Exchange of Phosphatidylcholine between Rabbit Erythrocytes and Plasma in vivo N O R M A N B. S M I T H * and D A V I D R U B I N S T E I N 1, Department of Biophysics, Health Sciences Centre, University of Western Ontario. London, Ontario, Canada N6A 5Cl ABSTRACT

Phosphatidylcholine exchange between rabbit erythrocytes and plasma was studied in vivo. The erythrocyte phosphatidylcholine was labeled by exchange in vivo with [32p] phosphatidylcholine and in vitro by acylation with [3tl] 16:0 and [t4C] 18:2. The erythrocytes were then injected into rabbits and the loss of labeled phosphatidylcholine from the ceils by exchange was followed. The rate constants for the exchange of [mp]-, [31t] 16:0-, and [t4C118: 2-phosphatidylcholine were .0131 • .0010, .0093 • .0014 and .0074 • .0013 h-t , and the exchange rates of the labels relative to that of [32p] were 1.0, 0.71 • .16, and 0.56 • . 14, respectively. These results confirm our earlier in vitro findings and represent the first in vivo demonstration of the dependency of the exchange rate of erythrocyte phosphatdylcholines on their metabolic prehistory. INTRODUCTION

M A T E R I A L S A N D METHODS

The exchange of phospholipid in vitro between mammalian erythrocytes and various acceptors such as whole plasma (1-4), isolated lipoproteins (3,5) phospholipid vesicles (6,7) and liver microsomes (8,9) has been investigated intensively for many years, In some of these studies, intracellular phospholipid exchange proteins from various organs were used to facilitate the transfer of phospholipid between donor and acceptor (6-9). The exchange of phosphatidylcholine (PC) between the outside of the erythrocyte membrane and the acceptor in the medium is rapid only in the presence of added intracellular (from heart or liver) phospholipid exchange protein (7-9). In other more physiological experimental systems, such as those in which only whole serum or plasma is used as the medium, the rate of exchange is slow, i.e., about l%/hr (2,3). Thus, while phospholipid exchange protein is useful as a model system probe, it has little physiological significance in relation to the erythrocyte. Indeed, with few exceptions (2), the physiological importance of all of the in vitro investigations remain to be demonstrated in vivo. Earlier we showed in vitro that the rates of exchange of PC between rabbit erythrocytes and serum depended on the origin of the PC. Those PC formed in the cell by acylation of 9,12-octadecadienoic (linoleic) and hexadecanoic (palmitic) acids with endogenous lysophosphatidylcholine exchanged at rates only 53 and 64%, respectively, of the rate of a2p. labeled PC which originally entered the erythrocyte by exchange (3). In this report, we provide results of a corresponding in vivo study of these phenomena.

[ 9, l 0 -a H ] Palmitic acid, [ 1J4C ] linoleic acid and SlCrO4 were obtained from New England Nuclear Corp., Boston, MA, and Naaa2PO4 was from Charles E. Frosst and Co., Montreal, Quebec. New Zealand White rabbits were supplied by Canadian Breeding Farms, St. Constant, Quebec. The PC of rabbit erythrocytes were labeled in vivo by exchange with [a2P]PC from the plasma, and in vitro by acylation of erythrocyte lysophosphatidylcholine with [ aH] palmitic acid and [14C] linoleic acid. The methods used have been described in detail before (3), but are outlined briefly here. Na332po4 was administered intravenously or intraperitoneally to donor rabbits and, after 3-4 days, the blood was collected. The ~P-labeled erythrocytes, freed of the buffy coat and repeatedly washed, were incubated with a mixture of [9,10-all] palmitic acid and [ 1-t4C]linoleic acid in Krebs-Ringer bicarbonate buffer for 2 hr at 37 C. During the last half hour of this acylation incubation, the erythrocytes were additionally labeled with SlCrO4 by the method of Mager et al. (10) in order to allow correction for loss of radioactivity due to removal of erythrocytes from the circulation. Two/aCi of SlCrO4/ml incubation mixture was added to the incubation medium. Incorporation of radioactive chromate was stopped 5 min before the end of the incubation by addition of ascorbic acid to a final concentration of 1 mg/ml. The erythrocytes were then separated from the medium and washed as before (3). Thus, all the erythrocytes prepared for infusion had been subjected to the same labeling conditions and were tagged with all 4 isotopes. Fifteen to 20 ml of the labeled erythrocytes suspended in saline at a hematocrit of 75-80%

*To whom all correspondence should be sent. 1Deceased.

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was infused over a period of a few minutes into recipient rabbits via ~ marginal ear vein. The first sample was taken 3/4 hr later, in order to allow sufficient time for mixing of the labeled ceils in the circulation, and was considered the "zero time" control. The samples were taken via a femoral catheter or by cardiac puncture. An aliquot of each blood sample was taken for the "/-counting of SZCr radioactivity. The erythrocytes and plasma of the remainder of each sample were then separated and the cells washed as before (3). Hemoglobin determinations on the aliquots of each sample were performed by the method of Brownstone and Denstedt (11). Using procedures described earlier (3), the lipids of the cells and plasma were extracted, separated by column and thin layer chromatography, and the PC fraction recovered and quantitated. Decay of 32p radioactivity over the experimental period was corrected back to the time of infusion of the labeled erythrocytes into the animals.

lO0 8O

~6o c

~40 = m E m

10,

3'o

6'o

9'o

Time (h)

FIG. 1. Exchange of labeled phosphatidylcholine (PC) from rabbit erythrocytes in vivo. Data points are the means from 3 rabbits + SEM: % [14C]linoleoyl PC; s [~H] palmitoyl PC; o, [s~p] PC. Labeled erythrocytes were injected into rabbits and samples of blood were taken as shown. The data were corrected for erythrocyte loss from the circulation and for reverse-exchange from the plasma.

RESULTS AND DISCUSSION

The loss of labeled erythrocytes from the circulation following infusion of the cells into the rabbits as monitored by SlCr/mg Hb was found to be logarithmic for each animal over the course of the experiments, but varied from one animal to another. Therefore, for each sample, fractional corrections for the corresponding losses of PC radioactivity were made by calculation, respectively, of 3H/S]Cr, 14C/ SlCr, and a2p/Cr. Appropriate correction, based on PC concentrations in cell and plasma, was also made for back exchange from the low levels of radioactive PC appearing in the plasma during the experiment (12). The data for each sample were normalized to 100% for the "zero time" control of each animal. The means of the normalized results from 3 rabbits are shown in Figure 1. With each label, there appeared to be an initial rapid, and as yet unexplained, drop in radioactivity between the first and second time points. After that, the

decay of radioactivity was exponential for the remainder of the experiment. From the linear portion of the curves, the rate constants for the exchange of the labeled PC were determined and the relative exchange rates of the acyl-labeled PC were calculated. These are compared in Table 1 with the corresponding data from our previous in vitro investigation (3). The rate constants for the exchange of the PC in vivo are similar to those calculated from th~ in vitro data. The exchang~e rates of the acyl-labeled PC relative to the ~ PC are also in very good agreement. Both the in vivo and in vitro data show that the rates of exchange of erythrocyte PC derived originally from the acylation of fatty acid with endogenous lysophosphatidylcholine are significantly lower than that of PC which entered the erythrocyte membrane originally by exchange. Also, in both studies, the PC labeled with [14C]linoleate exchanged at a rate which was

TABLE l Comparison of the Rate of Exchange of Acyl- and 32P-Labeled Phosphatidylcholines between Erythrocytes and Plasma in vitro (3) and in vivo R a d i o a c t i v e label

in phosphatidylcholine [aap] [aH]Palmitate [ Z4C] L i n o l e a t e

aMean + SEM (n). LIPIDS, VOL. 16, NO. 12 (1981)

Rate c o n s t a n t X tO a (h - t )

in vivo 13.1

• 1.0 (3) a

9 . 3 3 • 1.4 ( 3 ) 7 . 3 7 • 1.3 ( 3 )

in vitro 16.6

• .64 (14)

9.95 + .17 (10) 8.78 • .10 (10)

Relative exchange rates in vivo in vitro 1.0

1.0

.71 • . 1 6 .56 • .14

.64 • .12 . 5 3 -+ . 0 5

COMMUNICATIONS s o m e w h a t l o w e r t h a n t h a t labeled w i t h [ 3 H ] palmitate. The d i f f e r e n c e s o b s e r v e d in the e x c h a n g e rates f o r the d i f f e r e n t labeled PC p r o b a b l y is related to variation in their l o c a t i o n in the e r y t h r o c y t e m e m b r a n e , w h i c h p r o b a b l y arose f r o m d i f f e r e n c e s in t h e i r individual m e t a b o l i c origins. The palmitate- and linoleatelabeled PC p r o b a b l y were generated~ in p a r t , in t h e i n t e r i o r side o f the plasma m e m b r a n e and m a y have o c c u p i e d slightly d i f f e r e n t pools in t h e m e m b r a n e . On the o t h e r h a n d , the [32p] PC p r o b a b l y resided mainly on t h e e x t e r n a l side o f the erythrocyte membrane. This in vivo s t u d y t h e r e f o r e c o n f i r m s our earlier in vitro c o n c l u s i o n s t h a t the rate o f e x c h a n g e o f e r y t h r o c y t e PC varies with the m e t a b o l i c origin o f the m o l e c u l a r species o f the p h o s p h o l i p i d being e x c h a n g e d . F u r t h e r m o r e , it provides t h e first in vivo evidence o f this d e p e n d e n c y o f the rate o f e x c h a n g e o f PC on t h e acylation r e a c t i o n in the e r y t h r o c y t e and p r o b a b l y on the m o l e c u l a r species o f PC t h u s formed. ACKNOWLEDGMENTS The authors are grateful to Donald Lyles for assistance with the animal handling. The experimental work was supported by a grant from the Medical Research Council of Canada to D. Rubinstein (Mc-

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Gill University, Montre'al), and the data analysis by a grant from the Ontario Heart FoundatiOn to M.R. Roach (University of Western Ontario, London). REFERENCES 1. Hahn, L., and Hevesey, G. (1939) Nature 144, 72-74. 2. Reed, C.F. (1968) J. Clin. Invest. 47, 749-760. 3. Smith, N.B., and Rubinstein, D. (1974) Can:J. Biochem. 52, 706-717. 4. Renooij, W., and Van Golde, L.M.G. (1977) Biochim. Biophys. Acta 470,465-474. 5. Sakagami, T., Minari, O., and Orii, T. (1965) Biochim. Biophys. Acta 98, l I l-116. 6. Hellings, J.A., Kamp, H.H., Wirtz, K.W.A., and Van Deenen, L.L.M. (1974) Eur. J. Biochem. 47, 601-605. 7. Van Meer, G., Lange, L.G., Op den Kamp, J.A.F., and Van Deenen, L.L.M. (1980) Biochim. Biophys. Acta 598, 173-177. 8. Crain, R.C., and Zilversmit, D.B. (1980) Biochemistry 19, 1440-1447. 9. Van Meer, G., Poorthius, B.J.H.M., Wirtz, K.W.A., Op den Kamp, J.A.F., and Van Deenen, L.L.M. (1980) Eur. J. Biochem. 103,283-288. 10. Mager, J . , Hershko, A., Zeintlin-Beck, R., Shoshani, T., and Razin, A. (1967) Biochim. Biophys. Acta 149, 50-58. 11. Brownstone, Y.S., and Denstedt, O.F. (1961) Can. J. Biochem. Physiol. 39, 527-532. 12. McLean, L.R., and Phillips, M.C. (1981) Biochemistry 20, 2893-2900. [Received July 20, 1981 ]

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