The photodynamic action of riboflavin is generally considered to involve the generation of reactive oxygen species, whose production is enhanced when Cu(II) is present in the reaction. In the present study we report that photoactivated riboflavin causes K+ loss from fresh human red blood cells (RBC) in a time dependent manner. Addition of Cu(II) further enhances the K+ loss and also leads to significant hemolysis. Riboflavin in a 2:1 stoichiometry with Cu(II) leads to maximum K+ loss and up to 45% hemolysis. Bathocuproine, a specific Cu(I)-sequestering agent, when present in the reaction, inhibits the hemolysis completely. Free radical scavengers like superoxide dismutase, potassium iodide and mannitol inhibited the hemolysis up to 55% or more. However, thiourea was the most effective scavenger showing 90% inhibition. These results suggest that K+ leakage and hemolysis of human RBC are basically free radical mediated reactions.
The effect of aminophylline on human red blood cells (RBC) has been studied. Under in vitro condition, aminophylline alone does not hemolyse RBC. However, in the presence of riboflavin and visible light, aminophylline causes hemolysis of RBC. This hemolysis depends on the concentration of both riboflavin and aminophylline. Using different free radical scavengers we show that RBC hemolysis is caused by reactive oxygen species. Studies using bovine serum albumin show that riboflavin-aminophylline combination can also cause protein degradation in vitro.
Photoactivated riboflavin in the presence of Cu(II) generates reactive oxygen species (ROS) which can hemolyze human red blood cells (RBC). In the present work we examined the effect of sodium azide (NaN3) on RBC in the presence of riboflavin and Cu(II). The addition of NaN3 to the riboflavin-Cu(II) system enhanced K+ loss and hemolysis. The extent of K+ loss and hemolysis were time and concentration dependent. Bathocuproine, a Cu(I)-sequestering agent, inhibited the hemolysis completely. Among various free radical scavengers used to identify the major ROS involved in the reaction, thiourea was found to be the most effective scavenger. Thiourea caused almost 85%inhibition of hemolysis suggesting that ·OH is the major ROS involved in the reaction. Using spectral studies and other observations, we propose that when NaN3 is added to the riboflavin-Cu(II) system, it inhibits the photodegradation of riboflavin resulting in increased ·OH generation. Also, the possibility of azide radical formation and its involvement in the reaction could not be ruled out.