Contribution to Zn-speciation in human breast milk: Fractionation of organic compounds by HPLC and subsequent Zn-determination by DCP-AES.

Human milk was collected between the 2nd and 7th day after delivery from different women, pooled and separated into fat, proteins and low molecular weight (LMW) substances by centrifugation. The fatty share was rejected, the remaining two fractions were further separated by size exclusion chromatography (SEC) as described in (1), and analyzed for zinc (Zn). The HPLC-method was checked for stability of organo-metal complexes. Only bi-distilled water served as mobile phase during HPLC in order to maintain the Zn/organic molecule-complex intact and to avoid unnecessary contamination sources. Among milk proteins, zinc was associated with casein, albumin, lactoferrin, and metallothionein, whereas among LMW substances a zinc peak could be observed exclusively with citrate. The identity of citrate and proteins was verified with comparable HPLC runs of standard solutions, by citrate-specific examination of HPLC fractions and by isoelectric focusing (IEF) of collected HPLC fractions. Furthermore, native human milk, as well as fractions of proteins and LMW substances (with and without HPLC separation), were quantified with regard to total content of zinc, protein and citrate. No loss of substances was found. In human milk, zinc is primarily bound to citrate (~ 3200 μg/L of Zn in pooled human milk = 95% ), and only about 5% of the total amount of zinc is attached to proteins (~ 150 μg/L of Zn in pooled human milk). Determination of citrate content in human milk used in this study yielded values approximately twice as high as data cited in the literature (325-655 mg/L compared with 95 -270 mg/L, (2,3)). Total Zn content and total protein content however, only differed negligibly from results gained by other authors (~ 3400 μg/L of Zn compared with 1500-7500 μg/L of Zn (2,4,5,6) and ~ 10 g/L of protein compared with 8-12 g/L protein (7,8,9,10))