A-356 Development of postpartum-specific reference intervals for serum vitamin B12 and holotranscobalamin for reliable assessment of vitamin B12 status

Abstract Background Maternal vitamin B12 levels fluctuate during pregnancy and postpartum due to dynamic changes in holotranscobalamin and holohaptocorrin driven by physiochemical shifts. Total B-12 does not reliably indicate active B-12 adequacy for cellular metabolism. Additionally, postpartum holoTC variations with total B-12 necessitate further exploration, as recent research questions Herbert*s B-12 deficiency model. Current total B-12 and holoTC cutoffs for the non-pregnant population may not be applicable to postpartum women, as breastfeeding, nutrition, and hormonal factors remain distinct. This could lead to misclassifying their B-12 status as insufficient or deficient. Therefore, the aim of this study is to evaluate and establish specific reference intervals for total B-12 and holoTC in postpartum women, as no previous studies, to the best of our knowledge, have explored this possibility. Methods This study included 427 women at 6 weeks postpartum who had a normal vaginal delivery with no postpartum feto-maternal complications. Females with a history of hepatic/renal disease, a strict vegetarian diet, or B12 supplementation were excluded. Serum total vitamin B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), homocysteine (HCY), and 5-methyl tetrahydrofolate (5-methylTHF) were measured using commercially available ELISA kits. Reference intervals for serum total B-12 and holoTC concentrations were determined in accordance with the CLSI EP28-A3c guidelines. Medcalc version 23.1.6 was used to calculate the central 95% reference limits by parametric approach for total B-12 reference values after log transformation. A non-parametric approach was applied to estimate the reference limits for holoTC reference values. Results Using the existing total B-12 cutoffs for the non-pregnant population (<148 pmol/L = deficient; <194 pmol/L = insufficient), 23 of 427 postpartum women (5.4%) were classified as deficient, while 91 (21.3%) had insufficient B12 levels. Women with insufficient total B12 levels exhibited significantly higher holoTC levels than those with sufficient B12 levels (p < 0.05). There was no significant difference in the levels of 5-methylTHF, homocysteine and methylmalonic acid between the two groups. Based on the CLSI EP28-A3c guidelines, the central 95% reference interval for total B12 was determined to be 133 – 428pmol/L (90% CI for Lower Reference Limit: 127 – 138 pmol/L; 90% CI for Upper Reference Limit: 411 – 446 pmol/L). The reference interval for holoTC was found to be 24.5 – 158.4pmol/L (90% CI for Lower Reference Limit: 18.4 – 29.4 pmol/L; 90% CI for Upper Reference Limit: 151.0 – 172.8 pmol/L). With the new reference interval, 11 out of 427 postpartum women (2.6%) were deficient in total B-12 levels. Conclusion The existing total B-12 cutoffs falsely overestimate deficiency and insufficiency in postpartum women, as evidenced by normal levels of 5-methylTHF, homocysteine, and MMA. The elevated holoTC levels in women with insufficient total B-12 suggest that physiochemical changes during pregnancy and postpartum may have influenced holoTC dynamics to sustain cellular metabolism. This study led to the development of novel postpartum-specific reference intervals for total B-12 and holoTC, resulting in a significantly lower number of women classified as deficient. Future research should validate these postpartum-specific reference limits and determine a threshold for subclinical B12 deficiency.