Mechanisms of Ageing and Development, 4 (1975) 137-145
137
© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
C H O L E S T E R O L E X C H A N G E B E T W E E N T H E R E D B L O O D CELLS A N D P L A S M A OF Y O U N G A N D O L D RATS
SAROJ MALHOTRA and DAVID KRITCHEVSKY The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. 19104 (U.S.A.)
(Received July 3, 1974; in revised form January 17, 1975)
SUMMARY The exchange of unesterified cholesterol between plasma and red blood cells of young (1.5 month) and old (1-2 years) rats was studied using [3H]-cholesterol as label. Using labeled plasma and unlabeled red blood cells, no equilibration of specific activity of unesterified cholesterol with the specific activity of cholesterol of red blood cells occurred in either young or old rats. Using labeled red blood cells and unlabeled plasma, equilibration of radioactivity of cholesterol was observed in both young and old rats. However, a more rapid exchange of free cholesterol was observed when labeled old red blood cells were incubated with unlabeled plasma. This apparent paradox may be explained by the existence of a larger, more readily exchangeable pool of free cholesterol in red blood cells of old rats than of young rats. Red blood cells and plasma of old rats contained significantly more free cholesterol than those of young rats but phospholipid levels were comparable.
INTRODUCTION There have been suggestions that the rates of cholesterol metabolism i.e. synthesis, absorption, excretion, degradation and metabolic interconversions, slow with age. Tissue cholesterol is known to be constantly exchanged with blood cholesterol, but apparently, the exact parity of this exchange is different between young and old animals 1-3. It was of interest therefore, to determine if cholesterol exchange between red blood cells (RBC) and plasma also slows witb age and, if so, what role in the exchange is played by RBC and plasma, or its component, the lipoproteins. This study presents a quantification of the in vitro exchange of free cholesterol between RBC and plasma of young and old male Wistar rats. That an isotopic exchange of labeled unesterified cholesterol between RBC and plasma occurs both in vivo and in vitro has been well established 4-11. Two types of experiments were done using blood from young (40-45 days) and old (1-2 years) male Wistar rats: (1) Labeled RBC were incubated with labeled sera and unlabeled RBC were incubated with labeled sera from animals of the same age; (2) Labeled RBC from young rats were incubated with unlabeled sera of old rats and
138 unlabeled RBC of young rats were incubated with labeled sera of old rats and vice versa. The exchange of unesterified cholesterol between cells and sera was measured by following changes in the specific activity of unesterified cholesterol of RBC and serum. The data were derived and presented from a series of three experiments. In many animal tissues, a positive correlation exists between the levels of cholesterol and total lipids and phospholipids 12. Since cholesterol and phospholipids constitute a major part of the total lipids in RBC, it was of interest to evaluate the cholesterol-phospholipid relationship in plasma and RBC of young and old rats. MATERIALS AND METHODS Animals and labeling technique RBC and sera were obtained from young and old male Wistar rats. Blood was labeled in vivo by injecting 2~, #Ci per 10 g of [G-~H]cholesterol in propylene glycol intraperitoneally. The rats were fasted and killed 16 h after injection. Preparation o f R B C and plasma The rats were starved 20 h prior to being killed but were permitted free access to water. The blood was drawn into a tube containing an anticoagulant, acid citrate dextrose (ACD) and maintained at a blood: A C D ratio of 5:1. The RBC were separated from the plasma by centrifugation at 350 × g for 10 min in a refrigerated centrifuge, and then washed three times with a calcium-free Tyrode solution (pH 7.4) containing l g glucose per liter. RBC obtained in this manner were checked under the light microscope and were found to have retained normal morphology. Incubation procedure An aliquot of the plasma and RBC thus obtained was set aside as the zero-time control. Labeled plasma was then incubated with unlabeled RBC and unlabeled plasma was incubated with labeled RBC at 37°C, with gentle agitation. Aliquots were removed at intervals of 1 h up to 6 h, and plasma and RBC were separated by centrifugation (350 × g, 10 min) and the interfacial layer was discarded. Total lipid extracts were prepared from the aliquots of plasma and RBC by extraction with chloroform: methanol (1:1) 13. Aliquots of the lipid extracts were analyzed for unesterified and esterified cholesterol by the method of Kritchevsky et al. 14 and radioactivity was measured according to the procedure of Kritchevsky and Malhotra 15 in a Beckman liquid scintillation spectrometer. All specific activities are expressed in terms of disintegrations per min per mg of cholesterol. Phospholipids were separated by thin layer chromatography according to the method of Abramson and Blecher 16 and visualized by exposure to iodine vapour. After sublimation of the iodine, the marked spots were scraped off the plate into 25 ml testtubes and phospholipids were eluted from Silica gel according to procedure of De Bohner et al. 17 with 1 N methanolic HCl. Phosphorus was determined according to the modified method of Bartlett is. Results were calculated per ml packed RBC volume or plasma.
139
PL ~ YCE = LABELED PLASMA CHOLESTEROL ESTER, YOUNG RATS PL ! YC
• LABELED PLASMA CHOLEBTEROL~ YOUN8 RATS
RBC YC
= UNLABELED RED BLOOD CELLS CHOLESTEROL, YOUNG RATS
60 SO-~
\
~' 4 0 ~ ~ * ~ ' ~ • " ~ * -
~ 30o
20"
PL'* YCE
o~..o~.•
PL~ YC
I0"
RBC YC I
2
3
4
S
6
HOURS
Fig. 1. The exchange of cholesterol between unlabeled RBC (RBC YC) and labeled plasma (PL* YC) of young rats during incubation of blood at 37°C.
RESULTS AND DISCUSSION All exchange data (Figs. 1-8) represent averages of three separate experiments; sera were pooled for each experiment. The exchange of unesterified cholesterol between [aH]-cholesterol-labeled plasma and unlabeled RBC of young rats was demonstrated by the increase in the specific activity of the unesterified cholesterol of the RBC (Fig. 1). The specific activities of the cholesterol of both fractions stabilized at about 4 h but never equilibrated. Similar results were obtained with labeled plasma and unlabeled RBC of old rats (Fig. 2). The specific activities of cholesterol of plasma and red blood cells stabilized about 3 h after incubation. N o hemolysis of RBC was observed in these experiments.
PL ~ OC
• LABELED PLASMA CHOLESTEROL m OLD RATB
PL w ODE • LABELED PLASMA CHOLESTEROL EBTER~ OLD RATS RBD OC • UNLABELED RED BLOOD CELLS CHOLESTEROLt OLD RATS 60'
E
40'
20'50' ~ • e . ~•, ~ = : : ~ l a ~ SO"
Q
•
PL ~ OCE
I O ' [ w ~ m ~ . ~ , ~ l ~ g ~ m RSC OC I
2
3
4
5
6
HOURS
Fig. 2. The exchange of cholesterol between unlabeled RBC (RBC OC) and labeled plasma (PL* OC) of old rats during incubation of blood at 37 °C.
140
PL YC
= UNLABELED PLASMA CHOLESTEROL, YOUNG RATS
PL YCE
= U N L A B E L E D PLASMA CHOLESTEROL EBTER~ YOUNG RATS
BBC m YC = L A B E L E D RED BLOOD CELLS C H O L E S T E R O L , YOUNG BATS
6050-
' ~
/ / / ~
I1::~=
40-
RBC ~ YC
30=
/
2010
PL YC
~/
.~A~A~A~* I
2
5
4
PL YCE 5
6
HOURS
Fig. 3. Changes in specific activities of cholesterol of erythrocytes (RBC* YC) and plasma (PL YC) of young rats during incubation at 37°C.
The exchange in vitro of cholesterol from labeled RBC to unlabeled plasma of young rats, is shown by an increase in specific activity of the unesterified cholesterol in plasma (Fig. 3). Equilibration of isotopic cholesterol between unlabeled plasma and labeled RBC took place at about 4 h after incubation. The specific activity of unesterified cholesterol of plasma at the end of 6 h of incubation did not appreciably exceed the specific activity of unesterified cholesterol of RBC. Figure 4 shows the exchange of cholesterol between labeled RBC and unlabeled plasma of old rats. The specific activities of cholesterol of plasma and of RBC equilibrated at about 2 h after incubation. The specific activity of unesterified cholesterol of plasma exceeded that of cholesterol of RBC at about 2 h and continued to exceed it for the 6 h incubation period studied. In both young and old rats, the mass ratio of unesterified cholesterol of plasma to unesterified cholesterol of RBC was 1:3.
PL OC
:
UNLABELED P L A S M A CHOLESTEROL, OLD RATS
PL OCE
= UNLABELED P L A S M A CHOLEBTEROL ESTER, OLD RATS
RBC e OC = L A B E L E D REO BLOOD CELLS CHOLESTEROL, OLD RATS
60'
e•
50' o, E
1a
40' SO"
PL OC
20" I0" •
•
2
•
*
3 4 HOURS
5
6
Fig. 4. Changes in specific activities of RBC (RBC* OC) and plasma (PL OC) free cholesterol of old rats during incubation of blood at 37 °C.
141
l
PL~t YC
160 1404=
PL~YCE = LABELED PLASMA CHOLESTEROL ESTER, YOUNG RATS
i 120.a .~ 100'
O
B0
a.
RBC OC = UNLABELED RED BLOOD CELLS CHOLESTEROL, OLD RATS
~
:E
= LABELED PLASMA CHOLESTEROL, YOUNG RATS
-
•
s * ~
60.
•
PL
YCE
~e
40, I'~'~e
PL~ YC
20 .s=.....m~m~e~=~=
RBC 0C
HOURS
Fig. 5, Changes in specific activities of plasma free cholesterol (PL* YC) of young rats and erythrocyte cholesterol (RBC OC) of old rats during incubation of blood at 37°C.
These data impIy the presence of one or more non-exchangeable pools in the system. That such a non-exchangeable pool may reside in RBC has also been indicated by Bell and Schwartz 9 and Bell et al. 19. These workers have suggested that the distinction between exchangeable and non-exchangeable cholesterol may be that the less readily exchangeable cholesterol is part o f the structure of the membrane and the readily exchangeable cholesterol is bound to the membrane surface. Hence, the specific activity of the unesterified cholesterol of the RBC is less than the specific activity of the readily exchangeable pool of cholesterol in the RBC. Our data show that the exchangeable pool o f cholesterol in the RBC o f old animals was larger than that of young animals. Figure 5 shows specific activity curves as a function of incubation time, where labeled plasma o f young rats is incubated with unlabeled RBC of old rats. The specific activities of unesterified cholesterol of plasma and RBC nearly leveled off. When old
PL w OC
= LABELED PLASMA CHOLESTEROL, OLD RATS
PL w OCE = LABELEO PLASMA CHOLESTEROL ESTER, OLD RATS; RBC YC
= UNLABELED RED BLOOD CELLS CHOLESTEROL, YOUNG RATS
50"
40£ 30 20 'A,....A
~'~--'~ •
m...,m~m__m__,m~m.~l PL~ DeE L . . . . . '," RBC YC I
2
3
4
5
6
HOURS
Fig. 6. Changes in specific activities of plasma free cholesterol (PL* OC) of old rats and RBC cholesterol (RBC YC) of young rats during incubation of blood at 37°C.
142
PL YC
= UNLABELED PLASMA C H O L E S T E R O L , YOUNG RATS
PL YCE
:
UNLABELED PLASMA CHOLESTEROL ESTER= YOUNG RATS
RBC • OC = L A B E L E D RED BLOOD C E L L S C H O L E S T E R O L = OLD RATS
40 E '~
30'
.e'-~-¢ m q ~ l l ~ "
~ ' "
20"
R R C * OC
e/
P L YC
:E
== , 0 . / *
4
•
•
*
•
I
2
3
4
5
6
PL YCE
HOURS
Fig. 7. The exchange of cholesterol between cholesterol of RBC (RBC* OC) of old rats and cholesterol of plasma (PL YC) of young rats during incubation of blood at 37 °C. rat plasma is labeled (Fig. 6) specific activities of the unesterified cholesterol of plasma and RBC leveled off, although the specific activity of RBC cholesterol appeared to be increasing. When RBC of old rats were labeled and incubated with plasma of young rats, there was an equilibration of the specific activities of cholesterol at about 3 h, and the two specific activities remained about the same for the 6 h incubation period studied (Fig. 7). On the other hand, when " y o u n g " RBC were labeled and incubated with unlabeled " o l d " plasma the equilibration of specific activities of cholesterol occurred at about 4 h, and then the specific activity of cholesterol of " o l d " plasma tended to increase (Fig. 8). Unesterified cholesterol of plasma has generally been considered to be a single pool system 11. Our results could be explained by assuming either that plasma cholesterol behaves as at least a two pool system, or that the lipoproteins of the plasma of old rats have greater avidity for cholesterol i.e. contain a larger number of cholesterol binding sites.
PL OC
= UNLABELED PLASMA CHOLESTEROL, OLD RATS
PL OCE
= UNLABELED PLASMA CHOLESTEROL E S T E R , OLD RATS
180,
160 J
RBC m YC • LABELED RED BLOOD CELLS CHOLESTEROL, YOUNG RATS
140 120I00•
R B C ~ YC
80-
o 60-
/
4020' ~ . ¢ A l ~ l ~ l ~ ' I
' 2
' 3
' 4
' 5
PL OCE ' 6"
HOURS
Fig. 8. The exchange of cholesterol between cholesterol of RBC (RBC* YC) of young rats and cholesterol of plasma (PL OC) of old rats during incubation of blood at 37°C.
143 TABLE I CHOLESTEROL AND LIPID PHOSPHOROUS IN RBC AND PLASMA OF YOUNG 1 AND OLD 2 RATS. (mg/ml -E SEM; 6 DETERMINATIONS) Young rats RBC
Free cholesterol Lipid phosphorus C/P Ester cholesterol CE/P
Old rats Plasma
0.334 ~ 0.020 0.077 ± 0.004 4.34 ---
0.106 ± 0.015 0.029 ~ 0.002 3.67 0.370 ~ 0.027 12.81
RBC
Plasma
0.598 i 0.038* 0.077 ± 0.004 7.72 ---
0.191 ± 0.014"* 0.027± 0.005 7.06 0.605 ± 0.068** 22.36
* p < 0.001 ; ** p < 0.01 ; 1 1.5 months; 2 12-24 months. O u r results indicating t h a t esterified cholesterol is n o t exchanged to any appreciable extent between R B C and p l a s m a o f either y o u n g or old rats, confirms similar o b s e r v a t i o n s by H a g e r m a n a n d G o u l d 4 an d Bell et al. 9,19. Th er e is a slight change in p l a s m a cholesterol ester, however, which m i g h t be the result o f transesterification between labeled free and unlabeled ester cholesterol. Since cholesterol is associated with p h o s p h o l i p i d s in m e m b r a n e s , we t h o u g h t t h a t elucidation o f the lipid c o m p o s i t i o n o f p l a s m a and R B C m i g h t offer chemical evidence to explain o u r observations. T h e chemical c o m p o s i t i o n o f the lipids o f R B C an d p l a s m a is given in T a b l e I. T h e r e is no difference in the p h o s p h o l i p i d c o n t e n t between y o u n g a n d old rats. H o w e v e r , a highly significant difference was seen in the cholesterol an d cholesterol ester c o n t e n t o f b l o o d o f y o u n g an d old rats, with the ratio o f cholesterol to p h o s p h o l i p i d higher in the R B C an d p l a s m a o f the older rats. Since cholesterol is k n o w n to be a m e m b r a n e stabilizing c o m p o u n d in c o m p l e x with phos-
TABLE II PHOSPHOLIPIDS OF PLASMA AND RBC OF YOUNG 1 AND OLD 2 RATS (RESULTS AS LIPID PHOSPHORUS, /tg/ml ~- SEM; AVERAGE OF 6 DETERMINATIONS) Phospholipids*
Young rats Plasma
PI LPC + PS Sph PC PE PGP + PA Total
Old rats RBC
6.06 ~ 0.7 7.89 ~z 1.0 2.43 ~_ 0.4 8.86-L 1.0 2.83 ± 0.5 13.64 :~ 2.0 15.29 ± 2.0 36.50 ± 2.0 1.65 :~ 0.8 8.48 ~ 2.0 0.65 5_ 0.1 1.31 ± 0.2 28.91 76.68
RBC/ Plasma
Plasma
RBC
1.30 3.65 4.82 2.39 5.14 2.00 2.65
4.20 ~_ 0.6 8,80 ± 2.0 2.64 ~ 0.5 11.37 ± 0.8 3.91 ~ 0.8 12,00 + 0.7 14.54 ± 3.0 36,51 ± 2.0 1.12 ± 0.2 7,46 ± 2.0 0.65 ± 0.2 1,30 ± 0.3 27.06 77,44
RBC/ Plasma
2.10 4.31 3.07 2.51 6.66 2.00 2.86
1 1.5 months; 2 12-24 months; *PI = phosphatidyl inositol; LPC = lysophosphatidyl choline; PS = phosphatidyl serine; Sph ~ sphingomyelin; PC = phosphatidyl choline; PE ~ phosphatidyl ethanolamine; PGP = phosphoglycerol phosphate; PA -- phosphatidic acid.
144 p h o l i p i d s 20, the possibility t h a t this increase in r a t i o m a y p l a y some role in cholesterol exchange c a n n o t be ruled out. Table I I gives the p e r c e n t d i s t r i b u t i o n o f various p h o s p h a t i d e s in the p l a s m a a n d R B C o f y o u n g a n d old rats. There were differences in levels o f p l a s m a a n d R B C p h o s p h a t i d e s between y o u n g a n d old cells. W h i l e none o f the differences were statistically significant several were appreciable. P h o s p h a t i d y l inositol levels were higher in y o u n g p l a s m a (31 ~ ) a n d lower in R B C ( - - 1 2 ~ ) . Lysolecithin plus serine levels were lower in b o t h y o u n g p l a s m a ( - - 9 ~ ) a n d R B C ( - - 2 8 ~ ) . S p h i n g o m y e l i n levels were lower in y o u n g p l a s m a ( - - 3 8 700)a n d higher in y o u n g R B C (12 ~ ) a n d p h o s p h a t i d y l e t h a n o l a m i n e was present at a higher level in b o t h y o u n g p l a s m a (32 ~ ) a n d R B C (12 ~ ) . There were no differences in lecithin or p h o s p h a t i d i c acid plus cardiolipin levels. The involvement o f these p h o s p h a t i d e s in cholesterol exchange is under investigation. In view o f d a t a presented above, it is evident that the cholesterol exchange in b l o o d o f old rats does n o t follow the slower cholesterol t u r n o v e r p a t t e r n r e p o r t e d by H r u z a a n d his colleagues 1-3 in the other tissues o f old animals. ACKNOWLEDGEMENTS This investigation was s u p p o r t e d , in part, by U S P H S research grants HL-03299 a n d HL-05209 a n d research Career A w a r d HL-00734 f r o m the N a t i o n a l Institutes o f Health.
REFERENCES 1 Z. Hruza, A. Babick)~ and V. Hlavfi.~kovfi.,Binding of cholesterol to the rat aorta and collagen fibres during ageing and atherosclerosis in vitro, Exp. Gerontol., 2 (1967) 101-107. 2 Z. Hruza and M. Wachtlovft, Decrease of cholesterol turnover in old rats, Exp. Gerontol., 4 (1969) 245-250. 3 Z. Hruza, Increase of cholesterol turnover of old rats connected by parabiosis with young rats. Exp. GerontoL, 6 (1971) 103-107. 4 J. S. Hagerman and R. G. Gould, The in vitro interchange of cholesterol between plasma and red cells. Proc. Soc. Exp. Biol. Med., 78 (1951) 329-332. 5 N. E. Eckles, C. B. Taylor, D. J. Campbell and R. G. Gould, The origin of plasma cholesterol and the rates of equilibration of liver, plasma, and erythrocyte cholesterol, J. Lab. Clin. Med., 46 (1955) 359-371. 6 J. Avigan, A method for incorporating cholesterol and other lipides into serum lipoproteins in vitro, J. Biol. Chem., 234 (1959) 787-790. 7 J. R. Murphy, Erythrocyte metabolism. IV. Equilibration of cholesterol-4-C14 between erythrocytes and variously treated sera, J. Lab. Clin. Med., 60 (1962) 571-578. 8 S. H. Quarfordt and H. L. Hilderman, Quantitation of the in vitro free cholesterol exchange of human red cells and lipoproteins, J. Lipid Res., 11 (1970) 528-535. 9 F. P. Bell and C. J. Schwartz, Exchangeability of cholesterol between swine serum lipoproteins and erythrocytes, in vitro, Biochim. Biophys. Acta, 231 (1971) 553-557. 10 C. Sardet, H. Hansma and R. Ostwald, Effects of plasma lipoproteins from control and cholesterol-fed guinea pigs on red cell morphology and cholesterol content: an in vitro study, J. Lipid Res., 13 (1972) 705-715. 11 F. d'Hollander and F. Chevallier, Movement of cholesterol in vitro in rat blood and quantitation of the exchange of free cholesterol between plasma and erythrocytes. 3". Lipid Res., 13 (1972) 733-744. 12 S. Patton, Correlative relationship of cholesterol and sphingomyelin in cell membranes, J. Theoret. BioL, 29 (1970) 489-491.
145 13 J. T. Dodge, C. Mitchell and D. J. Hanahan, The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes, Arch. Biochem. Biophys., 100 (1963) 119-130. 14 D. Kritchevsky, L. M. Davidson, H. K. Kim and S. Malhotra, Quantitation of serum lipids by a simple TLC-charring method, Clin. Ck,im. Acta, 46 (1973) 63-68. 15 D. Kritchevsky and S. Malhctra, Recovery of lipids from thin-layer chromatography for radioassay, J. Chromatogr., 52 (1970) 498~J,99. 16 D. Abramson and M. Rlecher, Quantitative two dimensional thin-layer cbrcmatcgraphy of naturally occurring phospholipids, J. Lipid Res., 5 (1964) 628. 17 L, S. De Bohner, E. F. Soto and T. De Cohan, Quantitative analysis of phospholipids by thinlayer chromatography, J. Chromatogr., 17 (1965) 513-519. 18 G. R. Bartlett, Phosphorus assay in column chromatography, J. Biol. Chem., 234 (1959) 466-468. 19 F. P. Bell, J. B. Somcr, I. H. Craig and C. J. Schwartz, Membrane-lipid exchange: Exchange of cholesterol between porcine serum lipoproteins and erythrecytes, Pathology, 4 (1972) 205-214. 20 L. L. M. Van Deenen and J. De Gier, Chemical composition and metabolism of lipids in red cells of various animal species. In C. Bishop and D. M. Surgenor (eds.). The Red Blood Cell, Academic Press, New York, 1964, p. 243-307.
137
© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
C H O L E S T E R O L E X C H A N G E B E T W E E N T H E R E D B L O O D CELLS A N D P L A S M A OF Y O U N G A N D O L D RATS
SAROJ MALHOTRA and DAVID KRITCHEVSKY The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. 19104 (U.S.A.)
(Received July 3, 1974; in revised form January 17, 1975)
SUMMARY The exchange of unesterified cholesterol between plasma and red blood cells of young (1.5 month) and old (1-2 years) rats was studied using [3H]-cholesterol as label. Using labeled plasma and unlabeled red blood cells, no equilibration of specific activity of unesterified cholesterol with the specific activity of cholesterol of red blood cells occurred in either young or old rats. Using labeled red blood cells and unlabeled plasma, equilibration of radioactivity of cholesterol was observed in both young and old rats. However, a more rapid exchange of free cholesterol was observed when labeled old red blood cells were incubated with unlabeled plasma. This apparent paradox may be explained by the existence of a larger, more readily exchangeable pool of free cholesterol in red blood cells of old rats than of young rats. Red blood cells and plasma of old rats contained significantly more free cholesterol than those of young rats but phospholipid levels were comparable.
INTRODUCTION There have been suggestions that the rates of cholesterol metabolism i.e. synthesis, absorption, excretion, degradation and metabolic interconversions, slow with age. Tissue cholesterol is known to be constantly exchanged with blood cholesterol, but apparently, the exact parity of this exchange is different between young and old animals 1-3. It was of interest therefore, to determine if cholesterol exchange between red blood cells (RBC) and plasma also slows witb age and, if so, what role in the exchange is played by RBC and plasma, or its component, the lipoproteins. This study presents a quantification of the in vitro exchange of free cholesterol between RBC and plasma of young and old male Wistar rats. That an isotopic exchange of labeled unesterified cholesterol between RBC and plasma occurs both in vivo and in vitro has been well established 4-11. Two types of experiments were done using blood from young (40-45 days) and old (1-2 years) male Wistar rats: (1) Labeled RBC were incubated with labeled sera and unlabeled RBC were incubated with labeled sera from animals of the same age; (2) Labeled RBC from young rats were incubated with unlabeled sera of old rats and
138 unlabeled RBC of young rats were incubated with labeled sera of old rats and vice versa. The exchange of unesterified cholesterol between cells and sera was measured by following changes in the specific activity of unesterified cholesterol of RBC and serum. The data were derived and presented from a series of three experiments. In many animal tissues, a positive correlation exists between the levels of cholesterol and total lipids and phospholipids 12. Since cholesterol and phospholipids constitute a major part of the total lipids in RBC, it was of interest to evaluate the cholesterol-phospholipid relationship in plasma and RBC of young and old rats. MATERIALS AND METHODS Animals and labeling technique RBC and sera were obtained from young and old male Wistar rats. Blood was labeled in vivo by injecting 2~, #Ci per 10 g of [G-~H]cholesterol in propylene glycol intraperitoneally. The rats were fasted and killed 16 h after injection. Preparation o f R B C and plasma The rats were starved 20 h prior to being killed but were permitted free access to water. The blood was drawn into a tube containing an anticoagulant, acid citrate dextrose (ACD) and maintained at a blood: A C D ratio of 5:1. The RBC were separated from the plasma by centrifugation at 350 × g for 10 min in a refrigerated centrifuge, and then washed three times with a calcium-free Tyrode solution (pH 7.4) containing l g glucose per liter. RBC obtained in this manner were checked under the light microscope and were found to have retained normal morphology. Incubation procedure An aliquot of the plasma and RBC thus obtained was set aside as the zero-time control. Labeled plasma was then incubated with unlabeled RBC and unlabeled plasma was incubated with labeled RBC at 37°C, with gentle agitation. Aliquots were removed at intervals of 1 h up to 6 h, and plasma and RBC were separated by centrifugation (350 × g, 10 min) and the interfacial layer was discarded. Total lipid extracts were prepared from the aliquots of plasma and RBC by extraction with chloroform: methanol (1:1) 13. Aliquots of the lipid extracts were analyzed for unesterified and esterified cholesterol by the method of Kritchevsky et al. 14 and radioactivity was measured according to the procedure of Kritchevsky and Malhotra 15 in a Beckman liquid scintillation spectrometer. All specific activities are expressed in terms of disintegrations per min per mg of cholesterol. Phospholipids were separated by thin layer chromatography according to the method of Abramson and Blecher 16 and visualized by exposure to iodine vapour. After sublimation of the iodine, the marked spots were scraped off the plate into 25 ml testtubes and phospholipids were eluted from Silica gel according to procedure of De Bohner et al. 17 with 1 N methanolic HCl. Phosphorus was determined according to the modified method of Bartlett is. Results were calculated per ml packed RBC volume or plasma.
139
PL ~ YCE = LABELED PLASMA CHOLESTEROL ESTER, YOUNG RATS PL ! YC
• LABELED PLASMA CHOLEBTEROL~ YOUN8 RATS
RBC YC
= UNLABELED RED BLOOD CELLS CHOLESTEROL, YOUNG RATS
60 SO-~
\
~' 4 0 ~ ~ * ~ ' ~ • " ~ * -
~ 30o
20"
PL'* YCE
o~..o~.•
PL~ YC
I0"
RBC YC I
2
3
4
S
6
HOURS
Fig. 1. The exchange of cholesterol between unlabeled RBC (RBC YC) and labeled plasma (PL* YC) of young rats during incubation of blood at 37°C.
RESULTS AND DISCUSSION All exchange data (Figs. 1-8) represent averages of three separate experiments; sera were pooled for each experiment. The exchange of unesterified cholesterol between [aH]-cholesterol-labeled plasma and unlabeled RBC of young rats was demonstrated by the increase in the specific activity of the unesterified cholesterol of the RBC (Fig. 1). The specific activities of the cholesterol of both fractions stabilized at about 4 h but never equilibrated. Similar results were obtained with labeled plasma and unlabeled RBC of old rats (Fig. 2). The specific activities of cholesterol of plasma and red blood cells stabilized about 3 h after incubation. N o hemolysis of RBC was observed in these experiments.
PL ~ OC
• LABELED PLASMA CHOLESTEROL m OLD RATB
PL w ODE • LABELED PLASMA CHOLESTEROL EBTER~ OLD RATS RBD OC • UNLABELED RED BLOOD CELLS CHOLESTEROLt OLD RATS 60'
E
40'
20'50' ~ • e . ~•, ~ = : : ~ l a ~ SO"
Q
•
PL ~ OCE
I O ' [ w ~ m ~ . ~ , ~ l ~ g ~ m RSC OC I
2
3
4
5
6
HOURS
Fig. 2. The exchange of cholesterol between unlabeled RBC (RBC OC) and labeled plasma (PL* OC) of old rats during incubation of blood at 37 °C.
140
PL YC
= UNLABELED PLASMA CHOLESTEROL, YOUNG RATS
PL YCE
= U N L A B E L E D PLASMA CHOLESTEROL EBTER~ YOUNG RATS
BBC m YC = L A B E L E D RED BLOOD CELLS C H O L E S T E R O L , YOUNG BATS
6050-
' ~
/ / / ~
I1::~=
40-
RBC ~ YC
30=
/
2010
PL YC
~/
.~A~A~A~* I
2
5
4
PL YCE 5
6
HOURS
Fig. 3. Changes in specific activities of cholesterol of erythrocytes (RBC* YC) and plasma (PL YC) of young rats during incubation at 37°C.
The exchange in vitro of cholesterol from labeled RBC to unlabeled plasma of young rats, is shown by an increase in specific activity of the unesterified cholesterol in plasma (Fig. 3). Equilibration of isotopic cholesterol between unlabeled plasma and labeled RBC took place at about 4 h after incubation. The specific activity of unesterified cholesterol of plasma at the end of 6 h of incubation did not appreciably exceed the specific activity of unesterified cholesterol of RBC. Figure 4 shows the exchange of cholesterol between labeled RBC and unlabeled plasma of old rats. The specific activities of cholesterol of plasma and of RBC equilibrated at about 2 h after incubation. The specific activity of unesterified cholesterol of plasma exceeded that of cholesterol of RBC at about 2 h and continued to exceed it for the 6 h incubation period studied. In both young and old rats, the mass ratio of unesterified cholesterol of plasma to unesterified cholesterol of RBC was 1:3.
PL OC
:
UNLABELED P L A S M A CHOLESTEROL, OLD RATS
PL OCE
= UNLABELED P L A S M A CHOLEBTEROL ESTER, OLD RATS
RBC e OC = L A B E L E D REO BLOOD CELLS CHOLESTEROL, OLD RATS
60'
e•
50' o, E
1a
40' SO"
PL OC
20" I0" •
•
2
•
*
3 4 HOURS
5
6
Fig. 4. Changes in specific activities of RBC (RBC* OC) and plasma (PL OC) free cholesterol of old rats during incubation of blood at 37 °C.
141
l
PL~t YC
160 1404=
PL~YCE = LABELED PLASMA CHOLESTEROL ESTER, YOUNG RATS
i 120.a .~ 100'
O
B0
a.
RBC OC = UNLABELED RED BLOOD CELLS CHOLESTEROL, OLD RATS
~
:E
= LABELED PLASMA CHOLESTEROL, YOUNG RATS
-
•
s * ~
60.
•
PL
YCE
~e
40, I'~'~e
PL~ YC
20 .s=.....m~m~e~=~=
RBC 0C
HOURS
Fig. 5, Changes in specific activities of plasma free cholesterol (PL* YC) of young rats and erythrocyte cholesterol (RBC OC) of old rats during incubation of blood at 37°C.
These data impIy the presence of one or more non-exchangeable pools in the system. That such a non-exchangeable pool may reside in RBC has also been indicated by Bell and Schwartz 9 and Bell et al. 19. These workers have suggested that the distinction between exchangeable and non-exchangeable cholesterol may be that the less readily exchangeable cholesterol is part o f the structure of the membrane and the readily exchangeable cholesterol is bound to the membrane surface. Hence, the specific activity of the unesterified cholesterol of the RBC is less than the specific activity of the readily exchangeable pool of cholesterol in the RBC. Our data show that the exchangeable pool o f cholesterol in the RBC o f old animals was larger than that of young animals. Figure 5 shows specific activity curves as a function of incubation time, where labeled plasma o f young rats is incubated with unlabeled RBC of old rats. The specific activities of unesterified cholesterol of plasma and RBC nearly leveled off. When old
PL w OC
= LABELED PLASMA CHOLESTEROL, OLD RATS
PL w OCE = LABELEO PLASMA CHOLESTEROL ESTER, OLD RATS; RBC YC
= UNLABELED RED BLOOD CELLS CHOLESTEROL, YOUNG RATS
50"
40£ 30 20 'A,....A
~'~--'~ •
m...,m~m__m__,m~m.~l PL~ DeE L . . . . . '," RBC YC I
2
3
4
5
6
HOURS
Fig. 6. Changes in specific activities of plasma free cholesterol (PL* OC) of old rats and RBC cholesterol (RBC YC) of young rats during incubation of blood at 37°C.
142
PL YC
= UNLABELED PLASMA C H O L E S T E R O L , YOUNG RATS
PL YCE
:
UNLABELED PLASMA CHOLESTEROL ESTER= YOUNG RATS
RBC • OC = L A B E L E D RED BLOOD C E L L S C H O L E S T E R O L = OLD RATS
40 E '~
30'
.e'-~-¢ m q ~ l l ~ "
~ ' "
20"
R R C * OC
e/
P L YC
:E
== , 0 . / *
4
•
•
*
•
I
2
3
4
5
6
PL YCE
HOURS
Fig. 7. The exchange of cholesterol between cholesterol of RBC (RBC* OC) of old rats and cholesterol of plasma (PL YC) of young rats during incubation of blood at 37 °C. rat plasma is labeled (Fig. 6) specific activities of the unesterified cholesterol of plasma and RBC leveled off, although the specific activity of RBC cholesterol appeared to be increasing. When RBC of old rats were labeled and incubated with plasma of young rats, there was an equilibration of the specific activities of cholesterol at about 3 h, and the two specific activities remained about the same for the 6 h incubation period studied (Fig. 7). On the other hand, when " y o u n g " RBC were labeled and incubated with unlabeled " o l d " plasma the equilibration of specific activities of cholesterol occurred at about 4 h, and then the specific activity of cholesterol of " o l d " plasma tended to increase (Fig. 8). Unesterified cholesterol of plasma has generally been considered to be a single pool system 11. Our results could be explained by assuming either that plasma cholesterol behaves as at least a two pool system, or that the lipoproteins of the plasma of old rats have greater avidity for cholesterol i.e. contain a larger number of cholesterol binding sites.
PL OC
= UNLABELED PLASMA CHOLESTEROL, OLD RATS
PL OCE
= UNLABELED PLASMA CHOLESTEROL E S T E R , OLD RATS
180,
160 J
RBC m YC • LABELED RED BLOOD CELLS CHOLESTEROL, YOUNG RATS
140 120I00•
R B C ~ YC
80-
o 60-
/
4020' ~ . ¢ A l ~ l ~ l ~ ' I
' 2
' 3
' 4
' 5
PL OCE ' 6"
HOURS
Fig. 8. The exchange of cholesterol between cholesterol of RBC (RBC* YC) of young rats and cholesterol of plasma (PL OC) of old rats during incubation of blood at 37°C.
143 TABLE I CHOLESTEROL AND LIPID PHOSPHOROUS IN RBC AND PLASMA OF YOUNG 1 AND OLD 2 RATS. (mg/ml -E SEM; 6 DETERMINATIONS) Young rats RBC
Free cholesterol Lipid phosphorus C/P Ester cholesterol CE/P
Old rats Plasma
0.334 ~ 0.020 0.077 ± 0.004 4.34 ---
0.106 ± 0.015 0.029 ~ 0.002 3.67 0.370 ~ 0.027 12.81
RBC
Plasma
0.598 i 0.038* 0.077 ± 0.004 7.72 ---
0.191 ± 0.014"* 0.027± 0.005 7.06 0.605 ± 0.068** 22.36
* p < 0.001 ; ** p < 0.01 ; 1 1.5 months; 2 12-24 months. O u r results indicating t h a t esterified cholesterol is n o t exchanged to any appreciable extent between R B C and p l a s m a o f either y o u n g or old rats, confirms similar o b s e r v a t i o n s by H a g e r m a n a n d G o u l d 4 an d Bell et al. 9,19. Th er e is a slight change in p l a s m a cholesterol ester, however, which m i g h t be the result o f transesterification between labeled free and unlabeled ester cholesterol. Since cholesterol is associated with p h o s p h o l i p i d s in m e m b r a n e s , we t h o u g h t t h a t elucidation o f the lipid c o m p o s i t i o n o f p l a s m a and R B C m i g h t offer chemical evidence to explain o u r observations. T h e chemical c o m p o s i t i o n o f the lipids o f R B C an d p l a s m a is given in T a b l e I. T h e r e is no difference in the p h o s p h o l i p i d c o n t e n t between y o u n g a n d old rats. H o w e v e r , a highly significant difference was seen in the cholesterol an d cholesterol ester c o n t e n t o f b l o o d o f y o u n g an d old rats, with the ratio o f cholesterol to p h o s p h o l i p i d higher in the R B C an d p l a s m a o f the older rats. Since cholesterol is k n o w n to be a m e m b r a n e stabilizing c o m p o u n d in c o m p l e x with phos-
TABLE II PHOSPHOLIPIDS OF PLASMA AND RBC OF YOUNG 1 AND OLD 2 RATS (RESULTS AS LIPID PHOSPHORUS, /tg/ml ~- SEM; AVERAGE OF 6 DETERMINATIONS) Phospholipids*
Young rats Plasma
PI LPC + PS Sph PC PE PGP + PA Total
Old rats RBC
6.06 ~ 0.7 7.89 ~z 1.0 2.43 ~_ 0.4 8.86-L 1.0 2.83 ± 0.5 13.64 :~ 2.0 15.29 ± 2.0 36.50 ± 2.0 1.65 :~ 0.8 8.48 ~ 2.0 0.65 5_ 0.1 1.31 ± 0.2 28.91 76.68
RBC/ Plasma
Plasma
RBC
1.30 3.65 4.82 2.39 5.14 2.00 2.65
4.20 ~_ 0.6 8,80 ± 2.0 2.64 ~ 0.5 11.37 ± 0.8 3.91 ~ 0.8 12,00 + 0.7 14.54 ± 3.0 36,51 ± 2.0 1.12 ± 0.2 7,46 ± 2.0 0.65 ± 0.2 1,30 ± 0.3 27.06 77,44
RBC/ Plasma
2.10 4.31 3.07 2.51 6.66 2.00 2.86
1 1.5 months; 2 12-24 months; *PI = phosphatidyl inositol; LPC = lysophosphatidyl choline; PS = phosphatidyl serine; Sph ~ sphingomyelin; PC = phosphatidyl choline; PE ~ phosphatidyl ethanolamine; PGP = phosphoglycerol phosphate; PA -- phosphatidic acid.
144 p h o l i p i d s 20, the possibility t h a t this increase in r a t i o m a y p l a y some role in cholesterol exchange c a n n o t be ruled out. Table I I gives the p e r c e n t d i s t r i b u t i o n o f various p h o s p h a t i d e s in the p l a s m a a n d R B C o f y o u n g a n d old rats. There were differences in levels o f p l a s m a a n d R B C p h o s p h a t i d e s between y o u n g a n d old cells. W h i l e none o f the differences were statistically significant several were appreciable. P h o s p h a t i d y l inositol levels were higher in y o u n g p l a s m a (31 ~ ) a n d lower in R B C ( - - 1 2 ~ ) . Lysolecithin plus serine levels were lower in b o t h y o u n g p l a s m a ( - - 9 ~ ) a n d R B C ( - - 2 8 ~ ) . S p h i n g o m y e l i n levels were lower in y o u n g p l a s m a ( - - 3 8 700)a n d higher in y o u n g R B C (12 ~ ) a n d p h o s p h a t i d y l e t h a n o l a m i n e was present at a higher level in b o t h y o u n g p l a s m a (32 ~ ) a n d R B C (12 ~ ) . There were no differences in lecithin or p h o s p h a t i d i c acid plus cardiolipin levels. The involvement o f these p h o s p h a t i d e s in cholesterol exchange is under investigation. In view o f d a t a presented above, it is evident that the cholesterol exchange in b l o o d o f old rats does n o t follow the slower cholesterol t u r n o v e r p a t t e r n r e p o r t e d by H r u z a a n d his colleagues 1-3 in the other tissues o f old animals. ACKNOWLEDGEMENTS This investigation was s u p p o r t e d , in part, by U S P H S research grants HL-03299 a n d HL-05209 a n d research Career A w a r d HL-00734 f r o m the N a t i o n a l Institutes o f Health.
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145 13 J. T. Dodge, C. Mitchell and D. J. Hanahan, The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes, Arch. Biochem. Biophys., 100 (1963) 119-130. 14 D. Kritchevsky, L. M. Davidson, H. K. Kim and S. Malhotra, Quantitation of serum lipids by a simple TLC-charring method, Clin. Ck,im. Acta, 46 (1973) 63-68. 15 D. Kritchevsky and S. Malhctra, Recovery of lipids from thin-layer chromatography for radioassay, J. Chromatogr., 52 (1970) 498~J,99. 16 D. Abramson and M. Rlecher, Quantitative two dimensional thin-layer cbrcmatcgraphy of naturally occurring phospholipids, J. Lipid Res., 5 (1964) 628. 17 L, S. De Bohner, E. F. Soto and T. De Cohan, Quantitative analysis of phospholipids by thinlayer chromatography, J. Chromatogr., 17 (1965) 513-519. 18 G. R. Bartlett, Phosphorus assay in column chromatography, J. Biol. Chem., 234 (1959) 466-468. 19 F. P. Bell, J. B. Somcr, I. H. Craig and C. J. Schwartz, Membrane-lipid exchange: Exchange of cholesterol between porcine serum lipoproteins and erythrecytes, Pathology, 4 (1972) 205-214. 20 L. L. M. Van Deenen and J. De Gier, Chemical composition and metabolism of lipids in red cells of various animal species. In C. Bishop and D. M. Surgenor (eds.). The Red Blood Cell, Academic Press, New York, 1964, p. 243-307.
