PHOTODYNAMIC RELEASE OF PROTOPORPHYRIN FROM INTACT ERYTHROCYTES IN ERYTHROPOIETIC PROTOPORPHYRIA: THE EFFECT OF SMALL REPETITIVE LIGHT DOSES

Abstract— Erythrocytes from patients with erythropoietic protoporphyria contain large amounts of protoporphyrin bound to (hemo)globin. Irradiation of these cells causes a shift in fluorescence emission maximum and a decreased fluorescence intensity which is consistent with transfer of protoporphyrin from (hemo)globin to the cell membrane. When the erythrocytes were irradiated intermittently, nearly 70% of the protoporphyrin was released and the hemolysis was less than 3%. Giving the total light dose as a single pulse, resulted in 84% protoporphyrin release and 16% hemolysis.
In vivo the erythrocytes obtain small, repetitive light doses when circulating in the dermal capillaries. We suggest the possibility that in patients with erythropoietic protoporphyria these small light pulses could be sufficient to photodamage the binding place of protoporphyrin on (hemo)globin. In the dark, protoporphyrin can then move from (hemo)globin through the cell membrane and bind to albumin in the serum. Our findings indicate that if protoporphyrin is not present in the cell membrane during irradiation, no photohemolysis will occur. This may explain why patients with erythropoietic protoporphyria have no abnormal hemolysis. The effect of intermittent light pulses may also contribute to the understanding of the protoporphyrin release from erythrocytes in patients with erythropoietic protoporphyria.     

HEMOGLOBIN OXYGEN AFFINITY IS INCREASED IN ERYTHROPOIETIC PROTOPORPHYRIA

Abstract— Whole blood and hemolysates from seven normal and three erythropoietic protoporphyria patients were compared in terms of their hemoglobin function. The oxygen affinity (P₅₀) of the erythropoietic protoporphyria hemolysates compared to normals (13.1 ± 0.2 vs 17.5 ± 0.3 mmHg; P < 0.001) and erythropoietic protoporphyria erythrocytes compared to normals (23.4 ± 0.6 vs 27.1 ± 0.5 mmHg; P < 0.001) were increased while oxygen-binding cooperativity (n-value of the Hill equation) were similar. We conclude that hemoglobin function in erythropoietic protoporphyria patients is altered, but without pathophysiologic consequences. Because hemoglobin in which protoporphyrin IX substitutes for heme has a low oxygen affinity, our findings of a higher than normal affinity in erythropoietic protoporphyria red cells and hemolysates may indirectly support the findings by others that protoporphyrin IX binds to hemoglobin at non-heme-binding sites. In addition, based on the effect of other abnormal hemoglobins, this shift in P₅₀ will decrease any tendency for anemia in erythropoietic protoporphyria patients.     

Mechanisms of photosensitivity in porphyric patients with special emphasis on erythropoietic protoporphyria

In erythropoietic protoporphyria, protoporphyrin overproduction occurs mainly in erythroid tissue. Protoporphyrin can be released from erythrocytes in the dark, but the release is greatly increased if the erythrocytes are exposed to small amounts of light. Protoporphyrin can be bound in plasma either to albumin or to low density or high density lipoprotein. The cutaneous symptoms in erythropoietic protoporphyria are primarily elicited by protoporphyrin-sensitized photodamage of endothelial cells due to the presence of protoporphyrin in lipid structures. Which structures are damaged first in endothelial cells is unknown. Endothelial cells probably accumulate protoporphyrin from albumin or lipoproteins present in the plasma. A direct transfer from the erythrocyte membrane to the endothelial cell membrane can also occur. The transfer processes are probably facilitated by light exposure.

Degranulation of mast cells, invasion of neutrophus into interstitial tissue and complement activation seem to be of less importance than endothelial cell injury in the pathogenesis of erythropoietic protoporphyria. These processes may, however, participate in the final expression of the cutaneous symptoms.

Uroporphyrin and coproporphyrin are hydrophilic and are probably unbound in plasma, although weak binding to plasma proteins cannot be excluded. In the hepatic porphyrias and in erythropoietic porphyria, the clinical symptoms are probably evoked by uroporphyrin and coproporphyrin present in the interstitial tissue. Very little is known about the primary targets of uroporphyrin and coproporphyrin photodamage in these disorders, but photodamage to intercellular structures probably represents the initial event. Activation of complement may contribute to the final expression of the cutaneous symptoms.     

ERYTHROPOIETIC PROTOPORPHYRIA: PHOTODYNAMIC TRANSFER OF PROTOPORPHYRIN FROM INTACT ERYTHROCYTES TO OTHER CELLS

Erythrocytes in patients with erythropoietic protoporphyria (EPP) contain large amounts of protoporphyrin and are regarded as the main source of protoporphyrin in this disease. Cells in the skin of EPP patients accumulate protoporphyrin released from the erythrocytes and upon sun exposure endothelial cells are photodamaged. In the present study a light-induced transfer of protoporphyrin directly from EPP erythrocytes to cultured cells is demonstrated. Erythrocytes were layered upon cultured cells and irradiated. The nearness of erythrocyte and cultured cell membranes potentiated the transfer of protoporphyrin between these cells. This transfer was rapid and preceded the release of protoporphyrin to proteins in the medium. Further irradiation of the protoporphyrin-enriched cultured cells, after removal of the erythrocytes, caused severe photodamage to the cells and survival was dependent on both the amount of protoporphyrin transferred and on the light fluence. Clinical observations and the results of this study indicate that light energy may be involved in two steps in the pathophysiology of EPP: (A) light-induced release of protoporphyrin from erythrocytes to endothelial cells and (B) photodynamic damage to protoporphyrin-enriched endothelial cells.     

Light-induced redistribution and photobleaching of protoporphyrin in erythrocytes from patients with erythropoietic protoporphyria: an explanation of the rapid fading of fluorocytes

Erythrocytes from patients with erythropoietic protoporphyria (EPP) contain large amounts of protoporphyrin. By fluorescence microscopy it has been found that the erythrocytes show red fluorescence which fades very rapidly. During irradiation of erythrocytes from patients with EPP, a decrease in the fluorescence intensity and a red shift in the fluorescence emission maximum is observed. Since the human eye has a diminishing sensitivity in the red part of the spectrum, the red shift will augment the fluorescence decrease observed in the microscope. The decrease in fluorescence intensity is greater than what would be expected by photobleaching of protoporphyrin alone. We suggest that the rapid fading of fluorocytes observed in a fluorescence microscope can be explained both by photoinduced detachment of protoporphyrin from hemoglobin followed by a redistribution to the erythrocyte membrane and by protoporphyrin photobleaching.     

PROTOPORPHYRIN-INDUCED PHOTODAMAGE: STUDIES USING CULTURED SKIN FIBROBLASTS

A new system is described for the study of protoporphyrin-induced photodamage of cells. This system differs from those previously described in that fibroblasts are induced to synthesize protoporphyrin from its precursor δ-aminolevulinic acid.
Fibroblasts were cultured from skin biopsies of 6 normal individuals and 6 patients with protoporphyria. All cell lines in both groups accumulated protoporphyrin when incubated with δ-aminolevulinic acid in the absence of iron. Irradiation for 2 min with long-wave UV light caused death of cells which had accumulated protoporphyrin, but not of cells which had been incubated without δ-aminolevulinic acid. Cell damage could be quantitatively assessed by the release of chromium-51 into the medium.
Examination of irradiated protoporphyrin-rich fibroblasts by electron microscopy revealed no significant differences between lines from patients with protoporphyria and normal individuals. The earliest indications of photodamage were rarifaction of the mitochondrial matrix with dilatation of cristae. dilatation of endoplasmic reticulum, and loss of plasma membrance integrity. Condensation of the cells correlated with cell death. These structural alterations suggest a generalized cellular injury.     

Transfer of protoporphyrin IX between cells

Human adenocarcinoma cells of the line WiDr and human leukemia T cells of the line Jurkat were incubated with 5-aminolevulinic acid and found to produce protoporphyrin IX (PpIX). They were able to transfer a fraction of the sensitizer to neighboring control cells. The transfer took place through direct membrane contact. Light exposures, inactivating about 20% of the sensitized cells, did not result in any acceleration of the transfer of PpIX. This is in contrast to what has been reported for PpIX in erythrocytes from patients with erythropoietic protoporphyria. In these cells light exposure transfers PpIX from the binding sites on hemoglobin to the plasma membrane and further to neighboring cells. The lack of light-induced transfer in the WiDr and Jurkat cells may be related to the binding sites of PpIX, supposedly membrane lipids and proteins embedded therein. Light exposure slightly increased the rate of loss of PpIX from WiDr cells.     

Isolation of Hemoglobin from Bovine Erythrocytes by Controlled Hemolysis in the Membrane Bioreactor

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2–7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.     

Antioxidant inhibition of porphyrin-induced cellular phototoxicity.

Porphyrins such as protoporphyrin IX (PP IX) and uroporphyrin I (UP I) can be phototoxic to human cells. To study the protective ability of antioxidants (beta-carotene, lycopene, ascorbic acid and alpha-tocopherol), against such porphyrin phototoxicity, membrane destruction experiments (Jurkat cells) and human cell cultures (fibroblasts) were performed. Both beta-carotene and lycopene and also the combination of beta-carotene, ascorbic acid and alpha-tocopherol offered cell protection against PP IX phototoxicity. Investigations of both cell membrane protection and of cell growth showed differences in terms of the protection afforded by the anti-oxidants. Thus, for PP IX, carotenoids alone, and in combination with ascorbic acid and alpha-tocopherol, showed higher protection factors in general than UP I. However, for membrane protection there was significant protection against UP I by the combination of beta-carotene, ascorbic acid and alpha-tocopherol but not by any of these anti-oxidants alone. The membrane protection against PP IX by beta-carotene, and especially lycopene, is significant presumably because of the high lipophilicity of all these molecules. However, the hydrophilic UP I will cause phototoxicity mainly via H(2)O(2), radical or singlet oxygen production in the aqueous phase, and these reactive species may be generated some distance from the cell membrane. This may lead to the little or no protection observed for UP I by the individual antioxidants. Nevertheless, a combination of beta-carotene, ascorbic acid and alpha-tocopherol offers membrane protection against the phototoxicity of both porphyrins. This is believed to occur as a result of synergistic processes. Our results suggest that the treatment of porphyria cutanea tarda and erythropoietic protoporphyria may be improved by the use of a combination of the antioxidants studied.     

INHIBITION OF PHTHALOCYANINE-SENSITIZED PHOTOHEMOLYSIS OF HUMAN ERYTHROCYTES BY QUERCETIN

Abstract— Photohemolysis of erythrocytes in the presence of aluminum phthalocyanine tetrasulfonate as a sensitizer is inhibited by quercetin. D₂O (98.5%) stimulated photohemolysis regardless of quercetin presence, suggesting the participation of singlet oxygen in the process. Since it has been shown that this flavonoid reacts with singlet oxygen, the protective effect might be attributed, at least partially, to its competitive reaction with singlet oxygen. At the molecular level, the alterations of membrane proteins that escort the process of photohemolysis, such as cross-linking of spectrin monomers and of other membrane proteins, were selectively inhibited by quercetin. This effect was qualitatively similar to that induced by NaF, suggesting that quercetin may, like NaF, also inhibit type I photooxidations, which contribute to hemolysis. The lipophilicity of quercetin seems to be an essential factor in the inhibition process; rutin, a water-soluble 3-rutinoside of quercetin, had only a negligible protective effect on photohemolysis.     

A NEW HYPOTHESIS FOR THE TARGET IN PHOTOHEMOLYSIS: DIMERS OF THE BAND 3 PROTEIN

Abstract— Photohemolysis has been investigated extensively during the past 20 years, but the membrane target has eluded identification. It is proposed here that the target is the dimer of the band 3 anion exchange protein. This new hypothesis is based on three observations from the literature: (1) the kinetics of photohemolysis sensitized by exogenous sensitizers or induced by UV light obey a power dependence with light dose, with an exponent near two. From target theory this implies a two-component target. (2) Band 3 molecules exist in the erythrocyte membrane as dimers. (3) Binding of eosin to band 3 via an isothiocyano group markedly increases its potency to sensitize hemolysis. Experiments with eosin isothiocyanate bound to band 3 show that lytic rate varies as the 1.45 power of light dose, and that the bound sensitizer bleaches considerably, thereby blunting the effectiveness of higher light doses.     

STUDY OF THE EFFECTS OF ULTRAVIOLET LIGHT ON BIOMEMBRANES—IV. THE EFFECT OF OXYGEN ON UV-INDUCED HEMOLYSIS AND LIPID PHOTOPEROXIDATION IN RAT ERYTHROCYTES AND LIPOSOMES

Abstract— Hemolysis induced by irradiation with ultraviolet (UV) light at 254 nm showed a pronounced oxygen effect: under irradiation in vacuum, the rate of hemolysis was decreased by an order of magnitude. Irradiation at 254 nm in air but not under vacuum caused the peroxidation of erythrocyte membrane lipids. These results suggest that membrane lipid photoperoxidation is one of the causative factors of UV hemolysis. Irradiation at different wavelengths showed that UV-induced lipid photoperoxidation in erythrocyte membranes developed while the antioxidant α-tocopherol was directly photooxidized. It is shown that the process of lipid photolysis in erythrocyte membranes involves sensitization, possibly by protoporphyrin, whose presence in liposomes accelerates the photoperoxidation at 254 and 365 nm of unsaturated fatty acid residues in lecithin. Possible mechanisms of photochemical damage to erythrocyte membranes are discussed.     

CROSS-LINKING OF MEMBRANE PROTEINS AND PROTOPORPHYRIN-SENSITIZED PHOTOHEMOLYSIS*

Abstract— Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).
Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3ß-hydroxy-5α-hydroperoxy-△⁶-cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.     

Saccharomyces cerevisiae Mutants Defective in Heme Biosynthesis as a Tool for Studying the Mechanism of Phototoxicity of Porphyrins

Abstract— Mutants of Saccharomyces cerevisiae accumulating uroporphyrin (UP) or protoporphyrin (PP) were used as a model for the in vivo phototoxic effect of porphyrins observed in the human skin photosensitivity associated with porphyrias (porphyria cutanea tarda and erythropoietic protoporphyria). We have found that UP is localized in vacuoles and PP is present in all compartments except vacuoles in yeast cells. Endogenous PP is much more effective as a photosensitizer of yeast cells than UP. Protoporphyrin action is strictly dependent on the presence of oxygen. In contrast, UP displays a phototoxic effect even if oxygen is not present in the suspension, implicating a free radical mechanism that operates in anaero-biosis upon photosensitization by UP. Catalase or superoxide dismutase deficiency affects photosensitization by UP. A possible mechanism of UP photosensitizing activity is discussed.     

AMIODARONE PHOTOTOXICITY TO HUMAN ERYTHROCYTES AND LYMPHOCYTES

Amiodarone (AD) therapy for cardiac arrhythmia frequently leads to cutaneous phototoxicity. Amiodarone and its metabolite, desethylamiodarone (DEA), photosensitized hemolysis of red blood cells (RBC) and were phototoxic to lymphocytes. Hemolysis photosensitized by 3.3 μ M AD was partially oxygen dependent and was partially quenched 5 m M sodium azide, 50 m M mannitol, superoxide dismutase (251 U/me e ) and catalase (1500 U/m e ), but was unaffected when H₂O was replaced by D₂O. These results suggest that membrane damage may be important in the in vivo phototoxicity to AD, that both oxygen dependent and oxygen independent mechanisms may operate, and that active oxygen species such as O₂ and hydrogen peroxide may be involved. Photohemolysis was more rapid in the presence of DEA than of AD. However, this may be due to the greater fragility of the cell membrane in the presence of DEA. The greater phototoxicity of DEA than AD towards lymphocytes was not due to greater membrane fragility.     

LONG-WAVELENGTH UVA RADIATION INDUCES OXIDATIVE STRESS,CYTOSKELETAL DAMAGE and HEMOLYSIS*

Abstract— We investigated the ability of the different wavelength regions of UV radiation, UVA(320–400 nm), UVB(290–320 nm) and UVC(200–290 nm), to induce hemolysis. Sheep erythrocytes were exposed to radiation from either a UVA1 (>340 nm) sunlamp, a UVB sunlamp, or a UVC germicidal lamp. The doses used for the three wavelength regions were approximately equilethal to the survival of L5178Y murine lymphoma cells. Following exposure, negligible hemolysis was observed in the UVB- and UVC-irradiated erythrocytes, whereas a decrease in the relative cell number (RCN), indicative of hemolysis, was observed in the UVA 1-exposed samples. The decrease in RCN was dependent on dose(0–1625 kj/m²), time(0–78 h postirradiation) and cell density (10⁶-10⁷ cells/mL). Hemolysis decreased with increasing concentration of glutathione, hemoglobin or cell number, while the presence of pyruvate drastically enhanced it. Because scanning spectroscopy(200–700 nm) showed that hemoproteins and nicotinamide adenine dinucleotides were oxidized, cytoplasmic oxidative stress was implicated in the lytic mechanism. Further evidence of oxidation was obtained from electron micrographs, which revealed the formation of Heinz bodies near the plasma membrane. The data demonstrate that exposure of erythrocytes to UVA1, but not UVB or UVC, radiation causes oxidation of cytoplasmic components, which results in cytoskeletal damage and hemolysis.     

Photohemolysis Sensitized by the Furocoumarin Derivative Alloimperatorin and its Hydroperoxide Photooxidation Product

The dark and photosensitized effects of alloimperatorin methyl ether 1 (hereafter simply alloimperatorin) and its photooxygenation product alloimperatorin hydroperoxide 2 were investigated on human erythrocytes. The results reveal that the furocoumarin 1 photosensitizes efficiently the hemolysis of erythrocytes. The rate of photohemolysis increases on raising the temperature of the postirradiated incubation from 4°C to 37°C. Thermal activation of the photohemolysis and inhibition by 2,6‐di‐tert‐butyl‐p‐cresol (BHT) suggest that the furocoumarin 1 photosensitizes lipid peroxidation, increasing permeability in the erythrocyte membrane. The hydroperoxide 2 induces dark and photosensitized hemolysis more efficiently than the furocoumarin 1. The rate of hemolysis induced by 2 increases with the incubation temperature and decreases in the presence of tert‐butanol and BHT. The hydroperoxide 2 photosensitizes the formation of lipid peroxidation products as shown by the reaction with thiobarbituric acid. This process is diminished by BHT. Our data imply that the photohemolysis sensitized by the furocoumarin 1 is caused by the in situ‐formed photooxygenation product 2. Such hydroperoxides are potent hemolytic agents in the dark and especially on photosensitization.     

Red cell zinc protoporphyrin and protoporphyrin by HPLC with fluorescence detection

A high performance liquid chromatographic (HPLC) method was developed for the determination of zinc protoporphyrin (ZnPP) and protoporphyrin (PP) in whole blood. After adding the blood to dilute acetic acid, ZnPP and PP were extracted with dimethyl sulfoxide-acetone containing mesoporphyrin as internal standard. Following evaporation of the acetone, the haemin-free extract was analysed by HPLC. ZnPP and PP were separated on a reversed-phase column and quantitated by measuring fluorescence peak areas. The extraction method is simple, and applicable to batch analysis, and the HPLC separation is rapid and repoducible. The coefficient of variation for ZnPP was 5.6% and 3.3% for total red cell porphyrin levels of 3.5 and 10.2 mumol per litre RBC respectively. Results are discussed in patients with erythrohepatic protoporphyria, lead exposure, iron-deficiency and nonspecifically elevated total red cell porphyrins.     

THE EFFECT OF PROTOPORPHYRIN ON HISTAMINE SECRETION BY RAT PERITONEAL MAST CELLS: A DUAL PHOTOTOXIC REACTION

Abstract— Protoporphyrin-induced phototoxicity in rat peritoneal mast cells was manifested either by inhibition of 48/80-stimulated histamine secretion or by cell lysis. At a protoporphyrin concentration of 100ng/m/ (0.17 μM), histamine secretion was completely inhibited after 30min illumination. After initiation, the inhibited state progressed in the dark, and was irreversible, however, it did not develop into cell lysis. More severe phototoxic reactions in mast cells could not be produced by increasing the PP concentration or the incubation time; however, cell lysis was evoked by increasing the light intensity between 180–950W/m², using a light source with emission maxima in the 350–470nm region. Dual phototoxic effects could also be demonstrated in erythrocytes by manipulating the illumination conditions. Increased resistance to osmotic lysis was seen under moderate conditions, and decreased resistance and cell lysis were seen under severe conditions. In the absence of protoporphyrin, the effect of light alone on mast cells was similar to protoporphyrin-phototoxicity, although the light intensities required were higher both for inhibition (60–130W/m²) and lysis (280–950W/m²). The data therefore indicate that certain cell functions can be specifically disrupted by phototoxic reactions that are not cytotoxic; however, phototoxic reactions that lead to severe membrane protein denaturation and cell lysis also occur. The manifestation of these dual effects depends on the intensity of illumination in the 350–470nm region.     

RECOVERY FROM DAMAGE INDUCED BY ACRIDINE PLUS NEAR-ULTRAVIOLET LIGHT IN ESCHERICHIA COLI

Abstract— Escherichia coli cells treated with sublethal doses of acridine plus near-UV light exhibit an effective split-dose recovery response that requires an incubation period of about 30–45 min. Studies of the metabolic requirements for split-dose recovery revealed the following: (a) DNA synthesis is not required for split-dose recovery; (b) inhibition of electron transport or protein synthesis reduces the efficiency of split-dose recovery by about one-half; (c) inhibition of phospholipid synthesis or cell wall synthesis completely eliminates the split-dose recovery response. These results suggest an involvement of membrane repair mechanisms in response to damage by acridine plus near-UV light. Additional evidence for such a process was provided by more direct assays for membrane recovery. It was found that cells treated with sublethal doses of acridine plus near-UV light are sensitive to low concentrations of detergents, and lose that sensitivity upon incubation. Likewise, treated cells are susceptible to lethal osmotic shock, but can recover from this susceptibility if incubated after treatment but prior to exposure to low osmotic conditions. Based on accumulating evidence, we propose that E. coli cells are capable of repairing membrane damage resulting from exposure to acridine plus near-UV light.