An unusual photohaemolytic property of riboflavin

Riboflavin (RF) is a known photoreactive and phototoxic molecule. However, unlike other photosensitizers, it does not induce photohaemolysis of erythrocytes by itself. On the other hand, illuminated RF caused haemolysis but in the presence of serum or plasma. The kinetics of photohaemolysis in the presence of serum/plasma has been studied by monitoring the rate of haemolysis spectrophotometrically and morphological changes at erythrocytes membrane by scanning electron microscopy. We found that the extent of mammalian RBC membrane damage was dependent on the concentration of RF or hematoporphyrin (HP) (0-20 microgram/ml) and dose of sunlight (0-20 min). The RBC membrane-damaging potential of illuminated HP was not affected by the presence of plasma in the reaction system. Furthermore, RF showed a protective role against photohaemolysis caused by photoexcited HP if erythrocytes were preincubated with RF in the absence of serum/plasma. For mechanistic studies, biochemical parameters such as acetylcholinesterase activity (AChE) and formation of TBA-reactive substance (TBA-RS) were analysed in RBC and RBC+plasma under a similar set of experimental conditions. We observed about a 25% decrease in AchE activity as a synergistic action of RF or HP (20 microgram/ml) and sunlight (30 min) in both cases (RBC or RBC+plasma). Interestingly, illuminated RF caused about a 125% increase of TBA-RS in a reaction system consisting of RBC+plasma. On the other hand, an increase in the production of TBA-RS by illuminated RF was not observed in the absence of plasma/serum, in the reaction system. These results suggested that photooxidation of RBC membrane lipids by illuminated RF, under the influence of plasma/serum, may be one of the causes of membrane modification leading to RBC lysis.