Fraher L J, Adami S, Clemens T L, Jones G, O'Riordan J L
Clin Endocrinol (Oxf). 1983 Aug;19(2):151-65. doi: 10.1111/j.1365-2265.1983.tb02977.x.
A sensitive radioimmunoassay for 1,25-dihydroxy vitamin D2 was developed using a sheep antiserum which preferentially reacts with 1-hydroxylated forms of vitamin D. An improved isolation procedure was also developed using acetonitrile for the initial extraction of serum followed by chromatography on cartridges of C18 silica and high pressure liquid chromatography eluted with a ternary solvent system to separate 1,25-dihydroxy vitamin D2 and 1,25-dihydroxy vitamin D3. 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 were separated by further reverse phase high pressure liquid chromatography prior to competitive protein binding assay. The limits of detection were 4.3 pmol/1 (2.0 pg/ml) for the 1,25-dihydroxy metabolites and 1.25 nmol/1 (0.5 ng/ml) for both 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. 25-hydroxy vitamin D2 ranged from 2.0 to 11.3 nmol/1 (0.8-4.5 ng/ml) with a mean of 4.75 nmol/1 (1.9 ng/ml) in thirteen healthy British adults and this accounted for 9.0% of the mean total 25-hydroxy vitamin D. 1,25-dihydroxy vitamin D2 was detected in the sera of only one of these subjects whereas 1,25-dihydroxy vitamin D3 was present in all ranging from 48 to 163 pmol/1 (20-65 pg/ml) with a mean of 100 pmol/1 (42 pg/ml). Both 1,25-dihydroxy vitamin D2 and 1,25-dihydroxy vitamin D3 were detected in the sera of hypoparathyroid patients treated with vitamin D2 but the relationship between 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D was complex. For example, when an excess of 25-hydroxy vitamin D2 was present the serum concentration of 1,25-dihydroxy vitamin D3 was disproportionately high. Conversely, in patients who had previously been treated with vitamin D2 but were receiving only vitamin D3 at the time of study, the major 25-hydroxy metabolite was in the vitamin D3 form and there was a disproportionately high amount of 1,25-dihydroxy vitamin D2. Total 1,25-dihydroxy vitamin D ranged from 110 to 400 pmol/1 (45-165 pg/ml) and was above the upper limit of normal for 1,25-dihydroxy vitamin D3 in half of these hypoparathyroid patients treated with pharmacological doses of vitamin D.
利用一种优先与维生素D的1-羟基化形式发生反应的羊抗血清，开发了一种用于检测1,25-二羟基维生素D2的灵敏放射免疫分析法。还开发了一种改进的分离程序，先用乙腈对血清进行初步提取，然后在C18硅胶柱上进行色谱分离，再用三元溶剂系统进行高压液相色谱洗脱，以分离1,25-二羟基维生素D2和1,25-二羟基维生素D3。在竞争性蛋白结合分析之前，通过进一步的反相高压液相色谱法分离25-羟基维生素D2和25-羟基维生素D3。1,25-二羟基代谢物的检测限为4.3 pmol/1（2.0 pg/ml），25-羟基维生素D2和25-羟基维生素D3的检测限均为1.25 nmol/1（0.5 ng/ml）。在13名健康英国成年人中，25-羟基维生素D2的含量范围为2.0至11.3 nmol/1（0.8 - 4.5 ng/ml），平均为4.75 nmol/1（1.9 ng/ml），占总25-羟基维生素D平均值的9.0%。在这些受试者中，仅1名受试者的血清中检测到1,25-二羟基维生素D2，而所有受试者的血清中均存在1,25-二羟基维生素D3，含量范围为48至163 pmol/1（20 - 65 pg/ml），平均为100 pmol/1（42 pg/ml）。在用维生素D2治疗的甲状旁腺功能减退患者的血清中，检测到了1,25-二羟基维生素D2和1,25-二羟基维生素D3，但25-羟基维生素D与1,25-二羟基维生素D之间的关系很复杂。例如，当存在过量的25-羟基维生素D2时，1,25-二羟基维生素D3的血清浓度会异常高。相反，在之前用维生素D2治疗但在研究时仅接受维生素D3治疗的患者中，主要的25-羟基代谢物为维生素D3形式，且1,25-二羟基维生素D2的含量异常高。总1,25-二羟基维生素D的含量范围为110至400 pmol/1（45 - 165 pg/ml），在这些接受药理剂量维生素D治疗的甲状旁腺功能减退患者中，有一半患者的总1,25-二羟基维生素D高于1,25-二羟基维生素D3的正常上限。
