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Cobalamin-saturated transcobalamin, also called holotranscobalamin (holoTC), constitutes only between 6% and 20% of total plasma B12. Serum concentration of holoTC is a new marker in laboratory diagnosis of cobalamin deficiency. We tested the utility of holoTC in assessing vitamin B12 status.We measured concentrations of holoTC and methylmalonic acid (MMA) in 1018 serum samples that were referred to our laboratory for total cobalamin testing.Concentrations of MMA were lower in females compared to males and this difference was no more significant after adjusting for serum creat-inine. Moreover, age was associated with higher concentrations of serum MMA, higher holoTC and slightly higher concentrations of total cobalamin. Higher concentrations of serum creatinine were associated with higher concentrations of MMA and holoTC. However, no association between serum creatinine and total cobalamin was observed. Only subjects with normal serum creatinine showed a negative correlation between serum holoTC and MMA (r = −0.36, p<0.001). In subjects with MMA ≥ 300 nmol/L and holoTC ≤ 35 pmol/L, concentrations of total cobalamin were well within the normal range (median; 25th/75th percentiles = 212; 171/272 pmol/L). Receiver operating characteristic (ROC) curve analysis displayed a higher sensitivity and specificity for holoTC compared with vitamin B12 for detecting concentrations of MMA ≥ 300 nmol/L in individuals with normal renal function.Compared to total cobalamin, we observed a better performance of holoTC assay in detecting elevated concentrations of MMA in subjects with normal renal function. The majority of subjects with combined low holoTC and elevated MMA had normal concentrations of total cobalamin. HoloTC can be used as a first line parameter in detecting cobalamin deficiency.