137
Biochimica et Biophysica Acla, 467 (1977) 137--145 © Elsevier/North-Holland Biomedical Press
BBA 77702 EFFECTS OF TEMPERATURE AND BOVINE SERUM ALBUMIN ON LYSIS OF ERYTHROCYTES INDUCED BY DILAUROYLGLYCEROPHOSPHOCHOLINE AND DIDECANOYLGLYCEROPHOSPHOCHOLINE TAKAYUKI KITAGAWA *, YUTAKA TANAKA, KEIZO INOUE and SHOSHICHI NOJIMA Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 (Japan)
(Received September 16th, 1976) (Revised manuscript received January 31st, 1977)
Summary The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, mad rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5--10°C and above 40°C, but slow at around 25°C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the a m o u n t of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10°C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the a m o u n t of phosphatidylcholine required for lysis. Gottfried and Rapport [1] studied the hemolytic actions of a series of lysophospholipids on human, rabbit and sheep erythrocytes at 37°C and found that the nature of the linkage of the hydrocarbon chain to glycerol did not influence their hemolytic actions. Reman et al. [2] showed that the acyl chain had a great influence on the lyric actions of phosphatidylcholine, lysophosphatidylcholine and deoxylysophosphatidylcholine on beef erythrocytes, using a series of chemically well-defined derivatives with different fatty acid chains. Among the phosphatidylcholines with chain lengths of 8 to 12 carbon atoms (per acyl I
* P r e s s e n t address: D e p a r t m e n t of V i r o l o g y , I n s t i t u t e of Medical Sciences, U n i v e r s i t y of T o k y o , M i n a t o - k u , T o k y o 108, J a p a n .
138 chain) that th ey examined, di undecanoyl gl ycerophosphochol i ne had the highest activity and dilauroylglycerophosphocholine the least. Arnold and Weltzien [3] also investigated the lytic activities o f the hom ol ogous series o f lysophosphatidylcholine, modified lysophosphatidylcholine and short-chain phosphatidylcholine on human e r y t h r o c y t e s ; t h e y found that didecanoylglycerophosphocholine was lytic but dilauroyl- and dipelargonoylglycerophosphocholine were not. It is interesting that among the various phospholipids tested only d i d e c a n o y l g l y c e r o p h o s p h o c h o l i n e caused m ore rapid lysis o f human erythrocytes at 10 than at 37°C. We are interested in the relation between the structure of the membranes of e r y t h r o c y t e s and the lytic effects of various phospholipids. We studied the lysis of h u man e r y t h r o c y t e s by dilauroylglycerophosphocholine at various temperatures and f o u n d that the rate of lysis showed a biphasic dependence on temperature. We also studied the effects of bovine serum albumin and shift-down of the incubation t e m p e r a t u r e on the lysis of hum an red cells by dilauroylglycerophosphocholine. Preliminary results of these studies have been published [4]. Materials and Methods
Buffer. Veronal-buffered saline free of Ca 2+and Mg 2+was used t hroughout . It contained 0.375 g of sodium 5,5-diethylbarbiturate, 0.575 g of 5,5-diethylbarbituric acid, 8.5 g of sodium chloride per 1 of distilled water, pH 7.5. Erythrocytes. The human e r y t h r o c y t e s used were from freshly drawn, heparinized blood of healthy donors. The blood was centrifuged at 300 X g for 5 rain and the plasma and buf f y coat were discarded. The precipitated cells were then washed three times with veronal buffered saline and used for experiments within 24 h. E r y t h r o c y t e s of ot he r animals were obtained in a similar way. Lipids. Didecanoyl- and dilauroylglycerophosphocholines were obtained from Serdary Research Laboratories Inc., Ontario, Canada and were used witho u t further purification. Vesicles of didecanoyl-, and dilauroylglycerophosphocholine were prepared by suspending the dried sample o f lipid in veronal buffered saline and sonicating the suspensions in a bath-type sonicator for 30 min at r o o m temperature. Bovine serum albumin (crystalline) and f a t t y acid-free bovine albumin were obtained from A r m o u r Pharmaceutical Co., U.S.A. and Nutritional Biochemicals Co., Cleveland, Ohio, xespectively. Measurement of hemolysis. Hemolysis was measured using cells labeled with radioactive c h r o m a t e by the m e t h o d of Inoue et al. [5]. Veronal buffered saline solution containing an appropriate a m o u n t of phosphatidylcholine (0.4 ml) was preincubated for 10 min at the required t em perat ure and the reaction was started by adding 0.1 ml of S'Cr-labeled e r y t h r o c y t e suspension (5- 107 cells/ml) which had been kept in ice-water. The reaction m i xt ure was gently shaken occasionally. After incubation the mixture was centrifuged at 200 X g for 5 min and 250 pl of the supernatant was carefully removed for counting in an auto-gamma c ount er (Aloka Auto-well Gamma Counter). The percentage hemolysis was then calculated as follows: counts in 250 pl of the supernata,~.t X 2 X 100 counts in 2.5 - 106 e r y t h r o c y t e s
139
Results
Effects of temperature on hemolysis of human and cow erythrocytes by dilauroylglycerophosphocholine. When human erythrocytes were incubated with excess dilauroylglycerophosphocholine (21 nmol/ml) at various temperatures for 90 min, the rate of hemolysis was found to depend on the incubation temperature. The rate of lysis was very high above 40 ° C, but with decrease in temperature the rate decreased to a minimum at 30--25°C, and then increased again to a maximum at around 5--10°C, as reported in the previous paper [4]. The amount of phosphatidylcholine required for lysis of human erythrocytes was least at 10°C, and more at 45, 40 and 0°C, in this order. In contrast, the rate of lysis of cow erythrocytes by excess dilauroylglycerophosphocholine (17 nmol/ml) did not show a biphasic dependence on temperature: it was slow at 0--10°C and rapid above 15°C.
Effects of temperature on hemolysis of erythrocytes from various animals by dilauroylglyeerophosphocholine. The sensitivities of erythrocytes of various animals to dilauroylglycerophosphocholine were examined at temperatures between 0 and 40°C and classified into two types on the basis of the results. Monkey, rabbit and rat erythrocytes were of the human type, their hemolysis being rapid at 10 and 40°C but slow at 0 and 25°C (Fig. 1A). The lysis of chicken erythrocytes, was slightly different but it was also more rapid at 0 and 40°C than at 10 or 25°C. In contrast, the lysis of calf, sheep, pig, dog, and cat erythrocytes, like that of cow erythrocytes (Fig. 1B) increased with increase in the incubation temperature.
Effect of temperature on hemolysis of human erythrocytes by didecanoylglycerophosphocholine. Hemolysis of humun erythrocytes by didecanoylglyc-
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Incubation t e m p e r a t u r e (°C) F i g . 1. E f f e c t o f t e m p e r a t u r e o n t h e rate o f h e m o l y s i s o f e r y t h r o c y t e s of various animals by dilauroylglycerophosphocholine. ( A ) H u m a n ( J ) ; m ( m k e y (.'), r a b b i t ( o ) , rat ( 4 ) a n d c h i c k e n (A) e r y t h r o c y t e s w e r e incubated with 21 nmol/ml of dilauroylglycerophosphocholine f o r v a r i o u s p e r i o d s at d i f f e r e n t t e m p e r a t u r e s . ( B ) C o w ('~.), s h e e p ( o ) , d o g ( o ) , c a l f ( a ) , p i g ( i ) a n d c a t (A) e r y t h r o c y t e s w e r e t r e a t e d w i t h d i l a u r o y l glyeerophosphocholine as d e s c r i b e d in A . T h e t i m e s r e q u i r e d f o r 5 0 % h e m o l y s i s are p l o t t e d a g a i n s t t h e incubation temperatures.
140 erophosphocholine also showed a biphasic change with temperature, being faster at 10 and 40°C than at 25°C. Effect of bovine serum albumin on hemolysis of human erythrocytes by dilauroyl- and didecanoylglycerophosphocholine. The lyric activity of lysophosphatidylcholine is known to be inhibited by bovine serum albumin. When dilauroylglycerophosphocholine and didecanoylglycerophosphocholine were preincubated with bovine serum albumin their lytic activities were also inhibited; Table I shows the effects of preincubating dilauroylglycerophosphocholine with bovine serum albumin at 10 or 40°C for 30 min before adding it to erythrocytes. These phosphatidylcholine may combine with bovine serum albumin as lysophosphatidylcholine does. The lysis of human red cells by dilauroylglycerophosphocholine at either 40 or 10°C showed a "lag t i m e " of 30 min, as reported previously. Bovine serum albumin was found to shorten this lag time. Addition of bovine serum albumin to human erythrocytes that had been preincubated with dilauroylglycerophosphocholine for 15 min at 40°C, resulted in rapid and complete hemolysis (Fig. 2). The rate of hemolysis due to addition of phosphatidylcholine plus bovine serum albumin depended on the amounts of both bovine serum albumin (Fig. 3A) and phosphatidylcholine (Fig. 3B). Fig. 3B shows that the presence of bovine serum albumin decreased the concentration of phosphatidylcholine required for complete lysis from 16 nmol/ml to 10 nmol/ml. The presence of bovine serum albumin also reduced the concentration of didecanoylglycerophosphocholine required for lysis of human erythrocytes. Since fatty acid-free albumin had a similar effect of bovine serum albumin, the effect was not be due to contaminating f a t t y acids. Effect of pre-incubation temperature on shortening by bovine serum albumin of the "lag phase" in lysis of human erythrocytes with dilauroylglycerophosphocholine. Bovine serum albumin shortened the lag phase in hemolysis when erythrocytes were preincubated with dilauroylglycerophosphocholine for more than 5 min at 40°C (Fig. 4). At 25°C, about 60 min preincubation was required for complete lysis with bovine serum albumin. However, when the erythrocytes had been preincubated with dilauroylglycerophosphocholine at
TABLE I INHIBITION BY BOVINE SERUM ALBUMIN DILAUROYLGLYCER OPHOSPHOCHOLINE
OF
LYSIS
OF
HUMAN
ERYTHROCYTES
BY
H u m a n e r y t h r o c y t e s ( 1 0 7 c e l l s / m l ) w e r e i n c u b a t e d w i t h 21 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e , 4 m g o f b o v i n e s e r u m a l b u m i n o r m i x t u r e s o f 21 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e a n d 4 m g / m l o f b o v i n e s e r u m a l b u m i n f o r 9 0 m i n a t 1 0 o r 4 0 ° C . H e m o l y s i s w a s m e a s u r e d as d e s c r i b e d i n t e x t . H e m o l y s i s p e r c e n t at i n c u b a t i o n temperature
None Dilauro ylgly cerophosphocholine Dilauroylglycerophosphocholine Bovine serum albumin
+ bovine serum albumia
IO°C
4ff~C
6.5 94.0 1.7 5.0
13.5 98.0 3.0 6.5
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30
Fig. 2. E f f e c t of b o v i n e s e r u m a l b u m i n o n t h e " l a g p h a s e " in lysis o f h u m a n e r y t h r o c y t e s b y d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e , E r y t h r o c y t e s (107 c e l l s / m l ) w e r e i n c u b a t e d w i t h 21.1 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e at 4 0 ° C f o r 15 m i n . T h e n s a m p l e s w e r e i n c u b a t e d f u r t h e r w i t h ( ' ) or w i t h o u t (o) 4 mg/ml of bovine serum albumin.
10°C for 30 min no lysis was observed on addition of bovine serum albumin and shift of the temperature to 25°C. Osmotical fragility of human erythrocytes treated with dilauroylglycerophosphocholine. The osmotical fragility of erythrocytes that had been incubated with dilauroylglycerophosphocholine at 25°C for 30 min was less than that of untreated human erythrocytes (Fig. 5). Effect of shift-down in temperature on lysis of human red blood cells pretreated with dilauroylglycerophosphocholine. The lag time in lysis of human erythrocytes treated with dilauroylglycerophosphocholine was also shortened by shifting the temperature from 40 to 10 or 0°C but not from 40 to 25°C during incubation (Fig. 6). The rate of hemolysis was also increasing by shifting the temperature from 30 to 0°C during incubation, but hemolysis was not com-
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Fig. 3. ( A ) E f f e c t o f t h e a m o u n t o f b o v i n e s e r u m a l b u m i n o n lysis o f h u m a n e r y t h r o c y t e s b y d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e . H u m a n e r y t h r o c y e s ( 1 0 7 c e l l s / m l ) w e r e i n c u b a t e d w i t h 21 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e at 4 0 c C f o r 15 m i n . T h e n v a r i o u s a m o u n t s of b o v i n e s e r u m a l b u m i n w e r e a d d e d a n d h e m o l y s i s w a s m e a s u r e d a f t e r f u r t h e r i n c u b a t i o n f o r 15 r a i n at 2 5 ° C . (B) E f f e c t o f t h e a m o u n t o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e o n b o v i n e s e r u m a l b u m i n - d e p e n d e n t lysis o f h u m a n e r y t h r o c y t e s . H u m a n c r y t h r o c y t e s (107 cells/ml) were i n c u b a t e d with various a m o u n t s of d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e for 15 m i n at 4 0 ° C . H e m o l y s i s w a s m e a s u r e d a f t e r f u r t h e r i n c u b a t i o n at 25~'C f o r 15 rain w i t h (o) or w i t h o u t (o) 4 m g / m l o f b o v i n e s e r u m a l b u m i n .
142
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Fig. 4, E f f e c t of p r e i n c u b a t i o n t e m p e r a t u r e on b o v i n e s e r u m a l b u m i n - d e p e n d e n t lysis of h u m a n e r y t h r o c y t e s t r e a t e d with d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e . H u m a n e r y t h r o c y t e s (107 cells/roll w e r e i n c u b a t e d w i t h d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e (21.1 n m o l / m l ) for v a r i o u s t i m e s at 4 0 ° C ( ' ) , 2 5 ' C (:,), and 10'~C (~). I t e m o l y s i s was m e a s u r e d a f t e r f u r t h e r i n c u b a t i o n for 15 rain at 2 5 ° C w i t h 4 m g / m l of b o v i n e s e r u m albumin.
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Fig. 5. I n c r e a s e in o s m o t i c fragility of h u m a n e r y t h r o c y t e s on t r e a t m e n t with d i l a u r o y l g l y c e r o p h o s p h o choline. E r y t h r o c y t e s w e r e i n c u b a t e d w i t h ( ) a n d w i t h o u t ( e l 21.2 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e for 30 rain at 25~'C. T h e n v a r i o u s a m o u n t s of v c r o n a l - b u f f e r e d saline w e r e a d d e d , the m i x t u r e s w e r e i n c u b a t e d f u r t h e r for 30 m i n a t 25 °C, and h e m o l y s i s was m e a s u r e d . H e m o l y s i s is p l o t t e d against the final o s m o l a r i t y of the r e a c t i o n m i x t u r e .
1 ¢,
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Fig. 6. E f f e c t of s h i f t - d o w n in t e m p e r a t u r e on lysis of h u m a n e r y t h r o c y t e s p r e t r e a t c d with d i l a u r o y l g l y c e r o p h o s p h o e h o l i n e , H u m a n e r y t h r o c y t e s (107 cells]ml) w e r e i n c u b a t e d w i t h 21 n m o l / m l o f d i l a u r o y l glycerophosphocholine at 4 0 " C for 15 m i n . T h e n the r e a c t i o n m i x t u r e w e r e s h i f t e d f r o m 40 t o 0 ° C (X), 1 0 ° C ( ' ) a n d 2 5 ° C (~) a n d i n c u b a t e d f u r t h e r for 15 min. T h e c o n t r o l was i n c u b a t e d at 4 0 ° C w i t h o u t s h i f t - d o w n of the t e m p e r a t u r e ( ' ) .
143
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2'2
Fig. 7. E f f e c t of shift d o w n in t e m p e r a t u r e on t h e d e p e n d e n c e of h e m o l y s i s on d i l a u r o y l g l y e e r o p h o s p h o c h o l i n e c o n c e n t r a t i o n . H u m a n e r y t h r o c y t e s (107 c e l l s / m l ) w e r e i n c u b a t e d w i t h v a r i o u s a m o u n t s of d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e for 10 m i n at 4 0 ° C . T h e n h e m o l y s i s was m e a s u r e d 5 rain a f t e r shifting the t e m p e r a t u r e to 0°C (o) or f u r t h e r at 4 0 ° C (o).
plete within 5 min. Fig. 7 shows the effect of the dilauroylglycerophosphocholine concentration on lysis of erythrocytes incubated first at 40°C for 10 min and then at 0°C for 5 rain: the a m o u n t of phosphatidylcholine required for lysis was less when the temperature was shifted down than when it was not. It should be noted that after the shift-down of temperature lysis was again not complete. Discussion
Reman et al. [2] studied the lysis of beef erythrocytes by a series of shortchain phosphatidylcholines at 20°C; they showed that the minimum concentration of the most active phosphatidylcholine, diundecanoylglycerophosphocholine, was of the same order as that of stearoylglycerophosphocholine. However, with all the phosphatidylcholines tested, the "lag time", that is the time from the beginning of the incubation to the start of lysis, was much longer than with lysocompounds. Arnold and Weltzien [3] also examined the lysis of human erythrocytes by didecanoylglycerophosphocholine; they found that lysis by this phosphatidylcholine had a shorter lag time and was more rapid at 10 than at 37°C. We found that hemolysis of human erythrocytes by dilauroylglycerophosphocholine showed pronounced temperature-dependence, shiftdown in temperature increasing lysis. It is u n k n o w n why temperature affects the rate of hemolysis by phosphatidylcholines. The receptor for lysophosphatidylcholine or short-chain phosphatidylcholines on natural membranes have not been identified, though they could be lipid constituents of the membrane [2]. Liposomal membranes were found to be sensitive to lysophosphatidylcholine when they were in a state of phase separation [6,7]. Short-chain phosphatidylcholine may also interact mainly with parts of erythrocyte membranes showing phase separation. Shiftdown in the incubation temperature was reported to result in damage by lysophosphatidylcholine of liposomal membranes prepared from egg phosphatidyl-
144 choline [6]. It has been suggested that shift-down of the t em perat ure might cause t e m p o r a r y phase separation of lipid c o m p o n e n t s of the bilayer, and this could result in increased hemolysis on shift-down of temperature. In the present investigation on the t e m p e r a t u r e d e p e n d e n c e o f lysis o f erythr o c y tes o f various animals by dilauroylglycerophosphocholine, two types of e r y t h r o c y t e s were distinguished. The rate of lysis of the e r y t h r o c y t e s of cat, dog, cow, calf, pig and sheep increased linearly with increase in the incubation temperature. In contrast, the rate of lysis of human, rat, rabbit, m o n k e y and chicken e r y t h r o c y t e s showed a biphasic relation with the incubation temperature. Previously de Gier et al. [8] showed that the red cells of various animals could be divided into two similar groups on the basis of the t em perat ure dependence o f p en etr at i on of glycerol into the cells. The e r y t h r o e y t e s from the first group described above showed a glycerol permeability which is strongly depend e n t on temperature. On the ot her hand glycerol permeation into the red cells of the second group was f ound to be much less d e p e n d e n t on temperature. Red cells can also be classified into two groups on the basis of their contents of Na ÷ and K + [9]. These classification, however, do not necessarily correspond to the present one. Red cells of calf and pig, which are grouped to the cow t ype either by the glycerol permeability observation or by the present experiment, are involved in the cells of the human group which contains low Na + and high K*. It was shown that after n o n h e m o l y t i c hydrolysis of m em brane phospholipid by purified cobra venom phospholipase A2, addition of bovine plasma albumin (Fraction V) results in hemolysis w i t h o u t any furt her hydrolysis of phospholipid [10,11]. The lysis by albumin does not seem to be entirely due to the ability of albumin to remove cleaved f a t t y acid from the membrane, but may result f r o m its binding to and removal of lyso-derivatives of the m em brane produced by the enzyme. It has also been r e por t ed that 1 tool of albumin binds 1 mol of lysophosphatidylcholine [12,13]. In this study we found that the "lag times" in hemolysis of e r y t h r o c y t e s by didecanoyl- or dilauroylglycerophosphocholine were shortened by addition of albumin to the medium. Hemolysis may occur as follows: first phosphatidyleholine is adsorbed to the surface of the me m br a ne ; then there is a " d e e p e r i n t e r a c t i o n " of the molecules with the membrane, resulting in disorganization of the structural array and change in permeability. During the "lag t i m e " in the hemolysis by phosphatidylcholine, some step in these events may proceed w i t hout causing lysis. In the case of human e r y t h r o c y t e s , the rate of some early step may increase on increasing the incubation temperature, since the state of e r y t h r o c y t e which was sensitive to albumin or to a shift-down of temperature, was reached more rapidly at a higher temperature. Addition of albumin or shift-down of the temperature may stimulate the step inducing a change of organization. It was calculated that when e r y t h r o c y t e s has been treated with 21 nmol of dilauroylglycerophosphoeholine or 6.4 nmol of didecanoylglycerophosphocholine about 30--45 nmol or 7.5 nmol of bovine serum albumin (mol. wt. 67 000), respectively, were required for complete lysis. The binding of dilauroyl- or dideeanoylglyee r o p h o s p h o c h o l i n e to albumin seems to be stoiehiometric, 1 : 1 or 1 : 2, like the binding of lysophosphatidylcholine to albumin. Quantitative experiments are required to confirm that similar values were estimated from experiments on the interaction of short-chain phosphatidylcholine liposomes [14] and of lipo-
145
somes containing lysophosphatidylcholine [15] with albumin. It is interesting that the a m o u n t of dilauroylglycerophosphocholine required for 50% lysis of erythrocytes in the presence of bovine serum albumin is the same as that for 50% lysis of erythrocytes at 10°C. This a m o u n t of dilauroylglycerophosphocholine (4 • 10 -~6 mol/cell), which presumably reacts with erythrocytes in some way, is more than the a m o u n t of phospholipids in erythrocytes (about 2 • 10 -16 mol/cell) [16]. Using benzylated lysophosphatidylcholine (rac-l-alkyl-2-benzylglycero-3-phosphocholine) derivatives, Weltzien [17] showed that at the beginning of hemolysis the molar amounts of three lysophosphatidyleholine derivatives, including stearoylglycerophosphocholine, bound to the red cells were similar (1.5 • 10 -16 mol/cell). This a m o u n t is similar to the a m o u n t required for 50% hemolysis, and is of the same order as the minimum a m o u n t of dilauroylglycerophosphocholine required for 50% lysis, in spite of the fact that lysophosphatidyleholine and dilauroylglycerophosphoeholine exist in different aggregated forms, as spherical mieelles and liposomes, respectively. Recently, Weltzien et al. [18] also reported the aggregation state of the slow reacting lysophosphatidyleholine analog, 1-octadecyl-2-benzytglyeero-3-phosphoeholine, in aqueous solution. Aggregates of this were most similar to those of phosphatidylcholine liposomes than to usual lysophosphatidylcholine spherical micelles. They concluded that this liposomal structure is responsible for the slow adsorption of this lysophosphatidylcholine analog to erythroeytes and they proposed that the rate of adsorption may be determined by the rate of escape of single lysophospholipid molecules from the liposomes. These valuable experiments, together with the present study on dilauroylglycerophosphocholine which also has liposomal structures, will u n d o u b t e d l y stimulate furter investigations. Acknowledgements This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. We thank Dr. T. Abe, National Institute of Animal Industry, for supplying fresh samples of sheep blood. References 1 G o t t f r i e d , E.L. a n d R a p p o r t , M.M. ( 1 9 6 3 ) J. Lipid Res. 4, 5 7 - - 6 2 2 R e m a n , F.C., D e m e l , R . A . , de Gier, J., v a n D e e n e n , L . L . M . , Eibl, H. a n d Westphal, O. ( 1 9 6 9 ) C h e m . Phys. Lipids 3, 2 2 1 - - 2 3 3 3 A r n o l d , D. a n d Weltzien, H . U . ( 1 9 6 8 ) Z. N a t u r f o r s c h g . 2 3 b , 6 7 5 - - 6 8 3 4 K i t a g a w a , T., I n o u e , K. a n d N o j i m a , S. ( 1 9 7 5 ) J. B i o c h e m . 78, 4 3 1 - - 4 3 4 5 I n o u e , K., G r a f , L. and R a p p o r t , M.M. ( 1 9 7 2 ) J. Lipid Res. 13, 1 1 9 - - 1 2 7 6 I n o u e , K. a n d K i t a g a w a , T. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 6 3 , 361- 372 7 I n o u e , K. and K i t a g a w a , T. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 2 6 , 1 - - 1 6 S de Gier, J., v a n D e e n e n , L.L.M. a n d v a n S e n d e n , K . G . ( 1 9 6 6 ) E x p e r i e n t i a 22, 2 0 - - 2 1 9 P r a n k e r d , T . A . J . ( 1 9 6 1 ) in T h e R e d Cell, A n A c c o u n t of its C h e m i c a l P h y s i o l o g y and P a t h o l o g y , pp. 1 6 4 - - 1 6 5 , Blackwell Scientific P u b l i c a t i o n s , O x f o r d 10 Gul, S. a n d S m i t h , A.D. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2SS, 2 3 7 - - 2 4 0 11 Gul, S. a n d S m i t h , A.D. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 367, 2 7 1 - - 2 8 1 12 S w i t z e r , S. a n d E d e r , H.A. ( 1 9 6 5 ) J. L i p i d Res. 6, 5 0 6 - - 5 1 1 13 K l o p f e n s t e i n , W.E. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 8 1 , 3 2 3 - - 3 2 5 14 K i t a g a w a , T., I n o u e , K. a n d N o j i m a , S. ( 1 9 7 6 ) J. B i o c h e m . 79, 1 1 4 7 - - 1 1 5 5 15 K i t a g a w a , T., I n o u e , K. a n d N o j i m a , S. ( 1 9 7 6 ) J. B i o c h e m . 79, 1 1 3 5 - - 1 1 4 5 16 v a n D c e n c n , L.L.M. and de Gier, J. ( 1 9 7 4 ) in T h e Red B l o o d Cell ( S u r g e n o r , D.M., cd.) 2nd e(ln., pp. 1 5 0 - - 1 5 1 , A c a d e m i c Press, N e w Y o r k 17 Weltzien, H . U . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 1 , 6 - - 1 4 18 Wcltzien, H . U . , A r n o l d , B., B l u m e , A. and K a l k o f f , H.G. ( 1 9 7 6 ) C h e m . Phys. Lipids 16, 2 6 7 - - 2 7 5
Biochimica et Biophysica Acla, 467 (1977) 137--145 © Elsevier/North-Holland Biomedical Press
BBA 77702 EFFECTS OF TEMPERATURE AND BOVINE SERUM ALBUMIN ON LYSIS OF ERYTHROCYTES INDUCED BY DILAUROYLGLYCEROPHOSPHOCHOLINE AND DIDECANOYLGLYCEROPHOSPHOCHOLINE TAKAYUKI KITAGAWA *, YUTAKA TANAKA, KEIZO INOUE and SHOSHICHI NOJIMA Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 (Japan)
(Received September 16th, 1976) (Revised manuscript received January 31st, 1977)
Summary The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, mad rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5--10°C and above 40°C, but slow at around 25°C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the a m o u n t of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10°C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the a m o u n t of phosphatidylcholine required for lysis. Gottfried and Rapport [1] studied the hemolytic actions of a series of lysophospholipids on human, rabbit and sheep erythrocytes at 37°C and found that the nature of the linkage of the hydrocarbon chain to glycerol did not influence their hemolytic actions. Reman et al. [2] showed that the acyl chain had a great influence on the lyric actions of phosphatidylcholine, lysophosphatidylcholine and deoxylysophosphatidylcholine on beef erythrocytes, using a series of chemically well-defined derivatives with different fatty acid chains. Among the phosphatidylcholines with chain lengths of 8 to 12 carbon atoms (per acyl I
* P r e s s e n t address: D e p a r t m e n t of V i r o l o g y , I n s t i t u t e of Medical Sciences, U n i v e r s i t y of T o k y o , M i n a t o - k u , T o k y o 108, J a p a n .
138 chain) that th ey examined, di undecanoyl gl ycerophosphochol i ne had the highest activity and dilauroylglycerophosphocholine the least. Arnold and Weltzien [3] also investigated the lytic activities o f the hom ol ogous series o f lysophosphatidylcholine, modified lysophosphatidylcholine and short-chain phosphatidylcholine on human e r y t h r o c y t e s ; t h e y found that didecanoylglycerophosphocholine was lytic but dilauroyl- and dipelargonoylglycerophosphocholine were not. It is interesting that among the various phospholipids tested only d i d e c a n o y l g l y c e r o p h o s p h o c h o l i n e caused m ore rapid lysis o f human erythrocytes at 10 than at 37°C. We are interested in the relation between the structure of the membranes of e r y t h r o c y t e s and the lytic effects of various phospholipids. We studied the lysis of h u man e r y t h r o c y t e s by dilauroylglycerophosphocholine at various temperatures and f o u n d that the rate of lysis showed a biphasic dependence on temperature. We also studied the effects of bovine serum albumin and shift-down of the incubation t e m p e r a t u r e on the lysis of hum an red cells by dilauroylglycerophosphocholine. Preliminary results of these studies have been published [4]. Materials and Methods
Buffer. Veronal-buffered saline free of Ca 2+and Mg 2+was used t hroughout . It contained 0.375 g of sodium 5,5-diethylbarbiturate, 0.575 g of 5,5-diethylbarbituric acid, 8.5 g of sodium chloride per 1 of distilled water, pH 7.5. Erythrocytes. The human e r y t h r o c y t e s used were from freshly drawn, heparinized blood of healthy donors. The blood was centrifuged at 300 X g for 5 rain and the plasma and buf f y coat were discarded. The precipitated cells were then washed three times with veronal buffered saline and used for experiments within 24 h. E r y t h r o c y t e s of ot he r animals were obtained in a similar way. Lipids. Didecanoyl- and dilauroylglycerophosphocholines were obtained from Serdary Research Laboratories Inc., Ontario, Canada and were used witho u t further purification. Vesicles of didecanoyl-, and dilauroylglycerophosphocholine were prepared by suspending the dried sample o f lipid in veronal buffered saline and sonicating the suspensions in a bath-type sonicator for 30 min at r o o m temperature. Bovine serum albumin (crystalline) and f a t t y acid-free bovine albumin were obtained from A r m o u r Pharmaceutical Co., U.S.A. and Nutritional Biochemicals Co., Cleveland, Ohio, xespectively. Measurement of hemolysis. Hemolysis was measured using cells labeled with radioactive c h r o m a t e by the m e t h o d of Inoue et al. [5]. Veronal buffered saline solution containing an appropriate a m o u n t of phosphatidylcholine (0.4 ml) was preincubated for 10 min at the required t em perat ure and the reaction was started by adding 0.1 ml of S'Cr-labeled e r y t h r o c y t e suspension (5- 107 cells/ml) which had been kept in ice-water. The reaction m i xt ure was gently shaken occasionally. After incubation the mixture was centrifuged at 200 X g for 5 min and 250 pl of the supernatant was carefully removed for counting in an auto-gamma c ount er (Aloka Auto-well Gamma Counter). The percentage hemolysis was then calculated as follows: counts in 250 pl of the supernata,~.t X 2 X 100 counts in 2.5 - 106 e r y t h r o c y t e s
139
Results
Effects of temperature on hemolysis of human and cow erythrocytes by dilauroylglycerophosphocholine. When human erythrocytes were incubated with excess dilauroylglycerophosphocholine (21 nmol/ml) at various temperatures for 90 min, the rate of hemolysis was found to depend on the incubation temperature. The rate of lysis was very high above 40 ° C, but with decrease in temperature the rate decreased to a minimum at 30--25°C, and then increased again to a maximum at around 5--10°C, as reported in the previous paper [4]. The amount of phosphatidylcholine required for lysis of human erythrocytes was least at 10°C, and more at 45, 40 and 0°C, in this order. In contrast, the rate of lysis of cow erythrocytes by excess dilauroylglycerophosphocholine (17 nmol/ml) did not show a biphasic dependence on temperature: it was slow at 0--10°C and rapid above 15°C.
Effects of temperature on hemolysis of erythrocytes from various animals by dilauroylglyeerophosphocholine. The sensitivities of erythrocytes of various animals to dilauroylglycerophosphocholine were examined at temperatures between 0 and 40°C and classified into two types on the basis of the results. Monkey, rabbit and rat erythrocytes were of the human type, their hemolysis being rapid at 10 and 40°C but slow at 0 and 25°C (Fig. 1A). The lysis of chicken erythrocytes, was slightly different but it was also more rapid at 0 and 40°C than at 10 or 25°C. In contrast, the lysis of calf, sheep, pig, dog, and cat erythrocytes, like that of cow erythrocytes (Fig. 1B) increased with increase in the incubation temperature.
Effect of temperature on hemolysis of human erythrocytes by didecanoylglycerophosphocholine. Hemolysis of humun erythrocytes by didecanoylglyc-
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Incubation t e m p e r a t u r e (°C) F i g . 1. E f f e c t o f t e m p e r a t u r e o n t h e rate o f h e m o l y s i s o f e r y t h r o c y t e s of various animals by dilauroylglycerophosphocholine. ( A ) H u m a n ( J ) ; m ( m k e y (.'), r a b b i t ( o ) , rat ( 4 ) a n d c h i c k e n (A) e r y t h r o c y t e s w e r e incubated with 21 nmol/ml of dilauroylglycerophosphocholine f o r v a r i o u s p e r i o d s at d i f f e r e n t t e m p e r a t u r e s . ( B ) C o w ('~.), s h e e p ( o ) , d o g ( o ) , c a l f ( a ) , p i g ( i ) a n d c a t (A) e r y t h r o c y t e s w e r e t r e a t e d w i t h d i l a u r o y l glyeerophosphocholine as d e s c r i b e d in A . T h e t i m e s r e q u i r e d f o r 5 0 % h e m o l y s i s are p l o t t e d a g a i n s t t h e incubation temperatures.
140 erophosphocholine also showed a biphasic change with temperature, being faster at 10 and 40°C than at 25°C. Effect of bovine serum albumin on hemolysis of human erythrocytes by dilauroyl- and didecanoylglycerophosphocholine. The lyric activity of lysophosphatidylcholine is known to be inhibited by bovine serum albumin. When dilauroylglycerophosphocholine and didecanoylglycerophosphocholine were preincubated with bovine serum albumin their lytic activities were also inhibited; Table I shows the effects of preincubating dilauroylglycerophosphocholine with bovine serum albumin at 10 or 40°C for 30 min before adding it to erythrocytes. These phosphatidylcholine may combine with bovine serum albumin as lysophosphatidylcholine does. The lysis of human red cells by dilauroylglycerophosphocholine at either 40 or 10°C showed a "lag t i m e " of 30 min, as reported previously. Bovine serum albumin was found to shorten this lag time. Addition of bovine serum albumin to human erythrocytes that had been preincubated with dilauroylglycerophosphocholine for 15 min at 40°C, resulted in rapid and complete hemolysis (Fig. 2). The rate of hemolysis due to addition of phosphatidylcholine plus bovine serum albumin depended on the amounts of both bovine serum albumin (Fig. 3A) and phosphatidylcholine (Fig. 3B). Fig. 3B shows that the presence of bovine serum albumin decreased the concentration of phosphatidylcholine required for complete lysis from 16 nmol/ml to 10 nmol/ml. The presence of bovine serum albumin also reduced the concentration of didecanoylglycerophosphocholine required for lysis of human erythrocytes. Since fatty acid-free albumin had a similar effect of bovine serum albumin, the effect was not be due to contaminating f a t t y acids. Effect of pre-incubation temperature on shortening by bovine serum albumin of the "lag phase" in lysis of human erythrocytes with dilauroylglycerophosphocholine. Bovine serum albumin shortened the lag phase in hemolysis when erythrocytes were preincubated with dilauroylglycerophosphocholine for more than 5 min at 40°C (Fig. 4). At 25°C, about 60 min preincubation was required for complete lysis with bovine serum albumin. However, when the erythrocytes had been preincubated with dilauroylglycerophosphocholine at
TABLE I INHIBITION BY BOVINE SERUM ALBUMIN DILAUROYLGLYCER OPHOSPHOCHOLINE
OF
LYSIS
OF
HUMAN
ERYTHROCYTES
BY
H u m a n e r y t h r o c y t e s ( 1 0 7 c e l l s / m l ) w e r e i n c u b a t e d w i t h 21 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e , 4 m g o f b o v i n e s e r u m a l b u m i n o r m i x t u r e s o f 21 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e a n d 4 m g / m l o f b o v i n e s e r u m a l b u m i n f o r 9 0 m i n a t 1 0 o r 4 0 ° C . H e m o l y s i s w a s m e a s u r e d as d e s c r i b e d i n t e x t . H e m o l y s i s p e r c e n t at i n c u b a t i o n temperature
None Dilauro ylgly cerophosphocholine Dilauroylglycerophosphocholine Bovine serum albumin
+ bovine serum albumia
IO°C
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6.5 94.0 1.7 5.0
13.5 98.0 3.0 6.5
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Fig. 2. E f f e c t of b o v i n e s e r u m a l b u m i n o n t h e " l a g p h a s e " in lysis o f h u m a n e r y t h r o c y t e s b y d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e , E r y t h r o c y t e s (107 c e l l s / m l ) w e r e i n c u b a t e d w i t h 21.1 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e at 4 0 ° C f o r 15 m i n . T h e n s a m p l e s w e r e i n c u b a t e d f u r t h e r w i t h ( ' ) or w i t h o u t (o) 4 mg/ml of bovine serum albumin.
10°C for 30 min no lysis was observed on addition of bovine serum albumin and shift of the temperature to 25°C. Osmotical fragility of human erythrocytes treated with dilauroylglycerophosphocholine. The osmotical fragility of erythrocytes that had been incubated with dilauroylglycerophosphocholine at 25°C for 30 min was less than that of untreated human erythrocytes (Fig. 5). Effect of shift-down in temperature on lysis of human red blood cells pretreated with dilauroylglycerophosphocholine. The lag time in lysis of human erythrocytes treated with dilauroylglycerophosphocholine was also shortened by shifting the temperature from 40 to 10 or 0°C but not from 40 to 25°C during incubation (Fig. 6). The rate of hemolysis was also increasing by shifting the temperature from 30 to 0°C during incubation, but hemolysis was not com-
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Fig. 3. ( A ) E f f e c t o f t h e a m o u n t o f b o v i n e s e r u m a l b u m i n o n lysis o f h u m a n e r y t h r o c y t e s b y d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e . H u m a n e r y t h r o c y e s ( 1 0 7 c e l l s / m l ) w e r e i n c u b a t e d w i t h 21 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e at 4 0 c C f o r 15 m i n . T h e n v a r i o u s a m o u n t s of b o v i n e s e r u m a l b u m i n w e r e a d d e d a n d h e m o l y s i s w a s m e a s u r e d a f t e r f u r t h e r i n c u b a t i o n f o r 15 r a i n at 2 5 ° C . (B) E f f e c t o f t h e a m o u n t o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e o n b o v i n e s e r u m a l b u m i n - d e p e n d e n t lysis o f h u m a n e r y t h r o c y t e s . H u m a n c r y t h r o c y t e s (107 cells/ml) were i n c u b a t e d with various a m o u n t s of d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e for 15 m i n at 4 0 ° C . H e m o l y s i s w a s m e a s u r e d a f t e r f u r t h e r i n c u b a t i o n at 25~'C f o r 15 rain w i t h (o) or w i t h o u t (o) 4 m g / m l o f b o v i n e s e r u m a l b u m i n .
142
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Fig. 4, E f f e c t of p r e i n c u b a t i o n t e m p e r a t u r e on b o v i n e s e r u m a l b u m i n - d e p e n d e n t lysis of h u m a n e r y t h r o c y t e s t r e a t e d with d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e . H u m a n e r y t h r o c y t e s (107 cells/roll w e r e i n c u b a t e d w i t h d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e (21.1 n m o l / m l ) for v a r i o u s t i m e s at 4 0 ° C ( ' ) , 2 5 ' C (:,), and 10'~C (~). I t e m o l y s i s was m e a s u r e d a f t e r f u r t h e r i n c u b a t i o n for 15 rain at 2 5 ° C w i t h 4 m g / m l of b o v i n e s e r u m albumin.
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Fig. 5. I n c r e a s e in o s m o t i c fragility of h u m a n e r y t h r o c y t e s on t r e a t m e n t with d i l a u r o y l g l y c e r o p h o s p h o choline. E r y t h r o c y t e s w e r e i n c u b a t e d w i t h ( ) a n d w i t h o u t ( e l 21.2 n m o l / m l o f d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e for 30 rain at 25~'C. T h e n v a r i o u s a m o u n t s of v c r o n a l - b u f f e r e d saline w e r e a d d e d , the m i x t u r e s w e r e i n c u b a t e d f u r t h e r for 30 m i n a t 25 °C, and h e m o l y s i s was m e a s u r e d . H e m o l y s i s is p l o t t e d against the final o s m o l a r i t y of the r e a c t i o n m i x t u r e .
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(m~n)
Fig. 6. E f f e c t of s h i f t - d o w n in t e m p e r a t u r e on lysis of h u m a n e r y t h r o c y t e s p r e t r e a t c d with d i l a u r o y l g l y c e r o p h o s p h o e h o l i n e , H u m a n e r y t h r o c y t e s (107 cells]ml) w e r e i n c u b a t e d w i t h 21 n m o l / m l o f d i l a u r o y l glycerophosphocholine at 4 0 " C for 15 m i n . T h e n the r e a c t i o n m i x t u r e w e r e s h i f t e d f r o m 40 t o 0 ° C (X), 1 0 ° C ( ' ) a n d 2 5 ° C (~) a n d i n c u b a t e d f u r t h e r for 15 min. T h e c o n t r o l was i n c u b a t e d at 4 0 ° C w i t h o u t s h i f t - d o w n of the t e m p e r a t u r e ( ' ) .
143
~ 0
6O
/
J~E° ~ 4020 / ~,o 01
~
1'o 1~
14 1'6 1'8 ~o
~,mountof dlluuroylglycerophosphochohne (nmol/ml)
2'2
Fig. 7. E f f e c t of shift d o w n in t e m p e r a t u r e on t h e d e p e n d e n c e of h e m o l y s i s on d i l a u r o y l g l y e e r o p h o s p h o c h o l i n e c o n c e n t r a t i o n . H u m a n e r y t h r o c y t e s (107 c e l l s / m l ) w e r e i n c u b a t e d w i t h v a r i o u s a m o u n t s of d i l a u r o y l g l y c e r o p h o s p h o c h o l i n e for 10 m i n at 4 0 ° C . T h e n h e m o l y s i s was m e a s u r e d 5 rain a f t e r shifting the t e m p e r a t u r e to 0°C (o) or f u r t h e r at 4 0 ° C (o).
plete within 5 min. Fig. 7 shows the effect of the dilauroylglycerophosphocholine concentration on lysis of erythrocytes incubated first at 40°C for 10 min and then at 0°C for 5 rain: the a m o u n t of phosphatidylcholine required for lysis was less when the temperature was shifted down than when it was not. It should be noted that after the shift-down of temperature lysis was again not complete. Discussion
Reman et al. [2] studied the lysis of beef erythrocytes by a series of shortchain phosphatidylcholines at 20°C; they showed that the minimum concentration of the most active phosphatidylcholine, diundecanoylglycerophosphocholine, was of the same order as that of stearoylglycerophosphocholine. However, with all the phosphatidylcholines tested, the "lag time", that is the time from the beginning of the incubation to the start of lysis, was much longer than with lysocompounds. Arnold and Weltzien [3] also examined the lysis of human erythrocytes by didecanoylglycerophosphocholine; they found that lysis by this phosphatidylcholine had a shorter lag time and was more rapid at 10 than at 37°C. We found that hemolysis of human erythrocytes by dilauroylglycerophosphocholine showed pronounced temperature-dependence, shiftdown in temperature increasing lysis. It is u n k n o w n why temperature affects the rate of hemolysis by phosphatidylcholines. The receptor for lysophosphatidylcholine or short-chain phosphatidylcholines on natural membranes have not been identified, though they could be lipid constituents of the membrane [2]. Liposomal membranes were found to be sensitive to lysophosphatidylcholine when they were in a state of phase separation [6,7]. Short-chain phosphatidylcholine may also interact mainly with parts of erythrocyte membranes showing phase separation. Shiftdown in the incubation temperature was reported to result in damage by lysophosphatidylcholine of liposomal membranes prepared from egg phosphatidyl-
144 choline [6]. It has been suggested that shift-down of the t em perat ure might cause t e m p o r a r y phase separation of lipid c o m p o n e n t s of the bilayer, and this could result in increased hemolysis on shift-down of temperature. In the present investigation on the t e m p e r a t u r e d e p e n d e n c e o f lysis o f erythr o c y tes o f various animals by dilauroylglycerophosphocholine, two types of e r y t h r o c y t e s were distinguished. The rate of lysis of the e r y t h r o c y t e s of cat, dog, cow, calf, pig and sheep increased linearly with increase in the incubation temperature. In contrast, the rate of lysis of human, rat, rabbit, m o n k e y and chicken e r y t h r o c y t e s showed a biphasic relation with the incubation temperature. Previously de Gier et al. [8] showed that the red cells of various animals could be divided into two similar groups on the basis of the t em perat ure dependence o f p en etr at i on of glycerol into the cells. The e r y t h r o e y t e s from the first group described above showed a glycerol permeability which is strongly depend e n t on temperature. On the ot her hand glycerol permeation into the red cells of the second group was f ound to be much less d e p e n d e n t on temperature. Red cells can also be classified into two groups on the basis of their contents of Na ÷ and K + [9]. These classification, however, do not necessarily correspond to the present one. Red cells of calf and pig, which are grouped to the cow t ype either by the glycerol permeability observation or by the present experiment, are involved in the cells of the human group which contains low Na + and high K*. It was shown that after n o n h e m o l y t i c hydrolysis of m em brane phospholipid by purified cobra venom phospholipase A2, addition of bovine plasma albumin (Fraction V) results in hemolysis w i t h o u t any furt her hydrolysis of phospholipid [10,11]. The lysis by albumin does not seem to be entirely due to the ability of albumin to remove cleaved f a t t y acid from the membrane, but may result f r o m its binding to and removal of lyso-derivatives of the m em brane produced by the enzyme. It has also been r e por t ed that 1 tool of albumin binds 1 mol of lysophosphatidylcholine [12,13]. In this study we found that the "lag times" in hemolysis of e r y t h r o c y t e s by didecanoyl- or dilauroylglycerophosphocholine were shortened by addition of albumin to the medium. Hemolysis may occur as follows: first phosphatidyleholine is adsorbed to the surface of the me m br a ne ; then there is a " d e e p e r i n t e r a c t i o n " of the molecules with the membrane, resulting in disorganization of the structural array and change in permeability. During the "lag t i m e " in the hemolysis by phosphatidylcholine, some step in these events may proceed w i t hout causing lysis. In the case of human e r y t h r o c y t e s , the rate of some early step may increase on increasing the incubation temperature, since the state of e r y t h r o c y t e which was sensitive to albumin or to a shift-down of temperature, was reached more rapidly at a higher temperature. Addition of albumin or shift-down of the temperature may stimulate the step inducing a change of organization. It was calculated that when e r y t h r o c y t e s has been treated with 21 nmol of dilauroylglycerophosphoeholine or 6.4 nmol of didecanoylglycerophosphocholine about 30--45 nmol or 7.5 nmol of bovine serum albumin (mol. wt. 67 000), respectively, were required for complete lysis. The binding of dilauroyl- or dideeanoylglyee r o p h o s p h o c h o l i n e to albumin seems to be stoiehiometric, 1 : 1 or 1 : 2, like the binding of lysophosphatidylcholine to albumin. Quantitative experiments are required to confirm that similar values were estimated from experiments on the interaction of short-chain phosphatidylcholine liposomes [14] and of lipo-
145
somes containing lysophosphatidylcholine [15] with albumin. It is interesting that the a m o u n t of dilauroylglycerophosphocholine required for 50% lysis of erythrocytes in the presence of bovine serum albumin is the same as that for 50% lysis of erythrocytes at 10°C. This a m o u n t of dilauroylglycerophosphocholine (4 • 10 -~6 mol/cell), which presumably reacts with erythrocytes in some way, is more than the a m o u n t of phospholipids in erythrocytes (about 2 • 10 -16 mol/cell) [16]. Using benzylated lysophosphatidylcholine (rac-l-alkyl-2-benzylglycero-3-phosphocholine) derivatives, Weltzien [17] showed that at the beginning of hemolysis the molar amounts of three lysophosphatidyleholine derivatives, including stearoylglycerophosphocholine, bound to the red cells were similar (1.5 • 10 -16 mol/cell). This a m o u n t is similar to the a m o u n t required for 50% hemolysis, and is of the same order as the minimum a m o u n t of dilauroylglycerophosphocholine required for 50% lysis, in spite of the fact that lysophosphatidyleholine and dilauroylglycerophosphoeholine exist in different aggregated forms, as spherical mieelles and liposomes, respectively. Recently, Weltzien et al. [18] also reported the aggregation state of the slow reacting lysophosphatidyleholine analog, 1-octadecyl-2-benzytglyeero-3-phosphoeholine, in aqueous solution. Aggregates of this were most similar to those of phosphatidylcholine liposomes than to usual lysophosphatidylcholine spherical micelles. They concluded that this liposomal structure is responsible for the slow adsorption of this lysophosphatidylcholine analog to erythroeytes and they proposed that the rate of adsorption may be determined by the rate of escape of single lysophospholipid molecules from the liposomes. These valuable experiments, together with the present study on dilauroylglycerophosphocholine which also has liposomal structures, will u n d o u b t e d l y stimulate furter investigations. Acknowledgements This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. We thank Dr. T. Abe, National Institute of Animal Industry, for supplying fresh samples of sheep blood. References 1 G o t t f r i e d , E.L. a n d R a p p o r t , M.M. ( 1 9 6 3 ) J. Lipid Res. 4, 5 7 - - 6 2 2 R e m a n , F.C., D e m e l , R . A . , de Gier, J., v a n D e e n e n , L . L . M . , Eibl, H. a n d Westphal, O. ( 1 9 6 9 ) C h e m . Phys. Lipids 3, 2 2 1 - - 2 3 3 3 A r n o l d , D. a n d Weltzien, H . U . ( 1 9 6 8 ) Z. N a t u r f o r s c h g . 2 3 b , 6 7 5 - - 6 8 3 4 K i t a g a w a , T., I n o u e , K. a n d N o j i m a , S. ( 1 9 7 5 ) J. B i o c h e m . 78, 4 3 1 - - 4 3 4 5 I n o u e , K., G r a f , L. and R a p p o r t , M.M. ( 1 9 7 2 ) J. Lipid Res. 13, 1 1 9 - - 1 2 7 6 I n o u e , K. a n d K i t a g a w a , T. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 6 3 , 361- 372 7 I n o u e , K. and K i t a g a w a , T. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 2 6 , 1 - - 1 6 S de Gier, J., v a n D e e n e n , L.L.M. a n d v a n S e n d e n , K . G . ( 1 9 6 6 ) E x p e r i e n t i a 22, 2 0 - - 2 1 9 P r a n k e r d , T . A . J . ( 1 9 6 1 ) in T h e R e d Cell, A n A c c o u n t of its C h e m i c a l P h y s i o l o g y and P a t h o l o g y , pp. 1 6 4 - - 1 6 5 , Blackwell Scientific P u b l i c a t i o n s , O x f o r d 10 Gul, S. a n d S m i t h , A.D. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2SS, 2 3 7 - - 2 4 0 11 Gul, S. a n d S m i t h , A.D. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 367, 2 7 1 - - 2 8 1 12 S w i t z e r , S. a n d E d e r , H.A. ( 1 9 6 5 ) J. L i p i d Res. 6, 5 0 6 - - 5 1 1 13 K l o p f e n s t e i n , W.E. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 8 1 , 3 2 3 - - 3 2 5 14 K i t a g a w a , T., I n o u e , K. a n d N o j i m a , S. ( 1 9 7 6 ) J. B i o c h e m . 79, 1 1 4 7 - - 1 1 5 5 15 K i t a g a w a , T., I n o u e , K. a n d N o j i m a , S. ( 1 9 7 6 ) J. B i o c h e m . 79, 1 1 3 5 - - 1 1 4 5 16 v a n D c e n c n , L.L.M. and de Gier, J. ( 1 9 7 4 ) in T h e Red B l o o d Cell ( S u r g e n o r , D.M., cd.) 2nd e(ln., pp. 1 5 0 - - 1 5 1 , A c a d e m i c Press, N e w Y o r k 17 Weltzien, H . U . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 1 , 6 - - 1 4 18 Wcltzien, H . U . , A r n o l d , B., B l u m e , A. and K a l k o f f , H.G. ( 1 9 7 6 ) C h e m . Phys. Lipids 16, 2 6 7 - - 2 7 5
