Tatsuo Suda

The isolation of metallothionein and its protective role in cadmium poisoning

Abstract People that have been subjected to cadmium poisoning show marked calcified tissue and kidney disturbances. In rats fed a cadmium-containing, low-calcium-vitamin D-deficient diet, the major portion of the cadmium accumulated in the liver and kidneys. Despite the fact that only a small amount (2.8 ppm) of cadmium completely inhibits the in vitro enzymic 1-hydroxylation reaction of 25-hydroxycholecalciferol, the in vivo 1-hydroxylation proceeded without appreciable inhibition even in the rats loaded with large amounts of oral cadmium. No light-microscopic morphological changes could be found in the kidneys of cadmium-fed rats. Most of the cadmium that accumulated in the kidneys was in a form bound to the protein, metallothionein, and therefore was not toxic to that organ. On the other hand, only 20% of the cadmium present in bone appears to be protein bound. The data strongly suggest that the protective effect of metallothionein in the kidney is serendipitous when involved in cadmium poisoning and that cadmium ion acts directly on bone rather than by an indirect action through a functional disturbance of the kidney.

Prevention by metallothionein of cadmium-induced inhibition of vitamin D activation reaction in kidney

Several observations have indicated preferential accumulation of cadmium in the liver and kidney when animals were administered the metal by either peroral or parenteral route [ 1,2]. Most of the cadmium thus accumulated in the organs exists bound to a low molecular weight protein [3,4] . The cadmium binding protein has several characteristics which suggest the protein to be closely related to metallothionein originally detected in the horse kidney [5] . It is also remarkable that the protein appeared to be induced rapidly in the liver upon cadmium loads [6] . However, the biological significance of those observations has not been established yet. It is generally accepted that vitamin D must be hydroxylated on C-25 position in the liver and subsequently on C-l position in the kidney before it can function on bone and intestine [7]. We found that I-hydroxylation reaction of 25OHDs in chick kidney mitochondria was completely inhibited in the presence of 0.1 mM of cadmium in vitro [S] , but that the I-hydroxylation reaction occurred in vivo even in the animals loaded with large amounts of oral cadmium [ 14,151.

Biological activity of 24,24-difluoro-1α,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3-26,23-lactone in inducing Differentiation of human myeloid leukemia cells

The biological activity of 24,24-difluoro-1,25-dihydroxyvitamin D3 was compared with 1,25-dihydroxyvitamin D3 in the rat. The 24,24-difluoro-1,25-dihydroxyvitamin D3 has a potency of approximately 5-10 times that of 1,25-dihydroxyvitamin D3 in the known in vivo vitamin D responsive systems. These systems include intestinal calcium transport, bone calcium mobilization, calcification of epiphyseal plate cartilage, and elevation of plasma calcium and phosphorus concentrations. Thus, 24,24-difluoro-1,25-dihydroxyvitamin D3 is the first known analogue with higher potency than 1,25-dihydroxyvitamin D3 in vivo.

Metabolism of 1α-hydroxyvitamin D3 to 1α,25-dihydroxyvitamin D3 in perfused rat liver

Abstract The metabolism of 1α-hydroxyvitamin D3 (1α-OH-D3) was studied in rat liver perfused with [3H]-1α-OH-D3. [3H]-1α-OH-D3 was converted very rapidly to a more polar metabolite, which was identified as 1α,25-dihydroxy-vitamin D3 [1α,25-(OH)2-D3] by co-chromatography with synthetic 1α,25-(OH)2-D3 as well as by gas chromatography-mass spectrometry. [3H]-1α,25-(OH)2-D3 appeared in the perfusate as early as 20 min after addition of [3H]-1α-OH-D3, and its level in the perfusate increased linearly for at least 120 min. These data strongly indicate that 1α-OH-D3 is metabolized to 1α,25-(OH)2-D3, which exerts biological effects on bone and intestine.

The role of vitamin D in the mineralization of dentin in rats made rachitic by a diet low in calcium and deficient in vitamin D

A study was undertaken to ascertain whether vitamin D has a direct action on the mineralization of the dentin in rats made rachitic by a diet low in calcium and deficient in vitamin D. Physiological amounts of vitamin D, given orally to rachitic rats, increased their serum calcium from 4.8±0.5 mg/100 ml to 7.5±0.4 mg/100ml. The mineralization of dentin recovered markedly, although osteoporosis occurred in bone trabeculae. The results suggest that vitamin D increases serum calcium by accelerating bone resorption and that the increased serum calcium level acts directly to mineralize the dentin. When calcium was given to rachitic rats by subcutaneous injection, the serum calcium level increased and mineralization of dentin recovered to the same extent as that observed in rats given vitamin D. These results indicate that recovery of mineralization of rachitic dentin depends primarily on recovery of the serum calcium level and that vitamin D is an indirect factor in the mineralization process.

Direct control by calcium of 25-hydroxycholecalciferol-1-hydroxylase activity in chick kidney mitochondria

Abstract Kidney mitochondria were isolated from rachitic chicks and their activity in the metabolism of 25-OH-D3 was studied in relation to the amount of calcium added in vitro . The addition of 0.050.2 mM calcium to a mitochondrial suspension caused a marked and dose-related stimulation of 1-hydroxylation. A sharp decline in the activity was induced by higher concentrations (0.3-0.5 mM) of calcium. The rate of 24-hydroxylation was not influenced by calcium. In these effects, calcium was relatively specific among various divalent cations. These data strongly suggest that calcium is directly involved in the regulation of the vitamin D activation in kidney mitochondria.

1α,25-Dihydroxyvitamin D3-24-Hydroxylase (CYP24) Hydroxylates the Carbon at the End of the Side Chain (C-26) of the C-24-fluorinated Analog of 1α,25-Dihydroxyvitamin D3

The sequential oxidation and cleavage of the side chain of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1α,25(OH)2D3, 24,24-difluoro-1α,25-dihydroxyvitamin D3(F2-1α,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1α,25(OH)2D3-supplemented rats were incubated with F2-1α,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1α,25,26-trihydroxyvitamin D3(F2-1α,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1α,25,26(OH)3D3. Metabolism of F2-1α,25(OH)2D3into F2-1α,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1α,25(OH)2D3 into rats. The C-24 oxidation of 1α,25(OH)2D3 in renal homogenates was inhibited by F2-1α,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1α,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1α,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1α,25(OH)2D3 but also at C-26 of F2-1α,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.

Synthesis and biological activity of 1α-hydroxyvitamin D3

Abstract Hydroboration of cholesta-1,5-diene-3β-ol followed by alkaline-peroxide oxidation resulted in the formation of 1α- and 2α-hydroxy derivatives of cholesterol in nearly equal amounts. 1α-Hydroxycholesterol was then transformed to 1α-hydroxyvitamin D 3 , via 1α-hydroxycholest-5,7-diene-3β-ol. 1α-Hydroxyvitamin D 3 was as active as 25-hydroxyvitamin D 3 in the stimulation of intestinal calcium transport and bone mineral mobilization in intact rats, and moreover was able to produce both response in anephric rats similar to 1α,25-dihydroxyvitamin D 3 , the active metabolite of vitamin D 3 , as reported originally by DeLucas group.

25-hydroxylation of 1α-hydroxyvitamin D3 in vivo and in perfused rat liver

The demonstration that la-hydroxyvitamin D3 (la-OH-Ds) can replace the metabolically active form of vitamin Ds, lcy,25dihydroxyvitamin Ds (la,25(OH)z-Ds) in the treatment of various vitamin D refractory syndromes [l-3] has prompted investigations on the metabolism of this analog. In 1975, we synthesized [2-3H]-1~-OH-D3 and demonstrated that if was metabolized very rapidly to [3H]-lar,25-(OH)zD3 in perfused rat liver [4]. [3H]-1a,25-(OH)Z-D3 was found to be the only metabolite of [3H]-lcu-OHD3 produced by the liver [4]. Almost simultaneously, Holick et al. synthesized [6-3H]-la-OH-D3 and reached the same conclusion from in vivo studies in rats [5] . This paper reports in vivo and liver perfusion studies showing that 25-hydroxylation of l&OH-D3 by the liver is not under metabolic control.

A new synthetic method of 1α-hydroxy-7-dehydrocholesterol

Abstract Cholesta - 1,4,6 - trien - 3 - one ( 1 ) was converted to 3β - hydroxycholesta - 1,5,7 - triene ( 3 ) via the deconjugation procedure using t-BuOK in DMSO followed by the subsequent reduction with Ca(BH 4 ) 2 . The compound ( 3 ) readily reacted with 4-phenyl-1,2,4-triazoline-3,5-dione to yield the corresponding 1,4-addition product ( 4 ). Epoxidation of 4 with m -chloroperbenzoic acid resulted in the formation of the 1α,2α-epoxide ( 5 ) and the 1β,2β-epoxide ( 6 ) in the ratio 2:3. Reduction of 5 with LAH under reflux in THF afforded the titled compound ( 7 ). The same reduction of 6 gave 2β-hydroxy- and 1β - hydroxy - 7 - dehydrocholesterol ( 8 and 9 ) in the ratio 8:1. The compound ( 4 ) can be obtained in 25% yield from 1 without any purification of the intermediate compounds; cholesta - 1,5,7 - trien - 3 - one ( 2 , a very unstable compound) and 3 . Since 1 is obtained readily from cholesterol in high yield, the present study provides a simple and efficient synthetic method of 1α-hydroxycholecalciferol and is reasonably expected to be applicable in the synthesis of 12,25-dihydroxycholecalciferol and the other metabolites of vitamin D 3 .