Drug-induced Hemolysis Research Articles

Drug binding to human erythrocytes in the process of ionic drug-induced hemolysis: Flow microcalorimetric approaches

Erythrocyte hemolysis induced by cationic phenothiazine derivatives and anionic non-ster-oidal anti-inflammatory drugs was compared, by flow microcalorimetry, with respect to thermodynamic characteristics for drug binding to intact human erythrocytes. Phenothiazines having high hemolytic activities bound strongly to erythrocyte cells, inducing an immediate hemolytic action characterized by an endothermic heat effect prior to saturating available binding sites. The thermodynamic observable Δ H and ΔS fell within the ranges of −119 to −65.1 kJ/mol and −308 to −128J/mol/K, respectively, for these cationic species. There was a linear relationship between the hemolytic activity and the degree of exothermicity of ΔH which was enhanced significantly by the presence of a halogen atom(s) at the C-2 position of the phenothiazine nucleus in the order of H < Cl < CF 3. Anti-inflammatory drugs, however, bound to quite different sites in the erythrocytes with lower affinities and higher capacities than cationic drugs. The latter was characterized by small negative ΔH (−17.3 to −7.1 kJ/mol) and positive ΔS (10 to 41 J/mol/K). In the calorimetric profiles observed during hemolysis by anionic drugs, two stages were seen: the first, an exothermic process, arising from drug binding to the erythrocytes; the second, an endothermic process, corresponding to the heat of dilution of hemoglobin released from erythrocytes. Hemolysis occurred after the binding sites on the erythrocytes were saturated with drugs. Our data suggest that the binding activities of ionic drugs, such as the amounts of the bound drug and their binding energies to erythrocytes, contribute to the hemolysis.

Drug-induced haemolysis and renal failure in children with glucose-6-phosphate dehydrogenase deficiency in Afghanistan

Twenty children (18 boys and 2 girls) with a proven or presumptive diagnosis of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency developed intravascular haemolysis following administration of antimalarials in 9, chloroquine and chloramphenicol in 1, chloroquine, chloramphenicol and aspirin in 1, chloramphenicol and aspirin in 3, and aspirin alone in 4. Eleven of these children developed acute renal insufficiency. All were managed with supportive care, including blood transfusion, forced diuresis and peritoneal dialysis wherever indicated. Only 16 children recovered completely. The occurrence of G-6-PD deficiency is being reported for the first time in Afghanistan.

Drug-induced red cell dyscrasias

Major advances have been made in recent years in our understanding of the pathogenetic mechanisms of drug-induced blood dyscrasias, particularly those involving the red cell. Among the latter, hemolytic anemia is the most common. Drug-induced red cell destruction may occur on an immune basis or through disruption by the drug of red cell metabolism. The immunological basis of drug-induced hemolysis is reviewed with emphasis on the clinical and laboratory manifestations, differential diagnosis and the major mechanisms involved. Drug-induced oxidative hemolysis both in normal individuals and in those with certain enzymopathies, notably glucose-6-phosphate dehydrogenase deficiency, is summarized. Drugs may also produce red cell dyscrasias by acting on the immature erythroid compartment. Some of these inhibit erythroid growth by as yet poorly understood mechanisms. Others exert more specific metabolic effects in erythroid precursors. These include drugs which interfere with DNA synthesis causing megaloblastic erythropoiesis and those which disrupt mitochondrial function and the synthesis of heme manifested by sideroblastic erythropoiesis. A brief consideration of heme biosynthesis and the action of drugs which are associated with sideroblastic anemia, including the antituberculous agents, lead, alcohol and chloramphenicol is presented. Finally, where pertinent, an updated listing of drugs involved in red cell dyscrasias is included.

Mechanism of Ranitidine Associated Anemia

Ranitidine, an H2 receptor antagonist, has been associated with hematotoxicity. The mechanism(s) underlying the toxicity is not well understood. The authors studied the mechanism of anemia in a patient with ranitidine associated anemia and thrombocytopenia. Clinical evaluation suggested drug-induced Coombs' positive reticulocytopenic hemolysis. In vitro, with the patient off ranitidine, the authors were able to induce Coombs' positivity by incubating patient's red cells with ranitidine and his serum. This process was inhibited by prior exposure of his red cells to histamine. In vitro studies using clonal assays for hematopoietic progenitors revealed that while the patient's serum or ranitidine alone did not affect the patient's or normal bone marrow hematopoiesis, the simultaneous presence of both agents significantly suppressed both patient's and normal erythroid progenitor (BFU-E) colony development. This suppressive effect was prevented by the prior exposure of marrow to histamine and was not observed when the patient's serum was heat inactivated. These studies suggest that the anemia may have resulted from complement-dependent autoimmune destruction/inhibition of progenitor/mature erythroid cells by a process critically dependent on the presence of ranitidine and possibly acting at or near the histamine receptor.

Drug-induced erythrocyte haemolysis

Drug-induced erythrocyte haemolysis

Glomerular disease in hypercholesterolemia guinea pigs: A pathogenetic study

Recent evidence suggests a role for lipid deposition in the pathogenesis of some forms of glomerular disease. To gain further insight into this phenomenon guinea pigs (GP) were fed a 2% cholesterol (HC) diet and compared to GP on a normal diet (C). Serial observations were made 5, 10, 30 and 70 days after the initiation of the experiment. HC gained less weight than C (P less than 0.001) and developed hemolytic anemia after 30 days. At all time periods serum total cholesterol (TC) was significantly elevated in HC (P less than 0.001). High density lipoprotein-cholesterol and total phospholipids (PL) were significantly higher in HC at days 30 and 70. Lipoprotein-X was detected in HC serum. The relative proportion (%) of cholesteryl ester (CE) at day 70 was significantly higher in HC than in C when renal cortical lipids were analyzed (P less than 0.017). Renal function was normal in both groups throughout the 70 days. The HC group developed proteinuria and hematuria (proteinuria, HC = 22.1 +/- 7.2 mg/24 hr; C, 6.4 +/- 2.3 mg/24 hr), which was detected at day 70 but not at day 30. HC developed significant progressive mesangial expansion which was first evident at day 30. In HC only oil red 0 material was first detected in glomeruli at day 5 and was very conspicuous at day 70. Increased intraglomerular monocyte numbers were detected at day 70 (P less than 0.017) but not at day 30 in HC. No glomerulosclerosis was observed in GP's with drug-induced hemolysis on a normal diet. To see the effect of high protein intake on HC GP's, a group of GP's was put on a HC diet for 30 days followed by a 2% cholesterol-high protein (HCHP) diet for 40 days. Compared to HC GP's, the HCHP group showed significantly higher serum TC and PL (P less than 0.017), mesangial expansion (P less than 0.01) and proteinuria (P less than 0.01). The results indicate that hypercholesterolemia plays an important role in the pathogenesis of glomerulosclerosis in this model and that the process appears to be mediated, at least in part in the later stages, by monocytes. The addition of protein to the HC diet augments these effects.

Hydroxylamines and hemolytic anemia.

Hemolytic anemia, the uncompensated loss of red blood cells from the circulation, has been recognized as a side effect of drugs and other chemicals for over 50 years (Muelens, 1926; Cordes, 1926). This response is commonly associated with the aminoquinoline drugs, pamaquine and primaquine (Beutler, 1959); indeed the extensive studies carried out with these drugs in the 1940’s and 50’s by Alving, Carson, Beuiler, and others still serve as the basis for most of our knowledge in the clinical sequelae of drug-induced hemolysis.

Free-Radical Production and Oxidative Reactions of Hemoglobin

Mechanisms of autoxidation of hemoglobin, and its reactions with H2O2, O2-, and oxidizing or reducing xenobiotics are discussed. Reactive intermediates of such reactions can include drug free radicals, H2O2, and O2-, as well as peroxidatively active ferrylhemoglobin and methemoglobin-H2O2. The contributions of these species to hemoglobin denaturation and drug-induced hemolysis, and the actions of various protective agents, are considered.

Free-radical production and oxidative reactions of hemoglobin.

Mechanisms of autoxidation of hemoglobin, and its reactions with H2O2, O2-, and oxidizing or reducing xenobiotics are discussed. Reactive intermediates of such reactions can include drug free radicals, H2O2, and O2-, as well as peroxidatively active ferrylhemoglobin and methemoglobin-H2O2. The contributions of these species to hemoglobin denaturation and drug-induced hemolysis, and the actions of various protective agents, are considered.

The Effects of Cisplatin on Normal Human Erythrocytes in Vitro

Therapeutic administration of cisplatin is often followed by anaemia which may not be due entirely or even largely to myelosuppression but rather to drug-induced haemolysis. Similar experience with other cytotoxic agents prompted earlier investigations which yielded evidence of drug-induced changes in RBC shape in vitro. Current studies of cisplatin and bleomycin, which have been used together in clinical practice, revealed a significant alteration in the profile of RBC morphology; specifically a reduction in the number of 'bowl' shaped cells. However, this finding is in contrast to the increase in 'bowls' which was observed previously with other drugs. So it appears that direct erythrocytotoxicity cannot explain the occurrence of anaemia in patients who receive cisplatin. An alternative mechanism, similar to that of penicillin induced haemolysis, is suggested by immunoradiometric assays of surface IgG on red cells.

Toxicological screening models: Drug-induced oxidative hemolysis

In an attempt to obtain a simple screening system for the assessment of toxic-hemolytic effects of chemical substances, a battery of hematological tests was used. Phenacetin served as reference substance. The drug caused reversible formation of methemoglobin and Heinz bodies and an increase in peripheral reticulocytes after 2 and 4 weeks of treatment. Furthermore, an increase in the mean corpuscular volume of red blood cells (RBC) and the volume of RBC ghosts in hypotonic solutions, and a decrease of the mean corpuscular fragility was observed. The latter changes are considered to be a consequence of regenerative RBC compensation rather than due to structural membrane alteration caused by the drug. The results suggest that only a combination of several hematological tests can provide comprehensive information about the hemolytic potential of chemical substances, and that for screening purposes small numbers of animals are often sufficient.

Influence of double genetic polymorphism on response to sulfamethazine.

Combined effects of a double genetic polymorphism on sulfamethazine-induced changes in erythrocyte-reduced glutathione (GSH) concentration were investigated in 16 healthy Ghanaian men. The subjects were divided into four subgroups on the basis of their glucose-6-phosphate dehydrogenase (G-6-PD) activity levels and acetylator phenotypes. Each received a single oral dose of sulfamethazine (1.5 gm). Erythrocyte concentration of GSH and plasma concentrations of drug were determined in samples taken at specific times after dosing. Sulfamethazine treatment induced a decrease in erythrocyte GSH concentration in all subjects. The degree of exposure to sulfamethazine was greater in slow acetylators, as evidenced by higher plasma concentrations, longer half-lifes, and greater area under the plasma concentration-time curves. The greatest decrease in GSH concentration occurred in subjects who where both G-6-PD deficient and phenotypically slow acetylators. In these subjects the effect of the double genetic defect was approximately additive. Drug-induced hemolysis is associated with a low erythrocyte GSH concentration. Our data suggest that slow acetylator status is likely to increase susceptibility to hemolysis in G-6-PD-deficient individuals exposed to potentially hemolytic arylamines.

Interaction between Chlorpromazine and Radiation Damage to Human Erythrocyte Membrane

MISHRA, K. P., SRINIVASAN, V. T., AND SINGH, B. B. Interaction between Chlorpromazine and Radiation Damage to Human Erythrocyte Membrane. Radiat. Res. 88, 413-419 (1981). The effect of y irradiation on human erythrocytes and the modification of this by chlorpromazine (CPZ) have been investigated, the extent of hemolysis induced being taken as a criterion of damage. Cells irradiated under isotonic conditions for doses up to 150 krad did not hemolyze to any significant extent when measured within a few minutes of irradiation. The hemolysis gradually increased on postirradiation incubation, and eventually complete hemolysis occurred suddenly within a span of 2 to 3 hr. The time taken for complete hemolysis depended on the radiation dose. The stability of these irradiated cells in 0.45% sodium chloride solution decreased with postirradiation incubation. The extent of hypotonic hemolysis was found to be linearly dependent on the radiaiton dose. Under isotonic conditions, 0.25 mM of CPZ did not induce any significant hemolysis while 0.5 mM of CPZ caused complete hemolysis in about 12 hr. However, treatment of irradiated cells with 0.25 mM CPZ showed a synergistic effect. Although the amount of CPZ bound to the irradiated cells did not change with dose, the extent of drug-induced hemolysis increased with dose. The present results may partly explain the synergistic effect of CPZ and radiation on tumors.

Protective effects of cyclodextrins on drug-induced hemolysis in vitro.

beta-Cyclodextrin (beta-CyD) significantly protected the human erythrocytes from hemolysis and shape changes induced with chlorpromazine and flufenamic acid in isotonic solution. A good correlation between the stability constants of inclusion complexes (beta- greater than gamma- greater than alpha-CyD) and the inhibitory effects on drug-induced hemolysis was found. From the observations of drug uptake into erythrocytes and changes in surface tension, the protective effects of CyDs appeared to be due to the decrease in effective concentration of drug through inclusion complexation rather than the direct interaction of CyDs with erythrocyte membrane.

6 - Drug-Induced Oxidative Haemolysis

Mesial temporal lobe epilepsy (mTLE) is a common type of drug-resistant epilepsy and secondarily generalized tonic–clonic seizures (sGTCS) have devastating consequences for patients' safety and quality of life. To probe the mechanism underlying the genesis of sGTCS, we investigated the structural differences between patients with and without sGTCS in a cohort of mTLE with radiologically defined unilateral hippocampal sclerosis. We performed voxel-based morphometric analysis of cortex and vertex-wise shape analysis of subcortical structures (the basal ganglia and thalamus) on MRI of 39 patients (21 with and 18 without sGTCS). Comparisons were initially made between sGTCS and non-sGTCS groups, and subsequently made between uncontrolled-sGTCS and controlled-sGTCS subgroups. Regional atrophy of the ipsilateral ventral pallidum (cluster size = 450 voxels, corrected p = 0.047, Max voxel coordinate = 107, 120, 65), medial thalamus (cluster size = 1128 voxels, corrected p = 0.049, Max voxel coordinate = 107, 93, 67), middle frontal gyrus (cluster size = 60 voxels, corrected p < 0.05, Max voxel coordinate = − 30, 49.5, 6), and contralateral posterior cingulate cortex (cluster size = 130 voxels, corrected p < 0.05, Max voxel coordinate = 16.5, − 57, 27) was found in the sGTCS group relative to the non-sGTCS group. Furthermore, the uncontrolled-sGTCS subgroup showed more pronounced atrophy of the ipsilateral medial thalamus (cluster size = 1240 voxels, corrected p = 0.014, Max voxel coordinate = 107, 93, 67) than the controlled-sGTCS subgroup. These findings indicate a central role of thalamus and pallidum in the pathophysiology of sGTCS in mTLE.

Effect of drugs on human erythrocytes. V. Inhibition of cellular metabolism and adenosine triphosphatase activity by drugs and relation of the inhibition to drug-induced hemolysis.

In an attempt to clarify the effects of drugs on the metabolism, transport mechanism and life time of human erythrocytes, the effects of chlorpromazine and clemastine on the ATP content, sodium pump and glucose-6-phosphate (G-6-P) dehydrogenase were studied. Chlorpromazine at 2 and 3×10-4M caused only a 5% reduction in the ATP level of erythrocytes and the drug at 8×10-4 and 10-3M induced 20 and 22% decreases in the amount of the ghost membrane, respectively. The incubation of erythrocytes in an ATP-generating system had only a slight protective effect against the drug-induced hemolysis. The Na+, K+-ATPase activity in the ghost membrane was markedly decreased by chlorpromazine at 6×10-4 to 10-3M without inhibition of Mg2+-ATPase activity, and inhibition of the Na+, K+-ATPase activity was observed a short time after the drug treatment. There was also a partial inhibition of G-6-P dehydrogenase activity by the drugs. Complete inhibition of Na+, K+-ATPase activity with ouabain resulted in no increment of the directly drug-induced hemolysis and no extreme shape changes. Therefore, the directly drug-induced hemolysis is not necessarily an energy-dependent process. However, the cells exposed to chlorpromazine at prelytic concentrations were gradually hemolyzed upon prolonged incubation in isotonic solution at 37°. Consequently, the drug treatment appears to shorten the survival time of the cells, probably due to inhibition of cellular metabolism and to membrane damage.

Glucose 6-phosphate dehydrogenase variants in Japan.

Fifty-four cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency have so far been reported in Japan. Among them, 21 G6PD variants have been characterized. Nineteen out of the 21 variants were characterized in our laboratory and G6PD Heian and "Kyoto" by others. G6PD Tokyo, Tokushima, Ogikubo, Kurume, Fukushima, Yokohama, Yamaguchi, Wakayama, Akita, Heian and "Kyoto" were classified as Class 1, because all these cases showed chronic hemolytic anemia and severe enzyme deficiency. All these variants showed thermal instability. G6PD Mediterranean-like, Ogori, Gifu and Fukuoka were classified as Class 2, whereas G6PD Hofu, B(-) Chinese, Ube, Konan, Kamiube and Kiwa belonged to Class 3. All the 6 Class 3 variants were found as the results of the screening tests. The incidence of the deficiency in Japanese seems to be 0.1-0.5% but that of the cases which may slow drug-induced hemolysis would be much less. G6PD Ube and Konan appear to be relatively common in Japan.

Investigation of poikilocytic normochromic normocytic anemia

Spiculated poikilocytes occur in several different forms. Each form is associated with certain types of diseases or conditions. Identification of a dominant form in a blood smear provides a clue to diagnosis. Helmet cells, triangular cells, and schistocytes most commonly suggest disease in the microvasculature. "Bite" cells are usually a product of drug-induced hemolysis. Keratocytes are variants of helmet cells and therefore occur in the same clinical circumstances as helmet cells. Echinocytes are usually artifacts; true echinocytosis is rare and associated mainly with uremia. Acanthocytosis occurs most commonly after splenectomy but may be encountered in severe liver disease.

Mechanism for drug-induced methemoglobin formation: Oxidation of human oxyhemoglobin by ethopropazine.

The mechanism for oxidation of human oxyhemoglobin induced by ethopropazine was studied. The rate of methemoglobin formation followed second-order kinetics and became faster with an increase of pH. The methemoglobin formation was not inhibited by either catalase or superoxide dismutase. The increase of absorbance at 520 nm, which was attributed to the reduction of ferrocytochrome c, was not inhibited by superoxide dismutase. On the other hand, the decrease of absorbance at 550 nm which corresponded to the co-oxidation of ferrocytochrome c was inhibited by catalase. The oxidation of human oxyhemoglobin induced by ethopropazine seemed to occur through two electron reduction mechanism. In the initial step, a single electron transfers from ethopropazine to the bound dioxygen of oxyhemoglobin giving rise to (Fe2+-OO-) and a semiquinone free radical. In the second step, a single electron transfers from the iron atom to the bound dioxygen producing hydrogen peroxide and methemoglobin. The two electron transfer mechanism giving rise to hydrogen peroxide and methemoglobin seems to give an important implication to the drug-induced methemoglobin formation and hemolysis of erythrocyte.

Effect of drugs on human erythrocytes—4: Protecting effect of dextran on drug-induced hemolysis

The protection of drug-induced hemolysis was designed in an attempt to prevent adverse reactions of drugs. The dextrans (mol. wt of 7,600, 18,500 and 23,200) were used for this purpose. These dextrans produced a protecting effect on drug-induced hemolysis in vitro. Electron microscopic observations indicated that addition of the dextrans at higher concentrations to the cells partially prevented the cell shrinking, most of the cells retained smooth spheres at 0.8 mM chlorpromazine, at which concentration 100% hemolysis was produced in the absence of the dextrans. Although the dextrans had no stabilizing effect on the cell membrane, they strongly inhibited the diffusion of hemoglobin and K + and decreased quantities of the drug molecules adsorbed to the cells. There was a good correlation between the viscosity of the dextrans and the protecting effect on drug-induced hemolysis. The protecting effect of the dextrans thus is due to inhibition of diffusion of hemoglobin and K + and the decrease in quantities of drug molecules adsorbed to the cells, probably ascribed to removing solvent volume accessible to the diffusing hemoglobin, the increased viscosity and the sheath of dextran molecules.