Fe-lactoferrin to its receptors. Lactoferrin (50 and 100 nm), similar to ferricyanide, increased the accumulation of lactate (respectively by 25% and 30%). These results support the assumption that ferricyanide and lactoferrin are final acceptors of a common electron transport chain connected with the regulation of glycolysis. We established an antioxidative effect of lactoferrin on erythrocytes, which was expressed as: a) an influence on content and on activity of intracellular antioxidants Ð namely an enhancement of the content of reduced glutathione; b) a decreased content both of products of lipid peroxidation (thiobarbituric acid reactive substances) and hemolysis under normal conditions and oxidative stress. Lactoferrin is capable to bind metal ions and thus to block their catalytic participation in the oxidative disturbances of the membrane. In most of our experiments there were no metal ions in the incubation mixtures (except those stimulating oxidative stress). Our results showed that Lf limited both the generation of thiobarbituric acid reactive substances and hemolysis in the absence of metal ions in the media, as well as in their presence. These facts suggest that probably the antioxidative property of lactoferrin is glycolysis stimulation, leading to increased formation of ATP, which is necessary to maintain the ion gradient, membrane potential and morphology of the erythrocyte.

No abstract

The effect of metal ions at a concentration of 10(-8) to 10(-5) M [using their salts: ZnCl2, CdCl2, LiCl, CuSO4, NiSO4, Al2(SO4)3, (NH4)2MoO4 on the lactoferrin (Lf) binding to the erythrocyte membrane receptors was studied. In the absence of metal ions, Scatchard's analysis showed the existence of two kinds of binding site: one with high affinity and low capacity, and the another with low affinity and high capacity. All these metals, excluding Zn2+ and Cd2+, at a concentration 10(-5) M decreased the affinity of Lf binding (Ka1) to the high-affinity receptors. In the presence of Zn2+ and Cd2+, only the low-affinity binding site was found. Significant inhibition on the affinity (Ka2) of the low-affinity class of receptors showed Zn2+, Al3+, and Mo6+. Depending on their concentration (10(-8)-10(-5) M), these ions enhanced to a different extent, the binding capacity of the both types receptors, but the effect did not correspond to the applied doses. Several explanations of the mechanism for influence of the metal ions on the Lf-receptor interaction is discussed.

The aim of the present study was to assess if some flavonic compounds (quercetin, piceatannol and apigenin) and ascorbic acid could interfere with the Lf stimulatory effect on the erythrocyte function. Quercetin (1.5 μm) and piceatannol (30 μm) showed an additive effect on Lf stimulation of Na+/K+-ATPase when used together with Lf. The enhancement of Lf stimulation on Na+/K+-ATPase in the presence of flavonoids was probably due to their antioxidative properties and/or to their involvement in the erythrocyte signaling. None of the estimated flavonoids showed an effect on Lf stimulation of the lactate production. Quercetin itself enhanced the ATPase activity but did not affect the lactate formation. Apigenin (1.5 μm) enhanced reliably the lactate generation, but it did not exert any effect on the ATPase activity. High concentration of ascorbic acid (60 mm) did not change the Lf stimulatory effect on Na+/K+-ATPase, but decreased the Lf-specific-binding. A significantly strong inhibitory effect on the Lf-specific binding exerted the electron acceptors NAD+ (2 mm) and FAD (2 mm). These effects concern most likely the competition with Lf for electron(s) which is (are) provided from the erythrocyte intercellular electron transport chain(s).

The kinetics of 59 FeTf binding to the neutrophil membrane receptors was evaluated. Results showed that polymorphonuclear leucocytes (PMN) possess membrane receptors not only for lactoferrin, but also for transferrin. Receptor binding experiments showed biphasic patterns with two peaks of saturation: 16% and 30% of the specific binding to the cell membrane receptors. The Scatchards analysis evaluated two types of binding sites: One -with high affinity and low capacity: Кa1 = 9.88 х 10 8 M -1 and 40 300 receptors/cell, (у 1 = 9. 92 -0.182.х, n=2, r= -1, p<0.001), and second -with low affinity and high capacity: Кa 2 = 12.54 х 10 9 M -1 and 129 000 receptors/cell (у 2 = 4. 04 -0.21.х, n =4, r = -0.999, p<0.001). It is probably that the transferrin receptors on the neutrophil membrane have the function to ensure a controlled pathway of Fe 2+ transfer to prevent an iron overload, thus to accomplish an antioxidative protection of the cell membrane.