical staining showed that BMP plays a role in bone fracture healing. The ability of BMP-McAb to detect BMP and to inhibit the generation of new bone also makes it potentially useful in diagnosing, treating, and providing a prognosis for osteosarcoma and other bone diseases.

Aluminium deposition in bone after contamination of drinking water supply.

Two healthy individuals who drank water accidentally contaminated at source with aluminium sulphate solution were investigated 6-7 months later. Bone biopsy specimens showed discrete lines of positive staining for aluminium, the distribution being compatible with acute exposure some months previously. These findings show that under certain conditions normal individuals can absorb aluminium via the gut, and that such aluminium can be deposited in bone.

A critical crosslink region in human-bone-derived collagen type I. Specific cleavage site at residue Leu95.

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [alpha 1s(I)] migrating between alpha 1(I) and alpha 2(I) as well as a band [alpha 2s(I)] migrating in front of alpha 2(I). The collagenous nature of the pepsin-stable alpha 1s(I) protein was clearly demonstrated by digestion with human-leucocyte-derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of alpha 1(I) and alpha 1s(I) identified alpha 1s(I) as a shortened alpha 1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine-derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened alpha 1(I) and alpha 2(I) chains, respectively. The melting temperature was found to be 36 +/- 0.5 degrees C as judged from circular dichroism and susceptibility to proteolysis. Our data provide clear evidence that a shortened alpha 1-derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.

Characterization of interfaces in human calcified tissue.

Thermostimulated current spectroscopy has been applied to the investigation of molecular mobility in human calcified tissue. A comparative study of extracts at various stages of demineralization is presented. The response of an organic-mineral complex interphase has been identified.

[Bone mineral content and quantitative computerized tomography]. / Contenuto minerale osseo e tomografia computerizzata quantitativa.

Many methods are used to determine bone mineral content (BMC). Quantitative computed tomography (QCT) appears to be the most reliable method also because it allows the trabecular and the cortical bone to be measured separately. QCT is usually performed on the first four lumbar vertebral bodies. BMC is expressed in mg/ml and a mean value is calculated. Three hundred and fifteen subjects were studied (281 women and 34 men). The patients affected with Paget's disease or malignancies, with or without bone metastases, were not included in this study. The measurements were performed by means of a General Electric 9800 tomograph with software and calibration phantom (QCT-Bone program by Image Analysis). Fractured vertebrae were not included when calculating the mean value since an increased density is caused by fracture. The BMC of any studied vertebral body is considered in comparison with the BMC of the other lumbar vertebrae of the same subject. When the BMC of a given vertebral body exceeds the others by 25 mg/ml or more, nodules and/or stripes are observed during multiple-slice scanning of the bone. Lytic areas or angiomas are observed when the BMC of a given vertebral body is -25 mg/ml or higher. In calculating the mean vertebral BMC, vertebrae with both +25 and -25 must be excluded. In this way the method reliability increases.

Parathyroid glands in chronic aluminum intoxication.

In chronic renal failure, aluminum overload may influence parathyroid function. In a study of possible aluminum-induced parathyroid abnormalities, parathyroid glands from nine parathyroidectomized patients on hemodialysis were examined by light and electron microscopy and by X-ray microanalysis. Aluminum overload was assessed by the presence of stainable aluminum (aluminum surface, 23.3% +/- 11% of total surface) in bone biopsy specimens. The mean plasma aluminum concentration was 7.7 +/- 1.9 mumol/L. All patients but one had elevated plasma concentrations of immunoreactive parathyroid hormone as well as osteitis fibrosa. The aluminum concentrations in bone and parathyroid gland from these patients were significantly higher than those in tissue from patients on hemodialysis without stainable bone aluminum. Abundant aluminum deposits were present in parathyroid chief cell cytoplasm in lipoid bodies, lipofuscin granules, and mitochondria. These cells exhibited features of active hormonal synthesis and contained numerous secretory granules. The data show that in the parathyroid glands of these aluminum-intoxicated patients the presence of aluminum deposits neither induced cellular damage or chief cell necrosis nor interfered with the production of parathyroid hormone.

Serum bone Gla protein as an indicator of parathyroidectomy in patients with secondary hyperparathyroidism.

Bone Gla protein (BGP) is a vitamin K-dependent protein which is a marker of bone turnover. To determine whether serum BGP is a useful indicator for parathyroidectomy in patients with secondary hyperparathyroidism, we measured serum BGP levels. Thirty-seven patients with secondary hyperparathyroidism who were followed up for more than 1 year after parathyroidectomy were studied. All patients underwent total parathyroidectomy and autotransplantation. Controls were 46 patients who were treated by chronic hemodialysis for more than 3 years. Serum BGP levels (normal: less than 6.5 ng/ml) were markedly increased in 37 patients with parathyroidectomy, ranging from 4.2 ng/ml to 645 ng/ml, with a mean value of 278.8 +/- 159.8 ng/ml (mean +/- standard deviation) versus 65.0 +/- 85.2 ng/ml in the 46 controls (p less than 0.001). Patients with a high BGP level had severe bone and joint pain. Serum BGP in patients with parathyroidectomy was significantly correlated with serum alkaline phosphatase and mPTH (p less than 0.001 for both). The total weight of resected parathyroid tumors was 2,152 +/- 1,368 mg, and tumor weights ranged from 200 mg to 5,600 mg. There was a highly significant correlation between BGP level and tumor weight (r = 0.656, p less than 0.001). The 2 patients who showed BGP levels below 10 ng/ml had tumor weights of only 470 mg and 240 mg, respectively, and revealed no improvement of pain postoperatively, although their mPTH levels were increased. These results suggested that BGP measurement is a sensitive method for detecting increased bone turnover and is possibly useful as an indicator for parathyroidectomy in patients with secondary hyperparathyroidism.

Prospective evaluation of aluminum loading from formula in infants with uremia.

Assessment of potential aluminum loading from regular ingestion of a commercial infant formula (Similac PM 60/40), as the only milk substitute, was made in 14 infants aged 9.6 +/- 4.4 months who were also undergoing continuous cycling peritoneal dialysis. Tissue aluminum accumulation was assessed by serial measurements of plasma aluminum levels, from the increment in plasma aluminum after a standardized deferoxamine infusion, and from quantitative histomorphometry of bone and measurements of total bone aluminum content. Initial mean plasma aluminum levels were 0.61 +/- 0.32 mumol/L, (normal 0.30 +/- 0.04 mumol/L), and values were less than 0.92 mumol/L during the follow-up of 20 +/- 8 months. Plasma aluminum levels increased from 0.59 +/- 0.18 to 0.88 +/- 0.22 mumol/L after a single dose of deferoxamine. The histochemical stain for bone aluminum was negative for all patients, and the bone aluminum content was 0.27 +/- 0.22 mmol/kg dry weight (normal 0.08 +/- 0.04 mmol/kg dry weight). Thus the infant formula Similac PM 60/40 can be safely used in infants with chronic renal failure.

Effect of aluminum on performance and mineral metabolism in young chicks and laying hens.

This study was performed to determine the long-term effects of dietary aluminum on egg production and reproductive parameters in the mature laying hen and on growth rate and feed efficiency in young chicks. The diets used in these studies were adequate in phosphorus and other essential nutrients. Aluminum added to constitute 0.30% of the diet severely depressed growth and reduced feed efficiency, bone ash and plasma phosphorus in male Ross x Leghorn chicks. At the same time, 0.15% added aluminum mildly depressed growth, feed efficiency and bone ash but had no effect on plasma phosphorus levels. The reduction in bone ash was relatively mild, and no clinical signs of rickets were observed. In laying hens, diets containing 0.15% added aluminum did not affect egg production, but 0.30% added aluminum reduced production significantly. Long-term exposure to aluminum increased percent shell in both groups receiving aluminum, whereas egg weight remained similar to that in controls. There were no changes in hatchability or bone ash associated with dietary aluminum. Although dietary aluminum influenced bone aluminum content, egg aluminum content was not affected. These studies indicate that dietary aluminum interferes with systems in addition to phosphorus metabolism.

[Bone disorders in children with chronic renal insufficiency exposed to high ingestion of aluminum]. / Alteraciones óseas en niños con insuficiencia renal crónica expuestos a ingestión elevada de aluminio.

Bone disorders in 28 children with chronic renal failure exposed to aluminum intoxication were studied. All of the children were in the dialysis program. Aluminum blood levels were higher than normal in all of the children and without any correlation to the magnitude of hypocalcemia or with the increase of the parathormone, which were found in different amounts in all of the children. All of the children had various degrees of skeletal retardation and only one had pathological fractures. The bone biopsy showed hypocellular marrow, decreased osteoclastic activity in the majority of the cases same as trabecular mineralization, although the amount of osteoid was lacking in the trabeculae in the majority of the cases. The deposit of aluminum was detected in a great number of them. It is concluded that osteodystrophy recognizes a number of factors as may be hypocalcemia due to a decrease in the production of 1,25-cholecalciferol, an increase in the parathyroid hormone and the deposit of aluminum, coming mainly from water, in the trabeculae which interfere with the incorporation of calcium in the formation of new bone.

Effects of dietary aluminum and phosphorus on magnesium metabolism in dairy calves.

The metabolism of Mg was studied in young dairy calves fed two levels of added Al (0 and .20% Al) and two levels of added P (0 and .22% P) for 7 wk. The four treatments were 1) normal P-low Al, 2) low P-low Al, 3) normal P-high Al and 4) low P-high Al. The basal diet (low P-low Al) contained, by analysis, .132% P, .021% Al and .17% Mg. Added Al did not affect (P greater than .10) serum Mg. An Al x P interaction on bone Mg was detected (P less than .01). Magnesium was reduced in tibia shaft (.34 vs .44%) and in tibia joint (.43 vs .53%) in calves fed high Al in the presence of normal dietary P, but Mg was not reduced in the calves fed low-P diets. Apparent absorption of Mg was reduced by approximately five-fold (.18 g/d vs -.84 g/d, P less than .01); urinary Mg excretion was reduced 31% (1.12 g/d vs .77 g/d, P less than .01); and Mg retention declined 41% (-95 g/d vs -1.61 g/d, P less than .01) in calves fed added A1. Compared with calves fed low-P diets, calves fed normal levels of P had a higher Mg concentration in tibia shaft (P less than .01) and tibia joint (P less than .05). The data indicate that supplemental Al may adversely affect Mg metabolism in calves.

[Lectin histochemistry of bone cells and bone tumors]. / Csontsejtek és csonttumorok lektin-hisztokémiája.

The use of lectins in histochemical research substantially contributed to the knowledge on carbohydrates. This paper is a preliminary report of findings with lectin histochemistry on normal and pathological bone tissue.

Staining of bone for aluminum: use of acid solochrome azurine.

Fourteen individuals on long term hemodialysis, with varying amounts of aluminum in bone from 11 to 296 mg/kg by flameless atomic absorption spectrophotometry, were examined to see whether the aluminon or acid solochrome azurine (ASA) staining reactions best approximated the chemical determination. Correlation coefficients were 0.78 for aluminon and 0.88 for ASA. Together with 11 additional patients morphometric parameters were compared with the two aluminum stains. The aluminon stain gave satisfactory results in the osteomalacic group but underestimated the amount of aluminum present in those with hyperparathyroid, mixed or aplastic disease. Some individuals showed a striking difference between the two techniques which could have led to an erroneous conclusion regarding the amount of aluminum present. The aluminon stain was pH dependent and together with ASA could be enhanced by prior heat treatment of the sections. It is recommended that ASA either replace aluminon for routine use or be used together with the aluminon stain, particularly for bones without osteomalacia or with mild to moderate aluminum storage.

Acute epidemic aluminium osteomalacia secondary to water supply contamination.

When the aluminium content of the water supply to our Haemodialysis Unit rose from less than 0.5 mumol/l to 6 mumol/l over a two month period, we carried out bone biopsies and desferrioxamine infusion tests on twelve (12) patients who had been on haemodialysis for less than one year (mean 8 months) and had normal serum aluminium levels. The patients had no bone symptoms. Eight patients had positive aluminium bone stains. The aluminium osteomalacia group (n = 8) had a mean PTH of 1.4 ng/ml s.e. 0.3 whereas the non-ALO group had a mean PTH of 2.9 ng/ml s.e. 0.7. The difference in mean PTH is significant (p less than 0.05). There was no evidence of encephalopathy, fractures or microcytic anaemia in the ALO positive group. The aluminium contamination of the water supply occurred because of a change in the reservoir purification system from sand-filtration to alum.

Aluminium osteomalacia in chronic renal failure patients neither on dialysis nor taking aluminium containing phosphate binders.

The incidence of aluminium osteomalacia (ALO) in patients with chronic renal failure neither on dialysis nor taking aluminium-containing phosphate binders (ACPB) is not well documented. Biochemical and histological bone investigations were performed in 35 patients fulfilling the above conditions, among whom we found an incidence of ALO of 17%. In the ALO group, salient findings were PTH level (mean +/- s.d.) of 3.1 +/- 1.4 ng/ml (normal less than 0.5 ng/ml); elevated home tap-water aluminium levels of 6.5 +/- 1.2 umol/l (normal less than 2 umol/l); and a GFR of 20.5 mls/min/1.73m, (range 2-50 mls/min/1.73m). We conclude that the aetiology of ALO in this group involves the absorption of toxic home water aluminium in the presence of an elevated PTH level and a GFR less than 50 mls/min/1.73m.

[Renal osteodystrophy: aluminium and secondary hyperparathyroidism]. / Ostéodystrophie rénale: aluminium et hyperparathyroïdie secondaire.

Aluminium-related osteodystrophy results in osteomalacia or in the so-called aplastic bone. In this particular bone disease bone cells activities are distinctly reduced but there is no disorder of bone mineralization. Aluminium exerts a direct toxic effect on bone tissue, notably on osteoblasts which are always strongly depressed in case of major aluminium overload. Aluminium-related osteopathy is regularly accompanied by low levels of parathormone due to accumulation of aluminium in the parathyroid glands. Parathormone modulates the bone aluminium overload: hyperparathyroidism "protects" bones against the deleterious effect of aluminium, whereas aluminium deposit in bone increase after parathyroidectomy. The respective roles played by low parathormone levels and by aluminium deposits in aplastic bone lesions is difficult to determine since hypoparathyroidism itself can probably cause the aplastic osteopathy. The role of parathormone stands out more clearly now that in patients under dialysis the bone aluminium overload has markedly decreased. Many patients with aplastic bone (initially described in aluminium poisoning) show no aluminium deposits in bones but have, for some unknown reason, a normal or even low parathormone level. The clinical course of this type of osteopathy remains to be determined, but there seems to be no reason to worry since numerous patient are asymptomatic. Preventing secondary hyperparathyroidism while refraining from prescribing aluminium hydroxide is the principal therapeutic objective in osteodystrophy of haemodialysis patients.

Bone marrow fat content in relation to bone remodeling and serum chemistry in intact and ovariectomized dogs.

It was previously shown that 11 months after ovariectomy the volume fraction of trabecular bone in the spine and 11th rib medullary canal of Beagle dogs (6 control, 9 ovariectomized) was significantly reduced. In this paper it is shown that these changes are accompanied by increased marrow fat volume in the 11th rib (59.0 +/- 9.5% vs. 44.3 +/- 10.0%). Conversely, the volume fraction of functional (hematopoietic) cells in the marrow was reduced by ovariectomy. Additionally, variations in marrow fat volume were tested for correlation with 22 other variables pertinent to bone physiology. Marrow fat volume was significantly positively correlated with serum osteocalcin, rib trabecular bone porosity, rib cross-sectional area, and gains in body weight. It was negatively correlated with serum estrogen concentrations and the extent of rib trabecular surfaces labeled with tetracycline.

Clinical experience with desferrioxamine in dialysis patients with aluminium toxicity.

A desferrioxamine (DFO) infusion test, using a DFO dose of 36.9 +/- 11.2 mg/kg (mean +/- SD), was performed in 50 consecutive dialysis patients undergoing diagnostic bone biopsy. In 30 patients whose bones stained positively for aluminium the serum aluminium level increased by an average of 373 +/- 250.4 ng/ml. The increase in 20 aluminium-negative patients was 231 +/- 179.2 ng/ml (p less than 0.05). Aluminium-positive patients had lower levels of immunoreactive parathyroid hormone (336 +/- 442 muleq/ml) than aluminium-negative patients (1278 +/- 1400 muleq/ml; p less than 0.05). A change in serum aluminium level of greater than 200 ng/ml after the administration of DFO was 73 percent sensitive and 50 percent specific, and had a positive predictive value of 69 percent for detecting positive bone aluminium staining. The combination of a baseline immunoreactive parathyroid hormone level less than 200 muleq/ml and a change in serum aluminium of greater than 200 ng/ml after DFO was 90 percent specific and had a positive predictive value of 85 percent. In the second phase of our study, 28 dialysis patients with aluminium toxicity received long-term therapy (11.0 +/- 4.3 months) with DFO at an average starting dose of 41.7 +/- 17.1 mg/kg, administered once weekly. The four deaths which occurred during this treatment involved the only patients who had advanced dialysis dementia. Seven patients with less severe neurological symptoms responded favourably. Fractures decreased from 1.7 fractures/patient/year to 0.1 fracture/patient/year. Muscular strength and overall functional class were improved or stable in 25 patients; myalgias and arthralgias were also stable or improved in 19 patients. After 5-7 months of treatment, serum aluminium levels decreased from 401 +/- 262 ng/ml to 245 +/- 217 ng/ml (p less than 0.01); erythrocyte mean corpuscular volume increased from 86.3 +/- 10.91 fl to 94.1 +/- 9.23 fl (p less than 0.02); and serum calcium decreased from 10.4 +/- 0.94 mg/dl to 9.9 +/- 0.70 mg/dl (p less than 0.02). Serum immunoreactive parathyroid hormone levels remained stable in 25 patients, but severe hyperparathyroidism developed rapidly in three patients. Eight patients with transfusional iron overload had no change in serum ferritin levels. Iron depletion developed in six patients, with a decrease in serum ferritin from 251 +/- 229.8 micrograms/l to 45 +/- 29.3 micrograms/l, and they required parenteral iron supplementation. Significant side-effects occurring during long-term DFO administration were hypotension (11 patients), gastrointestinal upset (seven patients), porphyria cutaneous tarda-like lesions (three patients), and transient visual disturbance (one patient). There was a decrease in stainable bone aluminium in all nine patients with paired bone biopsy specimens (pre- and post-DFO).(ABSTRACT TRUNCATED AT 400 WORDS)

Histochemical identification of oestrogen target cells in the medullary bone of laying hens.

1. Oestrogen target cells in the medullary bone of laying hens were examined using a histochemical method with fluorescein isothiocyanate labelled oestradiol. 2. Specific fluorescence was found to occur strongly in osteoblasts and weakly in osteoclasts on the medullary bone surface. 3. This study suggests that osteoblasts on the medullary bone surface are oestrogen target cells.