van Hoof H J, Swinkels L M, Ross H A, Sweep C G, Benraad T J
Department of Chemical Endocrinology, Academic Hospital Nijmegen, The Netherlands.
Anal Biochem. 1998 May 1;258(2):176-83. doi: 10.1006/abio.1998.2586.
Most of the total circulating 1,25-dihydroxyvitamin D (1,25(OH)2D) is bound to plasma proteins, mainly vitamin D-binding protein (DBP) and albumin. Only a small fraction in plasma exists in the free form. It is widely assumed that the non-protein-bound free hormone reflects the biologically active fraction. We describe a dialysis method for the determination of plasma free 1,25(OH)2D which is relatively easy to perform. In this symmetric or "rate" dialysis method, identical samples are placed at both sides of a membrane. At one side, tritiated 1,25(OH)2D is added and the rate of transfer of this tritiated 1,25(OH)2D through a dialysis membrane is directly related to the free fraction of plasma 1,25(OH)2D. This method is much less susceptible toward tracer impurities than indirect equilibrium dialysis and centrifugal ultrafiltration. Moreover, it requires much less tracer. The intraassay coefficient of variation for the determination of the free fraction is 1.0%; the interassay variation is 7.7%. Comparison of the free fraction of 23 samples assessed with both centrifugal ultrafiltration and symmetric dialysis showed much higher values using the former method. No significant correlation between the two methods was found. The free fraction of 1,25(OH)2D in normal subjects as assessed with symmetric dialysis ranges from 0.049 to 0.103%.
循环中的1,25 - 二羟维生素D（1,25(OH)₂D）大部分与血浆蛋白结合，主要是维生素D结合蛋白（DBP）和白蛋白。血浆中只有一小部分以游离形式存在。人们普遍认为，非蛋白结合的游离激素反映了生物活性部分。我们描述了一种相对容易操作的用于测定血浆游离1,25(OH)₂D的透析方法。在这种对称或“速率”透析方法中，将相同的样品置于膜的两侧。在一侧加入氚标记的1,25(OH)₂D，这种氚标记的1,25(OH)₂D通过透析膜的转移速率与血浆1,25(OH)₂D的游离部分直接相关。与间接平衡透析和离心超滤相比，该方法对示踪剂杂质的敏感性要低得多。此外，它所需的示踪剂要少得多。测定游离部分的批内变异系数为1.0%；批间变异为7.7%。用离心超滤和对称透析两种方法评估的23个样品的游离部分比较显示，使用前一种方法的值要高得多。两种方法之间未发现显著相关性。用对称透析评估的正常受试者中1,25(OH)₂D的游离部分范围为0.049%至0.103%。
