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Differential resorption rates of subcutaneous implants of [3H]cholesterol, various [3H]cholesterol esters and [3H]cholesterol-[1-14C]linolenate
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam -- Printed irt The Netherlands
D I F F E R E N T I A L R E S O R P T I O N RATES OF SUBCUTANEOUS IMPLANTS OF [aHJCHOLESTEROL, VARIOUS I3HtCHOLESTEROL E S T E R S AND I3H]CHOLESTEROL-'J-14C~LINOLENATE
Y. H. ABI)ULLA, C. W. M. ADAMS AND R. S. MORGAN Department of Pathology, Gu.v's Hospital Medical School, London S.E.1 (Great Britain)
(Received August 9th, 1968)
SUMMARY
Free [3H]cholesterol, various [all]cholesterol esters and [aH]cholesterol[_14C)linolenate were implanted subcutaneously in rats. Three weeks after implantation [all]cholesterol linolenate and arachidonate were resorbed to a significantly greater extent than free i3HJcholesterol, [3H]cholesterol linolenate and the more saturated labelled sternal esters, [aH [Cholesterol was more rapidly mobilized titan the saturated and monounsaturated fall]cholesterol esters. The resorption of esterified cholesterol did not appear to depend on rapid hydrolysis of the fatty acid moiety and slow removal of the sterol nucleus, for aH and a4C labels disappeared a t nearly equal rates from implants of ~3H]cholesterol-[ 1-14Qlinolenate. Addition of antioxidants to the polyunsaturated ester implants did not significantly accelerate resorption, even though it did reduce chromolipid formation. We infer that esterification of cholesterol with linolenic or arachidonic acids would accelerate its reifioval from the normal or atherosclerotic arterial wall.
INTRODUCTION
In previous communicationsI, 2 we showed b y histological means that polyunsaturated cholesterol esters are considerably less sclerogenic and appear to be resorbed more rapidly from subcutaneous implants than are free cholesterol and its saturated and monounsaturated esters. The purpose of this present study is to measure the speed of resorption of 3H-labelled cholesterol and cholesterol esters from such implants b y radioisotope counting techniques. The general aim of this line of in-
This work was supported in part by research funds from Chas. Pfizer Ltd., and the Tobacco Research Council Y. H. ASDULLA: l~esearch Associate supported by the British Heart Foundation. J. Atheroscler. Res,, 1969, 9:81-85
82
Y . H . ABDULLA, C. W. M. ADAMS, R. S. MORGAN
vestigation is to predict which forms of cholesterol would be most sclerogenic and the most resistant to mobilization from arterial connective tissues. METHODS
Preparation of labelled lipids Generally-labelled [ZHlcholesterol and [1-14C]linolenic acid were purchased from the Radiochemical Centre, Amersham, Bucks. Other fatty acids were obtained from Sigma (see Fig. 1). Cholesterol esters were prepared and purified as previously described t. The radiochemicaI purity of the [aH]cholesteroI was shown to be greater than 99.99 ~o as sterol by thin-layer chromatography/scintillation-counting 3. The radiochemical purity of labelled fatty acids from the Radiochemical Centre is in the range of 97 %, However, we did not specially confirm the radiochemical purity of the [ 1-14C]linolenic acid, because the precise degree of such purity is not particularly relevant to the general problem of establishing that cholesterol and fatty acid are not resorbed at different rates from implants of esterified cholesterol. The chemical purity of the unlabelled fatty acids and the synthesized labelled cholesterol esters was established by quantitative silver-nitratethin-layer chromatography (see ref. 1). The antioxidants a-tocophorol (Sigma) or 2,6-di-tert.-butyl-p-cresol (BDH) were added to some lipid samples (see Fig. 1) at 1 : 1000 molar ratios. Alliipidprep-arations were crystallized (in some cases at --20~ and were then freed of solvent by evaporating to dryness under reduced pressure immediately before implantation. Specific radioactivity was determined in aliquots of all lipid samples (see below),
Subcutaneous implantation Groups of albino rats were anaesthetized with ether, the hair was removed from their abdomens with "Nair" depilating cream and then radioactive lipids were implanted subcutaneously in each quadrant of the abdomen. Solid lipids were injected with a trochar and cannula, whilst liquid lipids were injected with a 1-ml "tuberculin" syringel, 4. The weight of the injecting instrument was recorded on an aperiodic electric balance (sensitive to 0.l mg) before and after implantation. The size of implants was intentionally varied between 1 and 20 rag. Exactly 3 weeks after implantation the lipid mass was carefully dissected out in toto and was subjected to isotope-counting (see below).
Isotope-counting technique The lipid implants were homogenized in chloroform-methanol (2 : 1, v/v) and subjected to the conventional FOLCH wash 5. Aliquots of the lower phase were evaporated to dryness, redissolved in 20 ml of SNYDER'S6 No. 1 toluene-based scintillation fluid and then counted in the "A" channel of a Beckman 3-channel liquid scintillation counter. Radioactive carbon was counted in the "C" channel. Quenching was measured b y the channels' ratio method. J. Atheroscler. Res., 1969, 9 : 8 1 - 8 5
DIFFERENTIAL RESORPTION RATES OF SUBCUTANEOUS IMPLANTS OF CHOLESTEROL 83
RESULTS The percentage resorption of the various lipids implanted was calculated from the residual tissue radioactivity: these results are presented in Fig. 1. The addition of antioxidants to the implants substantially reduced the amount of orange pigmentation (chromolipid) encountered in the implanted 18:2, 18:3 and 20:4 esters. Antioxidants showed a tendency to increase the resorption rate of these esters, but this effect was within the range of resorption observed without adding antioxidants (see Fig. 1). Resorption speed was not related to implant size over the range 1-20 rag. In 3 implants of [~Hlcholesterol-El-14C]linolenate, the resorption ratios of the 3H/14C labels were respectively 0.974, 0.977 and 0.983 (see Fig. 1). As these lipids were implanted over the period March-June, seasonal variation could conceivably effect the results (see ref. 7). For this reason the resorption rates of ESHlcholesterol were separately measured at the beginning (March) and end (June) of the experimental period. The rate slightly increased (from a mean of 36.3 to 41.0 %) between March and June (see Fig. 1). However, this change was not statistically significant (t = 1.88, P ~ 0.1) and was too small appreciably to affect the results obtained with the other lipid implants. Moreover, inspection of the results revealed no tendency for polyunsaturated esters to resorb faster when implanted on a second occasion, later in the year.
LIPIO CLASS 4 Free ChoLesteroL
75
100
I
I
I
I
p< o.ool
~m
3
IE,I,vr
20:6
RESORPTION (~ 50
TTT TT
Ester ChoLesteroL 2 18:0 16:0 18:1 t8:Z 18:2 A 18:3 18:3 A
25
HONTH
+ TIT TV
A
+
t = 5.18 p < o-ool
1
J
e
0
A = anti0xidant added
I = mean A =14C
i
|
!
j
=
1
~V,X
20:~ k
~ = 1"74 p --~ 0 - 0 4
-&
0
J
"
|
9 = )H
from [3H]:hoLesteroL [1-'4C]Lino(enate
Fig. 1. Resorption of free Jail]cholesterol, various [SH]cholesterol esters and [SH]cholesterol[1-1aC]linolenate from 3-week-old subcutaneous implants in rats. The means and probability levels refer to the sI-I label only. J. Atheroscler. Res., 1969, 9:81-85
84
Y . H . ABDULLA, C. W. M. ADAMS, R. S. MORGAN
DISCUSSION
The re:sults s u m m a r i z e d in Fig. 1 indicate t h a t trienoic and tetraenoic cholesterol es~ters are resorbed more r a p i d l y from s u b c u t a n e o u s connective tissues t h a n cholesterol (P ~ 0.04) or its m o r e s a t u r a t e d esters (P < 0.001), while free cholesterol is mobilized faster t h a n its s a t u r a t e d a n d m o n o u n s a t u r a t e d esters ( P < 0.001). However, i t is not clear w h y the range of r e s o r p t i o n rates for these p o l y u n s a t u r a t e d esters was so wide when c o m p a r e d w i t h those for the o t h e r esters a n d free cholesterol. This v a r i a b i l i t y could not be a t t r i b u t e d to seasonal v a r i a t i o n or different i m p l a n t sizes. If these results can be e x t r a p o l a t e d to the connective tissues of the a r t e r i a l wall, it can be inferred t h a t cholesterol esterification w i t h trienoic a n d t e t r a e n o i c f a t t y acids Would u s u a l l y increase t h e r a t e of s t e r o l ' r e m o v a l from atherosclerotic lesions. I n this connection, it is r e l e v a n t t h a t t h e a r t e r i a l wall contains m e c h a n i s m s for s y n t h e s i z i n g cholesterol esters s (see ref. 9), at least one of which transfers fl-polyu n s a t u r a t e d f a t t y acid from lecithin to free Cholesterol 10. It is curious t h a t cholesterol linoleate was resorbed so slowly from s u b c u t a n e o u s tissues (see Fig. 1). Linoleic acid is widely r e g a r d e d as "beneficial" in atherosclerosis, b u t such an e f f e c t - - w i t h respect to cholesterol m o b i l i z a t i o n - - m i g h t possibly d e p e n d u p o n m e c h a n i s m s f o r e x t e n d i n g linoleate's h y d r o c a r b o n chain a n d i n t r o d u c i n g f u r t h e r e t h y l e n e b o n d s to form a r a c h i d o n i c acid. A l t h o u g h c h o l e s t e r o l linolenate is' m o b i l i z e d from tissues more slowly t h a n free cholesterol, nevertheless this polyu n s a t u r a t e d ester is m a r k e d l y less sclerogenic t h a n the free sterol a n d its more s a t u r a t e d estersl, 2. Likewise, cholesterol linolenate a n d a r a c h i d o n a t e are r e l a t i v e l y non-sclerogenicl, 2 and, in addition, are u s u a l l y mobilized r a p i d l y (see Fig. 1). O u r r e s u l t s c a n n o t be e x p l a i n e d in t e r m s of fast h y d r o l y s i s of the f a t t y acid m o i e t y a n d slow resorption of tile sterol nucleus of cholesterol esters, for the r e m o v a l r a t e s of 3H a n d 14C labels wer~ p r a c t i c a l l y i d e n t i c a l in i m p l a n t s of E3H]cholesterol il-x4Cjlinolenate. On the c o n t r a r y , we p o s t u l a t e t h a t cholesterol esters with open n o n - c o m p a c t e d f a t t y acid chains 1 are in general more r e a d i l y t a k e n u p b y m a c r o phages a n d mobilized t h a n the more s a t u r a t e d esters w i t h their rigid t i g h t l y - p a c k e d f a t t y acid chains. REFERENCES 1 ABDULLA, Y. H., C. W. M. ADAMS AND R. S. MORGAN, Connective-tissue reactions to im-
plantation of purified sterol, sterol esters, phosphoglycerides, glycerides and free fatty acids, J. Path. Bact., 1967, 94: 63. 2 ADAMS,C. W. M., Vascular tlistochemistry, Lloyd-Luke, London, 1967, p. 160. 3 ADAMS C. W..'VI., S. VIR~.G, ~R. S. MORGANAND C. C. ORTON, Dissociation of [3If]cholesterol and l"5i-labelled plasma protein influx in normal and atheromatous rabbit aorta. A quantitative histochemical study, J. Atheroscler. Res., 1968, 8: 679. 4 ADAMS,C. W..-'~., O. B. BAYLISS,M. Z. M. IBRAHIMAND M. W. WEBSTER, JR., Phospholipids in atherosclerosis: the modification of'.the cholesterol granuloma by phospholipid, J..Path. Bacl., 1963, 86: 431. 5 Foi.cH, J., M. LEES AND G. II. SLOANE-S'rANLEY, A simple method for the isolation and purification of total lipids from animal tissues, J. biol. Chem., 1957, 226: 497. 6 SNYDER, F., Radioassay of thin-layer chromatograms: a high-resolution zonal scraper for quantitative Cx4 and H 3 scanning of thin-layer chromatograms, Analyt. Biochem., 1964, 9: 183. J. Atheroscler. Res., 1969, 9:81-85
DIFFERENTIAL RESORPTION RATES OF SUBCUTANEOUS IMPLANTS OF CHOLESTEROL
85
7 ADAMS, C. W. M. AND 1~. S. MORGAN,The effect of s a t u r a t e d and p o l y u n s a t u r a t e d lecithins on the resorption of 4-14C-cholesterol from s u b c u t a n e o u s implants, J. Path. Bact., 1967, 94: 73. 8 LOFLAND H. B., JR., D. M. MOURY, C. W. HOFFMANN AND T. B. CLARKSON,Lipid metabolism in pigeon aorta during atherogenesis, J. Lipid Res., 1965, 6:112. 9 PATELSKI, J., D. E. BOWYER, A. N. HOWARD AND G. A. GRESHAM, Changes in phospholipase A, lipase and cholesterol esterase activity in the aorta in experimental atherosclerosis in the rabbit a n d rat, J. Atheroscler. Res., 1968, 8: 221. i0 •BDULLA, Y. H., C. C. ORTON AND C. W. M. ADAMS, Cholesterol esterification by transacylation in h u m a n and experimental a t h e r o m a t o u s lesions, J. Atheroscler. Res., 1968, 8: 967-973.
j . Atheroscler. Res., 1969, 9 : 8 1 - 8 5
Elsevier Publishing Company, Amsterdam -- Printed irt The Netherlands
D I F F E R E N T I A L R E S O R P T I O N RATES OF SUBCUTANEOUS IMPLANTS OF [aHJCHOLESTEROL, VARIOUS I3HtCHOLESTEROL E S T E R S AND I3H]CHOLESTEROL-'J-14C~LINOLENATE
Y. H. ABI)ULLA, C. W. M. ADAMS AND R. S. MORGAN Department of Pathology, Gu.v's Hospital Medical School, London S.E.1 (Great Britain)
(Received August 9th, 1968)
SUMMARY
Free [3H]cholesterol, various [all]cholesterol esters and [aH]cholesterol[_14C)linolenate were implanted subcutaneously in rats. Three weeks after implantation [all]cholesterol linolenate and arachidonate were resorbed to a significantly greater extent than free i3HJcholesterol, [3H]cholesterol linolenate and the more saturated labelled sternal esters, [aH [Cholesterol was more rapidly mobilized titan the saturated and monounsaturated fall]cholesterol esters. The resorption of esterified cholesterol did not appear to depend on rapid hydrolysis of the fatty acid moiety and slow removal of the sterol nucleus, for aH and a4C labels disappeared a t nearly equal rates from implants of ~3H]cholesterol-[ 1-14Qlinolenate. Addition of antioxidants to the polyunsaturated ester implants did not significantly accelerate resorption, even though it did reduce chromolipid formation. We infer that esterification of cholesterol with linolenic or arachidonic acids would accelerate its reifioval from the normal or atherosclerotic arterial wall.
INTRODUCTION
In previous communicationsI, 2 we showed b y histological means that polyunsaturated cholesterol esters are considerably less sclerogenic and appear to be resorbed more rapidly from subcutaneous implants than are free cholesterol and its saturated and monounsaturated esters. The purpose of this present study is to measure the speed of resorption of 3H-labelled cholesterol and cholesterol esters from such implants b y radioisotope counting techniques. The general aim of this line of in-
This work was supported in part by research funds from Chas. Pfizer Ltd., and the Tobacco Research Council Y. H. ASDULLA: l~esearch Associate supported by the British Heart Foundation. J. Atheroscler. Res,, 1969, 9:81-85
82
Y . H . ABDULLA, C. W. M. ADAMS, R. S. MORGAN
vestigation is to predict which forms of cholesterol would be most sclerogenic and the most resistant to mobilization from arterial connective tissues. METHODS
Preparation of labelled lipids Generally-labelled [ZHlcholesterol and [1-14C]linolenic acid were purchased from the Radiochemical Centre, Amersham, Bucks. Other fatty acids were obtained from Sigma (see Fig. 1). Cholesterol esters were prepared and purified as previously described t. The radiochemicaI purity of the [aH]cholesteroI was shown to be greater than 99.99 ~o as sterol by thin-layer chromatography/scintillation-counting 3. The radiochemical purity of labelled fatty acids from the Radiochemical Centre is in the range of 97 %, However, we did not specially confirm the radiochemical purity of the [ 1-14C]linolenic acid, because the precise degree of such purity is not particularly relevant to the general problem of establishing that cholesterol and fatty acid are not resorbed at different rates from implants of esterified cholesterol. The chemical purity of the unlabelled fatty acids and the synthesized labelled cholesterol esters was established by quantitative silver-nitratethin-layer chromatography (see ref. 1). The antioxidants a-tocophorol (Sigma) or 2,6-di-tert.-butyl-p-cresol (BDH) were added to some lipid samples (see Fig. 1) at 1 : 1000 molar ratios. Alliipidprep-arations were crystallized (in some cases at --20~ and were then freed of solvent by evaporating to dryness under reduced pressure immediately before implantation. Specific radioactivity was determined in aliquots of all lipid samples (see below),
Subcutaneous implantation Groups of albino rats were anaesthetized with ether, the hair was removed from their abdomens with "Nair" depilating cream and then radioactive lipids were implanted subcutaneously in each quadrant of the abdomen. Solid lipids were injected with a trochar and cannula, whilst liquid lipids were injected with a 1-ml "tuberculin" syringel, 4. The weight of the injecting instrument was recorded on an aperiodic electric balance (sensitive to 0.l mg) before and after implantation. The size of implants was intentionally varied between 1 and 20 rag. Exactly 3 weeks after implantation the lipid mass was carefully dissected out in toto and was subjected to isotope-counting (see below).
Isotope-counting technique The lipid implants were homogenized in chloroform-methanol (2 : 1, v/v) and subjected to the conventional FOLCH wash 5. Aliquots of the lower phase were evaporated to dryness, redissolved in 20 ml of SNYDER'S6 No. 1 toluene-based scintillation fluid and then counted in the "A" channel of a Beckman 3-channel liquid scintillation counter. Radioactive carbon was counted in the "C" channel. Quenching was measured b y the channels' ratio method. J. Atheroscler. Res., 1969, 9 : 8 1 - 8 5
DIFFERENTIAL RESORPTION RATES OF SUBCUTANEOUS IMPLANTS OF CHOLESTEROL 83
RESULTS The percentage resorption of the various lipids implanted was calculated from the residual tissue radioactivity: these results are presented in Fig. 1. The addition of antioxidants to the implants substantially reduced the amount of orange pigmentation (chromolipid) encountered in the implanted 18:2, 18:3 and 20:4 esters. Antioxidants showed a tendency to increase the resorption rate of these esters, but this effect was within the range of resorption observed without adding antioxidants (see Fig. 1). Resorption speed was not related to implant size over the range 1-20 rag. In 3 implants of [~Hlcholesterol-El-14C]linolenate, the resorption ratios of the 3H/14C labels were respectively 0.974, 0.977 and 0.983 (see Fig. 1). As these lipids were implanted over the period March-June, seasonal variation could conceivably effect the results (see ref. 7). For this reason the resorption rates of ESHlcholesterol were separately measured at the beginning (March) and end (June) of the experimental period. The rate slightly increased (from a mean of 36.3 to 41.0 %) between March and June (see Fig. 1). However, this change was not statistically significant (t = 1.88, P ~ 0.1) and was too small appreciably to affect the results obtained with the other lipid implants. Moreover, inspection of the results revealed no tendency for polyunsaturated esters to resorb faster when implanted on a second occasion, later in the year.
LIPIO CLASS 4 Free ChoLesteroL
75
100
I
I
I
I
p< o.ool
~m
3
IE,I,vr
20:6
RESORPTION (~ 50
TTT TT
Ester ChoLesteroL 2 18:0 16:0 18:1 t8:Z 18:2 A 18:3 18:3 A
25
HONTH
+ TIT TV
A
+
t = 5.18 p < o-ool
1
J
e
0
A = anti0xidant added
I = mean A =14C
i
|
!
j
=
1
~V,X
20:~ k
~ = 1"74 p --~ 0 - 0 4
-&
0
J
"
|
9 = )H
from [3H]:hoLesteroL [1-'4C]Lino(enate
Fig. 1. Resorption of free Jail]cholesterol, various [SH]cholesterol esters and [SH]cholesterol[1-1aC]linolenate from 3-week-old subcutaneous implants in rats. The means and probability levels refer to the sI-I label only. J. Atheroscler. Res., 1969, 9:81-85
84
Y . H . ABDULLA, C. W. M. ADAMS, R. S. MORGAN
DISCUSSION
The re:sults s u m m a r i z e d in Fig. 1 indicate t h a t trienoic and tetraenoic cholesterol es~ters are resorbed more r a p i d l y from s u b c u t a n e o u s connective tissues t h a n cholesterol (P ~ 0.04) or its m o r e s a t u r a t e d esters (P < 0.001), while free cholesterol is mobilized faster t h a n its s a t u r a t e d a n d m o n o u n s a t u r a t e d esters ( P < 0.001). However, i t is not clear w h y the range of r e s o r p t i o n rates for these p o l y u n s a t u r a t e d esters was so wide when c o m p a r e d w i t h those for the o t h e r esters a n d free cholesterol. This v a r i a b i l i t y could not be a t t r i b u t e d to seasonal v a r i a t i o n or different i m p l a n t sizes. If these results can be e x t r a p o l a t e d to the connective tissues of the a r t e r i a l wall, it can be inferred t h a t cholesterol esterification w i t h trienoic a n d t e t r a e n o i c f a t t y acids Would u s u a l l y increase t h e r a t e of s t e r o l ' r e m o v a l from atherosclerotic lesions. I n this connection, it is r e l e v a n t t h a t t h e a r t e r i a l wall contains m e c h a n i s m s for s y n t h e s i z i n g cholesterol esters s (see ref. 9), at least one of which transfers fl-polyu n s a t u r a t e d f a t t y acid from lecithin to free Cholesterol 10. It is curious t h a t cholesterol linoleate was resorbed so slowly from s u b c u t a n e o u s tissues (see Fig. 1). Linoleic acid is widely r e g a r d e d as "beneficial" in atherosclerosis, b u t such an e f f e c t - - w i t h respect to cholesterol m o b i l i z a t i o n - - m i g h t possibly d e p e n d u p o n m e c h a n i s m s f o r e x t e n d i n g linoleate's h y d r o c a r b o n chain a n d i n t r o d u c i n g f u r t h e r e t h y l e n e b o n d s to form a r a c h i d o n i c acid. A l t h o u g h c h o l e s t e r o l linolenate is' m o b i l i z e d from tissues more slowly t h a n free cholesterol, nevertheless this polyu n s a t u r a t e d ester is m a r k e d l y less sclerogenic t h a n the free sterol a n d its more s a t u r a t e d estersl, 2. Likewise, cholesterol linolenate a n d a r a c h i d o n a t e are r e l a t i v e l y non-sclerogenicl, 2 and, in addition, are u s u a l l y mobilized r a p i d l y (see Fig. 1). O u r r e s u l t s c a n n o t be e x p l a i n e d in t e r m s of fast h y d r o l y s i s of the f a t t y acid m o i e t y a n d slow resorption of tile sterol nucleus of cholesterol esters, for the r e m o v a l r a t e s of 3H a n d 14C labels wer~ p r a c t i c a l l y i d e n t i c a l in i m p l a n t s of E3H]cholesterol il-x4Cjlinolenate. On the c o n t r a r y , we p o s t u l a t e t h a t cholesterol esters with open n o n - c o m p a c t e d f a t t y acid chains 1 are in general more r e a d i l y t a k e n u p b y m a c r o phages a n d mobilized t h a n the more s a t u r a t e d esters w i t h their rigid t i g h t l y - p a c k e d f a t t y acid chains. REFERENCES 1 ABDULLA, Y. H., C. W. M. ADAMS AND R. S. MORGAN, Connective-tissue reactions to im-
plantation of purified sterol, sterol esters, phosphoglycerides, glycerides and free fatty acids, J. Path. Bact., 1967, 94: 63. 2 ADAMS,C. W. M., Vascular tlistochemistry, Lloyd-Luke, London, 1967, p. 160. 3 ADAMS C. W..'VI., S. VIR~.G, ~R. S. MORGANAND C. C. ORTON, Dissociation of [3If]cholesterol and l"5i-labelled plasma protein influx in normal and atheromatous rabbit aorta. A quantitative histochemical study, J. Atheroscler. Res., 1968, 8: 679. 4 ADAMS,C. W..-'~., O. B. BAYLISS,M. Z. M. IBRAHIMAND M. W. WEBSTER, JR., Phospholipids in atherosclerosis: the modification of'.the cholesterol granuloma by phospholipid, J..Path. Bacl., 1963, 86: 431. 5 Foi.cH, J., M. LEES AND G. II. SLOANE-S'rANLEY, A simple method for the isolation and purification of total lipids from animal tissues, J. biol. Chem., 1957, 226: 497. 6 SNYDER, F., Radioassay of thin-layer chromatograms: a high-resolution zonal scraper for quantitative Cx4 and H 3 scanning of thin-layer chromatograms, Analyt. Biochem., 1964, 9: 183. J. Atheroscler. Res., 1969, 9:81-85
DIFFERENTIAL RESORPTION RATES OF SUBCUTANEOUS IMPLANTS OF CHOLESTEROL
85
7 ADAMS, C. W. M. AND 1~. S. MORGAN,The effect of s a t u r a t e d and p o l y u n s a t u r a t e d lecithins on the resorption of 4-14C-cholesterol from s u b c u t a n e o u s implants, J. Path. Bact., 1967, 94: 73. 8 LOFLAND H. B., JR., D. M. MOURY, C. W. HOFFMANN AND T. B. CLARKSON,Lipid metabolism in pigeon aorta during atherogenesis, J. Lipid Res., 1965, 6:112. 9 PATELSKI, J., D. E. BOWYER, A. N. HOWARD AND G. A. GRESHAM, Changes in phospholipase A, lipase and cholesterol esterase activity in the aorta in experimental atherosclerosis in the rabbit a n d rat, J. Atheroscler. Res., 1968, 8: 221. i0 •BDULLA, Y. H., C. C. ORTON AND C. W. M. ADAMS, Cholesterol esterification by transacylation in h u m a n and experimental a t h e r o m a t o u s lesions, J. Atheroscler. Res., 1968, 8: 967-973.
j . Atheroscler. Res., 1969, 9 : 8 1 - 8 5