Incorporation of fatty acids into phosphatidylcholine is reduced during ...

Molecular and Cellular Biochemistry 213: 137–143, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Incorporation of fatty acids into phosphatidylcholine is reduced during storage of human erythrocytes: Evidence for distinct lysophosphatidylcholine acyltransferases Alison Rusnak,1 Gail Coghlan,2 Teresa Zelinski2,3 and Grant M. Hatch1,3 1 3

Department of Pharmacology and Therapeutics; 2Department of Pediatrics and Child Health; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada

Received 11 April 2000; accepted 4 July 2000

Abstract The incorporation of [1-14C]palmitic or [1-14C]oleic acid into phosphatidylcholine and the effect on blood group antigen expression were examined in human erythrocytes stored at 4°C for 0–3 weeks. Blood drawn into EDTA was obtained by venepuncture from healthy volunteers. A 50% suspension of washed erythrocytes was incubated in buffer containing [1-14C]fatty acid for up to 60 min at 37°C with moderate shaking. Phosphatidylcholine was extracted and analyzed for uptake of radiolabelled fatty acid and phospholipid phosphorus content. Incorporation of [1-14C]palmitic or [1-14C]oleic acid into phosphatidylcholine was reduced during storage. The mechanism for the reduction in radiolabelled fatty acid incorporation into phosphatidylcholine was a 64% (p < 0.05) reduction in membrane phospholipase A 2 activity. Although human erythrocyte membranes isolated from freshly drawn blood are capable of reacylating lysophosphatidylcholine to phosphatidylcholine, with storage, a markedly different substrate preference between palmitoyl-Coenzyme A and oleoyl-Coenzyme A was observed. Lysophosphatidylcholine acyltransferase activity assayed with oleoyl-Coenzyme A was unaltered with storage. In contrast, lysophosphatidylcholine acyltransferase activity assayed with palmitoyl-Coenzyme A was elevated 5.5-fold (p < 0.05). Despite these changes, storage of erythrocytes for up to 3 weeks did not result in altered expression of the various blood group antigens investigated. We conclude that the incorporation of palmitate and oleate into phosphatidylcholine is dramatically reduced during storage of human erythrocytes. The observed differential in vitro substrate utilization suggests that distinct acyltransferases are involved in the acylation of lysophosphatidylcholine to phosphatidylcholine in human erythrocytes. (Mol Cell Biochem 213: 137–143, 2000) Key words: phosphatidylcholine deacylation-reacylation, human erythrocytes, antigen expression Abbreviations: LPC AT – acyl coenzyme A:1-acylglycerophosphorylcholine acyltransferase; PC – phosphatidylcholine; LPC – lysophosphatidylcholine; PLA2 – phospholipase A2

Introduction Phosphoglycerides are the most abundant class of lipids in all mammalian membranes (for review see [1, 2]). Structural studies of diacylphosphoglyceride revealed the asymmetri-

cal distribution of acyl groups (for review see [3]). The unique distribution of acyl molecular forms differs among animal species and tissues at both the cellular and subcellular levels, suggesting that mechanisms exist to facilitate and maintain the distinctive, non-random acyl distribution in membrane

Address for offprints: G.M. Hatch, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Room A307 Chown Building, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada, R3E OW3

138 phosphoglycerides. In the early 1960s, Lands defined a mechanism for the deacylation-reacylation of phosphoglycerides based on the presence of phospholipase and lysophosphoglyceride acyltransferase activities in mammalian tissues [4, 5]. This scheme has been verified over the years through the characterization of mammalian phospholipases and acyltransferases (for review see [6]). Recently it has been suggested that lysophosphoglyceride acyltransferases are members of a enzyme superfamily, with each form recognizing a specific fatty acyl donor [2]. Phosphatidylcholine (PC) is the major membrane phosphoglyceride of the human erythrocyte membrane [1]. Palmitic acid (C16:0, a saturated fatty acid) and oleic acid (C18:1, an unsaturated fatty acid) are the major fatty acids found within erythrocyte PC [1]. Although erythrocyte membranes have the ability to incorporate fatty acids into PC [7], it is not known whether one or more enzymes are responsible for the reacylation of lysophosphatidylcholine (LPC) to PC in the human erythrocyte. The major antigens (C, c, D, E, and e) of the Rh blood group system are carried on two distinct palmitoylated erythrocyte membrane proteins (for review see [8]). Previous studies have suggested that expression of Rh antigens and membrane phosphoglyceride composition are related [9]. Specifically, treatment of erythrocyte membranes with PLA 2 abolished Rh antigen expression [10]. Further, the trypsin mediated degradation of the D polypeptide required pre-treatment with PLA2, whereas the CE polypeptide was not altered under these conditions [11]. In this study we examined the ability of erythrocytes to incorporate fatty acids into PC with storage and determined whether this process altered the expression of red cell antigens. Although our results show that the incorporation of palmitate and oleate into PC is reduced during storage of human erythrocytes, no gross antigenic changes were detected. Based on the differential in vitro utilization of palmitoyl-Coenzyme A or oleoyl-Coenzyme A, we provide evidence for the existence of distinct LPC acyltransferases in human erythrocyte membranes.

wood Cliffs, NJ, USA. PLA2 (N. mocambique mocambique) and all other biochemicals were of analytical grade and were obtained from either Fisher Scientific, Edmonton, Canada, Sigma Chemical Co., St. Louis MO, USA or CanLab.

Incubation of intact human erythrocytes with [1-14C]palmitic or [1-14C]oleic acid Blood drawn into EDTA was obtained by venepuncture from healthy volunteers and used immediately or stored at 4°C until needed. Whole blood was centrifuged at 2,500 rpm for 10 min in a clinical centrifuge. The buffy coat was removed and 2 ml of packed cells were transferred to a screw cap tube using a serological pipette. Cells were incubated as described [12] with the following modifications. Cells were mixed with 8 ml of buffer A (140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 5 mM glucose, 10 mM Tris-HCl, pH 7.4) by carefully inverting the tube. After three separate washes, an equivalent volume of buffer B (buffer A + 1 mM CaCl2) was added to the packed cells to obtain a final ratio of 1:1 cells:buffer B. One ml aliquots of the suspension were removed and added to separate screw cap tubes. 0.5 ml of substrate buffer (0.16 g defatted albumin/100 ml buffer B) was added to each tube. In some experiments, the substrate buffer contained 3.6 µM fatty acid and 2 µCi [14C]fatty acid or 3.6 µM fatty acid alone. These produced fatty acid:albumin ratios falling within the normal range (0.15:1) for the individual fatty acids examined. The tubes were incubated in a shaking water bath for up to 60 min at 37°C. At the appropriate time interval, tubes were removed from the bath and immediately placed on ice. The tubes were then centrifuged for 10 min at 2,500 rpm in a clinical centrifuge and the supernatant was removed. Ten volumes (5 ml) of ice cold wash buffer (2% defatted albumin in buffer A) were added and the cells were washed three times. The resulting erythrocyte suspension was analyzed for radioactivity incorporated into PC or for determination of PC phosphorus. In some experiments, the radioactive PC was isolated and treated with phospholipase A2 (PLA2) and radioactivity in LPC determined.

Materials and methods [1-14C]Palmitic acid, [1-14C]oleic acid, [1-14C]palmitoyl-Coenzyme A, [1-14C]oleoyl-Coenzyme A and 1-[14C]palmitoyl2-palmitoyl-glycerophosphorylcholine were obtained from either DuPont Canada Inc. or Mandel Scientific, Mississauga, Ontario, Canada. Thin-layer plates (silica gel 60, 0.25 mm thickness) were obtained from BDH, Toronto, Canada. Ecolite scintillation cocktail was obtained from CanLab Division of Baxter Co., Winnipeg, Canada. Palmitoyl-Coenzyme A, oleoyl-Coenzyme A, egg LPC, palmitic acid and oleic acid were obtained from Serdary Research Laboratories, Engle-

Preparation of human erythrocyte membranes and assay of enzyme activities Blood drawn into EDTA was obtained by venepuncture from healthy volunteers and used immediately or stored at 4°C until needed. A 2 ml suspension of packed erythrocytes was homogenized using a tight fitting Dounce A homogenizer in 10 ml of 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 2 mM EDTA. The homogenate was centrifuged in a clinical centrifuge for 5 min and the resultant supernatant centrifuged at 150,000 × g for 60 min in a Beckman Model 1101 Ultracen-

139 trifuge. The resulting pellet was resuspended in 1 ml of homogenizing buffer and used as the enzyme source. All enzyme activities were assayed under optimum conditions. Acyl coenzyme A:1-acylglycerophosphorylcholine acyltransferase (LPC AT) activity was determined by measuring the production of [14C]PC through the incorporation of [1-14C]palmitoylCoenzyme A or [1-14C]oleoyl-Coenzyme A into LPC. The incubation mixture contained as final concentrations 75 mM Tris-HCl, pH, 8.5, 150 nmol egg LPC, 92 µM [1-14C]oleoylCoenzyme A (0.04 µCi/tube) or 92 µM [1- 14C]palmitoylCoenzyme A (0.04 µCi/tube) and 50 µg protein in a total volume of 0.7 ml. The reaction was initiated by the addition of [1-14C]acyl-Coenzyme A, followed by incubation for 30 min at 25°C in a shaking water bath. The reaction was stopped by the addition of 2 ml chloroform and 1 ml methanol. Phase separation was achieved by the addition of 0.8 ml of KCl (0.9%). PC was isolated by thin layer chromatography and radioactivity incorporated into PC determined as described previously [13]. PLA2 was assayed in membrane or cytosolic fractions as detailed in a previous report [14], except that 1-[14C]palmitoyl-2-palmitoyl-glycerophosphorylcholine was used as substrate. Cytosolic PLA2 activity was measured since some of the enzyme may have been released from membranes during the homogenization procedure. The reaction mixture contained as final concentrations 50 mM Tris-HCl, pH 8.5, 5 mM CaCl2, 0.2 mM 1-[14C]palmitoyl2-palmitoyl-glycerophosphorylcholine (specific radioactivity 0.3 µCi/pmol), and 0.1 mg protein in a total volume of 0.5 ml. The mixture was incubated at 37°C for 30 min, terminated by the addition of 2 ml chloroform. One ml of methanol and 1 ml of KCl (0.9%) was added to facilitate phase separation. LPC was isolated by thin-layer chromatography in a solvent system containing chloroform:methanol:water:NH4OH (70:30:4:1, by vol) and radioactivity incorporated into LPC determined.

Determination of blood group antigen expression An aliquot of the erythrocyte suspension prepared above was used to test for alterations in erythrocyte antigen expression. Hemagglutination tests on all samples were performed in parallel using the capillary method [15]. The time elapsed to visible agglutination in each positive test was taken as an indication of the level of antigen expression. The Rh typing reagents used were polyclonal human antisera known to be sensitive to alterations in antigen expression. The treated erythrocytes were also tested in parallel with one example of polyor monoclonal antisera to antigens (indicated in brackets) in the ABO (A, and B), MNS (M and S), Lutheran (Lub), Kell (Kpa and Kpb), Lewis (Leb), Duffy (Fya), Kidd (Jkb), Yt (Yta), Scianna (Sc:1), Dombrock (Gya), Colton (Cob), LandsteinerWiener (LWab) and Gerbich (Ge:2) blood group systems.

Other determinations PC phosphorus was determined as described [16]. For fatty acid analysis, PC or the total erythrocyte fatty acid fraction were extracted from the silica gel as described [17]. Preparation of fatty acid methylesters was as described [18]. Fatty acid separation and quantitation were performed on a Shimadzu Model GC-14A gas chromatograph. Peaks were analyzed by a Shimadzu Model CR501 integrator. Protein concentration was determined as described [19]. Student’s t-test was used for statistical analysis of data and the level of significance was defined as p < 0.05.

Results Storage of erythrocytes for three weeks results in reduced incorporation of radioactive fatty acid into PC Initially we characterized the incorporation of [1-14C]palmitate or [1-14C]oleate into erythrocyte PC. Blood drawn into EDTA was obtained by venepuncture from healthy volunteers. The blood was stored at 4°C for up to 3 weeks. A 50% suspension of washed erythrocytes prepared from freshly drawn or stored blood was incubated in buffer containing [1-14C]fatty acid for up to 60 min at 37°C with moderate shaking. PC was extracted and analyzed for either incorporation of radioactivity or for phosphorus mass. Radioactivity incorporated into PC was unaltered at 1 or 2 weeks of storage. The radioactivity (expressed as dpm/nmol phosphorus) of [1- 14C]palmitic (Fig. 1) or [1-14C] oleic acid (Fig. 2) incorporated into PC was reduced during storage for 3 weeks as compared to freshly drawn erythrocytes. We next examined the position of the radioactive fatty acid incorporated into PC. PC isolated from freshly drawn or stored erthyrocytes that had been incubated with [1-14C]fatty acid was treated with PLA2 and radioactivity in the resulting lysophospholipid determined. Less than 1% of the radioactivity was observed in LPC indicating that the [114 C]fatty acids were incorporated into the sn-2 position of PC. Thus, storage of human erythrocytes for 3 weeks results in reduced incorporation of labelled palmitate or oleate into PC. To determine if the reduction in [1-14C]palmitic or [114 C]oleic acid incorporated into PC was due to alterations in the amount of palmitic or oleic acid within PC, or the amount of these fatty acids within erythrocytes, we analyzed the relative amount of each fatty acid in PC and the total fatty acid pool from freshly drawn or stored human erythrocytes prior to, and subsequent to, incubation with palmitic or oleic acid for up to 60 min. There was no significant difference in the relative amounts of palmitic or oleic acid under any condition (Fig. 3). In addition, palmitate and oleate represented 29 and 24% of the total fatty acid pool, respectively, and these amounts were also unaltered during storage (data not shown).

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Fig. 1. Incorporation of [1-14C]palmitic acid into PC. Human erythrocytes stored at 4°C for 0 or 3 weeks were incubated for up to 60 min in the presence of [1-14C]palmitic acid as described in ‘Materials and methods’. PC was isolated and analyzed for radioactivity or phosphorus mass. Open bars, freshly drawn blood; Hatched bars, blood stored for 3 weeks. Vertical bars represent the mean ± S.D. of 6 individuals. *p < 0.05.

Finally, the total amount of PC in either freshly drawn or stored erythrocytes was 229 ± 34 nmol/0.5 ml packed cells. Thus, the reduction in [1-14C]palmitic or [1-14C]oleic acid incorporated into PC was not due to alterations in PC mass, the relative amounts of these fatty acids within PC, or the relative amounts of these fatty acids within erythrocytes. PLA2 activity is reduced during storage of human erythrocytes Since the reduced incorporation of [1-14C]palmitic or [1C]oleic acid into erythroctye PC could be due to alterations in the activity of the enzymes that deacylate-reacylate PC, we measured PLA2 activity in membrane and cytosolic fractions prepared from freshly drawn or stored erythrocytes. Blood drawn into EDTA was obtained by venepuncture from healthy

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Fig. 3. Relative amount of palmitate and oleate in PC. Erythrocytes stored at 4°C for 0–3 weeks were incubated for 60 min in the absence (open bars) or presence of palmitate (solid bars) or oleate (hatched bars) as described in ‘Materials and methods’. Methylesters from extracted PC were prepared and the relative amount of palmitate and oleate determined by gas chromatography. (A) freshly drawn blood. (B) blood stored for 3 weeks. Vertical bars represent the mean ± S.D. of 6 individuals.

volunteers. A 50% suspension of washed erythrocytes was homogenized and membrane or cytosolic fractions prepared. Endogenous membrane and cytosolic PLA2 activities were reduced 64% (p < 0.05) and 50% (p < 0.05), respectively, in stored as compared to freshly drawn erythrocytes (Table 1). Thus, the observed reduction in [1-14C]palmitic or [1-14C]oleic acid incorporated into erythrocyte PC with storage was due to the reduced ability of these cells to hydrolyze PC.

In vitro LPC AT activity with palmitoyl-Coenzyme A as substrate is increased in stored human erythrocyte membranes Fig. 2. Incorporation of [1-14C]oleic acid into PC. Human erythrocytes stored at 4°C for 0 or 3 weeks were incubated for up to 60 min in the presence of [1-14C]oleic acid as described in ‘Materials and methods. PC was isolated and analyzed for radioactivity or phosphorus mass. Open bars, freshly drawn blood; Hatched bars, blood stored for 3 weeks. Vertical bars represent the mean ± S.D. of 6 individuals. *p < 0.05.

We next examined the ability of freshly drawn or stored human erythrocyte membranes to reacylate LPC to PC. Blood drawn into EDTA was obtained by venepuncture from healthy volunteers. A 50% suspension of washed erythrocytes was

141 to membranes prepared from freshly drawn erthyrocytes. LPC represented 2.4% of the total erythrocyte phospholipid mass and was unaltered during storage. Thus, LPC AT activity, with [1-14C]palmitoyl-Coenzyme A as substrate, was elevated in stored erythrocyte membranes and this was not due to alterations in LPC levels.

Table 1. PLA2 activities in freshly drawn or stored human erythrocyte membranes and cytosol PLA2 (nmol/min·mg) Fresh Stored Membranes Cytosol

1.31 ± 0.35 0.89 ± 0.11

0.47 ± 0.17* 0.45 ± 0.18*

Erythrocyte membranes and cytosol were prepared from freshly drawn or stored erythrocytes and PLA2 activities were determined as described in ‘Materials and methods’. Each value represents the mean ± S.D. of 5 different individuals. *p < 0.05 compared to fresh erythrocytes.

Antigen expression is not affected during erythrocyte storage Since treatment of erythrocyte membranes with PLA2 is known to affect Rh D antigen expression [8], we examined the expression of various blood group antigens in freshly drawn erythrocytes compared to erythrocytes stored at 4°C for 3 weeks, the latter being the point where endogenous PLA2 activity was reduced. Our investigations focused on the major antigens of the Rh blood group system since these were shown to be covalently acylated with palmitic acid in vivo [20]. As depicted in Table 3, expression of the C, c, D, E and e antigens was unaltered in freshly drawn or stored erythrocytes, before or after incubation with palmitic or oleic acid in albumin for 60 min or with only albumin 60 min (control). Additionally, expression of the A, B, M, S, Lub, Kpa, Kpb, Leb, Fya, Jka, Yta, Sc:1, Gya, Cob, LWab and Ge:2 antigens was not effected under these experimental conditions (data not shown). Thus, storage of human erythrocytes for up to 3 weeks does not appear to grossly alter blood group antigen expression even though the incorporation of fatty acids into PC was dramatically reduced.

Table 2. LPC AT activities in freshly drawn or stored human erythrocyte membranes Substrate

LPC AT (nmol/min·mg) Fresh Stored

[1-14C]palmitoyl-Coenzyme A [1-14C]oleoyl-Coenzyme A

0.10 ± 0.02 0.66 ± 0.11

0.55 ± 0.12* 0.76 ± 0.18

Erythrocyte membranes were prepared from freshly drawn or stored erythrocytes as described in ‘Materials and methods’. LPC AT activities were determined using either [1-14C]palmitoyl-Coenzyme A or [1-14C]oleoylCoenzyme A as substrate. Each value represents the mean ± S.D. of 5 different individuals. *p < 0.05 compared to fresh erythrocyte membranes.

homogenized and membrane fractions prepared. LPC AT activities were determined in these membrane preparations in the presence of [1-14C]palmitoyl-Coenzyme A or [1-14C]oleoyl-Coenzyme A. As set out in Table 2, LPC AT activities were measureable with either [1-14C]palmitoyl-Coenzyme A or [1-14C]oleoyl-Coenzyme A substrates. Storage did not affect the ability of erythrocyte membranes to acylate LPC to PC when [1-14C]oleoyl-Coenzyme A was used as substrate. In contrast, when [1-14C]palmitoyl-Coenzyme A was used as substrate, LPC AT activity in membranes prepared from stored erythrocytes was elevated 5.5-fold (p < 0.05) compared

Discussion The objective of this study was to determine whether the incorporation of fatty acids into erythrocyte PC varied with

Table 3. Reactivity of the major antigens of the Rh blood group system with storage Probable Rh genotype

CDe/cde

CDe/CDe

CDe/cDE

Antisera Storage

Anti-C 0 week 3 week

Anti-c 0 week 3 week

Anti-D 0 week 3 week

Condition Control Palmitate Oleate Control Palmitate Oleate Control Palmitate Oleate

8 8 8 6 6 6 11 11 11

11 11 11

11 11 14

11 11 10

14 11 11

4 4 4 3 3 3 3 3 3

8 8 8 6 6 6 13 13 13

4 4 5 3 3 3 4 3 3

Anti-E 0 week 3 week

Anti-e 0 week 3 week

5 5 5

5 5 5 5 5 5 10 10 10

7 5 5

5 5 5 5 5 5 13 13 13

Freshly drawn (0 week) or stored (3 week) erythrocytes were incubated for 60 min in the absence (control) or presence of palmitate or oleate. A 30% suspension of washed erythrocytes was used to monitor alterations in antigen expression by the capillary method. Time required for agglutination with saline reactive sera is given in min. The results from 3 individuals are shown.

142 storage and if so, did this process result in altered antigen expression. The major findings of our study are as follows: (1) Storage of human erythrocytes for up to 3 weeks resulted in a decreased ability to deacylate-reacylate erythrocyte PC with palmitic or oleic acid; (2) The mechanism appeared to occur through a reduction in erythrocyte PLA2 activity; (3) Distinctive acyltransferases that reacylate LPC may exist in the human erythrocyte membrane; and (4) The differential incorporation of palmitate into erythrocyte PC upon storage did not affect the expression of antigens within the major human blood group systems (including Rh). Erythrocytes are unable to synthesize phosphoglycerides de novo but can readily take up long-chain fatty acids and incorporate them into membrane phosphoglycerides [7]. Once taken up they are converted to acyl-Coenzyme A by acylCoenzyme A ligase in the presence of ATP. Subsequent incorporation into membrane phosphoglycerides occurs by reacylation of a lysophosphoglyceride generated by a phospholipase A [21]. PC was shown to be the major erythrocyte phosphoglyceride substrate for the incorporation of fatty acids [22]. ATP depletion has been observed in aging erythrocytes [23]. In addition, lowered uptake of fatty acids was observed in ATP-depleted erythrocytes [24]. Since the incubation buffer used in our study contained glucose, it is unlikely that a decrease in erythrocyte ATP levels was ratelimiting and therefore responsible for the observed changes. In support of this was the observation that the relative amounts of erythrocyte fatty acid was unaltered with storage. The acyltransferases that reacylate LPC are thought to be members of a superfamily comprised of multiple molecular forms with each being specific for a particular fatty acyl donor [2]. The observed in vitro incorporation of [1-14C]oleoylCoenzyme A into PC was approximately 6-fold higher than the in vitro incorporation of [1-14C]palmitoyl-Coenzyme A into PC in freshly prepared isolated erythrocyte membranes. These data suggest that there are separate acyltransferases responsible for the incorporation of palmitate and oleate into erythrocyte PC. It could be argued that the differences in these in vitro enzyme activities may have been due to the differential solubility of these fatty acids within the erythrocyte membrane. However, the fact that storage resulted in a selective increase in LPC AT activity with palmitoyl-CoA but not oleoyl-CoA as substrate lends support for the existence of separate enzymes. The lipid environment has been shown to influence the affinity of LPC AT for its substrates [25]. The reason for the elevation in LPC AT activity, assayed with palmitoyl-CoA as substrate, in stored erythrocytes remains undetermined. It is possible that storage may have reduced the stability of a protein inhibitor. Clearly, changes in the level of LPC or PC, the PC fatty acid composition, or fatty acid content were not responsible for the observed differences in incorporation of radioactive palmitoyl-CoA or oleoyl-CoA into PC, as all were unaltered during erythrocyte storage.

The mechanism for the reduced incorporation of [1-14C]palmitic or [1-14C]oleic acid into PC in stored erythrocytes was likely due to a reduction in PLA2 activity. Despite the 5.5-fold increase in LPC AT activity with palmitoyl-CoA as substrate in stored erythrocytes, incorporation of palmitate into PC was reduced to a similar extent as for cells incubated with oleate. These data support the notion that PLA2 activity may be rate- limiting in the deacylation-reacylation of erythrocyte PC. This reduction in PLA2 activity may have been due to reduced stability of the protein with storage. Such a reduction in PLA2 activity and fatty acid incorporation into PC may reflect an alteration in membrane fragility. PLA2-treatment of erythrocytes eliminated Rh serological activity, suggesting that PC plays a potential role in the expression of Rh antigens [9–11]. Since the Rh antigens are palmitoylated proteins [20] it is reasonable to assume that a reduction in the amount of available membrane palmitate might effect Rh antigen expression. However, this observed reduction in PLA2 activity and subsequent reduction in the ability of the erythrocyte membrane to incorporate palmitate into PC with storage, did not appear to alter Rh antigen expression or that of the other blood group antigens examined. In addition, the relative amount of palmitate or oleate in PC from erythrocytes stored for 3 weeks was unaltered. Thus, the previously reported changes in Rh antigen expression in PLA2-treated erythrocytes [9–11] may be related to either a reduction in diacylphosphoglyceride mass or the consequent elevation in lysophosphoglyceride and fatty acid levels rather than the phosphoglyceride fatty acyl molecular composition. Lysophosphoglycerides generated by PLA2-treatment of biological membranes are potent membrane lytic compounds [26]. Previously, expression of human erythrocyte blood group antigens was examined subsequent to storage under various conditions [27–29]. No significant loss of expression of antigens belonging to the ABO or Rh systems was reported in blood stored for 21–35 days in various anticoagulants or preservatives [27]. In the present study, expression of Rh antigens and of other blood group antigens examined was unaltered in stored erythrocytes incubated for up to 60 min in the absence or presence of palmitate or oleate. These findings coupled with the observation that incorporation of palmitate or oleate into PC was reduced during storage suggests that expression of blood group antigens is not dependent on the acyl composition of erythrocyte PC.

Acknowledgements We would like to thank the individuals who participated in this study. This work was supported by grants from The Medical Research Council of Canada (MT-14261) to G.M.H. and from The Winnipeg Rh Institute Foundation Inc. and

143 The Children’s Hospital Foundation of Manitoba Inc. to T.Z. G.M.H. is an R.P.P. M.R.C. Scientist.

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