液相色谱串联质谱测定24,25(OH)2D方法的建立及其用于筛查CYP24A1突变导致的特发性高钙血症的效用评价

维生素D与骨骼及代谢性疾病密切相关，补充维生素D已非常普遍。24羟化酶CYP24A1是维生素D代谢的关键酶，CYP24A1突变时25OHD无法代谢为24,25(OH)2D，导致特发性高钙血症、高钙尿症等。CYP24A1突变患者补充维生素D极易诱发高钙血症，重者可致肾衰竭及死亡。基因测序可确诊该突变，然而其费时、昂贵，不适于临床常规筛查，国内尚无简便的检测方法，无法对病人进行及时诊治。最新研究发现血清25OHD/24,25(OH)2D比值可用于筛查CYP24A1突变。课题组将在已完成的25OHD方法建立及临床应用基础上，研究24,25(OH)2D的色谱质谱行为，建立基于LC-MS/MS的24,25(OH)2D检测方法，分析中国人群25OHD/24,25(OH)2D比值范围，确定筛查CYP24A1突变的切点，为早期预防、及时诊治CYP24A1突变导致的高钙血症及相关肾病提供简便可靠的检测方法。

Vitamin D supplementation is very popular among all age groups because vitamin D correlates with bone healthy and many other metabolism diseases. However, when mutation happened at the enzyme of CYP24A1 who catalyzes vitamin D into the form without biological activity: 24,25(OH)2D will result in idiopathic hypercalcemia, hypercalciuria, nephrolithiasis, even renal failure and death. Especially the development of symptomatic hypercalcemia may be triggered by vitamin D prophylaxis. Gene sequencing can be the method for diagnosis of CYP24A1 mutation, however, it is time consuming, expensive, and is not suitable for screening in routine clinical laboratory. The newest study reported that the ratio of 25OHD/24,25(OH)2D can be used for screening the mutation of CYP24A1. However, there is still no method for determination of 24,25(OH)2D, no method and cut-off for screening CYP24A1 mutation in China. Our laboratory has established and validated the liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of 25OHD, and investigated the chromatographic and mass spectrometric conditions for 24,25(OH)2D. Based on the preliminary experimental data, the purpose of this study was to develop a simple, rapid and accurate method for 24,25(OH)2D based on LC-MS/MS, investigate the reference range for the ratio of 25OHD/24,25(OH)2D, and establish and validate the cut-off for screening CYP24A1 mutations. The newly developed LC-MS/MS method will provide a potentially useful tool for screening and diagnosis of CYP24A1 mutation.