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Title:   Author  item
                                            Hampel, Danielaitem
                                            Shahab-Ferdows, Settiitem
                                            Domek, Josephitem
                                            SIDDIQUA, TOWFIDA - University Of California - Cooperative Extension Serviceitem
                                            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: 12/30/2014
                                    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. DOI: 10.1016/j_foodchem.2013.12.033.Interpretive Summary: Methods used for B12 analysis in human milk include microbiological and competitive protein binding assays developed for serum and plasma. The latter have emerged as the method of choice even though little has been reported concerning validation and adaptability of this method to the human milk matrix. Moreover, some competitive binding luminescence assays fail to accurately determine low B12 values even in serum samples. 
In human milk B12 is bound to its binding protein, haptocorrin (HC), and must be liberated prior to analysis. Recently, Lildballe et al. (2009) showed that large amounts of apo-HC in human milk interfere with the analysis and proposed removal of apo-HC by cbi-sepharose prior to analysis. An additional problem is that the vitamin B12 concentration of milk from women with poor B12 status is very low in the vitamin. 
In this study we examined two competitive protein binding assays for their suitability to analyze vitamin B12 in human milk. IMMULITE®/IMMULITE®1000 Vitamin B12 solid-phase, competitive chemiluminescent enzyme immunoassay can be used for B12 analysis in human milk without the need for additional pre-treatment while SimulTRAC SNB radioassay should not be used at this time. The overall recovery using IMMULITE® was 78.9 ± 9.1% and using diluted milk samples the assay revealed linearity at very low B12 concentrations (24 pM), well below the former analytical cut-off point of 50 pM.  Breast milk samples obtained from vitamin B12 supplemented and unsupplemented Bangladeshi women demonstrate that the method can detect the influence of maternal supplementation on B12 concentrations in milk from populations with poor vitamin B12 status.
                                Technical Abstract: BACKGROUND 
Few accurate data exist on the concentration of vitamin B12 in human milk. Binding of the vitamin to haptocorrin (HC) can interfere with the assay if not removed by pretreatment, and very low values can occur in women with poor B12 status. This study evaluated two competitive enzyme binding immunoassays for analysis of vitamin B12 in human milk.

METHODS
Vitamin B12 in human milk was determined by IMMULITE® and by SimulTRAC-SNB assay. Samples were subjected to cobinamide (cbi)-sepharose pre-treatment to examine possible interferences by apo-HC as previously described (1). Samples analyzed by IMMULITE® were capped prior to the initial heating step after which they were cooled in an ice/water bath for 5 min. Existing samples were analyzed from Bangladesh and California to illustrate the range of milk B12 concentrations. 

RESULTS
The overall recovery of B12 using IMMULITE® was 78.9 ± 9.1 % whereas with the SimulTRAC it was 225 ± 108 % (range 116 to 553 %). With the IMMULITE assay diluted human milk samples revealed linearity at low B12-concentrations (24 – 193 pM) with r2 > 0.985. Milk B12 concentrations from Bangladeshi women were significantly lower compared to women in California (p < 0.0001).

CONCLUSION
The IMMULITE® is the first validated method for analysis of vitamin B12 in human milk matrix without the need for additional pre-treatment of samples to remove HC. SimulTRAC-SNB analysis should not be used. The method can detect low human milk B12 concentrations and effects of maternal supplementation.


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