Functional vitamin B12 deficiency: Improving methylmalonic acid reference intervals in urine

•MMA is considered the best biomarker to detect functional deficiency of vitamin B12.•The age, sex, and cobalamin have little impact on MMA concentrations.•In subclinical B12 deficiency, MMA values could go unnoticed.•A cut-off value for vitamin B12 suggesting subclinical deficiency remains a challenge. Most laboratory requests focus on the detection of possible vitamin B12 deficiency. In this context, methylmalonic acid (MMA) is reported as the best biomarker. The aim of our study was to establish the biological reference interval for MMA in urine, and assess the influence of age, sex, and vitamin B12 status on MMA concentrations. This is a prospective observational study considering individuals with normal results for blood count and liver and kidney function. Individuals who presented supplementation, any pathology or treatment that could cause cobalamin metabolism disorders, and pregnant women were excluded. Likewise, individuals whose vitamin B12 result presented antibody-mediated interference were excluded. Individuals were grouped by age-group and sex. Reference intervals were determined by non-parametric calculation (percentiles 1–99). It was established a single reference interval [0.52 (CI90%: 0.50–0.54) – 5.75 (CI90%: 5.57–6.17) mmolMMA/mol creatinine], with 100 % of individuals with MMA above the upper limit of reference presenting a total vitamin B12 concentration ≤ 238 pmol/L. The establishment of optimal reference intervals for methylmalonic acid excretion in urine is crucial in individuals with a suspicion of functional vitamin B12 deficiency. However, the possibility of establishing a cut-off value for total vitamin B12 suggesting subclinical deficiency remains a challenge for this magnitude.