Hemolysis of Rat Erythrocytes by Replacement of the Natural Phosphatidylcholine by Various Phosphatidylcholines

Abstract

Vesicles of a variety of types of phosphatidylcholine have been shown to be suitable donors of phosphatidylcholine to intact rat erythrocytes in the presence of a specific phosphatidylcholine exchange protein. Coincident with the progression of phosphatidylcholine exchange is the onset of hemolysis, occurring at degrees of exchange characteristic for the type of phosphatidylcholine employed. During exchange with the dimyristoyl, dipalmitoyl and distearoyl species, hemolysis starts when 27 %, 25 % and 22 % of the native phosphatidylcholine is replaced by these disatured species, respectively. In the case of dielaidoylglycerophosphocholine and 1 -stearoyl-2-oleoylglycerophosphocholine hemolysis starts after the introduction of 30 % and 28 %, respectively. In contrast, the replacement of native erythrocyte phosphatidylcholine with egg phosphatidylcholine, and the dioleoyl species up to levels of 60% does not result in rapid hemolysis. Despite such functional consequences, overall contents of the individual phospholipids and cholesterol are normal. Accompanying the biochemical events are morphologic changes, including echinocyte and spherocyte formation. The structure-function implications and possible mechanisms of hemolysis are discussed Structure-function correlations in biological membranes have been studied successfully by the introduction of structural changes in one of the membrane constituents, e.g. alteration of fatty acid patterns of phospholipids in microorganisms. However, in intact mammalian membranes, studies of systematic alterations and their attendant functional consequences have been more difficult. In this regard the introduction of exchange proteins [l-41 offers the opportunity to conservatively alter the membrane structure in a systematic fashion. [12,13] demonstrated the exchange of phosphatidylcholine between intact human and rat erythrocytes and various donor systems such as microsomes, liposomes, vesicles and etherosomes. To generalize the capability of systematic structural alteration of a mammalian membrane, we have extended the use of phosphatidylcholine-exchange protein to vesicles prepared from a variety of phosphatidylcholine species with intact rat erythrocytes serving as acceptor. The preparation of rat erythrocytes variously modified with diverse phosphatidylcholines and some of their properties are reported in the present paper. MATERIALS AND METHODS Donor Vesicles1,2-Dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero -3 -phosphocholine, 1,2-dielaidoyl -sn -glycero-3 -phosphocholine and l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were synthesized following standard procedures [14] and were generously donated by colleagues

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

“…A similar observation has been made before with rat erythrocytes [16], although in this case hemolysis started already after 20% replacement. Rat erythrocyte PC contains, however, a high content (35%) of disaturated species, whereas in human erythrocytes only 13% of the total PC is present in the disaturated form.…”

“…Since extensive modifications are impossible to obtain in vivo, an in vitro approach has been applied. Recently, Lange et al [16] used the phosphatidylcholine specific transfer protein of bovine liver to modify the molecular species composition of PC in the rat erythrocyte membrane and it was observed that an increase in the degree of saturation of the phosphatidylcholine in this membrane resulted in hemolysis.…”

Topology and dynamics of phospholipids in membranes

“…Freeze-fracture electron micrographs show the evaginations of the membrane in detail [35]. When exchange was continued spherocytes were formed ( fig.(i).…”

Transbilayer movement of various phosphatidylcholine species in intact human erythrocytes

“…During the exchange reaction 10-20% of the native erythrocyte PC was replaced by donor PC. In order to prevent modification of the fatty acyl composition of the erythrocyte PC [15] in the transbilayer movement studies, the (14C)PC trace was embedded in most experiments in a matrix of egg PC, which has a fatty acyl composition that resembles that of the human erythrocyte. Because peroxidation of PC was reported to induce transbilayer movement of phospholipids in model membranes [29], we also checked the degree of peroxidation of the egg PC.…”

“…In previous studies [l 1,15, 161 it was shown that defined PC species can be introduced into the outer monolayer of intact erythrocytes by means of the PCspecific phospholipid exchange protein [17]. In the present study this technique was combined with the use of phospholipases to hydrolyze the outer monolayer [18].…”