C-3 Epimers Can Account for a Significant Proportion of Total Circulating 25-Hydroxyvitamin D in Infants, Complicating Accurate Measurement and Interpretation of Vitamin D Status

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We have recently introduced liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 25-hydroxyvitamin D2 (25OHD2) and 25OHD3 testing. During subsequent clinical use, we identified significantly elevated results in some infants. We hypothesized this might represent assay interference caused by C-3 epimers of 25OHD2 or 25OHD3.


Our aims were to 1) determine the prevalence of C-3 epimers of 25OHD2 or 25OHD3 in human serum, and 2) identify the patient populations that might be affected.

Study Design:

We modified our LC-MS/MS method to allow detection of C-3 epimers. We retested specimens from four patient groups with the new method and an extracted RIA: 1) children less than 1 yr old, 2) children 1-18 yr old, 3) adults aged 20-87 yr with liver disease, and 4) adults aged 19-91 yr without liver disease.


In 172 children from group 1 with detectable 25OHD2 or 25OHD3, we identified C-3 epimers in 39 (22.7%). The epimers contributed 8.7-61.1% of the total 25-OHD. There was an inverse relationship between patient age and epimer percentage (r = 0.48; P < 0.002). The RIA gave accurate 25-OHD results that correlated with the modified LC-MS/MS method. No C-3 epimers were detected in any of the other groups.


Significant concentrations of C-3 epimers of 25OHD2 or 25OHD3 are commonly found in infants. This can lead to overestimation of 25-OHD levels. Measurements in children less than 1 yr should therefore be performed with an assay that allows accurate detection of 25-OHD in the presence of its C-3 epimers.

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