J. Van Steveninck

Protoporphyrin-induced photohemolysis in protoporphyria and in normal red blood cells

Abstract Irradiation of erythrocytes of patients with erythropoietic protoporphyria with light of about 410 nm wavelength in the presence of oxygen, causes hemolysis. In a nitrogen atmosphere photohemolysis was inhibited completely, indicating that an oxidative step is involved in the process. Several redox reagents inhibited the photohemolytic response. Similar photohemolysis could be observed with normal red cells, after adding protoporphyrin-IX-dimethyl ester to the medium. The hemolytic process itself could be characterized as a colloid osmotic hemolysis.

Photodynamic damage of the erythrocyte membrane caused by protoporphyrin in protoporphyria and in normal red blood cells

Abstract The photodynamic effect of protoporphyrin on red blood cells of patients with erythropoietic protoporphyria and on sensitized normal erythrocytes leads primarily to increased passive cation permeability of the membrane. This increased permeability appeared to be caused by photo-oxidation of a cellular target, localized in the red cell membrane. Both unsaturated fatty acids and sulfhydryl, methionyl, histidyl, tyrosyl and tryptophan residues in membrane proteins appeared to be sensitive to protoporphyrin-induced photo-oxidation. It was shown that the increased cation permeability can not be attributed to peroxidation of unsaturated fatty acids in the membrane. Therefore the observed damage of the permeability barrier is presumably caused by photo-oxidation of amino acid residues in membrane proteins. The experimental results are discussed in relation to data in recent literature, concerning the significance of amino acid residues in the maintenance of functional and structural integrity of cellular membranes.

Photodynamic effects of protoporphyrin on the architecture of erythrocyte membranes in protoporphyria and in normal red blood cells

Protoporphyrin causes a photodynamic damage of the red blood cell membrane. After illumination of red blood cells in the presence of protoporphyrin three effects can be observed: 1. Red blood cell membranes show particle aggregation on the outer and inner fracture face, as seen in freeze-etch electron microscopy. 2. Electrophorograms of membrane proteins show an increasing protein association, not disrupted by sodium dodecyl sulfate. 3. The immunological response of A+ red cells to anti A serum is progressively retarded. It seems likely that these effects are interrelated consquences of the protoporphyrin-induced photodynamic membrane damage.

Metabolic aspects of the photodynamic effect of protoporphyrin in protoporphyria and in normal red blood cells

Abstract The photodynamic action of protoporphyrin on erythrocytes of patients with erythropoietic protoporphyria and on sensitized normal red blood cells leads to K + loss and Na + gain, resulting finally in colloid osmotic hemolysis. To elucidate the pathochemical background of this process, the ATP and glutathione-producing metabolic systems and the activity of other cellular enzymes was studied during irradiation, in the prelytic period. The glucose consumption, intracellular ATP level and catalase activity were not influenced by the photochemical process, whereas the cellular glutathione concentration decreased during irradiation. The activity of cellular acetylcholinesterase and of membrane ATPase decreased in the prelytic period, the (Na + + K + )-dependent ATPase being more sensitive than the Mg 2+ -stimulated ATPase. Measurements of 86 Rb + uptake demonstrated a decreasing active Rb + transport with a concomitant increase of passive Rb + permeability during irradiation. The results indicated that the cellular membrane is presumably the primary target of the protoporphyrin-induced photodynamic process.

Photodynamic effects of protoporphyrin on cholesterol and unsaturated fatty acids in erythrocyte membranes in protoporphyria and in normal red blood cells

Abstract The protoporphyrin-induced photodynamic damage of erythrocytes is caused by photooxidation of a cellular target, localized in the red cell membrane. In model experiments it appeared that some amino acid residues of membrane proteins, unsaturated fatty acid side chains of phospholipids and cholesterol are sensitive to photooxidation, induced by protoporphyrin. In previous studies it has been shown that the membrane damage cannot be attributed to photooxidation of unsaturated fatty acid side chains. Photosensitized peroxidation of cholesterol was studied in model systems, in red cell ghosts and in intact cells. Although cholesterol appeared to be sensitive to protoporphyrin-induced photooxidation in model systems and in red cell ghosts, it could be shown that photohemolysis of red blood cells of patients with erythropoietic protoporphyria cannot be rationalized on the basis of a cholesterol peroxidation.

Photodynamic modification of proteins in human red blood cell membranes, induced by protoporphyrin

Abstract Illumination of erythrocytes or erythrocyte membranes with visible light in the presence of protoporphyrin causes photodynamic damage of the cell membrane. This process is reflected a.o. by a mutilated ultrastructure and changes of the physical properties of the membrane proteins. Illumination in the presence of protoporphyrin causes association of membrane proteins, leading to blurring of the protein bands in electropheretograms, disappearance of bands and the appearance of protein aggregates on top of the gels. The formation of large protein aggregates is also indicated by Sephadex gel filtration of the solubilized membrane proteins. In kinetic studies it appeared that spectrin and the bands 2.1, 2.2, 2.3 and 6 are most susceptible and that band 3 is least susceptible to this cross-linking reaction. Experimental results indicate that this cross-linking is caused by direct photooxidation of membrane proteins. Peroxidation of unsaturated fatty acids is not involved in the process. The significance of this process for studies on membrane structure and on photodynamic membrane damage is discussed.

Binding of Protoporphyrin to Hemoglobin in Red Blood Cells of Patients with Erythropoietic Protoporphyria

Virtually all protoporphyrin in erythrocytes of patients with erythropoietic protoporphyria is bound to hemoglobin. The maximum of the fluorescence excitation spectrum of this protoporphyrin-hemoglobin complex shifted, with increasing concentration, from 405 nm to 389 nm. A similar shift was observed when titrating a solution of free protoporphyrin with hemoglobin. The Soret maximum of free protoporphyrin itself, on the other hand, was not concentration-dependent. These observations indicate that spectrofluorometric measurements do not allow conclusions concerning the mode of protoporphyrin binding to hemoglobin. Experiments on protoporphyrin exchange between the hemoglobins A, F and S reinforced the previously drawn conclusion that protoporphyrin is bound to hemoglobin at the heme-binding sites.

Determination of vitamin A in blood plasma of patients with carotenaemia.

Abstract High plasma carotene levels interfered with a fluorometric vitamin A determination described recently. A modification of the fluorometric procedure is described in which the carotene disturbance is eliminated.

Porphyrin synthesis in blood cells of patients with erythropoietic protoporphyria

The protoporphyrin accumulation observed in the red blood cells of patients with erythropoietic protoporphyria can be explained by decreased activity of the enzyme heme synthetase or by increased production of porphyrins in the affected cells. In literature experimental evidence both for a partial heme synthetase deficiency and for increased porphyrin biosynthesis has been presented. In a group of ten patients with erythropoietic protoporphyria the biosynthesis of porphyrins from delta-aminolevulinic acid and from glycine-succinic acid appeared to be normal in peripheral blood cells. These results are consistent with the partial heme synthetase deficiency previously found to be the basic defect of this disease.

Characterization of protoporphyrin in red blood cells of patients with erythropoietic protoporphyria

It was investigated whether the protoporphyrin that can be extracted from red blood cells of erythropoietic protoporphyria (E.P.P.) patients is present in the cells as free molecules or protein-bound. With isoelectric focusing and with starch gel electrophoresis it could be shown that virtually all protoporphyrin in the erythrocytes is protein-bound. It is very likely that the protoporphyrin is bound to hemoglobin at heme-binding sites. This was indicated by several observations: 1. With isoelectric focusing the protoporphyrin-protein complex is focused at a pH only slightly higher than the isoelectric point of hemoglobin. 2. With chromatography on Sephadex columns it appeared that hemoglobin and the protopotphyrin-protein complex have the same molecular weight. 3. A Heme-protoporphyrin exchange occurred when the heme-globin bond was labialized by conversion to hemiglobin. The resulting protoporphyrin-hemoglobin complex had the same electrophoretic mobility with starch gel electrophoresis as the protoporphyrin-protein complex, extracted from red blood cells of E.P.P. patients.