Characterization and Quantitation of a Zinc-Binding Ligand in Human Milk

Abstract

SummaryA low-molecular-weight zinc-binding ligand from human milk has been isolated and characterized. The ligand was isolated by chromatography on Dowex 50, Dowex 1, and Sephadex G-15 equilibrated with 0.153 mM Zn(N03)2. Mass spectroscopy, thinlayer chromatography, and infrared spectroscopy proved that the zinc-binding ligand isolated by this method from human milk is pyridine-2-carboxylic acid, commonly known as picolinic acid. The concentration of picolinic acid in human milk was 308 pM, the concentration in one brand of processed cow's milk was 20 pM, but picolinic acid was undetectable in a second brand of cow's milk and in four different infant formulas. Weanling rats fed supplemental picolinic acid absorbed significantly more dietary zinc and gained significantly more weight than rats fed an unsupplemented diet. The results suggest that the high bioavailability of zinc in human milk results from the presence of picolinic acid, a bidentate chelating ligand which facilitates zinc absorption from the intestine. SpeculationThe high bioavailability of zinc in human milk results from the presence of picolinic acid, a bidentate chelating ligand which facilitates zinc absorption from the intestine.Several clinical observations demonstrate that the bioavailability of zinc from human milk is much greater than that from cow's milk. In children affected with acrodermatitis enteropathica, an inherited disease that affects zinc metabolism (8), the symptoms characteristic of the disease do not generally appear until the child is weaned from the breast (1, 4, 5). Human milk has also been used successfully in the treatment of acrodermatitis enteropathica (15). Hambidge et al. (9) found that the plasma zinc concentration of breast-fed infants is not significantly different than that of healthy young adults. In contrast, the plasma zinc concentration of infants fed either zinc-supplemented formula or non-zinc-supplemented formula is significantly less than that of young adults. These observations suggest that human milk may contain a ligand or ligands which facilitate zinc absorption.To date, attempts to isolate zinc-binding ligands from biologic fluids have not been successful due to the fact that investigators have consistently used classical gel filtration chromatography. The problems encountered when attempting to detect and identify zinc-chelating ligands by the use of classical gel filtration have been discussed by Evans et al. (6). These authors have also described a technique, referred to as modified gel filtration chromatography, which can be used to reproducibly detect zinc-binding ligands in biologic fluids. This technique has now been adapted to isolate and quantitate a zinc-binding ligand in human milk. MATERIALS AND METHODS DETECTION OF ZINC-BINDING LIGANDSified gel filtration chromatography. A glass column 1.5 x 90 crr was packed with 44.4 g Sephadex G-15 (Pharmacia Fine Chemi. cals, Inc.) (18). The gel was equilibrated with 50 mM Tris acetate pH 7.4, that contained 0.1% NaN3 and 0.153 mM Zn(NO3)z. Thc buffer...

Advanced Dairy Chemistry Volume 3

“…The identity of this low molecular weight zincbinding ligand in human milk has been the subject of much controversy. Evans and Johnson (1980) suggested that picolinic acid is the ligand, but this has been discounted by Rebello et a/. (1983) who found that the concentration of picolinic acid in human milk is insufficient to bind a significant proportion of the zinc and by Hurley and Lonnerdal (1981) who found that picolinic acid does not bind zinc in human milk.…”

Pharmacology of the Skin II

“…Premature infants therefore have a considerable requirement for zinc. Although human milk contains less zinc than cow's milk, some of the zinc is associated with low molecular weight compounds picolinic acid (EVANS and JOHNSON 1980) or citrate, whereas it is entirely associated with high molecular weight compounds in cow's milk (COUSINS and SMITH 1980) from which zinc is less well absorbed by the neonatal gastrointestinal tract. Although human milk contains less zinc than cow's milk, some of the zinc is associated with low molecular weight compounds picolinic acid (EVANS and JOHNSON 1980) or citrate, whereas it is entirely associated with high molecular weight compounds in cow's milk (COUSINS and SMITH 1980) from which zinc is less well absorbed by the neonatal gastrointestinal tract.…”

Zum Einfluß von Pikolinsäure und Zitronensäure auf die intestinale Zink-Absorption in vitro und in vivo

“…Mit diesen Komplexeigenschaften dtirfte in Zusammenhang stehen, dab Krieger [20] die Zn-Dosis zur erfolgreichen Behandlung von Akrodermatitis enteropathica mit Zn-Dipikolinat im Vergleich zu einem Zn-Salz deutlich erniedrigen konnte. Die gleiche Arbeitsgruppe wies mit Hilfe der Isotopenverdiinnungsmethode ebenfalls nach, dab bereits durch die alleinige Zugabe yon 0,2 g Pikolins~iure pro kg Futter zuverschiedenen Rattendiiiten die Zn-Absorption signifikant gesteigert werden kann [5,6,7,9]. Die gleiche Arbeitsgruppe wies mit Hilfe der Isotopenverdiinnungsmethode ebenfalls nach, dab bereits durch die alleinige Zugabe yon 0,2 g Pikolins~iure pro kg Futter zuverschiedenen Rattendiiiten die Zn-Absorption signifikant gesteigert werden kann [5,6,7,9].…”

“…Als Hypothese ffir die Absorption kann angenommen werden, dab durch organische Liganden als ,,niedermolekulare, Zn-bindende Komponenten" die Aufnahme yon Zink aus dem Darmlumen fiber den Bfirstensaum der Mukosazellen erleichtert und/oder der Transport durch die Absorptionszelle und der Durchtritt durch die basolaterale Zellmembran beeinflugt werden. ~ber die Konzentration von Pikolins~iure in einzelnen Organen oder in der Humanmilch liegen erst wenige, sehr unterschiedliche Ergebnisse vor [6,9,27]. Forth und Rummel [11] bezeichnen dies als eine Frage der effektiven Komplexstabilitiit.…”

“…Dies Rihrte zu der Hypothese, dal3 Zink in der Humanmilch in einer absorptionsf/Srdernden Bindungsform vorliegen muB bzw. Demgegenfiber identifizierten Evans und Johnson [6] diesen Liganden als Pikolins~iure und legten mehrere Untersuchungen vor [7,8,9,16], die die Bedeutung yon Pikolins[iure ftir die Absorption und den Transport yon Zink unterstreichen. Nach mehreren Untersuchungen der Arbeitsgruppe um Hurley wurde dieser ,,Zink-bindende niedermolekulare Ligand" [3,4] in der Frauenmilch als Zitronens~iure charakterisiert [22], wiihrend das Zink in der Milch weiterer Species (z.…”