Utility and limitations of biochemical markers of vitamin B12 deficiency

Eur J Clin Invest. 2013 Mar;43(3):231-7. doi: 10.1111/eci.12034. Epub 2013 Jan 18.


Background: There is an urgent need for proper utilization of laboratory markers to diagnose vitamin B12 deficiency that should reduce false negative cases. We worked out a diagnostic algorithm that provides a two-step detection of vitamin B12 deficiency using holotranscobalamin as a first line marker and methylmalonic acid (MMA) as a second line marker.

Materials and methods: We tested 1359 serum samples sent to our laboratory for total vitamin B12 assay. Serum samples were used for the determination of holotranscobalamin, MMA and creatinine.

Results: Compared with total B12, holotranscobalamin showed a higher area under the receiver operating characteristic curve for detecting MMA levels > 300 nM. However, the distribution of holotranscobalamin in individuals with elevated creatinine irrespective of MMA and in individuals with elevated MMA irrespective of creatinine was shifted into the higher ranges. In the grey zone of holotranscobalamin between 23 and 75 pM (the range extending from the 90% diagnostic sensitivity to the 90% diagnostic specificity), MMA testing as a second line marker would help detecting 18% of deficient cases. Lowering MMA after vitamin B12 treatment may help setting the diagnosis of B12 deficiency in individuals with elevated creatinine.

Discussion: Testing for vitamin B12 deficiency should start with holotranscobalamin measurement. Holotranscobalamin between 23 and 75 pM should be followed by MMA testing that can filter substantial number of deficient cases in the grey range in individuals with normal renal function. This diagnostic strategy may significantly improve assessing vitamin B12 deficiency.

Publication types

  • Comparative Study

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Algorithms
  • Analysis of Variance
  • Biomarkers / metabolism
  • Creatinine / metabolism*
  • Humans
  • Methylmalonic Acid / metabolism*
  • Middle Aged
  • Transcobalamins / metabolism*
  • Vitamin B 12 Deficiency / diagnosis*


  • Biomarkers
  • Transcobalamins
  • Methylmalonic Acid
  • Creatinine