Inhibitory effect of bilirubin on complement-mediated hemolysis

We investigated the in vitro action of the bile pigments, unconjugated bilirubin (UB) and bilirubin monoglucuronide (BMG) on complement (C) cascade reaction. Both UB and BMG inhibited hemolysis in the classical pathway (CP) in a dose-dependent manner at low micromolar concentrations, UB showing a stronger effect than BMG. The analysis of the action of UB on the hemolytic activity of the C1, C4, C2 and C-EDTA components of the C cascade revealed that the C1 step was the most inhibited. An enzyme immunoassay was developed to evaluate the effect of UB on the binding of C1q, one of the subcomponents of C1, to human IgM and IgG. The study demonstrated that the unconjugated pigment interferes both the C1q-IgM and -IgG interactions, thus tentatively explaining the inhibitory action of UB on hemolytic activity of C1. We conclude that the anti-complement effect of UB is mainly exerted on the C1 component, the recognition unit of CP. The potential clinical implication of the reported effects in hyperbilirubinemia is discussed.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

A kinetics study of pig erythrocyte hemolysis induced by polyene antibiotics

The kinetics of the hemolysis induced by filipin is of the damage type, indicating the formation of large nonselective perforations of erythrocyte membranes. The process is relatively independent of the ionic composition of the incubation medium, and the differences between the hemolysis induced by filipin in pig and human erythrocytes are not significant. In a sucrose medium, filipin-induced hemolysis is inhibited in humans, whereas it is stimulated in pig erythrocytes. It is suggested that low ionic strength is the reason for the different modifications of complexation of filipin in pig and human erythrocyte membranes in a sucrose medium. The kinetics of the hemolysis induced in pig erythrocytes by amphotericin B and nystatin is of the permeability type, indicating the formation of selective channels in erythrocyte membranes and colloid osmotic hemolysis. The rate of the hemolysis, which is high in a KCl medium, is decreased in all the other media tested (CaCl2, MgCl2, potassium phosphate buffer, K2SO4, sucrose), although there are no changes in the kinetics of hemolysis. The results are interpreted as the formation of highly selective channels at a low concentration of the antibiotics. At increasing concentrations, channels of decreasing selectivity occur. The resistances of pig erythrocytes to amphotericin B and nystatin are lower than those of human erythrocytes.     

Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte hemolysis

Excessive peroxidation of biomembranes is thought to contribute to the initiation and progression of numerous degenerative diseases. The present study examined the inhibitory effects of a cocoa extract, individual cocoa flavanols (-)-epicatechin and (+)-catechin, and procyanidin oligomers (dimer to decamer) isolated from cocoa on rat erythrocyte hemolysis. In vitro, the flavanols and the procyanidin oligomers exhibited dose-dependent protection against 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis between concentrations of 2.5 and 40 microM. Dimer, trimer, and tetramer showed the strongest inhibitory effects at 10 microM, 59.4%, 66.2%, 70.9%; 20 microM, 84.1%, 87.6%, 81.0%; and 40 microM, 90.2%, 88.9%, 78.6%, respectively. In a subsequent experiment, male Sprague-Dawley rats (approximately 200 g; n = 5-6) were given a 100-mg intragastric dose of a cocoa extract. Blood was collected over a 4-hr time period. Epicatechin and catechin, and the dimers (-)-epicatechin-(4beta>8)-epicatechin (Dimer B2) and (-)-epicatechin-(4beta>6)-epicatechin (Dimer B5) were detected in the plasma with concentrations of 6.4 microM, and 217.6, 248.2, and 55.4 nM, respectively. Plasma antioxidant capacity (as measured by the total antioxidant potential [TRAP] assay) was elevated (P < 0.05) between 30 and 240 min following the cocoa extract feeding. Erythrocytes obtained from the cocoa extract-fed animals showed an enhanced resistance to hemolysis (P < 0.05). This enhanced resistance was also observed when erythrocytes from animals fed the cocoa extract were mixed with plasma obtained from animals given water only. Conversely, plasma obtained from rats given the cocoa extract improved the resistance of erythrocytes obtained from rats given water only. These results show cocoa flavanols and procyanidins can provide membrane protective effects.     

Oxidative damage by phenylhydrazine diminishes erythrocyte anion transport

Human erythrocytes were exposed to oxidative stress by treatment with the slowly hemolytic drug phenylhydrazine. Phenylhydrazine has been previously shown to trigger the production of toxic oxygen metabolites including O-2 and H2O2 and the formation of Heinz bodies. The concentration-dependent formation of Heinz bodies was confirmed using optical microscopy. Heinz body formation was accompanied by surface protuberances as shown by scanning electron microscopy. The formation of Heinz bodies was accompanied by inhibition of anion translocation. Anion translocation was measured using the anionic fluorescent substrate analog N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine). The efflux of NBD-taurine was measured by continuous monitoring of transport by fluorescence (CMTF). The mean value of the kinetic rate constant for transport, k, was found to be -0.090 +/- 0.017 min-1. Phenylhydrazine was found to decrease k to less than one-half of control values in a dose-dependent fashion. The disruption of anion translocation may be related to the oxidative effects of phenylhydrazine and to the generation of Heinz bodies, which bind to the N-terminal domain of band 3.     

Viscous macromolecules inhibit erythrocyte hemolysis induced by snake venom phospholipase A2

The effect of medium viscosity on lysis of red blood cells (RBC) induced by snake venom phospholipase A2 (PLA2) was examined. The medium viscosity was modified by the addition of various macromolecules which differ in their chemical nature and in their capacity to increase fluid viscosity. PLA2 and Ca++ were applied to cells suspended in viscous medium to induce hemolysis. It was found that the hemolysis is inhibited in direct proportion to increasing viscosity of the extracellular fluid. This phenomenon was observed with aggregated as well as disaggregated RBC. To examine whether the viscosity interferes with the accessibility of the enzyme to the cell, the medium viscosity was modified after binding of the enzyme to the cells; PLA2 was added to a RBC suspension in the presence of Ba++ which binds the enzyme to the cell membrane but does not activate it. The cell-enzyme complex was separated by gel filtration and suspended in viscous medium in the presence of Ca++ which activates the reaction. Also in this case RBC lysis was inhibited as the medium viscosity was increased. It is proposed that the action of PLA2 on RBC membrane is regulated by the viscosity of the cell surface aqueous environment.     

Inhibitory effect of mannose on erythrocyte defense against oxidants

The erythrocyte can phosphorylate a variety of hexoses. Since it can consume mannose and glucose equivalently in the hereditary deficiencies of hexokinase and phosphoglucose isomerase and since erythrocyte defense against oxidants is impaired in a variety of hereditary hemolytic anemias, we tested the hypothesis that mannose may be a significant alternative to glucose as a fuel for this defense system. Unexpectedly, mannose inhibited defense against oxidants as manifested by increased Heinz body formation when both normal and high-reticulocyte erythrocytes were incubated with acetylphenylhydrazine (APH). Using APH as the oxidant, mannose-incubated erythrocytes had decreased reduced glutathione stability and impaired hexose oxidation by the pentose shunt compared to glucose-incubated erythrocytes. After incubation with mannose and APH, normal erythrocytes showed a decrease in ATP content. Approximately 25% of the consumed mannose accumulated in the erythrocytes as mannose 6-phosphate. Erythrocytes incubated with mannose and APH displayed a significant loss of redox potential as manifested by decreased NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios. Since phosphomannose isomerase is the rate-limiting step for mannose metabolism, our results suggest that mannose impairs erythrocyte defense against oxidants by causing ATP depletion and by impairing the regeneration of reduced pyridine nucleotides by the Embden-Meyerhof and pentose phosphate pathways.     

Disulfiram may mediate erythrocyte hemolysis induced by diethyldithiocarbamate and 1,4-naphthoquinone-2-sulfonate.

The increase in 1,4-naphthoquinone-2-sulfonate (NQS)-induced hemolysis by the superoxide dismutase inhibitor diethyldithiocarbamate (DEDC) was formerly attributed to increased superoxide anion levels in the erythrocyte. Our results show that removal of DEDC after preincubation and prior to the addition of NQS did not produce a significant increase in hemolysis, which suggests that hemolysis is primarily caused by the reaction products of DEDC with NQS and not to the inactivation of superoxide dismutase. Disulfiram, the oxidized product of DEDC, was found to be the main product formed when excess DEDC was reacted with NQS. Oxygen uptake also occurred and hydrogen peroxide was formed. The latter caused the oxidation of DEDC to disulfiram as catalase prevented disulfiram formation. Disulfiram was found to readily hemolyze erythrocytes at low concentrations as well as to crosslink the proteins in the erythrocyte membrane. Furthermore, disulfiram-induced hemolysis was markedly enhanced in glutathione-depleted erythrocytes. Disulfiram was subsequently found to readily oxidize glutathione in red blood cells. When equimolar concentrations of DEDC and NQS were reacted, the major product formed was the diethyldithiocarbamate:1,4-naphthoquinone (DEDC:NQS) conjugate. However, the principal mediator of erythrocyte hemolysis when excess DEDC is reacted with 1,4-naphthoquinone-2-sulfonate is disulfiram, whose mode of action may be to modify membrane protein sulfhydryls.     

Inhibitory effect of the plant flavonoid galangin on rat vas deferens in vitro

Flavonoids are phenolic compounds that are widely distributed in higher plants and therefore are ingested by humans and animals with their regular foods, but also have various pharmacological properties. In the present study we have investigated the effect of galangin, a member of the flavonol class, on the contractile response elicited by electrical field stimulation (EFS) in the rat isolated vas deferens. Galangin (10(-8)-3 x 10(-4) M) produced a concentration- dependent inhibition of the EFS-evoked contractile response, with only a minimal inhibitory effect on phenylephrine-induced contractions. The inhibitory effect of galangin was unaffected by atropine (10(-6) M) plus hexamethonium (10(-4) M), a combination of the NK(1) receptor antagonist SR 140333 (10(-7) M), the NK(2) receptor antagonist SR 48968 (10(-6) M) and the NK(3) receptor antagonist SR 142801 (10(-7) M), L-NAME (3 x 10(-4) M), naloxone (10(-6) M) or yohimbine (10(-7) M). However, the vanilloid receptor antagonist capsazepine (10(-5) M) significantly reduced the inhibitory effect of galangin. It is concluded that the galangin inhibits excitatory transmission of the rat vas deferens with a mechanism involving, at least in part, vanilloid receptors.     

Inhibitory effect of PGE1 on the liberation of insulin induced by glibenclamide

Glycemia and insulinemia in rat blood samples have been determined at different times before and after administration of glibenclamide, PGE1, glibenclamide and PGE1, glibenclamide and glucose, PGE1 and glucose, and glibenclamide, PGE1 and glucose. PGE1 led to a partial inhibition of glibenclamide induced insulin release, with and without glucose administration, but a total inhibition did not occur. The inhibitory action of PGE1 on insulin secretion was also reflected on the glycemia curves. Defects in insulin release in diabetes could be due in part to an excessive production of PGs, that involve a failure in the beta-cells to respond to glucose signals. The present paper shows that glibenclamide secretory action was not cancelled out by PGE1. These results could explain the availability of glibenclamide in the treatment of diabetes mellitus.     

Inhibitory effect of hirudin on thrombosis induced by prothrombin complex concentrates

The influence of hirudin on thrombus formation induced by prothrombin complex concentrate (PCC) was studied in two different test series in rats. Within the first test series hirudin was i.v. administered to the animals 10 min before they received PCC. Complete prevention of thrombus formation required a hirudin dose of 0.2 mg/kg. Within the second test series hirudin was added to the transfusion unit of PCC before application of PCC was started. In this case complete prevention of thrombus formation was yielded by addition of 140 micrograms hirudin to the PCC transfusion unit. In comparison with heparin and the synthetic thrombin inhibitor N alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine piperidide, hirudin was most potent.     

Hemoglobin oxidation and erythrocyte hemolysis induced by a synthetic analog of alpha-tocopherol not containing an isoprenoid chain

The effect of alpha-tocopherol and its synthetic analogue which does not contain an isoprenoid chain, 2,2,5,7,8-pentamethyl-6-hydroxychroman (chromanol), on rat erythrocyte and hemoglobin has been studied. Chromanol, unlike alpha-tocopherol, induces oxidation of hemoglobin into aquomethemoglobin and causes erythrocyte hemolysis. A mechanism of the reaction has been established. It consists of two-electron reduction of haem-associated oxygen molecule. The products formed can cause oxidative membrane damage and subsequent hemolysis. The absence of similar activity of alpha-tocopherol seems to be connected with the inaccessibility of ligand sphere of hemin iron because of the presence of the isoprenoid chain. The oxidative activity of chromanol can explain the absence of E-vitamin activity in this compound.     

Opposite effect of albumin on the erythrocyte aggregation induced by immunoglobulin G and fibrinogen

The effect of albumin on the immunoglobulin G (IgG)-induced and fibrinogen-induced aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. As albumin concentration in the medium was increased, the IgG-induced erythrocyte aggregation was inhibited, while the fibrinogen-induced erythrocyte aggregation was accelerated (albumin itself was not able to aggregate erythrocytes). These relations were empirically expressed by the equations, v = aG1.8/A and v = a'F1.5 (A + b'), respectively (v, the velocity of erythrocyte aggregation; A, G and F, the concentrations of albumin, IgG and fibrinogen, respectively; a, a' and b', constant). The IgG-induced erythrocyte aggregation was remarkably inhibited by the addition of poly(glutamic acid), but the fibrinogen-induced erythrocyte aggregation was not. A mechanism for the interaction of immunoglobulin G and fibrinogen with the surface of erythrocytes was proposed.