Fatty acid induced hemolysis. Protective action of ceruloplasmin, albumins, thiols and vitamin C

The hemolytic effect of saturated fatty acids increased rapidly, when the number of carbon atoms in the chain exceeded 12. At low fatty acid concentrations (less than 60 microM) the hemolytic effect decreased with increasing number of double bonds in the carbon chain (cis-form fatty acids). A more complex pattern was observed at higher fatty acid concentrations. Trans-unsaturated fatty acids were more hemolytic than cis-analogs. Ceruloplasmin, a serum protein with no fatty acid binding capacity, reduced the hemolytic effect of fatty acids; possibly by interacting with the cell membrane. Reducing compounds (thiols, vitamin C) also protected against fatty acid induced hemolysis.     

Critical temperatures for the interaction of free fatty acids with the erythrocyte membrane

Non-esterified long-chain fatty acids reduce the extent of hypotonic hemolysis at a certain low concentration range but cause hemolysis at higher concentrations. This biphasic behavior was investigated at different temperatures (0-37 degrees C) for lauric (12:0), myristic (14:0), palmitoleic (16:1), oleic (cis-18:1) and elaidic (trans-18:1) acids. The results are summarized as follows: (A) the fatty acids examined exhibit a high degree of specificity in their thermotropic behavior; (B) oleic acid protects against hypotonic hemolysis even at the highest concentrations, up to 15 degrees C, when it becomes hemolytic, but only in a limited concentration range; (C) elaidic acid does not affect the osmotic stability of erythrocytes up to 20 degrees C, when it starts protecting: above 30 degrees C, it becomes hemolytic at the highest concentrations; (D) palmitoleic acid is an excellent protecting agent at all temperatures in a certain concentration range, becoming hemolytic at higher concentrations; (E) lauric acid protects up to 30 degrees C and becomes hemolytic only above this temperature; (F) myristic acid exhibits an extremely unusual behavior at 30 and 37 degrees C by having alternating concentration ranges of protecting and hemolytic effects; (G) there is a common critical temperature for hemolysis at 30 degrees C for saturated and trans-unsaturated fatty acids; (H) the initial slope of Arrhenius plots of percent hemolysis at the concentration of maximum protection is negative for cis-unsaturated fatty acids and positive for saturated and trans-unsaturated fatty acids.     

Reversible Binding of Long-chain Fatty Acids to Purified FAT,the Adipose CD36 Homolog

Transport of long-chain fatty acids into rat adipocytes was previously shown to be inhibited by the reactive derivative sulfosuccinimidyl oleate consequent to its binding to a membrane protein FAT, which is homologous to CD36. In this report, the ability of the purified protein to bind native fatty acids was investigated. CD36 was isolated from rat adipocytes by phase partitioning into Triton X-114 followed by chromatography on DEAE and then on wheat germ agglutinin. Fatty acid binding was determined by incubating CD36, solubilized in buffer containing 0.1 Triton X-100, with fatty acids at 37°C, and then by adsorbing the unbound ligand with Lipidex 1,000 at 0°C. Bovine serum albumin was used as a positive control and gelatin, a protein that does not bind fatty acids, as a negative control. Measurements with albumin yielded reproducible binding values which were not altered by the presence of 0.1% Triton X-100. Under the same conditions, gelatin yielded reproducibly negative measurements that did not differ significantly from zero. CD36 bound various long-chain fatty acids at low ligand to protein ratios. Warming the protein-FA-Lipidex mixture to 37°C removed the FA off the protein. Thus, binding was reversible and distinct from the palmitoylation of the protein known to occur on an extracellular domain. Comparison of the predicted secondary sequence of CD36 with that of human muscle fatty acid binding protein suggested that a potential binding site for the fatty acid on CD36 may exist in its extracellular segment between residues 127 and 279. Received: 17 January 1996/Revised: 8 May 1996     

The mixture of aldehydes and hydrogen peroxide produced in the ozonation of dioleoyl phosphatidylcholine causes hemolysis of human red blood cells

Dioleoyl phosphatidylcholine (PC) liposomes were ozonized and the ozonized liposomes were tested for their lytic potency on human red blood cells (RBC). Ozonation of PC liposomes generated approximately 1 mole equivalent of hydrogen peroxide (H2O2) and 2 mole equivalents of aldehydes, based on the moles of ozone consumed. The time necessary for 50% hemolysis induced by ozonized liposomes (a convenient measure of hemolytic activity) was found to depend on the extent of ozonation of the PC liposomes, indicating the formation and accumulation of hemolytic agents during ozonation. Hemolysis was also observed when RBC were incubated with nonanal, the expected product of the ozonation of oleic acid, the principle unsaturated fatty acid in the liposomes. Hydrogen peroxide, another product of PC ozonation, did not induce hemolysis; however, a combination of H2O2 and nonanal was significantly more hemolytic than nonanal alone. A ratio of 1:2 H2O2/nonanal (the ratio observed in the ozonized liposomes) provided hemolytic activity comparable to that observed with ozonized dioleoyl PC. Among different antioxidants tested, ascorbate, catalase, and glutathione peroxidase partially inhibited hemolysis induced by ozonized liposomes and by H2O2/nonanal mixtures, but they were not protective against the nonanal-induced hemolysis. Identification of H2O2 and aldehydes as cytotoxic chemical species generated from the ozonation of unsaturated fatty acids may have an important bearing on the in vivo toxicity of ozone on the lung as well as on extrapulmonary tissues.     

Possible involvement of red cell membrane proteins in the hemolytic action of Portuguese Man-of-War toxin.

Glycophorin and the fragments isolated from trypsinizing intact rat, dog, sheep and human red blood cells (rbc's) neutralize the hemolytic action of the Portuguese Man-of-War venom. This action can be blocked by rabbit antisheep hemolysin and phytohemagglutinin, a lectin which preferentially binds to glycophorin. Concanavalin A, which binds to band-3 protein of rbc membranes, does not block the neutralizing action of rbc tryptic fragments or glycophorin. The concentrations of rat, dog, human and sheep glycophorin which half neutralize venom induced hemolysis are inversely and linearly proportional to the hemolytic sensitivities of these rbc's to the venom. These data implicate glycophorin as a possible binding site for the hemolytic component of the Portuguese Man-of-War venom.     

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4. n - 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.     

Fatty acids bound to vitamin D-binding protein (DBP) from human and bovine sera

Human and bovine vitamin D-binding protein (DBP) have been isolated from serum by a method that does not involve denaturing steps. This method includes Cibacron Blue-Sepharose chromatography, gel filtration, DEAE-Sephadex chromatography and albumin immunoadsorption. Analysis of fatty acids bound to the isolated human and bovine DBP showed molar ratios of fatty acid to protein of 0.4 and 1.3 respectively meanwhile human and bovine albumin have bound 1.8 and 1.5 moles per mol respectively. Most of fatty acids bound to human and bovine DBP are monounsaturated and saturated, mainly oleic and palmitic acids, which together account for 50% of the total of fatty acids in both species. By contrast, polyunsaturated fatty acids represented a minor component, less than 5%.     

Comparative studies of the binding of some ligands to human serum albumin non-covalently attached to immobilized Cibacron Blue, or covalently immobilized on Sepharose, by column affinity chromatography.

A comparative study of the ligand-binding properties of human serum albumin was performed by the technique of affinity chromatography with the protein attached to immobilized Cibacron Blue F3GA (Blue Sepharose), or covalently immobilized on Sepharose. The binding strength of octanoate, decanoate and dodecanoate is much weaker when human serum albumin is attached to immobilized Cibacron Blue, indicating that the binding sites for fatty acids are involved in the attachment of human serum albumin to immobilized Cibacron Blue. The results revealed additional alterations of the ligand binding when human serum albumin was attached to immobilized Cibacron Blue, involving sites outside of the binding domains of fatty acids. Thus the stereoselective binding of L-tryptophan was abolished, and the resolution of the warfarin enantiomers was impaired. However, the binding strength of warfarin and salicylic acid was rather close to the values observed with human serum albumin covalently immobilized on Sepharose. It is suggested that the availability of the binding sites for L-tryptophan, warfarin and salicylic acid is partially blocked by the complex between albumin and the dye without direct participation in the complex-formation. An alternative interpretation involves an allosteric mechanism brought about by complex-formation between serum albumin and the immobilized Cibacron Blue.     

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k₁ = 0.003 hr^?1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k₁ = 0.145 and 0.162 hr^?1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A₂ may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley-Liss, Inc.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Fatty acids influence binding of cobalt to serum albumin in patients with fatty liver

Human serum albumin binds ligands such as fatty acids and metals in circulation. Oxidative stress can modify albumin and affect ligand binding. This study examines the role of oxidative stress and fatty acids in modulating cobalt binding to albumin in patients with fatty liver. Elevated levels of malondialdehyde and protein carbonyls, indicative of oxidative stress were evident in serum of patients with fatty liver. A significant decrease in albumin-cobalt binding was also observed. Albumin isolated from patient serum also showed an increase in bound fatty acids. In vitro experiments indicated that while oxidant exposure or removal of fatty acids independently decreased cobalt binding to albumin, removal of fatty acids from the protein prior to oxidant exposure did not influence the oxidant effect on albumin-cobalt binding. These results suggest that oxidative stress and fatty acids on albumin can influence albumin-cobalt binding in patients with fatty liver by independent mechanisms.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Partial characterization of a cell-free hemolytic factor produced by Helicobacter pylori

Abstract Maximum cell-free hemolytic activity of Helicobacter pylori cultured in broth containing 10% horse serum occurred only after the stationary phase of growth was reached, unlike many hemolysins produced by Gram-negative bacteria which are active during exponential growth. This characteristic of the H. pylori hemolytic factor suggested that it might also possess protease activity. However, because no evidence of albumin degradation was found, the hemolysis by cell-free concentrates of H. pylori appears to be due to a unique factor derived from the organism. Because variable hemolysis results were obtained with culture broths lacking albumin or serum, these proteins may act as carriers or stabilizers of the putative hemolysin.     

A cytolytic protein from the edible mushroom, Pleurotus ostreatus

Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lytic in vitro for mammalian erythrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists a monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate and sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.     

Conformational changes in human serum albumin studied by fluorescence and absorption spectroscopy. Distance measurements as a function of pH and fatty acids.

pH- and fatty acid-induced conformational changes in human serum albumin were investigated by fluorescence-energy transfer, determining the distance between Trp-214 and bound bilirubin at 25 degrees C. This distance changes significantly with the pH, being 2.52 +/- 0.01 nm at pH 6, 2.31 +/- 0.04 nm at pH 9, 2.13 +/- 0.07 nm at pH 11.0 and 2.77 nm at pH 11.9. The influence of different fatty acids on the distance was also determined. At pH 7.4 medium-chain fatty acids seem to increase this distance, whereas long-chain fatty acids, at low concentrations, decrease the distance between the two chromophores. The contraction of the protein carrying long-chain saturated fatty acids is even more pronounced at pH 9.     

Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity

BackgroundTlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis.MethodsTlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis.ResultsPurified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments.ConclusionsOligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence.General significanceOur results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.     

Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipids.

Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and alpha-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting.     

Serum albumin stimulates serotonin uptake into human blood platelets

Active uptake of serotonin (5-hydroxytryptamine, 5-HT) into blood platelets from healthy donors exhibited a lower Vmax value in buffer media than in plasma. Also in plasma ultrafiltrate Vmax was reduced, but it returned to the level measured in plasma upon addition of human serum albumin (40 milligrams) containing fatty acids. Fatty-acid-free albumin was even more stimulatory and when added to platelets in a phosphate-buffered medium, it increased Vmax beyond the value observed in plasma. Km values calculated on the basis of unbound 5-HT were not affected by the media except for a slight decrease in ultrafiltrate as compared to plasma. The fraction of 5-HT (0.5 mumol/l) bound to 40 milligrams albumin was 17% with the preparation containing fatty acids and 22% with fatty-acid-free albumin, while total plasma proteins dissolved in phosphate buffer bound 24%. The uptake of 1 mumol/l 5-HT was enhanced by both albumin preparations already at 1 milligram and near-maximal effects occurred at 10 milligrams.     

Peroxidation of liposomes in the presence of human erythrocytes and induction of membrane damage of erythrocytes by peroxidized liposomes

Hemolysis (Kobayashi, T., Takahashi, K., Yamada, A., Nojima, S. and Inoue, K. (1983) J. Biochem. 93, 675-680) and shedding of acetylcholinesterase-enriched membrane vesicles (diameter 150-200 nm) were observed when human erythrocytes were incubated with liposomes of phosphatidylcholine which contained polyunsaturated fatty acyl chains. These events occurring on erythrocyte membrane were inhibited by radical scavengers or incorporation of alpha-tocopherol into liposomes, suggesting that lipid peroxidation is involved in the process leading to membrane vesiculation and hemolysis. The idea was supported by findings that generation of chemiluminescence, formation of thiobarbituric acid reactive substance, accumulation of conjugated diene compounds in liposomes and decrease of polyunsaturated fatty acids in liposomes occurred concomitantly during incubation. Hemolysis was also suppressed by the addition of extra liposomes, insensitive to peroxidation, or of serum albumin even after the completion of peroxidation of liposomes. These results suggest that peroxidized lipids, responsible for vesiculation and hemolysis, may be formed first in liposomes and then gradually transferred to erythrocyte membranes. The accumulation of these lipids peroxides may eventually cause membrane vesiculation followed by hemolysis.     

A cytolytic protein from the edible mushroom, Pleurotus ostreatus

Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lytic in vitro for mammalian erthrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists as monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate adn sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.