Abstract
The physiological roles played by hepatocytes and nonparenchymal cells of rat liver in the metabolism of vitamin D3 have been investigated. Tritium-labeled vitamin D3 dissolved in ethanol was administered intravenously to two rats. Isolation of the liver cells 30 and 70 min after the injection showed that vitamin D3 had been taken up both by the hepatocytes and by the nonparenchymal liver cells. The relative proportion of vitamin D3 that accumulated in the nonparenchymal cells increased with time. Perfusion of the isolated rat liver with [3H] vitamin D3 added to the perfusate confirmed the ability of both cell types to efficiently take up vitamin D3 from the circulation. By a method based on high pressure liquid chromatography and isotope dilution-mass fragmentography it was found that isolated liver cells in suspension had a considerable capacity to take up vitamin D3 from the medium. About 2.5 fmol of vitamin D3 were found to be associated with each hepatocyte or nonparenchymal cell after 1 h of incubation. 25-Hydroxylation in vitro was found to be carried out only by the hepatocytes. The rate of hydroxylation was about the same whether the cells were isolated from normal or rachitic rats (3.5 and 4 pmol of 25-hydroxyvitamin D3 formed per h per 10(6) cells, respectively). The possibility that the nonparenchymal cells might serve as a storage site for vitamin D3 in the liver is discussed.
Highlights
The physiological roles played by hepatocytes and MATERIALS AND METHODSNonparenchymal cells of rat liver in the metabolismof Chemicals-Vitamin D, (cholecalciferol) and vitamin D2
Nonparenchymal cells of rat liver in the metabolismof Chemicals-Vitamin D, and vitamin D2
Ciferol) obtained from Sigma Chemical Co., 25-hydroxyvitamin D, Tritium-labeled vitamin D3dissolved in ethanol was from Philips-Duphar B.V., Veenendaal, The Netherlands, [la,2a(n)
Summary
Nonparenchymal cells of rat liver in the metabolismof Chemicals-Vitamin D, (cholecalciferol) and vitamin D2 Bovine serum albumin was omitted, from the medium used for preparing cells from rachitic rats. Animals were incubated in the same medium except that it contained Prior to extraction of the cell preparations a known amount (310 cytosolic fraction (4 mgof protein/ml)instead of bovine serum ng) of vitamin Dzwas added to correct for recovery during extraction albumin. From the tory effect observed by these cofactors can be explained by activation distribution of radioactivity in the collected fractions (l/min) the of the 25-hydroxylase activity in nonviable cells No such stimu- relative amount of tritium-labeled vitamin D3 (retention time 3.2 min) lation was observed with cultured rat hepatocytes.) The reaction was and 25-hydroxyvitamin Da (retention time 7.3 min) could be deterstarted by the addition of 171 nmol of vitamin DB(cells from normal mined. Entire sample was first chromatographed on a Zorbax-ODS column (4.6 x 250 mm, particle size 5 pm) with 7.5% H 2 0 in methanolas
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