Wei S, Tanaka H, Kubo T, Ichikawa M, Seino Y
Department of Pediatrics, Okayama University Medical School, Japan.
Anal Biochem. 1994 Nov 1;222(2):359-65. doi: 10.1006/abio.1994.1503.
We describe a multiple assay of the three main vitamin D metabolites, 25OHD, 24,25(OH)2D, and 1,25(OH)2D, in 0.5 ml of serum, which does not require high-performance liquid chromatography. The assay involves extracting the serum with acetonitrile, separation and purification on a C-18/OH cartridge and a Sep-Pak silica cartridge, and quantitation using 1,25(OH)2D receptors from calf mammary gland for 1,25(OH)2D, and vitamin D binding protein for 25OHD and 24,25(OH)2D. For 25OHD, 24,25(OH)2D, and 1,25(OH)2D, the method is sensitive to 0.125 ng/tube, 0.025 ng/tube, and 0.5 pg/tube, with the B50 occurring at 1 ng/tube, 0.2 ng/tube, and 8 pg/tube, respectively. The coefficients of variation (SD/mean x 100%) intraassay (n = 8) were 5.4, 12.8, and 6.6% and interassay (n = 6) 12.3, 10.8, and 8.6%. The overall recovery was 80.4 +/- 5.5, 58.0 +/- 6.3, and 77.4 +/- 5.6% (mean +/- SD, n = 40). The validity of the assay was confirmed by dilution test, analytical recovery of added vitamin D metabolites, and comparison with a standard assay using HPLC. This assay offers a simple, rapid, and precise method with which to determine the three main vitamin D metabolites in small serum samples, so it should be particularly useful in studies of pediatrics or small animals.
我们描述了一种在0.5毫升血清中对三种主要维生素D代谢物25-羟基维生素D（25OHD）、24,25-二羟基维生素D（24,25(OH)2D）和1,25-二羟基维生素D（1,25(OH)2D）进行多重检测的方法，该方法不需要高效液相色谱法。该检测方法包括用乙腈提取血清，在C-18/OH柱和Sep-Pak硅胶柱上进行分离和纯化，并使用来自小牛乳腺的1,25(OH)2D受体对1,25(OH)2D进行定量，使用维生素D结合蛋白对25OHD和24,25(OH)2D进行定量。对于25OHD、24,25(OH)2D和1,25(OH)2D，该方法的灵敏度分别为0.125纳克/管、0.025纳克/管和0.5皮克/管，半数结合浓度（B50）分别出现在1纳克/管、0.2纳克/管和8皮克/管。批内变异系数（标准差/平均值×100%，n = 8）分别为5.4%、12.8%和6.6%，批间变异系数（n = 6）分别为12.3%、10.8%和8.6%。总回收率分别为80.4±5.5%、58.0±6.3%和77.4±5.6%（平均值±标准差，n = 40）。通过稀释试验、添加维生素D代谢物的分析回收率以及与使用高效液相色谱法的标准检测方法进行比较，证实了该检测方法的有效性。该检测方法提供了一种简单、快速且精确的方法来测定小血清样本中的三种主要维生素D代谢物，因此在儿科或小动物研究中应该特别有用。
