NORMAL SERUM VITAMIN B12 LEVELS IN SYMPTOMATIC PATIENTS: DIAGNOSTIC VALUE OF METHYLMALONIC ACID AND HOMOCYSTEINE IN FUNCTIONAL DEFICIENCY
DOI:
https://doi.org/10.31435/ijitss.2(50).2026.5587Keywords:
Vitamin B12 Deficiency, Functional Vitamin B12 Deficiency, Methylmalonic Acid, Homocysteine, HolotranscobalaminAbstract
Background: Vitamin B12 deficiency is a clinically important condition associated with hematological, neurological, and neuropsychiatric manifestations. However, diagnosis may be difficult in symptomatic patients with normal total serum vitamin B12 concentrations, as normal circulating levels do not necessarily reflect adequate intracellular metabolic function. This has led to growing interest in the concept of functional vitamin B12 deficiency. The aim of this review was to evaluate the diagnostic value of methylmalonic acid (MMA) and homocysteine in symptomatic patients with normal or borderline serum vitamin B12 levels.
Methods: A narrative review was conducted using the PubMed database. English-language peer-reviewed articles published over the last decade were screened for relevance to vitamin B12 metabolism, laboratory assessment, functional deficiency, and the diagnostic utility of MMA, homocysteine, and holotranscobalamin. Eighteen publications were included in the qualitative synthesis.
Results: Total serum vitamin B12 has limited sensitivity for early or functional deficiency. MMA and homocysteine reflect metabolic consequences of impaired vitamin B12–dependent pathways and may improve diagnostic sensitivity in patients with normal or borderline serum levels. MMA is more specific for vitamin B12–related metabolic impairment but is influenced by renal function and age. Homocysteine is less specific and is affected by folate and vitamin B6 status, renal function, and age. Holotranscobalamin reflects the circulating biologically active fraction of vitamin B12 and may allow earlier detection of reduced availability. Biomarker interpretation is limited by physiological and inter-individual variability, as well as the lack of standardised cut-off values.
Conclusions: In symptomatic patients with inconclusive serum vitamin B12 results, MMA and homocysteine provide clinically relevant complementary information. A combined diagnostic approach integrating serum vitamin B12, functional biomarkers, and clinical assessment offers greater diagnostic accuracy than reliance on total serum vitamin B12 alone.
References
Mucha, P., Kus, F., Cysewski, D., Smolenski, R. T., & Tomczyk, M. (2024). Vitamin B12 metabolism: A network of multi-protein mediated processes. International Journal of Molecular Sciences, 25(15), Article 8021. https://doi.org/10.3390/ijms25158021
Wolffenbuttel, B. H., Owen, P. J., Ward, M., & Green, R. (2023). Vitamin B12. BMJ, 383, Article e071725. https://doi.org/10.1136/bmj-2022-071725
Sobczyńska-Malefora, A., Delvin, E., McCaddon, A., Ahmadi, K. R., & Harrington, D. J. (2021). Vitamin B12 status in health and disease: A critical review. Diagnosis of deficiency and insufficiency—Clinical and laboratory pitfalls. Critical Reviews in Clinical Laboratory Sciences, 58(6), 399–429. https://doi.org/10.1080/10408363.2021.1885339
Green, R., Allen, L. H., Bjørke-Monsen, A.-L., Brito, A., Guéant, J.-L., Miller, J. W., Molloy, A. M., Nexo, E., Stabler, S., Toh, B.-H., Ueland, P. M., Yajnik, C., & Yeates, A. J. (2017). Vitamin B12 deficiency. Nature Reviews Disease Primers, 3, Article 17040. https://doi.org/10.1038/nrdp.2017.40
Obeid, R., Andrès, E., Češka, R., Green, R., Hall, I., Herrmann, W., Humble, G., Hvas, A.-M., Karamitros, T., Kumar, S., Quadros, E. V., Toft-Petersen, A. P., & The Vitamin B12 Consensus Panelists Group. (2024). Diagnosis, treatment and long-term management of vitamin B12 deficiency in adults: A Delphi expert consensus. Journal of Clinical Medicine, 13(8), Article 2176. https://doi.org/10.3390/jcm13082176
Nexo, E., & Parkner, T. (2024). Vitamin B12-related biomarkers. Food and Nutrition Bulletin, 45(1_suppl), S28–S33. https://doi.org/10.1177/03795721241227114
Jarquin Campos, A., Risch, L., Nydegger, U., Wiesner, J., Vazquez Van Dyck, M., Renz, H., Stanga, Z., & Risch, M. (2020). Diagnostic accuracy of holotranscobalamin, vitamin B12, methylmalonic acid, and homocysteine in detecting B12 deficiency in a large, mixed patient population. Disease Markers, 2020, Article 7468506. https://doi.org/10.1155/2020/7468506
Harrington, D. J., Stevenson, E., & Sobczyńska-Malefora, A. (2025). The application and interpretation of laboratory biomarkers for the evaluation of vitamin B12 status. Annals of Clinical Biochemistry, 62(1), 22–33. https://doi.org/10.1177/00045632241292432
Murphy, M. J., Brandie, F., Ebare, M., Harrison, M., Dow, E., Bartlett, B., & Craig, D. (2021). Personalising laboratory medicine in the “real world”: Assessing clinical utility, by clinical indication, of serum total B12 and Active-B12® (holotranscobalamin) in the diagnosis of vitamin B12 deficiency. Annals of Clinical Biochemistry, 58(5), 445–451. https://doi.org/10.1177/00045632211003605
Hannibal, L., Lysne, V., Bjørke-Monsen, A.-L., Behringer, S., Grünert, S. C., Spiekerkoetter, U., Jacobsen, D. W., & Blom, H. J. (2016). Biomarkers and algorithms for the diagnosis of vitamin B12 deficiency. Frontiers in Molecular Biosciences, 3, Article 27. https://doi.org/10.3389/fmolb.2016.00027
Tiwari, A., Kumar Singh, R., Satone, P. D., Meshram, R. J., Jajoo, S., Patel, P., Goyal, S., & Wanjari, M. B. (2023). Metformin-induced vitamin B12 deficiency in patients with type-2 diabetes mellitus. Cureus, 15(10), Article e47771. https://doi.org/10.7759/cureus.47771
Huynh, D. T., Nguyen, N. T., & Do, M. D. (2024). Vitamin B12 deficiency in diabetic patients treated with metformin: A cross-sectional study. PLOS ONE, 19(4), Article e0302500. https://doi.org/10.1371/journal.pone.0302500
National Institute for Health and Care Excellence. (2024). Evidence review for diagnostic tests: Vitamin B12 deficiency in over 16s: Diagnosis and management. NICE.
Warendorf, J. K., van Doormaal, P. T. C., Vrancken, A. F. J. E., Verhoeven-Duif, N. M., van Eijk, R. P. A., van den Berg, L. H., & Notermans, N. C. (2022). Clinical relevance of testing for metabolic vitamin B12 deficiency in patients with polyneuropathy. Nutritional Neuroscience, 25(12), 2536–2546. https://doi.org/10.1080/1028415X.2021.1985751
Dastidar, R., & Sikder, K. (2022). Diagnostic reliability of serum active B12 (holo-transcobalamin) in true evaluation of vitamin B12 deficiency. BMC Research Notes, 15(1), Article 329. https://doi.org/10.1186/s13104-022-06224-8
Özdemir, S., & Demirtaş, S. (2025). Are vitamin B-12 measurements adequate for evaluating its deficiency in individuals? Asia Pacific Journal of Clinical Nutrition, 34(2), 232–239. https://pmc.ncbi.nlm.nih.gov/articles/PMC11937488/
Åsberg, A., Mikkelsen, G., & Lian, I. A. (2025). Three new tools to diagnose B12 deficiency: eGFR-adjusted methylmalonic acid (MMA100), a bivariate reference area for MMA100 and cobalamin, and a cobalamin deficiency index. Scandinavian Journal of Clinical and Laboratory Investigation, 85(2), 101–107. https://doi.org/10.1080/00365513.2025.2463084
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Copyright (c) 2026 Michalina Weronika Nieścioruk, Dominika Julia Kozdroń, Paulina Szczepańska, Jacek Kowalski, Dominika Dutkiewicz, Alia Echtay Yarbou, Rami Mallah, Adrian Mikołajuk
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