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Clinical Chemistry and Laboratory Medicine (CCLM)

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Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter

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Reference intervals for serum total vitamin B12 and holotranscobalamin concentrations and their change points with methylmalonic acid concentration to assess vitamin B12 status during early and mid-pregnancy

Theresa H. Schroder
  • Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, BC, Canada
  • British Columbia Children’s Hospital Research Institute, Vancouver, BC, Canada
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/ Amy Tan
  • Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, BC, Canada
  • British Columbia Children’s Hospital Research Institute, Vancouver, BC, Canada
  • Other articles by this author:
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/ Andre Mattman
  • Department of Clinical Chemistry and Laboratory Medicine, St Paul’s Hospital, Vancouver, BC, Canada
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/ Graham Sinclair
  • British Columbia Children’s Hospital Research Institute, Vancouver, BC, Canada
  • Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia, Vancouver, BC, Canada
  • BC Children’s Hospital and BC Women’s Health Centre, Vancouver, BC, Canada
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/ Susan I. Barr
  • Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, BC, Canada
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/ Hilary D. Vallance
  • British Columbia Children’s Hospital Research Institute, Vancouver, BC, Canada
  • Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia, Vancouver, BC, Canada
  • BC Children’s Hospital and BC Women’s Health Centre, Vancouver, BC, Canada
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/ Yvonne Lamers
  • Corresponding author
  • British Columbia Children’s Hospital Research Institute, 950 W 28th Ave, Vancouver, BC V5Z 4H4, Canada
  • Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, FNH 245, 2205 East Mall, Vancouver, BC V6T 1Z4, Canada
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Published Online: 2019-05-14 | DOI: https://doi.org/10.1515/cclm-2018-1337



Maternal vitamin B12 (B-12) adequacy is important for maternal health and optimal fetal growth. However, pregnancy-specific cut-offs for B-12 biomarkers are lacking.


Reference intervals for serum total B-12, holotranscobalamin (holoTC) and methylmalonic acid (MMA) concentrations were calculated following CLSI EP28-A3c guidelines in 723 pregnant women of European (50%) and South Asian (50%) ethnicity, residing in British Columbia, Canada, at median (range) 11.4 (8.3–13.9) and 16.1 (14.9–20.9) weeks of gestation. Change point analyses described relationships between log serum MMA concentration with serum total B-12 and holoTC concentrations, assuming linear-linear relationships.


The central 95% reference interval limits indicated that serum total B-12 <89.9 and <84.0 pmol/L, holoTC <29.5 and <26.0 pmol/L and MMA >371 and >374 nmol/L, in the first and second trimesters, respectively, may indicate B-12 deficiency in pregnant women. The lower limits of total B-12 and holoTC and the upper limits of MMA significantly differed by ethnicity in both trimesters. According to the change point analysis, total B-12 <186 and <180 pmol/L and holoTC <62.2 and <67.5 pmol/L in the first and second trimesters, respectively, suggested an increased probability of impaired intracellular B-12 status, with no difference between ethnicities.


We present novel reference limits and change points for B-12 biomarkers, which may be employed to identify possible B-12 deficiency in women during early and mid-pregnancy. Future research is needed to validate these cut-offs and determine the predictors and functional outcomes associated with impaired B-12 status in ethnically diverse populations.

Keywords: change point analysis; ethnicity; first trimester; holotranscobalamin; methylmalonic acid; pregnancy; reference interval; second trimester; vitamin B12


  • 1.

    Molloy AM, Kirke PN, Troendle JF, Burke H, Sutton M, Brody LC, et al. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics 2009;123:917–23.CrossrefGoogle Scholar

  • 2.

    Ray JG, Wyatt PR, Thompson MD, Vermeulen MJ, Meier C, Wong P-Y, et al. Vitamin B12 and the risk of neural tube defects in a folic-acid-fortified population. Epidemiology 2007;18:362–6.CrossrefGoogle Scholar

  • 3.

    Muthayya S, Kurpad A, Duggan CP, Bosch RJ, Dwarkanath P, Mhaskar A, et al. Low maternal vitamin B12 status is associated with intrauterine growth retardation in urban South Indians. Eur J Clin Nutr 2006;60:791–801.CrossrefPubMedGoogle Scholar

  • 4.

    Sukumar N, Rafnsson SB, Kandala N-B, Bhopal R, Yajnik CS, Saravanan P. Prevalence of vitamin B-12 insufficiency during pregnancy and its effect on offspring birth weight: a systematic review and meta-analysis. Am J Clin Nutr 2016;103:1232–51.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 5.

    Rogne T, Tielemans MJ, Chong MF, Yajnik CS, Krishnaveni GV, Poston L, et al. Associations of maternal vitamin B12 concentration in pregnancy with the risks of preterm birth and low birth weight : a systematic review and meta-analysis of individual participant data. Am J Epidemiol 2017;185:212–23.PubMedWeb of ScienceGoogle Scholar

  • 6.

    Schroder T, Sinclair G, Mattman A, Jung B, Barr SI, Vallance HD, et al. Pregnant women of South Asian ethnicity in Canada have substantially lower vitamin B12 status compared to pregnant women of European ethnicity. Br J Nutr 2017;118:454–62.PubMedCrossrefGoogle Scholar

  • 7.

    Jeruszka-Bielak M, Isman C, Schroder T, Li W, Green T, Lamers Y. South Asian ethnicity is related to the highest risk of vitamin B12 deficiency in pregnant Canadian women. Nutrients 2017;9:317.Web of ScienceCrossrefGoogle Scholar

  • 8.

    Carmel R, Mallidi PV, Vinarskiy S, Brar S, Frouhar Z. Hyperhomocysteinemia and cobalamin deficiency in young Asian Indians in the United States. Am J Hematol 2002;70:107–14.PubMedCrossrefGoogle Scholar

  • 9.

    Bindra GS, Gibson RS, Berry M. Vitamin B12 and folate status of East Indian immigrants living in Canada. Nutr Res 1987;7:365–74.CrossrefGoogle Scholar

  • 10.

    Gupta AK, Damji A, Uppaluri A. Vitamin B12 deficiency. Prevalence among South Asians at a Toronto clinic. Can Fam Physician 2004;50:743–7.Google Scholar

  • 11.

    Carlin A, Alfirevic Z. Physiological changes of pregnancy and monitoring. Crit Care Obstet 2008;22:801–23.Google Scholar

  • 12.

    Raghavan R, Ashour FS, Bailey R. A review of cutoffs for nutritional biomarkers. Adv Nutr 2016;7:112–20.Web of SciencePubMedCrossrefGoogle Scholar

  • 13.

    Murphy MM, Molloy AM, Ueland PM, Fernandez-Ballart JD, Schneede J, Arija V, et al. Longitudinal study of the effect of pregnancy on maternal and fetal cobalamin status in healthy women and their offspring. J Nutr 2007;137:1863–7.PubMedCrossrefGoogle Scholar

  • 14.

    Milman N, Byg KE, Bergholt T, Eriksen L, Hvas AM. Cobalamin status during normal pregnancy and postpartum: a longitudinal study comprising 406 Danish women. Eur J Haematol 2006;76:521–5.CrossrefPubMedGoogle Scholar

  • 15.

    Koebnick C, Heins UA, Dagnelie PC, Wickramasinghe SN, Ratnayaka ID, Hothorn T, et al. Longitudinal concentrations of vitamin B12 and vitamin B12-binding proteins during uncomplicated pregnancy. Clin Chem 2002;48:928–33.PubMedGoogle Scholar

  • 16.

    Quadros EV, Nakayama Y, Sequeira JM. The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin. Blood 2009;113:186–92.Web of SciencePubMedCrossrefGoogle Scholar

  • 17.

    Rasmussen K, Vyberg B, Pedersen KO, Brøchner-Mortensen J. Methylmalonic acid in renal insufficiency: evidence of accumulation and implications for diagnosis of cobalamin deficiency. Clin Chem 1990;36:1523–4.PubMedGoogle Scholar

  • 18.

    Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Lindenbaum J. Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry. J Clin Invest 1988;81:466–74.PubMedCrossrefGoogle Scholar

  • 19.

    Stabler SP. Vitamin B12 deficiency. N Engl J Med 2013;368:149–60.PubMedCrossrefGoogle Scholar

  • 20.

    Bae S, West AA, Yan J, Jiang X, Perry CA, Malysheva O, et al. Vitamin B-12 status differs among pregnant, lactating, and control women with equivalent nutrient intakes. J Nutr 2015;145:1507–14.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 21.

    Milman N, Bergholt T, Byg KE, Eriksen L, Hvas AM. Reference intervals for haematological variables during normal pregnancy and postpartum in 434 healthy Danish women. Eur J Haematol 2007;79:39–46.Web of ScienceCrossrefPubMedGoogle Scholar

  • 22.

    Henny J, Petitclerc C, Fuentes-Arderiu X, Petersen PH, Queraltó JM, Schiele F, et al. Need for revisiting the concept of reference values. Clin Chem Lab Med 2000;38:589–95.PubMedGoogle Scholar

  • 23.

    Petitclerc C. Normality: the unreachable star? Clin Chem Lab Med 2004;42:698–701.PubMedGoogle Scholar

  • 24.

    Clinical & Laboratory Standards Institute (CLSI). Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline. 3rd ed. Wayne, PA: CLSI, 2008.Google Scholar

  • 25.

    Bailey RL, Durazo-Arvizu RA, Carmel R, Green R, Pfeiffer CM, Sempos CT, et al. Modeling a methylmalonic acid-derived change point for serum vitamin B-12 for adults in NHANES. Am J Clin Nutr 2013;98:460–7.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 26.

    Selhub J, Jacques PF, Dallal G, Choumenkovitch S, Rogers G. The use of blood concentrations of vitamins and their respective functional indicators to define folate and vitamin B12 status. Food Nutr Bull 2008;29(2 Suppl):S67–73.CrossrefPubMedGoogle Scholar

  • 27.

    Vogiatzoglou A, Oulhaj A, Smith AD, Nurk E, Drevon CA, Ueland PM, et al. Determinants of plasma methylmalonic acid in a large population: implications for assessment of vitamin B12 status. Clin Chem 2009;55:2198–206.CrossrefGoogle Scholar

  • 28.

    Perinatal Services BC. BC Prenatal Genetic Screening Program [Internet]. 2018 [cited 2018 Dec 13]. Available from: http://www.perinatalservicesbc.ca/our-services/screening-programs/prenatal-genetic-screening-program.

  • 29.

    Perinatal Services BC. British Columbia Perinatal Data Registry. Years Provided: 2015 to 2016. Resource Type: Data Extract. Data Provided on 2018.Google Scholar

  • 30.

    Schroder TH, Quay TA, Lamers Y. Methylmalonic acid quantified in dried blood spots provides a precise, valid, and stable measure of functional vitamin B-12 status in healthy women. J Nutr 2014;144:1658–63.CrossrefWeb of SciencePubMedGoogle Scholar

  • 31.

    Allen RH, Stabler SP, Savage DG, Lindenbaum J. Elevation of 2-methylcitric acid I and II levels in serum, urine, and cerebrospinal fluid of patients with cobalamin deficiency. Metabolism 1993;42:978–88.PubMedCrossrefGoogle Scholar

  • 32.

    Harris EK, Boyd JC. On dividing reference data into subgroups to produce separate reference ranges. Clin Chem 1990;36:265–70.PubMedGoogle Scholar

  • 33.

    Lahti A, Petersen PH, Boyd JC, Rustad P, Laake P, Solberg HE. Partitioning of nongaussian-distributed biochemical reference data into subgroups. Clin Chem 2004;50:891–900.CrossrefPubMedGoogle Scholar

  • 34.

    Aparicio-Ugarriza R, Palacios G, Alder M, González-Gross M. A review of the cut-off points for the diagnosis of vitamin B12 deficiency in the general population. Clin Chem Lab Med 2015;53:1–11.Google Scholar

  • 35.

    Refsum H, Johnston C, Guttormsen AB, Nexo E. Holotranscobalamin and total transcobalamin in human plasma: determination, determinants, and reference values in healthy adults. Clin Chem 2006;52:129–37.CrossrefPubMedGoogle Scholar

  • 36.

    Hølleland G, Schneede J, Ueland PM, Lund PK, Refsum H, Sandberg S. Cobalamin deficiency in general practice: assessment of the diagnostic utility and cost-benefit analysis of methylmalonic acid determination in relation to current diagnostic strategies. Clin Chem 1999;45:189–98.PubMedGoogle Scholar

  • 37.

    Pfeiffer CM, Caudill SP, Gunter EW, Osterloh J, Sampson EJ. Biochemical indicators of B vitamin status in the US population after folic acid fortification: results from the National Health and Nutrition Examination Survey 1999–2000. Am J Clin Nutr 2005;82:442–50.PubMedCrossrefGoogle Scholar

  • 38.

    Carmel R. Biomarkers of cobalamin (vitamin B-12) status in the epidemiologic setting: a critical overview of context, applications, and performance characteristics of cobalamin, methylmalonic acid, and holotranscobalamin II. Am J Clin Nutr 2011;94:348S–358S.CrossrefGoogle Scholar

  • 39.

    Allen LH, Miller JW, Groot L De, Rosenberg IH, Smith AD, Refsum H, et al. Biomarkers of nutrition for development (BOND): vitamin B-12 review. J Nutr 2018;148(Suppl 4):1995S–2027S.Google Scholar

  • 40.

    Wu BTF, Innis SM, Mulder KA, Dyer RA, King DJ. Low plasma vitamin B-12 is associated with a lower pregnancy-associated rise in plasma free choline in Canadian pregnant women and lower postnatal growth rates in their male infants. Am J Clin Nutr 2013;98:1209–17.CrossrefPubMedWeb of ScienceGoogle Scholar

  • 41.

    Visentin CE, Masih SP, Plumptre L, Schroder TH, Sohn K-J, Ly A, et al. Low serum vitamin B-12 concentrations are prevalent in a cohort of pregnant Canadian women. J Nutr 2016;146:1035–42.Web of ScienceCrossrefGoogle Scholar

  • 42.

    Duggan C, Srinivasan K, Thomas T, Samuel T, Rajendran R, Muthayya S, et al. Vitamin B-12 supplementation during pregnancy and early lactation increases maternal, breast milk, and infant measures of vitamin B-12 status. J Nutr 2014;144:758–64.PubMedCrossrefGoogle Scholar

  • 43.

    Katre P, Bhat D, Lubree H, Otiv S, Joshi S, Joglekar C, et al. Vitamin B12 and folic acid supplementation and plasma total homocysteine concentrations in pregnant Indian women with low B12 and high folate status. Asia Pac J Clin Nutr 2010;19:335–43.PubMedGoogle Scholar

  • 44.

    Yajnik CS, Deshpande SS, Jackson AA, Refsum H, Rao S, Fisher DJ, et al. Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study. Diabetologia 2008;51:29–38.PubMedGoogle Scholar

  • 45.

    Stewart CP, Christian P, Schulze KJ, Arguello M, LeClerq SC, Khatry SK, et al. Low maternal vitamin B-12 status is associated with offspring insulin resistance regardless of antenatal micronutrient supplementation in rural Nepal. J Nutr 2011;141:1912–7.Web of ScienceCrossrefPubMedGoogle Scholar

  • 46.

    Masih SP, Plumptre L, Ly A, Berger H, Lausman AY, Croxford R, et al. Pregnant Canadian women achieve recommended intakes of one-carbon nutrients through prenatal supplementation but the supplement composition, including choline, requires reconsideration. J Nutr 2015;145:1824–34.Web of ScienceCrossrefPubMedGoogle Scholar

  • 47.

    Fayyaz F, Wang F, Jacobs RL, O’Connor DL, Bell RC, Field CJ. Folate, vitamin B12, and vitamin B6 status of a group of high socioeconomic status women in the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort. Appl Physiol Nutr Metab 2014;39:1402–8.CrossrefPubMedGoogle Scholar

  • 48.

    Henny J, Vassault A, Boursier G, Vukasovic I, Mesko Brguljan P, Lohmander M, et al. Recommendation for the review of biological reference intervals in medical laboratories. Clin Chem Lab Med 2016;54:1893–900.Web of SciencePubMedGoogle Scholar

  • 49.

    Lindenbaum J, Rosenberg IH, Wilson PW, Stabler SP. Prevalence of cobalamin deficiency in the Framingham elderly population. Am J Clin Nutr 1994;60:2–11.CrossrefPubMedGoogle Scholar

  • 50.

    Molloy AM, Pangilinan F, Mills JL, Shane B, O’Neill MB, McGaughey DM, et al. A common polymorphism in HIBCH influences methylmalonic acid concentrations in blood independently of cobalamin. Am J Hum Genet 2016;98:869–82.PubMedCrossrefWeb of ScienceGoogle Scholar

About the article

aTheresa H. Schroder and Amy Tan shared first authorship.

Received: 2018-12-17

Accepted: 2019-03-05

Published Online: 2019-05-14

Author contributions: THS and YL designed the study and led the sample analysis; AT led the data retrieval from the Perinatal Data Registry; AT and YL led the data analysis; AM, GS and HDV provided input on study design, execution and data interpretation. SIB contributed to data analysis and interpretation. THS wrote the first draft of the manuscript; AT and YL are main contributors to the manuscript writing; and AM, GS, SIB and HDV provided critical evaluation and input into the manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: This study was supported by the Canadian Institutes for Health Research/Canada Research Chair Program, and a grant of no charge materials from Abbott Laboratories.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Disclaimer: All inferences, opinions, and conclusions drawn in this publication are those of the authors, and do not reflect the opinions or policies of Perinatal Services BC.

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), 20181337, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2018-1337.

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