Localization of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the mammalian kidney.
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Biomedical subjects
Publications and source records attributed to T Suda.
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Decreased deformability, slightly increased suspension viscosity and retarded oxygen egrees were observed in cholesterol-loaded, human erythrocytes. These functional changes resulted from the decreased membrane fluidity induced by cholesterol.
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The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.
1. In order to study the kinetics os the oxygen egress from human red cells in the 50 sec-20 min time range, an apparatus for measuring the oxygen dissociation process was constructed, combining a spectrophotometer with an oxygen electrode of quick response. 2. Starting from air-saturated haemolysate or red cell suspensions, the velocity of oxygen dissociation from oxyhaemoglobin (Vdiss) and of oxygen disappearance in the medium (Vobs) after addition of bakers' yeast (consuming the dissolved oxygen at the velocity of Vconsump) were recorded. A parameter (r) was defined as the ratio of two velocities, Vegress (the velocity of oxygen egress into the medium) and Vdiss, r identical to Vegress/Vdiss = (Vconsump -Vobs)/Vdiss. Vcomsump could be calculated by the Michaelis-Menten equation as follows, Vconsump = Vmax [O2]/(Km + [O2]), where Vmax was the maximal velocity of oxygen consumption of bakers' yeast. 3. The r value was always 1.0 for the haemolysate, but it was less than 1.0 for the normal red cells. Further, the oxygen dissociation curve of red cells obtained at higher Vmax was distorted, due to the non-equilibration between intra- and extracellular oxygen concentrations. 4. The r value was (i) independent of the amounts of the allosteric effectors (2,3-diphosphoglycerate and H+) but (ii) dependent on the haemoglobin contents and (iii) dependent on the amounts of the membrane cholesterol. Therefore, the r value reflected only the process of the oxygen diffusion but not the "chemical reaction' rate. The "barrier' of the oxygen diffusion decreased at lower haemoglobin contents, but increased at higher cholesterol contents in the membrane.
Immunoreactive beta-endorphin in the third ventricular fluid was measured in response to electrical stimulation of the periaqueductal gray matter in 8 patients with intractable pain during rostral mesencephalic reticulotomy for pain relief. In all patients, marked increase of immunoreactive beta-endorphin was observed. On the other hand, in cases of electrical stimulation of the zona incerta performed during stereoencephalotomy, in 5 patients with involuntary movement, immunoreactive beta-endorphin in the third ventricular fluid did not show any significant change. The authors conclude that the increase of immunoreactive beta-endorphin on electrical stimulation of the periaqueductal gray matter is not a nonspecific response to brain stimulation but a specific response in regard to cerebral localization of endorphins. Direct correlation between pain relief and periaqueductal gray stimulation is also questioned.
Metabolism of 25-hydroxyvitamin D3 (25-OH-D3) was examined in chicks supplemented with vitamin D3. Kidney homogenates metabolized in vitro [3H]-25-OH-D3 to 3 new metabolites (peaks A, C and E) by way of 24,25-dihydroxyvitamin D3. The enzymes responsible for the synthesis of these metabolites appeared to be induced by 1 alpha,25-dihydroxyvitamin D3. Production of these metabolites was increased in parallel with the increase of the supplemented levels of vitamin D3, while recovery of the radioactivity in the chloroform phase was sharply decreased. The production of peak C was considered to be closely related to the transfer of the radioactive metabolites to the water-soluble phase. These results may indicate that 24-hydroxylation is a degradation step in the 25-OH-D3 metabolism.
Administration of indomethacin inhibited the appearance of osteoclasts and bone resorption induced by experimental tooth movement in rats. Effects were limited to groups injected within 12 h after treatment. Prostaglandin E1 or E2 solutions injected in gingiva lying near the upper first molar caused the appearance of osteoclasts and bone resorption.
A laboratory model of renal osteodystrophy was developed in rats by a single injection of glycopeptide isolated from renal cortical tissues of rats according to the method used by Shibata et al. to induce glomerulonephritis. Approximately 60-70 days after injection, severe proteinuria appeared and continued for at least 170 days at a rate of more than 1 g/day. Morphological changes in the kidney were typical of chronic glomerulonephritis. The plasma calcium concentration was lowered transiently by the 96th day after injection, but was restored to the normal range thereafter. Plasma parathyroid hormone levels, however, continued to rise in parallel with the degree of proteinuria. Marked secondary hyperparathyroidism was induced which led to severe bone atrophy. Histological examinations showed a marked increase of resorbing cavities, with a quantitatively larger number of osteoclasts in cortical bone tissues compared with the control animals. No spontaneous remission was observed. It is emphasized that all of the biochemical and morphological changes reported here were induced by a single injection of homologous renal glycopeptide, and they were highly reproducible.
The comparative effectiveness of vitamin D3 and its derivatives in curing hyperparathyroidism and osteodystrophic bone lesions was examined in a laboratory model of renal osteodystrophy associated with marked secondary hyperparathyroidism in rats. The experimental model was prepared by a single injection of homologous glycopeptide. Plasma levels of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] appeared to decrease in the rats receiving glycopeptide. Various doses of vitamin D3 derivatives [2 or 10 microgram/kg D3, 2 microgram/kg 25-hydroxyvitamin D3 (25OHD3), 0.1 microgram/kg 1 alpha,25(OH)2D3, and 0.1 or 0.2 microgram/kg 1 alpha-hydroxyvitamin D3 (1 alpha OHD3)] were daily administered orally to the nephritic rats for 23 days before sacrifice. 1 alpha,25(OH)2D3 and 1 alpha OHD3 were much more potent than 25OHD3 and D3 in reducing the hyperplasia of parathyroir glands. The potency of 1 alpha OHD3 in curing the histological changes of osteodystrophy appeared to be greater than that of 1 alpha,25(OH)2D3. The same dose level of 1 alpha OHD3 was more effective than 1 alpha,25(OH)2D3 in enhancing plasma 1 alpha,25(OH)2D3 levels.
A 12-yr-old female patient with an unusual form of vitamin D dependency and alopecia is described. She was a product of consanguineous mating and developed signs and symptoms suggesting vitamin D dependency early in life. Neither 150 microgram/day (6 microgram/kg.day) 1 alpha-hydroxyvitamin D3 nor 5 microgram/day (0.2 microgram/kg.day) 1,25-dihydroxyvitamin D3 proved to have an effect on her abnormal serum chemistry. Seven million international units per day (about 2 x 10(5) IU/kg.day) of native vitamin D restored her serum chemistry to normal and brought about marked improvement on skeletal radiographs, when her serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-di-hydroxyvitamin D were 4250, 4.8, and 35 ng/ml, respectively. Even with the high serum levels of vitamin D metabolites, her intestinal 47Ca absorption rate remained in the lower normal range and urinary calcium excretion was decidedly low. Association of hypoparathyroidism was ruled out. These results suggest that the patient has extreme and-organ (intestine) hyposensitivity, probably of congenital origin, to the biologically active metabolites of vitamin D.
Pituitary adenomas were obtained from eight of nine patients with Cushing's disease, and the surrounding tissues as well were obtained from six of nine patients. ACTH, beta-lipotropin (beta-LPH), beta-endorphin, GH, TSH, LH, and PRL concentrations in these tissues were determined by RIA. Immunoreactive ACTH and beta-endorphin (beta-endorphin + beta-LPH) were present in high concentrations in all adenomas, and low concentrations were found in the surrounding tissues, except for one patient. As compared to levels seen in normal pituitary tissue, the GH concentration in the surrounding tissues was suppressed in five of six cases. TSH and LH concentrations were suppressed in four and three cases, respectively. The PRL concentration was not suppressed in any of the six patients studied. These four hormones were not detected in any adenoma. Plasma GH, TSH, and LH responses to various stimuli which were suppressed preoperatively returned to normal in most of the patients after adenomectomy. Basal plasma cortisol concentrations were normal or subnormal and were suppressed by the administration of 1 mg dexamethasone after adenomectomy, in contrast to the lack of such suppression preoperatively. ACTH and beta-endorphin secretion were stimulated by lysine-8-vasopressin and suppressed by dexamethasone and cyproheptadine in vitro.
Serum concentrations of 25-hydroxyvitamin D (25-OH-D) and 24,25-dihydroxy-vitamin D [24,25-(OH)2-D] were measured in lipid extracts of 1 ml of human sera by a competitive protein binding assay. 25-OH-D and 24,25-(OH)2-D were isolated from th lipid extracts by Sephadex LH-20 column chromatography. The 24,25-(OH)2-D fraction on the Sephadex LH-20 columns were further purified by high pressure liquid chromatography prior to assay. Serum levels of 25-OH-D and 24,25-(OH)2-D in normal males were similar to those in normal females throughout each age group. Serum levels of 25-OH-D and 24,25 (OH)2-D in newborns were 8.28 +/- 1.68 ng/ml (mean +/- SD) and 0.55 +/- 0.16 ng/ml, respectively, which were significantly (p<0.001) lower than those in adults (21.3 +/- 4.8 ng/ml of 25-OH-D and 1.55 +/- 0.31 ng/ml of 24,25-(OH)2-D). A seasonal variation was demonstrated in serum levels of 24,25-(OH)2-D as well as those of 25-OH-D. Serum levels of 24,25-(OH)2-D were highly correlated (gamma=0.884, p<0.001) with those of 25-OH-D in normal human subjects, and the percentage ratio of 24,25-(OH)2-D/25-OH-D was 7.4 +/- 1.4% (mean +/- SD), irrespective of age or seasonal variations.
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In order to study the changes in erythroid precursor cells in primary acquired and secondary sideroblastic anemia, bone marrow cells from 4 patients with primary acquired sideroblastic anemia (PASA) and 3 patients with refractory anemia with excess of myeloblasts (RAEM) or erythroleukemia associated with an excess of ringed sideroblasts were cultured for erythroid colony-forming units (CFU-E). The number of CFU-E was markedly decreased in all 7 cases, and erythroid colonies formed consisted exclusively of normal-appearing erythroblasts, while ringed sideroblasts were observed in scattered single erythroblasts or in small aggregates of erythroblasts in primary as well as in secondary sideroblastic anemia. These findings may indicate the presence of 2 populations of erythroid progenitor cells in the bone marrow of patients with primary acquired and secondary sideroblastic anemia. A slight to moderate decrease in granulocyte-macrophage colony-forming units (GM-CFU) was observed in 3 cases of PASA. The decrease in GM-CFU, however, was marked in sideroblastic anemia associated with RAEM or erythroleukemia.
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