Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities.

Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of skeletal calcium deficiency in postmenopausal osteoporosis.

A previous study of iliac crest composition identified skeletal calcium deficiency in 25% of 56 postmenopausal osteoporotic patients evaluated prior to the use of stanozolol or calcitonin. This report is a follow-up of biopsy data after 2 years of treatment with drug or placebo in 31 patients, 11 of whom had skeletal calcium deficiency. The study diet, consisting of 1 g elemental calcium plus 400 U Vitamin D, repaired the skeletal calcium deficiency in all patients, treated and untreated alike. Total body calcium (TBC) results were influenced by separation into calcium deficient and normal mineral groups, apparent treatment response being observed largely in patients with calcium deficiency. It is suggested that if all postmenopausal women ingested 1 g elemental calcium plus 400 U Vitamin D daily, skeletal calcium deficiency in osteoporosis would disappear as a problem. Meanwhile, it is important to recognize that repair of calcium deficiency is an important variable capable of influencing bone response to therapy and the evaluation thereof.

Effects of dietary alteration of bicarbonate and magnesium on rat bone.

To study the effects of bicarbonate and magnesium on bone, mild acidosis and/or hypermagnesemia were produced in growing rats by feeding ammonium chloride and/or magnesium sulfate. Bone composition, quantitative histomorphometry, and mineral x-ray diffraction (XRD) characteristics were measured after 6 wk of treatment. The results demonstrated that both acidosis (decreased HCO3) and hypermagnesemia inhibited periosteal bone formation, and, when combined, results were summative; and the previously observed in vitro role of HCO3- and Mg2+ as inhibitors of crystal growth were confirmed in vivo. XRD measurements demonstrated that decreased plasma HCO3 resulted in larger crystals and increased Mg resulted in smaller crystals. However, the combined XRD effects of acidosis and hypermagnesemia resembled acidosis alone. It is postulated that the final composition and crystal structure of bone are strongly influenced by HCO3- and Mg2+, and the effects are mediated by the combined influence on both osteoblastic bone formation and the growth of hydroxyapatite.

Bone matrix and mineral abnormalities in postmenopausal osteoporosis.

Iliac crest biopsies from 56 postmenopausal osteoporotic females with spontaneous compression fractures and decreased total body Ca were compared to similar tissue from 48 normal controls. Biopsies were analyzed for bone density, Na, Ca, Mg, P, Co3, and hydroxyproline (OH-P). From the results OH-P/matrix, % mineral, and the ion content of the mineral were calculated. osteoporotic subjects showed decreased bone density, % mineral in bone, and OH-P in the bone matrix. Within the mineral, CO3 and Ca/P were decreased, while Na and Mg were increased. Statistical analysis showed that matrix OH-P and % mineral varied independently, and therefore the patients were separated into 4 subgroups: Group Ia: decreased matrix OH-P with normal % mineral (n = 9), Group Ib: decreased matrix OH-P with decreased % mineral (n = 5), Group IIa: normal matrix OH-P with normal % mineral (n = 33), Group IIb: normal matrix OH-P with decreased % mineral (n = 9). Decreased % mineral was associated with decreased bone density and an increase in Na and Mg in the mineral, which suggests skeletal Ca deficiency. Decreased matrix OH-P was associated with decreased bone density and, in the low % mineral group, with decreased mineral CO3 and Ca/P, suggesting a mineral of decreased mean crystal size. When both abnormalities coexisted (Group Ib), the greatest reduction in total body Ca was seen. Patients with normal matrix and normal % mineral (Group IIa) still had decreased bone density. The results suggest that in a large, clinically homogeneous population of postmenopausal osteoporotic women, 4 subgroups can be identified by differences in chemical composition of iliac crest biopsies.

Normal maturational changes in bone matrix, mineral, and crystal size in the rat.

Normal rat bone maturation has been studied using biochemical methods and hydrazine separation of matrix and mineral for X-ray diffraction. In the bone, the amount of mineral increases between 4 and 22 weeks of age, while in the matrix, the ratio of noncollagenous protein to collagen progressively decreases. In mineral, in the absence of serum ion changes, growth in mean crystal size appears to be the determinant of the changing ratios of calcium, magnesium, carbonate, and phosphorus, and of the increasing mineral density.

Surface properties of membrane systems: interaction of ketamine with monomolecular films of ganliosides and mitochondrial lipids.

Ketamine solutions did not form a film (pi=0) but had an appreciable surface potential (delta V=500 mv), indicating a significant array of +/- oriented charge dipoles at the air-water interface, as opposed to calcium chloride solutions whose delta V was zero. The delta V values of ganglioside films spread on the aqueous phase varied in the order water less than sodium chloride less than calcium chloride less than ketamine hydrochloride. At equivalent concentrations, calcium chloride was 500 times as effective as sodium chloride, and ketamine at the clinical concentrations of 10-20 microgram/ml (36-72 micrometer) was 6000 times as effective as calcium chloride in raising the surface potential of gangliosides; the delta V effect with mitochondrial lipid was in the reverse order; water less than sodium chloride = ketamine hydrochloride less than calcium chloride. This calcium-ketamine inversion indicates a unique specificity of ketamine for gangliosides. Since ketamine acts on the brain and did not affect mitochondrial respiration, the surface potential data suggest that part of the mechanism of action of ketamine could be its interaction with synaptic surfaces and, specifically, with the sialic acid of gangliosides and/or glycoproteins present on the synaptic membrane surface.

Muscle buffer values.

The in vitro skeletal and cardiac muscle buffer values have been determined for normal and potassium-depleted dogs. Depletion produced a statistically significant decrease in buffer value for both skeletal 66 vs. 61 sl, and cardiac muscle, 64 vs. 55 sl. The decreased cardiac muscle buffer value was obtained despite no statistically significant loss of cardiac muscle potassium.

The relationship of fatty acid composition and surface activity of lung extracts.

Male weanling rats were fed fat-free diets supplemented with 4% (w/w) safflower oil (control) or 4% tripalmitin (essential fatty acid (EFA) deficient) for 14 weeks. Whereas the amount of lecithin in lung lavage material remained unchanged, lung lavage lecithin from EFA-deficient rats contained significantly less palmitic acid (61.4 +/- 2.0% vs. 77.4 +/- 5.8%, P less than 0.01) than that from controls. Surface tension vs. area hysteresis loops were obtained for total lipid extracts (TLE) of lung lavage fluid, intra- and extra-cellular lipoprotein fractions (IBI and IBE) and lipid extracts of those lipoprotein fractions (LBI and LBE). A significant increase in minimal surface tension (gammamin) was found for all samples obtained from EFA-deficient rats as compared to controls. Refeeding of diets containing safflower oil for 7-14 days reversed these changes. Air pressure-volume curves on degassed, excised lungs indicated that greater pressure is required to maintain a given lung volume in EFA-deficient rats. These results support the hypothesis that the fatty acid composition of pulmonary surfactant lecithins is a major determinant of the surface activity of lung extracts.

The effects of crystal size distributions on the crystallinity analysis of bone mineral.

The mechanisms by which crystal size distributions affect the usual method of quantities X-ray diffraction analysis of bone mineral have been determined on synthetic crystals. It was observed that each component of a crystal-size distribution diffracts independently. This independence causes systematic nonlinear behavior in the plot of integrated intensity vs. broadening parameter curves. The nonlinearity resulted in an overestimation of the amount of nondiffracting material present in bone mineral. Because crystal size distributions may vary for different crystallographic directions, it is strongly suggested that the usual practice of adding the c-axis and a-axis integrated intensities to estimate the crystallinity of the sample be discontinued. Methods of understanding the crystal size distribution function in bone mineral are discussed and evaluated.

Role of calcium in bone maturation arrest after thyroparathyroidectomy in the rat.

Thyroparathyroidectomy in the rat results in decreased plasma calcium and magnesium and increased phosphorus. The associated bone changes are decreased calcium, hydroxyproline, carbonate, and wholebone density. Bone magnesium, sodium, mineral density, and percent crystallinity are increased. The delayed matrix formation and mineralization previously identified by histologic techniques are herein characterized by direct measurement as arrest of the normal increase of hydroxyproline/matrix and percent mineral. The bone mineral present is of high density and x-ray-diffraction crystallinity, suggesting a decrease in the mineralization front high in the amorphous phase and/or small nondiffracting crystalloids. The chemical studies reveal that in the absence of available Ca, Mg and Na are substituted, and CO3 is decreased. The restoration of these plasma and bone abnormalities to normal by a diet high in CaCO3 adds further emphasis to the essential role of Ca in bone cell function.

Evaluation in dogs of cross-circulation in the treatment of acute hepatic necrosis induced by yellow phosphorus.

The efficacy of cross-circulation in the treatment of acute liver failure has been evaluated in dogs. Four of 5 dogs administered a dose of yellow phosphorus that is lethal 90% of the time survived after treatment by cross-circulation of whole blood for between 1 and 8 hr with a normal dog. In 2 normal unmatched dogs plasma cross-circulations were performed over a period of 31 days without any clinical or laboratory manifestation of hypersensitivity except for lymphocytotoxic antibody titer rise. The results suggest that whole blood cross-circulation is effective and imply that a single donor could be utilized for prolonged periods of plasma cross-circulation with avoidance of immunological consequences of whole blood exchange.

Bone crystal maturation in renal osteodystrophy in humans.

Calcium, phosphorus, sodium, carbonate, magnesium, and hydroxyproline were measured in iliaccrest biopsies of 22 normal volunteers and 24 selected patients with renal osteodystrophy. Histologic classification revealed that 10 were mildly abnormal, 8 osteomalacic, and 6 osteofibrotic. Bone density measurements were performed on an additional 12 normal, 11 mildly abnormal, 6 osteomalacic, and 10 osteofibrotic subjects. The results revealed an increase in magnesium and adecrease in carbonate apparent in the minimal and osteomalacic lesions and a much greater change in osteofibrosis. The bone density was decreased in patients with osteofibrosis. These observations would appear to be explained by postulation of an impairment of the normal maturational process of bone whereby there is an increase in amorphous calcium phosphate and a decrease in apatite crystal. The data suggest that the maturational defect is present as soon as any abnormality can be identified histologically, is present to the same degree in osteomalacia, and is most severe in osteofibrosis. In comparison of two sets of six patients matched for age and duration of dialysis, neither acidosis nor vitamin D therapy appeared to influence the severity of the maturational defect.

Changes in bone sodium and carbonate in metabolic acidosis and alkalosis in the dog.

Metabolic acidosis and alkalosis were produced in adult dogs over 5- to 10-day periods. Midtibial cortical bone was analyzed for calcium, sodium, phosphorus, and carbonate. In acidosis bone CO(3)/Ca decreased 9.5% and bone Na/Ca decreased 6.3%. In alkalosis bone CO(3)/Ca increased 3.1% and bone Na/Ca increased 3.0%. Previous attempts to account for changes in net acid balance by summation of extra- and intracellular acid-base changes have uniformly resulted in about 40-60% of acid gained or lost being "unaccounted for." If it is assumed that changes in tibial cortex reflect changes in the entire skeletal system, changes in bone CO(3) (=) are sufficiently large to account for the "unaccounted for" acid change without postulating changes in cellular metabolic acid production.