Inhibition of hydroxyl radical formation by human tears

The effect of human tears on oxygen radical formation was investigated using xanthine-xanthine oxidase as the oxygen radical generating system. Superoxide (O2.-) and hydrogen peroxide (H2O2) were measured using ferricytochrome c as indicator. OH. formation was monitored by measuring the hydroxylation of salicylate. Addition of traces of iron (Fe3+) and chelator (EDTA) was a prerequisite for OH. formation in this system. Human tears did not detectably affect O2.- or H2O2 formation but markedly inhibited OH. formation. Tears obtained from eight different individuals all showed a marked inhibitory effect on OH. formation, whereby only a small individual variation was observed. During separation of human tears by gelfiltration on a Sephadex G75 column, three protein peaks eluted from the column. The first contained lactoferrin, the second as yet unidentified material, and the third lysozyme. Inhibitory activity on OH. formation coincided with the first protein peak and also with fractions eluting after the protein peak containing lysozyme. The major inhibition on OH. formation was seen in these latter fractions, which contain small organic and anorganic substances. The fact that ascorbic acid could not be detected in human tears and that it did not affect formation of OH. in this investigation's assay system indicates that this compound was not involved in the observed low molecular weight inhibitory effect. Analysis of various cations suggested that the low molecular weight inhibitory effect could largely be ascribed to tear calcium. Tear calcium binds to EDTA and thus possibly prevents formation of the essential catalytic iron-EDTA complex. Experiments using purified human milk lactoferrin showed, that this protein, which is abundantly present in human tears, can inhibit OH. formation in the model used here. The inhibitory effect of lactoferrin was counteracted by increasing the iron concentration in the reaction mixtures. These findings suggest that tear lactoferrin may play an important role in the protection of the ocular surface against OH. induced damage.