Kaufmann Martin, Morse Nicole, Molloy Billy Joe, Cooper Donald P, Schlingmann Karl Peter, Molin Arnaud, Kottler Marie Laure, Gallagher J Christopher, Armas Laura, Jones Glenville
Department of Biomedical & Molecular Sciences, Queen's University, Kingston, ON, Canada.
Health Sciences Research, Waters Corporation, Wilmslow, UK.
J Bone Miner Res. 2017 Jul;32(7):1589-1596. doi: 10.1002/jbmr.3135. Epub 2017 Apr 21.
CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D :24,25-(OH) D ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH) D remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH) D peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH) D as a contaminant of the 24,25-(OH) D peak. In normals, the concentration of 24,25-(OH) D greatly exceeds 25,26-(OH) D ; however, 25,26-(OH) D becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH) D levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH) D levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH) D from 25,26-(OH) D produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH) D in IIH patients appears to be multifactorial. © 2017 American Society for Bone and Mineral Research.
CYP24A1突变现已被确认为特发性婴儿高钙血症（IIH）的病因。一种基于快速液相色谱串联质谱（LC-MS/MS）的血液检测方法，能够测量25-羟基维生素D（25-OH-D）与24,25-二羟基维生素D（24,25-(OH)D）的比值（R），可以根据体内CYP24A1对25-OH-D的C24-羟化作用降低来识别IIH患者。尽管该比值在IIH患者中显著升高，但令人惊讶的是，血清24,25-(OH)D仍可检测到。本研究探讨了对此现象的可能解释，包括：某些CYP24A1基因型个体中残留的CYP24A1酶活性、替代C24-羟化酶的表达，以及LC-MS/MS上24,25-(OH)D峰的等压污染可能性。我们在LC-MS/MS上采用了延长至20分钟的运行时间，以研究因CYP24A1或SLC34A1突变导致的IIH患者、未受影响的杂合子和透析患者、维生素D缺乏患者以及25-OH-D水平范围广泛的正常受试者的血清维生素D代谢产物。我们鉴定出25,26-(OH)D是24,25-(OH)D峰的污染物。在正常人群中，24,25-(OH)D的浓度大大超过25,26-(OH)D；然而，在CYP24A1突变的IIH患者和透析患者中，25,26-(OH)D变得更为显著，在这些患者中，当CYP24A1功能受损时，24,25-(OH)D水平较低。30例IIH-CYP24A1患者的平均R值为700（范围为166至2168；临界值 = 140），而163例对照者的平均R值为31。此外，携带CYP24A1 L409S等位基因的患者比其他突变患者表现出更高的24,25-(OH)D水平和更低的R值（平均R = 268；n = 8）。我们得出结论，一种能将24,25-(OH)D与25,26-(OH)D分离的色谱方法能产生更准确的R值，可用于区分CYP24A1活性改变的病理状态。IIH患者血清中残留的24,25-(OH)D的来源似乎是多因素的。© 2017美国骨与矿物质研究学会。
