There is an urgent need for optimizing laboratory markers to diagnose vitamin B12 deficiency. Measurement of total serum B12 has a relatively poor sensitivity and specificity in predicting B12 status especially in the low and intermediate ranges (1). The insensitivity of the total B12 test cannot be resolved by shifting the cut-off value towards a higher level, because more subjects with normal B12 status will be falsely diagnosed as deficient. There is no conventional test that can reliably differentiate between B12 deficient and non-deficient cases. The distributions of serum concentrations of total serum B12 in deficient and non-deficient people overlap (2). In a large population based study, different cut-offs for total B12 (<148; <200; <258 pmol/L) and MMA (>271; >376 nmol/L) resulted in very different prevalence estimates for low B12 status (3 – 26% for low serum B12 and 2.3 – 5.8% for high MMA) (3). Two percent of young women showed low B12 and normal MMA, which might falsely reflect low B12 when looking at B12 as a sole marker. Furthermore, in older adults about 40% of the highest MMA group (>376 nmol/L) had cognitive impairment (3). MMA was suggested as a better biomarker for cognitive function than serum B12. Neither renal function, nor holoTC were estimated in the study by Bailey et al. (3).
A major limitation of serum concentrations of MMA and tHcy are their increase with decreasing renal function. Elevated concentrations of MMA are common in renal patients and this can be partly corrected after treatment with B12, indicating a pre-treatment deficiency (4).
In a previous study by our group (5) and in a similar one (6), receiver operating characteristic (ROC)-curve analysis revealed that the area under the curve (AUC) was higher for holoTC as compared to total B12. The cut-off value for holoTC of 35 pmol/L has been suggested in several publications (7). In a study the diagnostic sensitivity for holoTC (< 35 pmol/L) in detecting subjects with MMA >300 nmol/L was 72% and the diagnostic specificity was 60% (n >1,300 samples). The corresponding total B12 level at 72% sensitivity was 243 pmol/L and the resulting specificity was only 45%. In this study, the predictive value for a positive test (holoTC < 35 pmol/L) was 29%, and for a negative test (holoTC ≥ 35 pmol/L) the predictive value was 90%. Within a gray range for holoTC a second marker like MMA might be useful to set the diagnosis. For this set of analyses the study data from 1,034 samples with serum creatinine ≤ 97.2 µmol/L was applied. A cut-off value for MMA of > 300 nmol/L was suggestively utilized to define deficiency. At a diagnostic sensitivity level of 90%, the corresponding diagnostic specificity was 27%. This corresponded to a holoTC concentration of 22 pmol/L. In addition, at a diagnostic specificity of 90%, the sensitivity was 21% and the holoTC concentration at this point was 76 pmol/L. Thus, the gray zone spans the holoTC concentrations between 22 and 76 pmol/L. Samples falling within this range are suggested to be tested for MMA. Utilizing this algorithm it was found that 66% (n = 681) of all samples screened with holoTC are in the gray zone (8). Further testing of MMA as a second line marker, will identify 18% (n = 121 of the 681) as B12 deficient. Of special interest is the group displaying low holoTC < 23 pmol/L but normal MMA (81 of 133 subjects). This condition is in agreement with B12 depletion or negative balance (9) where B12 stores are still able keeping the cellular B12 dependent metabolism normal.
The holoTC immunoassay is available as an automated test. With regard to the cost-benefit effect of early detection of B12 deficiency by using holoTC, this test will become established as first line parameter to measure B12 status. The holoTC test is replacing total B12 as marker for screening for B12 deficiency.
1. Herrmann W, Schorr H, Bodis M, Knapp JP, Muller A, Stein G, Geisel J. Role of homocysteine, cystathionine and methylmalonic acid measurement for diagnosis of vitamin deficiency in high-aged subjects. Eur J Clin Invest 2000;30:1083-9.
2. Obeid R, Herrmann W. Holotranscobalamin in laboratory diagnosis of cobalamin deficiency compared to total cobalamin and methylmalonic acid. Clin Chem Lab Med 2007;45:1746-50.
3. Bailey RL, Carmel R, Green R, Pfeiffer CM, Cogswell ME, Osterloh JD et al. Monitoring of vitamin B-12 nutritional status in the United States by using plasma methylmalonic acid and serum vitamin B-12. Am J Clin Nutr 2011;94:552-61.
4. Obeid R, Kuhlmann MK, Kohler H, Herrmann W. Response of homocysteine, cystathionine, and methylmalonic acid to vitamin treatment in dialysis patients. Clin Chem 2005;51:196-201.
5. Herrmann W, Obeid R, Schorr H, Geisel J. Functional vitamin B12 deficiency and determination of holotranscobalamin in populations at risk. Clin Chem Lab Med 2003;41:1478-88.
6. Lloyd-Wright Z, Hvas AM, Moller J, Sanders TA, Nexo E. Holotranscobalamin as an indicator of dietary vitamin B12 deficiency. Clin Chem 2003;49:2076-8.
7. Herrmann W, Obeid R, Schorr H, Geisel J. The usefulness of holotranscobalamin in predicting vitamin B12 status in different clinical settings. Curr Drug Metab 2005;6:47-53.
8. Herrmann W, Obeid R. Utility and limitations of biochemical markers of vitamin B12 deficiency. Eur J Clin Invest 2013;43:231-7.
9. Herbert V. Staging vitamin B-12 (cobalamin) status in vegetarians. Am J Clin Nutr 1994;59:1213S-22S.