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In the absence of vitamin E deficiency, red cell lipid peroxidation has not been clearly demonstrated in freshly drawn blood obtained from patients with various hemolytic anemias despite indirect evidence that oxidative decomposition of cell membrane unsaturated fatty acids occurs in these particular hemolytic states. Recent studies have indicated that malonaldehyde, a decomposition product of oxidized polyunsaturated fatty acids, is able to covalently cross-link the amino groups of protein or lipid resulting in a fluorescent compound. In the present study we have utilized spectrofluorescent technique to assess whether such fluorescence is present in red cell lipid extracts in association with lipid peroxidation. In vitro red cell lipid peroxidation produced by ultraviolet radiation or the oxidant gas ozone was associated with the development of a fluorescent peak (excitation maximum 360 nm; emission maximum 440 nm) in lipid-containing red cell extracts Similar fluorescence was observed after incubation of red cells with malonaldehyde or with malonaldehyde-containing extracts of peroxidized red cell lipid. Spectrofluorescent evaluation of chloroform: isopropanol extracts obtained from the freshly drawn red cells of six patients receiving the oxidant hemolytic drug diaminodiphenylsulfone also revealed a peak at 440 nm which ranged from 39 to 78 U. In contrast, the levels in samples obtained from 11 hematologically normal subjects were 17-27 fluorescence U. No evidence for an increase in blood levels of free malomaldehyde was observed using the 2-thiobarbituric acid test which is the most commonly performed assay of lipid peroxidation. Serum vitamin E levels were within the normal range. Density separation indicated that the bulk of the fluorescence was present in older red cells. A similar fluorescent peak was also observed in lipid-containing extracts of red cells obtained from rabbits repeatedly injected with phenylhydrazine. The finding of fluorescent spectra consistent with the cross-linking of aminolid by malonaldehyde in the red cells of patients receiving diaminodiphenylsulfone indicates that in vivo red cell lipid peroxidation does occur in the absence of vitamin E deficiency.

Spectrofluorescent detection of in vivo red cell lipid peroxidation in patients treated with diaminodiphenylsulfone.