Parviainen M T, Savolainen K E, Alhava E M, Mäenpää P H
Ann Clin Res. 1981 Feb;13(1):26-33.
An assay procedure capable of determining 25(OH)D2 (25-hydroxyvitamin D2), 25(OH)D3 (25-hydroxyvitamin D3) and total 24,25(OH)2D (24,25-dihydroxyvitamin D) in a single 2-5 ml sample of human serum is described. The assay involves methanol/chloroform extraction of serum lipids followed by separation of the mono- and dihydroxyvitamin D metabolites and purification from interfering contaminants by chromatography on Sephadex LH-20 and by high-performance liquid chromatography (HPLC). 25(OH)D2 and 25(OH)D3 are quantified by HPLC using UV detection. Total 24,25(OH)2D is routinely measured by a competitive protein-binding (CPB) assay with human serum as a source of the binding protein. Mean values (+/- SD) in 16 normal human serum samples taken in October-November were: 25(OH)D2 5.1 +/- 2.2 ng/ml (12.8 +/- 5.5 nmol/l); 25(OH)D3 16.6 +/- 4.4 ng/ml (41.5 +/- 11.0 nmol/l); total 25(OH)D 21.7 +/- 4.9 ng/ml (54.3 +/- 12.3 nmol/l); total 24,25(OH)2D 1.6 +/- 0.6 ng/ml (3.8 +/- 1.4 nmol/l) and the ratio 24,25(OH)2D/25(OH)D 0.066 +/- 0.024. The ratio was 0.048 +/- 0.009 in women and 0.081 +/- 0.022 in men (p less than 0.002). Values in three serum samples taken from patients receiving vitamin D3 therapy were: 25(OH)D2 4.1 +/- 2.7 ng/ml (10.3 +/- 6.8 nmol/l); 25(OH)D3 46.6 +/- 20.9 ng/ml (116.5 +/- 52.3 nmol/l); total 24,25(OH)2D 3.4 +/- 1.9 ng/ml (8.2 +/- 4.6 nmol/l); 24,25(OH)2D/25(OH)D 0.075 +/- 0.022.
本文描述了一种检测方法，该方法能够在2 - 5毫升人血清样本中同时测定25(OH)D2（25 - 羟基维生素D2）、25(OH)D3（25 - 羟基维生素D3）以及总24,25(OH)2D（24,25 - 二羟基维生素D）。该检测方法包括用甲醇/氯仿提取血清脂质，随后分离单羟基和二羟基维生素D代谢物，并通过Sephadex LH - 20柱色谱和高效液相色谱（HPLC）从干扰污染物中纯化。25(OH)D2和25(OH)D3通过使用紫外检测的HPLC进行定量。总24,25(OH)2D通常通过竞争性蛋白结合（CPB）测定法进行测量，以人血清作为结合蛋白的来源。10月至11月采集的16份正常人血清样本的平均值（±标准差）为：25(OH)D2 5.1±2.2 ng/ml（12.8±5.5 nmol/l）；25(OH)D3 16.6±4.4 ng/ml（41.5±11.0 nmol/l）；总25(OH)D 21.7±4.9 ng/ml（54.3±12.3 nmol/l）；总24,25(OH)2D 1.6±0.6 ng/ml（3.8±1.4 nmol/l），24,25(OH)2D/25(OH)D的比值为0.066±0.024。女性该比值为0.048±0.009，男性为0.081±0.022（p＜0.002）。从接受维生素D3治疗的患者采集的三份血清样本中的值为：25(OH)D2 4.1±2.7 ng/ml（10.3±6.8 nmol/l）；25(OH)D3 46.6±20.9 ng/ml（116.5±52.3 nmol/l）；总24,25(OH)2D 3.4±1.9 ng/ml（8.2±4.6 nmol/l）；24,25(OH)2D/25(OH)D 0.075±0.022。
