Cunningham J, Griffin G, Avioli L V
Division of Bone and Mineral Metabolism, Jewish Hospital, St. Louis, Missouri.
Calcif Tissue Int. 1987 Dec;41(6):342-5. doi: 10.1007/BF02556674.
Cultured mouse kidney cells grown in serum-free medium were used to assess the metabolism of 25-hydroxyvitamin D3 in the presence of simulated metabolic acidosis. Kidney epithelial cells isolated from 4-6 week old mice were grown to confluence in a defined serum-free medium at pH 7.4. The confluent monolayers were incubated with tritiated 25-hydroxyvitamin D3 for 6 hours, the samples were extracted, and vitamin D metabolites were separated and quantitated by high pressure liquid chromatography (HPLC). The pH of the incubation medium was set at 6.9, 7.1, 7.4, or 7.7 by adjusting the bicarbonate concentration, using chloride as the balancing anion at constant Pco2. When pH was altered at the beginning of the 6 hour assay, production of 1,25-dihydroxyvitamin D3 was the same at each pH. More prolonged pH perturbation for a total of 30 hours likewise had no influence on 1,25-dihydroxyvitamin D3 production. These results confirm that intact mammalian kidney cells in serum-free culture possess an active 25-hydroxyvitamin D3-1-hydroxylase and that the activity of the enzyme is unaffected by pH over the range 6.8-7.7. In experiments where acidosis has been shown to alter 1,25-dihydroxyvitamin D3 production, the mechanism was probably indirect.
在无血清培养基中培养的小鼠肾细胞被用于评估在模拟代谢性酸中毒情况下25-羟基维生素D3的代谢。从4至6周龄小鼠分离出的肾上皮细胞在pH值为7.4的特定无血清培养基中生长至汇合状态。将汇合的单层细胞与氚标记的25-羟基维生素D3孵育6小时，对样品进行提取，然后通过高压液相色谱法（HPLC）分离并定量维生素D代谢物。通过在恒定Pco2条件下以氯离子作为平衡阴离子来调节碳酸氢盐浓度，将孵育培养基的pH值设定为6.9、7.1、7.4或7.7。当在6小时测定开始时改变pH值时，在每个pH值下1,25-二羟基维生素D3的产生量相同。总共30小时的更长时间的pH值扰动同样对1,25-二羟基维生素D3的产生没有影响。这些结果证实，无血清培养中的完整哺乳动物肾细胞具有活跃的25-羟基维生素D3-1-羟化酶，并且该酶的活性在6.8 - 7.7范围内不受pH值的影响。在已证明酸中毒会改变1,25-二羟基维生素D3产生的实验中，其机制可能是间接的。
