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Title:   Author  item
                                            HAMPEL, DANIELA - University Of Californiaitem
                                            Shahab-Ferdows, Settiitem
                                            Domek, Josephitem
                                            SIDDIQUA, TOWFIDA - University Of Californiaitem
                                            RAQIB, RUBHANA - International Centre For Diarrhoeal Disease Researchitem
                                            Allen, Lindsay

                                                         - *A*
  Submitted to: Journal of Food Chemistry
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 12/7/2013
                                    Publication  Date: 12/14/2013
                                    Citation: Hampel, D., Shahab-Ferdows, S., Domek, J.M., Siddiqua, T., Raqib, R., Allen, L.H. 2013. Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk. Journal of Food Chemistry. 153:60-65. https://doi.org/10.1016/j.foodchem.2013.12.033.DOI: https://doi.org/10.1016/j.foodchem.2013.12.033                                                              
                                Interpretive Summary: The concentration of vitamin B12 binding protein, haptocorrin, is very high in human milk and in serum of some patients with myeloproliferative disorders. Vitamin B12 in serum and milk is often measured with competitive protein binding assays which require the release of B12 from its binding proteins. Previous work showed that B12 release may be incomplete when haptocorrin is high, resulting in erroneous values for B12 content. In order to obtain accurate measurements of vitamin B12 in human milk, we compared two competitive enzyme-binding immunoassays generally used on serum/plasma (IMMULITE and SimulTRAC-SNB). Recovery of added B12 was 78.9 ± 9.1% with IMMULITE, but 225 ± 108% (range 116-553%) with the SimulTRAC-SNB most likely due to excess haptocorrin. The lack of haptocorrin interference in the IMMULITE assay means that previously reported mandatory removal of haptocorrin is not necessary for measuring vitamin B12 in human milk. Moreover the assay was linear down to low B12 concentrations (24-193 pM). This enabled quantitative measurement of B12 in milk from Bangladeshi women (72-959 pM) which was significantly lower than that in milk of Californian women (154-933 pM, p <0.001), showing the robustness of the IMMULITE assay for measuring breast milk B12 in deficient populations.
                                Technical Abstract: Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past
analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al.,
2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for
serum/plasma (IMMULITE and SimulTRAC-SNB) for B12 analysis in human milk. B12-recovery rates
(United States Environmental Protection Agency, 2007) were determined to be 78.9 ± 9.1% with
IMMULITE and 225 ± 108% (range 116–553%) using SimulTRAC-SNB, most likely due to the presence of
excess HC. HC-interferences were not observed with the IMMULITE assay, rendering previously reported
mandatory HC-removal (Lildballe et al., 2009) unnecessary. Linearity continued at low B12-concentrations
(24–193 pM; r2 > 0.985). Milk B12 concentrations from Bangladeshi women (72–959 pM) were
significantly lower than those from California (154–933 pM; p < 0.0001) showing IMMULITE’s robustness
against the complex milk matrix and its ability to measure low milk B12 concentrations.


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