Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points

Clin Chem Lab Med. 2015 Jul;53(8):1215-25. doi: 10.1515/cclm-2014-0818.


Background: A novel approach to determine vitamin B12 status is to combine four blood markers: total B12 (B12), holotranscobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy). This combined indicator of B12 status is expressed as cB12=log10[(holoTC·B12)/(MMA·Hcy)]-(age factor). Here we calculate cB12 in datasets with missing biomarkers, examine the influence of folate status, and revise diagnostic cut-points.

Methods: We used a database with all four markers (n=5211) plus folate measurements (n=972). A biomarker Z (assumed missing) was plotted versus X (a combination of other markers) and Y (age). Each chart was approximated by a function Ztheor, which predicted the potentially absent value(s). Statistical distributions of cB12 were aligned with physiological indicators of deficiency and used to determine cut-offs.

Results: The predictive functions Ztheor allowed assessment of the "incomplete" indicators, 3cB12 (three markers known) and 2cB12 (two markers known). Predictions contained a systematic deviation associated with dispersion along two axes Z and X (and unaccounted by the least squares fit). Increase in tHcy at low serum folate was corrected (cB12+Δfolate) based on the function of Δfolate=log10(Hcyreal/Hcytheor) versus folate. Statistical distributions of cB12 revealed the boundaries of groups with B12 deficiency, i.e., cB12<-0.5.

Conclusions: We provide equations that combine two, three or four biomarkers into one diagnostic indicator, thereby rescaling unmatched data into the same coordinate system. Adjustment of this indicator is required if serum folate is <10 nmol/L and tHcy is measured. Revised cut-points and guidelines for using this approach are provided.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biomarkers / blood
  • Folic Acid / blood*
  • Healthy Volunteers
  • Homocysteine / blood*
  • Humans
  • Methylmalonic Acid / blood*
  • Transcobalamins / analysis*
  • Vitamin B 12 / blood*


  • Biomarkers
  • Transcobalamins
  • Homocysteine
  • Methylmalonic Acid
  • Folic Acid
  • Vitamin B 12