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

J Clin Endocrinol Metab. 2006 Aug;91(8):3055-61. doi: 10.1210/jc.2006-0710. Epub 2006 May 23.


Context: We have recently introduced liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 25-hydroxyvitamin D(2) (25OHD(2)) and 25OHD(3) 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 25OHD(2) or 25OHD(3).

Objective: Our aims were to 1) determine the prevalence of C-3 epimers of 25OHD(2) or 25OHD(3) 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.

Results: In 172 children from group 1 with detectable 25OHD(2) or 25OHD(3), 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.

Conclusions: Significant concentrations of C-3 epimers of 25OHD(2) or 25OHD(3) 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.

MeSH terms

  • 25-Hydroxyvitamin D 2 / blood*
  • 25-Hydroxyvitamin D 2 / chemistry*
  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Aging*
  • Calcifediol / blood*
  • Calcifediol / chemistry*
  • Child
  • Child, Preschool
  • Chromatography, High Pressure Liquid
  • Female
  • Humans
  • Infant
  • Isomerism
  • Male
  • Mass Spectrometry
  • Middle Aged
  • Nutritional Status*
  • Radioimmunoassay
  • Reference Values
  • Sensitivity and Specificity


  • 25-Hydroxyvitamin D 2
  • Calcifediol