Picolinic acid as a zinc-binding ligand in human milk: an unconvincing case.

In the paper entitled "Characterization and Quantitation of a Zinc-Binding Ligand in Human Milk," Evans and Johnson (4) claim to have identified as picolinic acid a low-molecular-weight ligand binding zinc in human milk. This claim is in conflict with our report identifying this ligand as citrate (13). We now present some experimental data which we hope will clarify the issue. We also question some of the techniques, as well conclusions, described in the paper by Evans and Johnson. To evaluate the possible presence of piwlinic acid in human milk, we subjected to gel filtration samples of (1) human milk, (2) picolinic acid plus zinc, and (3) human milk plus picolinic acid plus zinc. A column (1.6 x 90 cm) was packed with Sephadex G15 (Pharmacia Fine Chemicals, Piscataway, NJ) that had been treated with NaBH, to remove surface charges originally present on the gel matrix (1 1). Under these conditions, recovery of zinc from the column was 95 to 100%; in contrast, when untreated gels are used, recovery of zinc is variable and often lower than 60% (3, 12). The column was equilibrated with 0.1 M ammonium acetate buffer (pH 6.5) at a flow rate of 60 ml/hr. The fractions were analyzed for zinc by flame atomic absorption spectrophotometry (Perkin-Elmer 370) and for aromatic absorption at 280 nm (Varian, Cary 2 19). As shown in Figure IA, zinc in human milk eluted in two peaks. The first peak consisted of high-molecular-weight zinc-binding protein@), and the second was a low-molecular-weight peak coeluting with citrate. This was demonstrated by a specific enzymatic assay for citrate (2), confirming our previous report (13). Figure I B shows the elution behavior of picolinic acid plus zinc I300 pM picolinic acid + 0.3 pg/ml of zinc in the form of Zn (NOR)2]. It is clear that picolinic acid coelutes with zinc, but at a position considerably different from that of the low-molecularweight zinc complex of human milk seen in Figure IA. No such zinc peak was observed in the elution pattern of human milk. It can also be noted that this peak is positioned after the total volume of the column. This is in accordance with the elution behavior of aromatic compounds on Sephadex gels (8); such compounds are retarded because of hydrophobic interaction and thus elute after the total volume of the column. When picolinic acid plus zinc (in the same concentrations as above) was added to human milk and this combination was subjected to gel filtration, three zinc-binding peaks were observed (Figure 1C). The first two peaks were the same as those shown in Figure 1A for human milk; the third peak appeared in the same elution position as that shown for picolinic acid plus zinc in Figure 1B. Therefore, it is evident that if there were a zinc picolinate complex in human milk, this complex would elute after the total volume of the column. Our results demonstrate that zinc picolinate cannot be a significant naturally occurring zinc complex in human milk. Furthermore, examination of Figure 1 in Evans and Johnson's paper shows that their low-molecular-weight zinc complex eluted before the total volume of the wlumn. This result is, in fact, in accordance with the elution pattern we observed for zinc citrate.