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Lipase, esterase and triglyceride in the ageing human aorta
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
LIPASE, E S T E R A S E AND T R I G L Y C E R I D E IN T H E A G E I N G HUMAN AORTA A QUANTITATIVE HISTOCHEMICAL AND BIOCHEMICAL STUDY C. W. M. ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER AND M. A. ROOT Departments of Pathology and Medicine, Guy's Hospital Medical School, London, and Department of Clinical Chemistry, Welsh National School of Medicine, Cardiff (Great Britain)
(Revised, received November 6th, 1968)
SUMMARY
Lipase (tributyrinase), esterase (fl-naphthyl laurate esterase), cholesterol, phospholipid and triglyceride were estimated in human aortas from the age of 20 years to the 9th decade. Overall activity of lipase and esterase tended to decrease on a wet weight basis with advancing age, but aortic lipase activity in women remained nearly constant up to the end of the 6th decade. When such decreased activity was compared with the reciprocal of the age increase in aortic weight (i.e. inferred surface area), neither enzyme showed this age-dependent tendency to decline. On this basis aortic lipase in women became more active up to the end of the 6th decade. Quantitative histochemical estimation of these two enzymes in consecutive layers through the aorta revealed a midmedial decline in esterase activity with advancing age, but no similar decline in lipase. Increments in enzyme activity in the atherosclerotic or hyperplastic intima probably counterbalance the midmedial loss of esterase activity in the ageing aorta in both sexes and the lower medial lipase activity in the ageing male aorta. Aortic cholesterol shows a marked increase on a wet weight basis with advancing age, while the increment in triglycerides is only trivial. Phospholipid occupies an intermediate position in this respect. When lipid values are compared with the reciprocal of aortic weight, the increase in triglyceride remains trivial. These results suggest t h a t residual medial activity together with intimal augmentation of lipase and esterase in the ageing human aortic wall prevent any substantial accumulation of triglycerides therein.
Key words: H u m a n aorta - E n z y m e s - L i p a s e - E s t e r a s e - E x p r e s s i o n o f aortic e n z y m e activity -
Quantitative histochemistry - Aortic ageing - Triglycerides -
P h o s p h o l i p i d s - Cholesterol
J. Atheroscler. Res., 1969, 9:87-102
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C . W . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
INTRODUCTION
Previous biochemical studies have shown that lipolytic activity declines with advancing age in the rat and rabbit's aorta 1-3. Likewise, our previous studies have shown that tributyrin lipase and fl-naphthyl laurate esterase activities decline in the ageing human aorta, when results are expressed on a simple wet weight basis 4. ZE~PL~NYIS, 6 has suggested that the lipolytic activity of the arterial wall protects the vessel against the deposition of lipids (triglycerides) therein. In this connection S.~IT~!Sv studies showed that triglycerides increase only slightly in the ageing human aorta, in contrast to esterified cholesterol which shows a far more spectacular increase. Histochemical results confirm the relative absence of triglycerides from hunmn atherosclerotic lesions 8. The purpose of this paper is to report further studies on lipolytic activity, esterolytic activity and triglyceride concentration in the ageing human aorta. A problem that arises from these studies is how best to express these results in view of the comple~:- alterations in arterial structure and mural volume that are caused b y lipid accumulation, cellular degeneration and sclerosis. The hypothesis to be tested is whether the apparently trivial triglyceride accumulation in human aortic atherosclerosis can be explained by an actual maintenance of lipolytic activity, when the age-dependent increase in aortic weight is discounted. METHODS
Preparation of samples Aortic samples from 125 Caucasian subjects, not suffering from congenital heart disease, were obtained at necropsy from the junction of the thoraci9 and abdominal portions of the vessel. This region was chosen because it is accessible at an early stage of the necropsy procedure and it is less ~0mmonly involved by severe calcific atherosclero~is. The severity of aortic atherosclerosis was graded by the WHO method 9 "['issue for enzyme estimation from 70 cases varied from grade 0 in. a few subjects under 40 years to grade III (ulcerated or slightly calcified plaques) in some subiects aged over 50 years. Atherosclerotic and grossly normal areas were not separately dissected and assayed; areas involved by severe calcification or mural thrombus were excluded. Further tissue from 55 cases was used to establish the extent of aortic weight increase with age (see below). The incidence and severity of atherosclerosis in ~elation to age were similar in this material to t h a t used for enzyme estimation. The necropsy diagnoses of subjects used for one or the other study showed no bias or "weighting" towards a particular disease category. The tunica adventitia was carefully stripped off a 2 cm • 2 cm sample of tim vessel; the specirpen was than either we.ighed and homogenized in an Ultraturrex blender or, alternatively, was used for quantitative histochemical estimation. Serial consecutive aortic layers were prepared from.the intima to the outer media by a histological technique. The unfixed specimen of aorta was flattened by j . Atheroscler. Res.,. 1969, 9 : 8 7 - 1 0 2
L I P A S E ESTERASE AND T R I G L Y C E R I D E IN T H E A G E I N G HUMAN AORTA
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means of a glass microscope slide onto the chuck of a De Ira Rue Frigistor thermoelectric microtome. The microtome knife was adjusted so as to be perfectly parallel with the surface of the aorta. Frozen sections were cut at 50 # and were arranged in groups of 6 or 7 equally divided zones (i.e. 2--4 sections in each zone), 'from the,in.~ide to the outside of the vessel. The superficial (inner) layers of plaque tissue (incomplete sections) were grouped together to form part of layer 1. More sections were included in each individual layer from atherosclerotic aortas. Thus, the total number of layers obtained from " n o r m a l " and atherosclerotic aortas was often the same. The zonal samples were weighed in tubes of known tare and then homogenized. Tiffs procedure is a slight modification of our previous technique for eytochemical analysis of the aortal0,tt. Homogenates of whole aortic wall (intima + media) or homogenized serial zones of the aorta were estimated for lipase and esterase activities and analysed for their lipid and total protein content.
Age increase in aortic weight. Aortic weight increase with advancing age was determined in a second series of vessels (see above). For this purpose a circle o[ tissue was cut from each quadrant of the thoraco-lumbar aortic junction by means of a No. 6 cork borer (12 m m internal diameter). After removing surface fluid, these 4 aortic circles were then weighed on an aperiodic electric balance sensitive to 0.1 rag.
Analytical methods Samples for lipase estimation were homogenised in either 0.15 M Krebs-Ringer phosphate buffer (pH 7.4) or distilled water. Lipolytic activity against tributyrin was estimated b y MAHLER'S method 21. One-ml duplicate aliquots (each - 50 mg tissue) of supernatants (derived by centrifuging the homogenate at 2000 rev./min for 5 min) were incubated with 1 ml 0.4 % tributyrin (BDH) in 0.15 M phosphate buffer for 1 h at 37~ After incubation protein was precipitated b y adding 0.5"ml of 50 % trichloroacetic acid; 0.2-ml aliquots of the supernatant were used for estimating the released glyceroP 3. Esterase activity against/5-naphthyl laurate was estimated b y a modification t4 o[ SELIGMAN AND NACHLAS' method 15. One-half-ml duplicate aliquots (each - 5 mg tissue) of whole aqueous homogenates (see above) were incubated with 2.5 ml 0.02 % fl-naphthyl laurate 15 in veronal-HC1 buffer (pH 7.4) for 2 11 at 37~ The liberated flnapththol was estimated at 0~ with diazo blue B (Sdgma) as coupling reagent: the resulting bis-azo dye was extracted into ethyl acetate and its absorption determined at 540 m/~. From this reading was subtracted the value obtained with a homogenate that had been boiled before incubation. The protein content of aliquots of some homogenates was determined b y the standard method using the Folin-Ciocalteau reagent for tyrosine. Total lipids were extracted from adjacent samples of aorta with chloroformmethanol (2 : 1, v/v) b y the conventional FOLCH method 16. Lipid classes were separ-
j. Atheroscler. Res,, 1969, 9:87-102
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C. W. M. ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
ated on thin layer chromatoplates by FREEMANAND WEST'S method 17. Phospholipids were estimated by MARINETTI'S18 modification of the Bartlett method, total cholesterol was determined bylLEFLER AND McDoUGALD'Smethod 19, while triglycerides were estimated by SKIDMORE AND ENTEMAN'S technique 20.
Kinetic, solubility and inhibitor studies Enzyme activity was estimated in aliquots of aortic homogenates after various periods of storage at 4~ the time interval between death and estimation varied from 36-168 h. Enzyme activity was also estimated at different homogenate dilutions and after varying the incubation time from 30-120 min (see Table 1). Aortic lipase and esterase activities were characterised by their responses to sodium taurocholate, protamine sulphate, sodium chloride, Cu 2+, Zn 2+, physostigmine (eserine) and diethyl-p-dinitrophenyl phosphate (E600). These inhibitors were TABLE 1 K I N E T I C S A N D C H A R A C T E R I S T I C S OF A O R T I C W A L L L I P A S E A N D E S T E R A S E
Procedure or inhibitor
% Activity compared with control aliquot lipase esterase
E s t i m a t i o n a t 48/36 h respectively E s t i m a t i o n at 72/60 h respectively E s t i m a t i o n at 96/84 h respectively E s t i m a t i o n at 168/156 h respectively Dilution 50 %, • 2 Dilution 25 %, • 4 Dilution 12.5 %, • 8 Dilution 6.25 %, • 16 I n c u b a t i o n 30 min, x 2 I n c u b a t i o n 60 m i n I n c u b a t i o n 90 min, x 0.67 I n c u b a t i o n 120 min, • 0.5
t00 117 42 29 97 100 121 124 134 100 114 109
100 96 88 77 94 98 98
1 % Sodium t a u r o c h o l a t e 1 % Protamine sulphate M s o d i u m chloride 0,01 M Cu 2+ 0.01 M Zn 2+ 10 -4 M eserine 10 -3 M E600 10 -5 M E600
100 81 81 65 62 lO0 64 67
100 a 34
Homogenized unfixed n o n - i n c u b a t e d sections Unfixed i n c u b a t e d sections tissue h o m o g e n a t e i n c u b a t i n g fluid Formalin-fixed incubated sections tissue h o m o g e n a t e i n c u b a t i n g fluid E t h a n o l - " f i x e d " incubated sections tissue h o m o g e n a t e i n c u b a t i n g fluid Acetone-"fixed" incubated sections tissue h o m o g e n a t e i n c u b a t i n g fluid
100 4 96 23
100 52 48
Interferes w i t h reaction.
J. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
1.3 38 2.5 60
145 100 115 80
8.5 100 53 95
LIPASE ESTERASE AND TRIGLYCERIDEIN THE AGEING HUMAN AORTA
91
used during incubation at the dilutions indicated in Table 1; preincubation inhibition was not used. In order to determine the solubility of aortic lipase and esterase under histochemical conditions, groups of 100 unfixed aortic sections were weighed and then fixed for 1 h with 4 % f o r m a l d e h y d e - 1 % calcium acetate, ethanol or acetone. The fixatives were removed b y washing the sections thoroughly with several changes of distilled water. All groups of fixed sections were then " d u m m y " incubated for 1 h at 37~ with 0.15 M phosphate buffer (pH 7.4.) A group of 100 unfixed sections was "incubated" likewise, while a further group of 100 unfixed sections was used as the non-incubated control. Enzyme activity was estimated in the "soaking fluid" (buffer), homogenised incubated tissue residue (sections) and in homogenised non-incubated unfixed sections. RESULTS
The effect on enzyme activity of storing the homogenate at 4~ is shown in Table 1. As activity of both lipase and esterase declined after 72 h from death, no aorta was used for such estimations when more than 3 days had lapsed between death and proposed analysis. The effects of homogenate dilution and length of incubation are summarised in Table 1; both aortic enzymes displayed approximately zero order kinetics. The effects of known lipase and esterase inhibitors are also presented in Table 1. The weight of the four standard circles cut from the thoraco-lumbar aortic j unction progressively increased with advancing age; these results are set out for men
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Fig. 1. Change with age of weight of thoraco-lumbar junction of male aorta. J. Atheroscler. Res,, 1969, 9:87-102
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c . w . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
in Fig. 1 and for women in Fig. 2. In the large sample studied, it was assumed that this weight increase was due mainly to atherosclerosis and slightly to diffuse intimal thickening. Naturally, such increases would be partly counterbalanced by atrophic changes in the tunica media. Quantitative histochemical estimations of lipase activity in serial zones from the blner intima to the outer media are presented in Fig. 3 (men) and Fig. 4 (women). Likewise, zonal esterase activity is shown in Fig. 5 (men) and Fig. 6 (women). Lipase activity undergoes no midzonal decline with advancing age in either sex; it shows a just significant overall decrease in older men (P ~- 0.05) but not in women. Esterase activity declines moderately in the midmedial zones of both sexes w~th~advancing years (men, P ~- 0.004; women, P -----0.01); it shows a significant overall decrease in women (P ~- 0.01) and a similar overall trend in men (P ~ 0.2). The change in overall lipase activity in whole aortic homogenates is illustrated in Fig. 7 (men) and Fig. 8 (women). Results for overall aortic esterase activity are presented in Fig. 9 (men) and Fig. 10 (women). With the exception of aortic lipase in women up to 60 years, these overall enzyme activities tended to decline with advanc.ing age when results were expressed on a wet weight basis. (Probability values are shovvn~in ~he legends to these figures.) However., tile reciprocal of the percentage weight increase in the aorta from age 20 years onwards is also shown as interrupted lines on these last four figures: it is clear.that the apparent decline in aortic enzyme activity on a wet weight,basis can largely be attributed to the increasing age-dependent weight of the vessel. In fact women's aortic lipolytic activity actually increases up to the 7th
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Fig. 2. As for Fig. 1, b~t in female aorta. J. Atheroscler. Res., 1969, 9:87-102
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LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
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Fig. 3. Zonal lipase a c t i v i t y in m a l e a o r t a . I , a y e r 1 = i n n e r i n t i m a ; l a y e r 6 = o u t e r m e d i a ; 9 ----0 ( O ) = s u b j e c t s u n d e r 40 yr.: 9 . . . . . . . 9 ( A ) - s u b j e c t s 40 y e a r s a n d over. O v e r a l l a c t i v i t y for s u b j e c t s u n d e r 40 vs_ o v e r 40 y e a r s , P -~ 0.05.
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Fig. 4. Z o n a l tip ase a c t i v i t y in f e m a l e a o r t a . L a y e r 1 = i n n e r i n t i m a ; l a y e r 6 or 7 = o u t e r m e d i a ; Q-----O ( O ) ~ s u b j e c t s u n d e r 60 y e a r s ; 9 . . . . . . 9 ( A ) -" s u b j e c t s 60 y e a r s a n d over.
J. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
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C . W . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
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Fig. 5. Zonal esterase activity in male aorta. L a y e r 1 = inner intima; layer 6 or 7 = o u t e r media; 9 9 ( O ) = subjects u n d e r 40 years; 9 . . . . . 9 (A) = subjects 40 years and over. Overall a c t i v i t y for subjects u n d e r 40 vs. over 40 years, P -"- 0.2. % activity in layer 3 vs. layer 6; subjects u n d e r 40 years, P -~- 0.3; over 40 years, P ~- 0.004.
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Fig. 6. Zonal esterase activity in female aorta. L a y e r 1 = inner intima; L a y e r 6 = o u t e r media. 9 -9 ( O ) = subjects u n d e r 60 years; 9 . . . . . 9 ( A ) = subjects 60 years a n d over; Overall a c t i v i t y for subjects u n d e r 60 vs. over 60 years. P m_ 0.01. % activity in layer 3 vs. layer 6 in s u b j e c t s over 60 years, P -"- 0.01. j . Atheroscler. R e s . , 1969, 9 : 8 7 - 1 0 2
LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
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&it ty~ar~} Fig. 7. Change with age of overall lipase activity in homogenate supernatants of male aorta. Continuous line = mean activity calculated for decades; interrupted line = reciprocal of aortic weight calculated from Fig. 1. Overall activity (wet wt. basis) for subjects under 40 vs. over 40 years, P ~-~0.005.
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Fig. 8. As for Fig. 7, but in female aorta. Interrupted line = reciprocal of aortic weight calculated from Fig. 2. decade, when results a r e c o m p a r e d w i t h t h e reciprocal of t h e age increase in aortic weight. I n a selected n u m b e r of vessels, aortic lipase a c t i v i t y was referred to b o t h wet weight a n d p r o t e i n nitrogen. T h e results of this s u b s i d i a r y e x p e r i m e n t showed une x p e c t e d l y high a c t i v i t y on a n i t r o g e n basis in 2 o u t of 8 severely atherosclerotic a o r t a s (2.S-3.75-fold t h a t e x p e c t e d from " w e t w e i g h t " a c t i v i t y ) . A m o n g 12 m i l d l y or m o d e r a t e l y diseased vessels, only one showed higher a c t i v i t y on a nitrogen basis (2.25fold) t h a n t h a t e x p e c t e d from t h e " w e t w e i g h t " results. T h e s o l u b i l i t y of aortic lipase a n d esterase u n d e r histochemical i n c u b a t i o n conditions is i n d i c a t e d in T a b l e 1. I t is a p p a r e n t t h a t t h e esterase is p a r t l y soluble (48 %) from unfixed a o r t i c sections. No significant a m o u n t of lipase would b e left in unfixed, e t h a n o l - " f i x e d " or a c e t o n e - " f i x e d " tissue sections. j . Atherosder. Res., 1969, 9:87-102
96
C. W. M. ADAM.S, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
4"0 I
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Fig. 9. C h a n g e with age of overall esterase a c t i v i t y in male aorta. C o n t i n u o u s line -- m e a n a c t i v i t y c a l c u l a t e d for decades; i n t e r r u p t e d line -- reciprocal of aortic w e i g h t calculated f r o m Fig. 1. Overall a c t i v i t y (wet wt. basis) for s u b j e c t s u n d e r 40 vs. over 40 years, P -"- 0.15.
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Fig. 10. As for Fig. 9, b u t in female aorta. I n t e r r u p t e d line = reciprocal of aortic w e i g h t c a l c u l a t e d from Fig. 2. Overall a c t i v i t y (wet wt. basis) for s u b j e c t s u n d e r 60 vs. over 60 years, P -~- 0.18. j . Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
20
30
40
50
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60
70
80
9 7
90
(ye,rs)
Fig. 11. C h a n g e w i t h age in c o n c e n t r a t i o n s of a o r t i c cholcsteroI, p h o s p h o l i p i d a n d t r i g l y c e r i d e . I n t e r r u p t e d lines -- r e c i p r o c a l s of a o r t i c w e i g h t s c a l c u l a t e d from Figs. 1 a n d 2. 9 = me n, Q = women.
However, a small part of aortic lipolytic activity (23 %) is retained within formalin-fixed tissue sections. It is not clear whether such residual activity is due to retention (fixation) of a small part of the enzyme or whether it represents reduced (inhibited) activity of fully retained enzyme. The quantitative estimations of cholesterol, total phospholipid and triglyceride in whole aortic wall are shown in Fig. 11. Thin-layer chromatography of consecutive serial zones of tile aorta showed only slight accumulation of triglycerides in layers 1, 2, 3 and 4 of the ageing vessel; these layers correspond to tile tunica intima, and the inner and inner-middle zones of ttie tuniea media. DISCUSSION
Characterisation of aortic enzymes The lipase studied in this investigation resembles the arterial wall enzyme characterised b y ZEMPLI~;NYIeta/. 2,21,22 but not the rabbit aortic lipase described by ZOLDOS AND HEINEMANN23. ZEMPLI~NYI'Sand our lipase are unaffected by taurocholate and eserine u, but are partly inhibited by 1 M sodium chloride, E600 and protamine. The/~-naphthyl laurate esterase differs from the arterial lipase in certain inhibiJ. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
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C . W . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
tion characteristics (see Table 1) and in its zonal distribution in the senescent arterial wall (see Figs. 3-6). KIRK24 reported that arterial /5-naphthyl laurate esterase is inhibited by taurocholate. However, we could not confirm this observation. Bile salts are reported to activate true lipase and to inhibit both lipoprotein lipase and esterases25-27; but they do not seem to influence the arterial wall lipase and esterase (present results and ref. 2). As the precise nature of these arterial wall enzymes remains somewhat uncertain, they should perhaps be defined operationally as "tributyrinase" and "fl-naphthyl laurate esterase" respectively.
Expression of results It has been argued that arterial wall enzyme activity should be referred to DNA (i.e. cell nuclei plus mitochondria) or to non-collagen protein nitrogen (i.e. elastic tissue plus cell contents). However, results expressed on this basis would indicate the level of enzyme activity within surviving cells, without revealing to what extent the cellular elements have atrophied or necrosed. Referring results to total protein nitrogen would be substantially biased by the considerable quantity of collagen that is formed in the atherosclerotic or hyperplastic intima and atrophied degenerating media (see refs. 5,6,3o). In one experimental series reported here, lipase activity of aortic homogenates was referred both to wet weight and protein nitrogen. In only 3 out of 20 cases were results expressed on these two bases grossly inconsistent with each other. In 2 of these lipid deposition had presumably diluted enzyme activity so that a low "wet weigh t result" was obtained. Enzyme results based on wet weight can take no account of the progressively increasing weight of the ageing aorta, due to lipid deposition and sclerosis. However, this difficulty was partly overcome in this study by comparing decreased wet weight activity with the reciprocal of the age increase in aortic weight from 20-80 years. This is equivalent to relating results to the original weight of the aorta at 20 years. (This age was chosen because extensional somatic growth has virtually stopped and relatively little atherosclerosis is found at this early period of adulthood.) This method of interpreting the results is essentially similar to using intimal surface area as the reference basis. The initial design of this present study did not provide for estimation of both enzyme activity and weight in the same aortic disc. If this has been done, both parameters could have been directly referred to surface area. In retrospect, it is only too clear that such a technique is simpler, more direct and more elegant than comparing the decline in enzyme activity (referred to wet weight) with the reciprocal of the age increase in aortic weight (inferred surface area or inferred weight at age 20 years). In contrast to the above methods of expressing results, quantitative histochemical results--as reported in this paper--reveal the level of enzyme activity in each sectioned zone of the flattened aortic wall. As these estimations were made on zones of approximately equal volume, they present an accurate picture of enzyme activity in each successive layer of the aortic wall from inside outwards.
j. Atheroscler. Res., 1969, 9:87-102
L I P A S E ESTERASE AND T R I G L Y C E R I D E IN T H E A G E I N G HUMAN AORTA
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We believe that such a combination of methods for preparing tissues and expressing results is considerably more informative than any one method in isolation.
Location of enzymes
When results are expressed on a wet weight basis, overall aortic lipolytic and esterolytic activities tend to decline with advancing age. However, when such reduced overall esterolysis and lipolysis are compared with the reciprocal of the age i~crease in aortic weight (see Figs. 1 and 2), the decline in these enzyme activities largely disappears (see Figs. 7-10). In fact on this basis aortic lipase in women "increases" up to the age of 60 years (Fig. 8). A priori, it could be concluded that these enzymes remain intact in the ageing human aorta. However, the quantitative hi.,;tochemical results disclose a moderate midmedial loss of esterolytic activity in the ageing human aorta. This medial loss is presumably counterbalanced by esterolysis in the thickened atherosclerotic or hypeiplastic tunica intima, as shown here by quantitative histochemistry and previously by "slide" histochemistry zs,29. This balance between midmedial enzyme loss and intimal gain may well explain how total esterolytic activity remains constant when compared with the reciprocal of aortic weight. LEVONEN el al.'s 29 "slide" histochemical studies showed increased esterolytic activity ill the tunica media in the severely atherosclerotic human aorta. Nevertheless, our quantitative histochemical results do not confirm this reported augmentation and, moreover, human aortic muscle fibres usually only display weak esterase activity in slide preparations2, ~~ The position concerning aortic lipolytic activity is more complex. Quantitative histochemical results reveal no specific decline in this enzyme in the midzone of the ageing human aortic media even though activity falls in each layer of the male aorta with advancing age. In "slide" histochemical studies Tween lipase activity has been reported to increase in the smooth muscle cells underlying human atherosclerotic plaques 31 but, using GOMORI'S Tween method, we have been unable to confirm this observation ~0. Tween lipase activity has also been reported to be present in the cells of the human atherosclerotic intima32, 33. However, it is not at all clear what relationship this Tween lipase bears to the arterial wall lipolytic enzyme studied here. Moreover, not more than 4 ~o of arterial lipase activity is retained in aortic tissue- sections after subjecting them to " d u m m y " histochemical incubation or to any other conventional fixative, except formalin (see Table 1). When overall lipolytie activity in whole aortic homogenates is compared with the reciprocal of aortic weight, lipase remains essentially unaffected in the male and actually "increases" in women up to the end of the 6th decade. As with esterase, these results must represent the sum of intimal and medial lipolytic activities. In men, total medial lipase activity falls with advancing age (Fig. 3), but activity gained from the thickened atherosclerotic intima keeps the overall activity constant when results are compared with the reciprocal of aortic weight. In women, however, medial lipolytic J. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
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activity is unaffected up to the age of 60 years (Fig. 4), so that overall activity increases when compared with the reciprocal of aortic weight.
Triglyceride content of arterial wall In general accord with SMITH'S7 observations, we found that the amount of triglyceride in the aortic wall only increases by a trivial amount with advancing age (see Fig. 11) : this contrasts with the greater increment in phospholipid and the striking increase in cholesterol. The above-mentioned results are expressed on a wet weight basis. When these estimations are compared with the reciprocal of aortic weight (interrupted lines in Fig. 11), triglyceride accumulation remains trivial, but the increments of cholesterol and phospholipid become even more substantial. These findings suggest either that triglycerides do not penetrate the arterial wall or that they are rapidly catabolized and do not accumulate to any great extent therein. The first explanation is not plausible, because radioactivity has been detected in the aortic wall after feeding rabbits with 125I-labelled triolein. It follows that triglycerides do enter the arterial wall and then must be actively catabolised by vascular lipolytic enzymes (see refs. 5, 6). Any triglyceride synthesized within the arterial wall35, 36 is presumably also rapidly catabolised. CONCLUSIONS
Cholesterol cannot apparently be significantly catabolised by the arterial wall. Apart from oxidation of the side chain and other minor biochemical but physiologically important modifications of the nucleus, the bodily economy is unable to catabolise sterols in the same sense that triglycerides can be broken down to their constitutent parts. Hence, it has been suggested that the arterial wall can only dispose of cholesterol (and perhaps cholestenones and cholestanol) by processes that promote dispersion and transport of sterols out of the tissue (see refs. 3o,37). The failure and relative inefficiency of such mechanisms may explain the peculiar rather selective accumulation of cholesterol in atherosclerotie lesions (see ref. a0). By contrast triglycerides can be degraded to relatively simple components by the action Of various tissue lipases. However, it must be admitted that it is not clear how the arterial wall disposes of the fatty acids thus released from triglycerides, unless they take part in acylation and transacylation reactions (see ref. 3s) or are conjugated with albumin. The relative absence of triglycerides from human atherosclerotic lesions can be attributed both to the efficiency of arterial lipolytic activityS, 6 and the relative persistence of lipase and esterase in the ageing aortic wall. The midmedial decline in esterolytic activity in both sexes and the overall medial decline in lipase activity in the male aorta appear to be counterbalanced by the gain in enzymic activity from cells in the thickened atherosclerotic intima. Thus, it is inferred that the overall lipolytic and esterolytic activity of the ageing aortic wall is not significantly lower than that which would have pertained in a similar segment of the vessel in early adulthood.
j. Atheroscler. Res., 1969, 9:87-102
LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
101
ACKNOWLEDGEMENTS The authors are indebted to the Department
of M e d i c a l I l l u s t r a t i o n , G u y ' s
H o s p i t a l M e d i c a l S c h o o l for p r e p a r i n g t h e figures. T h e a u t h o r s also w i s h t o a c k n o w l e d g e R e s e a r c h G r a n t s f r o m t h e B r i t i s h H e a r t Foundation
(Y.H.A.), t h e T o b a c c o R e s e a r c h Council (Y.H.A.), Chas. Pfizer L t d .
(C.W.M.A.)a n d
the U.S. Public H e a l t h Service (Grant H E - 0 9 4 5 7 (R.F.M.); Fellow-
s h i p A w a r d 1 - F 3 - A M - 2 9 (M.R.).)
REFERENCES I ZE~PL~NYI, T. AND D. GRAFNETTER, The lipolytic activity of the aorta, its relation to ageing and atherosclerosis, Gerontologia (Basel), 1959, 3: 55. 2 ZEMPL~NYI, T., Z. LOJDA AND O. I~RHOV~, Enzymes of the vascular wall in experimental atherosclerosis in the rabbit. In: M. SANDLER AND G. H. BOURNE (Eds.), Atherosclerosis and its Origin, Academic Press, New York, 1963, p. 459. 3 MALLOV, S., Aortic lipoprotein lipase activity in relation to species, age, Sex and blood pressure, Circular. Res., 1964, 14: 357. 4 ADAMS, C. W. M., Y. H. ABDULLA, O. B. BAYLISS, R. F. MAHLER AND M. A. ROOT, O u a n t i t a t i v e
histochemical observations on certain oxidative and lipolytic enzymes in human aortic wall, Progr. biochem. Pharmaeol., 1967, 4: 218. s ZZ~PL~NYI, T., Enzymes of the arterial wall, J. Atheroscler. Res., 1962, 2: 2. 8 ZEMvLgNYI, T., Enzyme Biochemistry of the Arterial Wall as related to Atherosclerosis, LloydLuke, London, 1968. 7 SMITH, E. B., The influence of age and atherosclerosis on.the chemistry of aortic intima, Part (The iipids), J. Atheroseler. Res., 1965, 5: 224. 8 ADAMS, C. W. M., Y. a . ABDULLA, O. B. BAYLISS AND R. 0 . WELLER, Histoehemieal detection of triglyceride esters with specific lipases and a calcium-lead sulphide technique, J. Histochem. Cytochem., 1966, 14: 385. 9 WORLD HEALTH ORGANIZATION, Classification of atherosclerotic lesions, Wld, Hlth. Org. techn. Rep. Ser., No. 143, 1958. 10 DAVIS, J. N., C. W. M. ADAMSAND O. B. BAYLISS, Gradient in cholesterol concentration across human aortic wall, Lancet, 1963, if: 1254. 11 ABDULLA, Y. H. AND C. W. IV[. ADAMS, The distribution and nature of phospholipids in the human aortic wall, J. Atheroscler. Res., 1965, 5: 504. 12 MAHLER, i . F., TO be published. la KORN, E. D., Clearing factor, a heparin activated lipoprotein Iipase, Part 1 (Isolation and characterization of the enzyme from normal rat heart), J. biol. Chem., 1955, 215: 1. 14 MAIER, N. AND H. HAIMOVICI, Metabolism of arterial tissue with special refelence to esterase and Iipase, Proe. Soe. exp. Biol. ( N . Y . ) , 1965, 118: 258. 15 SELIGMAN, A. M. AND N. M. NACHLAG, The coiorimetric determination of iipase and esterase in human serum, J. clin. Invest., 1950, 2 9 : 3 1 16 FoLey, J., M. LEES AND G. H. SLO.~cE-SrA~LEV, A simple method for the isolation and purification of total lipides from animal tissues, J. biol. Chem., 1987, 226: 497. 17 FREEMAN, C. P. AND D. WEST, Complete separation of lipid classes on a single thin layer plate, J. Lipid Res., 1966, 7: 324. 18 MARINETTI, G. V., Chromatographic separation, identification and analysis of phosphatides, J. Lipid Res., 1962, 3: 1. 19 LEFLER, H. H. AND C. I2[. McDOUGALD, Estimation of cholesterol in serum, Amer. J. clin. Path., 1963, 39: 311. 20 SI
j . Atheroscler. Res., 1969, 9:87-102
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C. W. M. ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
23 ZSOLDOS, S. J. AND H. O. HEINEMANN, Lipolytic a c t i v i t y of r a b b i t a o r t a
in vitro, Amer. J.
Physiol., 1964, 206: 615. 24 KIRK~ J. E., Aliesterase activities of n o r m a l a n d atherosclerotic h u m a n v a s c u l a r tissue, Lab. Invest., 1965, 14: 573. 25 GEORGE, J. C. AND P. M. AMBADKAR, H i s t o c h e m i c a l d e m o n s t r a t i o n of lipids a n d lipase a c t i v i t y in r a t testis, J. Histochem. Cytochem., 1963, 11: 420. 26 ABE, M., S. P. •RAMER, L. D. ARONSON, M. D. SULKIN, M. G. ROSENFELD AND A. M. SELIGMAN, A n e w m e t h o d for t h e d e m o n s t r a t i o n of p a n c r e a t i c lipase, J. Histochem. Cytochem., 1964, 12: 26. 27 MUNEAKI, M., S. P. KRAMER AND A. M. SELIGMAN, T h e h i s t o e h e m i c a l d e m o n s t r a t i o n of p a n c r e a t i c - l i k e lipase a n d c o m p a r i s o n w i t h t h e d i s t r i b u t i o n of esterase, J. Histochem. Cytochem., 1964, 12: 364. 28 DERIBAS, V. I., B. B. FUKS AND G. S. SCHISCHKIN, E s t e r a s e a c t i v i t y in h u m a n atheroselerotic lesions, Dokl. Akad. Nauk. SSSR, Otd. Biol., 1960, 134: 443. 29 LEVONErL E., J. RAEKALLIO AND U. UOTILA, H i s t o c h e m i e a l d e m o n s t r a t i o n of s o m e h y d r o l y t i c e n z y m e s in a t h e r o m a t o u s a o r t a s , Nature (Lond.), 1960, 188: 677. 3o ADAMS, C. W. M., Vascular Histochemistry, L l o y d - L u k e , L o n d o n , 1967, p. 19. sl LEITES, F. L., A c t i v i t y of lipolytic e n z y m e s in atherosclerosis, Fed. Proc., 1964, 23: T565. 82 GOMORI, G., D i s t r i b u t i o n of lipase in t h e t i s s u e s u n d e r n o r m a l a n d p a t h o l o g i c c o n d i t i o n s , Arch. Path., 1964, 41: 121. 33 TISCHENDORF, F. AND S. B. CURRI, ]:)as V e r h a l t e n der L i p a s e in a t h e r o s k l e r o t i s c h v e r a n d e r t e n A r t e r i e n v o m muskul~iren T y p , Acta histochem. (Jena), 1959, 8: 158. 84 FRIEDMAN, M., S. O. BY~RS, L. FENTON AND P. CADV, Localization a n d r e t e n t i o n of I TM f r o m fed triolein in t h e atherosclerotie infiltration of r a b b i t aortas, J. clin. Invest., 1959, 38: 539. a5 STEIN, O., Z. SELINGER AND Y. STEIN, I n c o r p o r a t i o n of [1-14C]liuoleic acid into lipids of h u m a n u m b i l i c a l arteries, J. Atheroscler. Res., 1963, 3: 189. 36 STEIN, Y., O. ST~IN AND B. SHAPIRO, E n z y m i c p a t h w a y s of glyceride a n d p h o s p h o l i p i d s y n t h e s i s in aortic h o m o g e n a t e s , Biochim. biophys. Acta (Amst.), 1963, 70: 33. 87 ADAMS, C. W . M., S. VIRAG, R. S. MORGAN AND C. C. ORTON, Dissociation of [SH]cholesterol a n d 125I-labelled p l a s m a p r o t e i n influx in n o r m a l a n d a t h e r o m a t o u s r a b b i t aorta. A q u a n t i t a t i v e h i s t o e h e m i c a l s t u d y , J. Atheroscler. Res., 1968, 8: 679-696. 38 ABDULLA, Y. H . , C. C. ORTON AND C. W . M. ADAMS, Cholesterol esterification b y t r a n s a c y l a tion in h u m a n a n d e x p e r i m e n t a l 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 7 - 1 0 2
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
LIPASE, E S T E R A S E AND T R I G L Y C E R I D E IN T H E A G E I N G HUMAN AORTA A QUANTITATIVE HISTOCHEMICAL AND BIOCHEMICAL STUDY C. W. M. ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER AND M. A. ROOT Departments of Pathology and Medicine, Guy's Hospital Medical School, London, and Department of Clinical Chemistry, Welsh National School of Medicine, Cardiff (Great Britain)
(Revised, received November 6th, 1968)
SUMMARY
Lipase (tributyrinase), esterase (fl-naphthyl laurate esterase), cholesterol, phospholipid and triglyceride were estimated in human aortas from the age of 20 years to the 9th decade. Overall activity of lipase and esterase tended to decrease on a wet weight basis with advancing age, but aortic lipase activity in women remained nearly constant up to the end of the 6th decade. When such decreased activity was compared with the reciprocal of the age increase in aortic weight (i.e. inferred surface area), neither enzyme showed this age-dependent tendency to decline. On this basis aortic lipase in women became more active up to the end of the 6th decade. Quantitative histochemical estimation of these two enzymes in consecutive layers through the aorta revealed a midmedial decline in esterase activity with advancing age, but no similar decline in lipase. Increments in enzyme activity in the atherosclerotic or hyperplastic intima probably counterbalance the midmedial loss of esterase activity in the ageing aorta in both sexes and the lower medial lipase activity in the ageing male aorta. Aortic cholesterol shows a marked increase on a wet weight basis with advancing age, while the increment in triglycerides is only trivial. Phospholipid occupies an intermediate position in this respect. When lipid values are compared with the reciprocal of aortic weight, the increase in triglyceride remains trivial. These results suggest t h a t residual medial activity together with intimal augmentation of lipase and esterase in the ageing human aortic wall prevent any substantial accumulation of triglycerides therein.
Key words: H u m a n aorta - E n z y m e s - L i p a s e - E s t e r a s e - E x p r e s s i o n o f aortic e n z y m e activity -
Quantitative histochemistry - Aortic ageing - Triglycerides -
P h o s p h o l i p i d s - Cholesterol
J. Atheroscler. Res., 1969, 9:87-102
88
C . W . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
INTRODUCTION
Previous biochemical studies have shown that lipolytic activity declines with advancing age in the rat and rabbit's aorta 1-3. Likewise, our previous studies have shown that tributyrin lipase and fl-naphthyl laurate esterase activities decline in the ageing human aorta, when results are expressed on a simple wet weight basis 4. ZE~PL~NYIS, 6 has suggested that the lipolytic activity of the arterial wall protects the vessel against the deposition of lipids (triglycerides) therein. In this connection S.~IT~!Sv studies showed that triglycerides increase only slightly in the ageing human aorta, in contrast to esterified cholesterol which shows a far more spectacular increase. Histochemical results confirm the relative absence of triglycerides from hunmn atherosclerotic lesions 8. The purpose of this paper is to report further studies on lipolytic activity, esterolytic activity and triglyceride concentration in the ageing human aorta. A problem that arises from these studies is how best to express these results in view of the comple~:- alterations in arterial structure and mural volume that are caused b y lipid accumulation, cellular degeneration and sclerosis. The hypothesis to be tested is whether the apparently trivial triglyceride accumulation in human aortic atherosclerosis can be explained by an actual maintenance of lipolytic activity, when the age-dependent increase in aortic weight is discounted. METHODS
Preparation of samples Aortic samples from 125 Caucasian subjects, not suffering from congenital heart disease, were obtained at necropsy from the junction of the thoraci9 and abdominal portions of the vessel. This region was chosen because it is accessible at an early stage of the necropsy procedure and it is less ~0mmonly involved by severe calcific atherosclero~is. The severity of aortic atherosclerosis was graded by the WHO method 9 "['issue for enzyme estimation from 70 cases varied from grade 0 in. a few subjects under 40 years to grade III (ulcerated or slightly calcified plaques) in some subiects aged over 50 years. Atherosclerotic and grossly normal areas were not separately dissected and assayed; areas involved by severe calcification or mural thrombus were excluded. Further tissue from 55 cases was used to establish the extent of aortic weight increase with age (see below). The incidence and severity of atherosclerosis in ~elation to age were similar in this material to t h a t used for enzyme estimation. The necropsy diagnoses of subjects used for one or the other study showed no bias or "weighting" towards a particular disease category. The tunica adventitia was carefully stripped off a 2 cm • 2 cm sample of tim vessel; the specirpen was than either we.ighed and homogenized in an Ultraturrex blender or, alternatively, was used for quantitative histochemical estimation. Serial consecutive aortic layers were prepared from.the intima to the outer media by a histological technique. The unfixed specimen of aorta was flattened by j . Atheroscler. Res.,. 1969, 9 : 8 7 - 1 0 2
L I P A S E ESTERASE AND T R I G L Y C E R I D E IN T H E A G E I N G HUMAN AORTA
89
means of a glass microscope slide onto the chuck of a De Ira Rue Frigistor thermoelectric microtome. The microtome knife was adjusted so as to be perfectly parallel with the surface of the aorta. Frozen sections were cut at 50 # and were arranged in groups of 6 or 7 equally divided zones (i.e. 2--4 sections in each zone), 'from the,in.~ide to the outside of the vessel. The superficial (inner) layers of plaque tissue (incomplete sections) were grouped together to form part of layer 1. More sections were included in each individual layer from atherosclerotic aortas. Thus, the total number of layers obtained from " n o r m a l " and atherosclerotic aortas was often the same. The zonal samples were weighed in tubes of known tare and then homogenized. Tiffs procedure is a slight modification of our previous technique for eytochemical analysis of the aortal0,tt. Homogenates of whole aortic wall (intima + media) or homogenized serial zones of the aorta were estimated for lipase and esterase activities and analysed for their lipid and total protein content.
Age increase in aortic weight. Aortic weight increase with advancing age was determined in a second series of vessels (see above). For this purpose a circle o[ tissue was cut from each quadrant of the thoraco-lumbar aortic junction by means of a No. 6 cork borer (12 m m internal diameter). After removing surface fluid, these 4 aortic circles were then weighed on an aperiodic electric balance sensitive to 0.1 rag.
Analytical methods Samples for lipase estimation were homogenised in either 0.15 M Krebs-Ringer phosphate buffer (pH 7.4) or distilled water. Lipolytic activity against tributyrin was estimated b y MAHLER'S method 21. One-ml duplicate aliquots (each - 50 mg tissue) of supernatants (derived by centrifuging the homogenate at 2000 rev./min for 5 min) were incubated with 1 ml 0.4 % tributyrin (BDH) in 0.15 M phosphate buffer for 1 h at 37~ After incubation protein was precipitated b y adding 0.5"ml of 50 % trichloroacetic acid; 0.2-ml aliquots of the supernatant were used for estimating the released glyceroP 3. Esterase activity against/5-naphthyl laurate was estimated b y a modification t4 o[ SELIGMAN AND NACHLAS' method 15. One-half-ml duplicate aliquots (each - 5 mg tissue) of whole aqueous homogenates (see above) were incubated with 2.5 ml 0.02 % fl-naphthyl laurate 15 in veronal-HC1 buffer (pH 7.4) for 2 11 at 37~ The liberated flnapththol was estimated at 0~ with diazo blue B (Sdgma) as coupling reagent: the resulting bis-azo dye was extracted into ethyl acetate and its absorption determined at 540 m/~. From this reading was subtracted the value obtained with a homogenate that had been boiled before incubation. The protein content of aliquots of some homogenates was determined b y the standard method using the Folin-Ciocalteau reagent for tyrosine. Total lipids were extracted from adjacent samples of aorta with chloroformmethanol (2 : 1, v/v) b y the conventional FOLCH method 16. Lipid classes were separ-
j. Atheroscler. Res,, 1969, 9:87-102
90
C. W. M. ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
ated on thin layer chromatoplates by FREEMANAND WEST'S method 17. Phospholipids were estimated by MARINETTI'S18 modification of the Bartlett method, total cholesterol was determined bylLEFLER AND McDoUGALD'Smethod 19, while triglycerides were estimated by SKIDMORE AND ENTEMAN'S technique 20.
Kinetic, solubility and inhibitor studies Enzyme activity was estimated in aliquots of aortic homogenates after various periods of storage at 4~ the time interval between death and estimation varied from 36-168 h. Enzyme activity was also estimated at different homogenate dilutions and after varying the incubation time from 30-120 min (see Table 1). Aortic lipase and esterase activities were characterised by their responses to sodium taurocholate, protamine sulphate, sodium chloride, Cu 2+, Zn 2+, physostigmine (eserine) and diethyl-p-dinitrophenyl phosphate (E600). These inhibitors were TABLE 1 K I N E T I C S A N D C H A R A C T E R I S T I C S OF A O R T I C W A L L L I P A S E A N D E S T E R A S E
Procedure or inhibitor
% Activity compared with control aliquot lipase esterase
E s t i m a t i o n a t 48/36 h respectively E s t i m a t i o n at 72/60 h respectively E s t i m a t i o n at 96/84 h respectively E s t i m a t i o n at 168/156 h respectively Dilution 50 %, • 2 Dilution 25 %, • 4 Dilution 12.5 %, • 8 Dilution 6.25 %, • 16 I n c u b a t i o n 30 min, x 2 I n c u b a t i o n 60 m i n I n c u b a t i o n 90 min, x 0.67 I n c u b a t i o n 120 min, • 0.5
t00 117 42 29 97 100 121 124 134 100 114 109
100 96 88 77 94 98 98
1 % Sodium t a u r o c h o l a t e 1 % Protamine sulphate M s o d i u m chloride 0,01 M Cu 2+ 0.01 M Zn 2+ 10 -4 M eserine 10 -3 M E600 10 -5 M E600
100 81 81 65 62 lO0 64 67
100 a 34
Homogenized unfixed n o n - i n c u b a t e d sections Unfixed i n c u b a t e d sections tissue h o m o g e n a t e i n c u b a t i n g fluid Formalin-fixed incubated sections tissue h o m o g e n a t e i n c u b a t i n g fluid E t h a n o l - " f i x e d " incubated sections tissue h o m o g e n a t e i n c u b a t i n g fluid Acetone-"fixed" incubated sections tissue h o m o g e n a t e i n c u b a t i n g fluid
100 4 96 23
100 52 48
Interferes w i t h reaction.
J. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
1.3 38 2.5 60
145 100 115 80
8.5 100 53 95
LIPASE ESTERASE AND TRIGLYCERIDEIN THE AGEING HUMAN AORTA
91
used during incubation at the dilutions indicated in Table 1; preincubation inhibition was not used. In order to determine the solubility of aortic lipase and esterase under histochemical conditions, groups of 100 unfixed aortic sections were weighed and then fixed for 1 h with 4 % f o r m a l d e h y d e - 1 % calcium acetate, ethanol or acetone. The fixatives were removed b y washing the sections thoroughly with several changes of distilled water. All groups of fixed sections were then " d u m m y " incubated for 1 h at 37~ with 0.15 M phosphate buffer (pH 7.4.) A group of 100 unfixed sections was "incubated" likewise, while a further group of 100 unfixed sections was used as the non-incubated control. Enzyme activity was estimated in the "soaking fluid" (buffer), homogenised incubated tissue residue (sections) and in homogenised non-incubated unfixed sections. RESULTS
The effect on enzyme activity of storing the homogenate at 4~ is shown in Table 1. As activity of both lipase and esterase declined after 72 h from death, no aorta was used for such estimations when more than 3 days had lapsed between death and proposed analysis. The effects of homogenate dilution and length of incubation are summarised in Table 1; both aortic enzymes displayed approximately zero order kinetics. The effects of known lipase and esterase inhibitors are also presented in Table 1. The weight of the four standard circles cut from the thoraco-lumbar aortic j unction progressively increased with advancing age; these results are set out for men
I
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.
400
200
20
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i
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~0 60 (years)
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Fig. 1. Change with age of weight of thoraco-lumbar junction of male aorta. J. Atheroscler. Res,, 1969, 9:87-102
92
c . w . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
in Fig. 1 and for women in Fig. 2. In the large sample studied, it was assumed that this weight increase was due mainly to atherosclerosis and slightly to diffuse intimal thickening. Naturally, such increases would be partly counterbalanced by atrophic changes in the tunica media. Quantitative histochemical estimations of lipase activity in serial zones from the blner intima to the outer media are presented in Fig. 3 (men) and Fig. 4 (women). Likewise, zonal esterase activity is shown in Fig. 5 (men) and Fig. 6 (women). Lipase activity undergoes no midzonal decline with advancing age in either sex; it shows a just significant overall decrease in older men (P ~- 0.05) but not in women. Esterase activity declines moderately in the midmedial zones of both sexes w~th~advancing years (men, P ~- 0.004; women, P -----0.01); it shows a significant overall decrease in women (P ~- 0.01) and a similar overall trend in men (P ~ 0.2). The change in overall lipase activity in whole aortic homogenates is illustrated in Fig. 7 (men) and Fig. 8 (women). Results for overall aortic esterase activity are presented in Fig. 9 (men) and Fig. 10 (women). With the exception of aortic lipase in women up to 60 years, these overall enzyme activities tended to decline with advanc.ing age when results were expressed on a wet weight basis. (Probability values are shovvn~in ~he legends to these figures.) However., tile reciprocal of the percentage weight increase in the aorta from age 20 years onwards is also shown as interrupted lines on these last four figures: it is clear.that the apparent decline in aortic enzyme activity on a wet weight,basis can largely be attributed to the increasing age-dependent weight of the vessel. In fact women's aortic lipolytic activity actually increases up to the 7th
1100
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LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
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J. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
94
C . W . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
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Fig. 6. Zonal esterase activity in female aorta. L a y e r 1 = inner intima; L a y e r 6 = o u t e r media. 9 -9 ( O ) = subjects u n d e r 60 years; 9 . . . . . 9 ( A ) = subjects 60 years a n d over; Overall a c t i v i t y for subjects u n d e r 60 vs. over 60 years. P m_ 0.01. % activity in layer 3 vs. layer 6 in s u b j e c t s over 60 years, P -"- 0.01. j . Atheroscler. R e s . , 1969, 9 : 8 7 - 1 0 2
LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
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&it ty~ar~} Fig. 7. Change with age of overall lipase activity in homogenate supernatants of male aorta. Continuous line = mean activity calculated for decades; interrupted line = reciprocal of aortic weight calculated from Fig. 1. Overall activity (wet wt. basis) for subjects under 40 vs. over 40 years, P ~-~0.005.
1,0
,~1.(
9
t 20
I $0
I 40
I 50
I RO
I 70
I 80
Fig. 8. As for Fig. 7, but in female aorta. Interrupted line = reciprocal of aortic weight calculated from Fig. 2. decade, when results a r e c o m p a r e d w i t h t h e reciprocal of t h e age increase in aortic weight. I n a selected n u m b e r of vessels, aortic lipase a c t i v i t y was referred to b o t h wet weight a n d p r o t e i n nitrogen. T h e results of this s u b s i d i a r y e x p e r i m e n t showed une x p e c t e d l y high a c t i v i t y on a n i t r o g e n basis in 2 o u t of 8 severely atherosclerotic a o r t a s (2.S-3.75-fold t h a t e x p e c t e d from " w e t w e i g h t " a c t i v i t y ) . A m o n g 12 m i l d l y or m o d e r a t e l y diseased vessels, only one showed higher a c t i v i t y on a nitrogen basis (2.25fold) t h a n t h a t e x p e c t e d from t h e " w e t w e i g h t " results. T h e s o l u b i l i t y of aortic lipase a n d esterase u n d e r histochemical i n c u b a t i o n conditions is i n d i c a t e d in T a b l e 1. I t is a p p a r e n t t h a t t h e esterase is p a r t l y soluble (48 %) from unfixed a o r t i c sections. No significant a m o u n t of lipase would b e left in unfixed, e t h a n o l - " f i x e d " or a c e t o n e - " f i x e d " tissue sections. j . Atherosder. Res., 1969, 9:87-102
96
C. W. M. ADAM.S, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
4"0 I
9
~
9
0
9O
""~
,=
1.0
I 30
20
I I 40 50 Age (years)
I 60
I 70
Fig. 9. C h a n g e with age of overall esterase a c t i v i t y in male aorta. C o n t i n u o u s line -- m e a n a c t i v i t y c a l c u l a t e d for decades; i n t e r r u p t e d line -- reciprocal of aortic w e i g h t calculated f r o m Fig. 1. Overall a c t i v i t y (wet wt. basis) for s u b j e c t s u n d e r 40 vs. over 40 years, P -"- 0.15.
4.0
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9
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9
-~
ee 9
t.0
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9 e",e 9
I 40
1 50
-.
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Age {years)
Fig. 10. As for Fig. 9, b u t in female aorta. I n t e r r u p t e d line = reciprocal of aortic w e i g h t c a l c u l a t e d from Fig. 2. Overall a c t i v i t y (wet wt. basis) for s u b j e c t s u n d e r 60 vs. over 60 years, P -~- 0.18. j . Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
20
30
40
50
~e
60
70
80
9 7
90
(ye,rs)
Fig. 11. C h a n g e w i t h age in c o n c e n t r a t i o n s of a o r t i c cholcsteroI, p h o s p h o l i p i d a n d t r i g l y c e r i d e . I n t e r r u p t e d lines -- r e c i p r o c a l s of a o r t i c w e i g h t s c a l c u l a t e d from Figs. 1 a n d 2. 9 = me n, Q = women.
However, a small part of aortic lipolytic activity (23 %) is retained within formalin-fixed tissue sections. It is not clear whether such residual activity is due to retention (fixation) of a small part of the enzyme or whether it represents reduced (inhibited) activity of fully retained enzyme. The quantitative estimations of cholesterol, total phospholipid and triglyceride in whole aortic wall are shown in Fig. 11. Thin-layer chromatography of consecutive serial zones of tile aorta showed only slight accumulation of triglycerides in layers 1, 2, 3 and 4 of the ageing vessel; these layers correspond to tile tunica intima, and the inner and inner-middle zones of ttie tuniea media. DISCUSSION
Characterisation of aortic enzymes The lipase studied in this investigation resembles the arterial wall enzyme characterised b y ZEMPLI~;NYIeta/. 2,21,22 but not the rabbit aortic lipase described by ZOLDOS AND HEINEMANN23. ZEMPLI~NYI'Sand our lipase are unaffected by taurocholate and eserine u, but are partly inhibited by 1 M sodium chloride, E600 and protamine. The/~-naphthyl laurate esterase differs from the arterial lipase in certain inhibiJ. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
98
C . W . M . ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
tion characteristics (see Table 1) and in its zonal distribution in the senescent arterial wall (see Figs. 3-6). KIRK24 reported that arterial /5-naphthyl laurate esterase is inhibited by taurocholate. However, we could not confirm this observation. Bile salts are reported to activate true lipase and to inhibit both lipoprotein lipase and esterases25-27; but they do not seem to influence the arterial wall lipase and esterase (present results and ref. 2). As the precise nature of these arterial wall enzymes remains somewhat uncertain, they should perhaps be defined operationally as "tributyrinase" and "fl-naphthyl laurate esterase" respectively.
Expression of results It has been argued that arterial wall enzyme activity should be referred to DNA (i.e. cell nuclei plus mitochondria) or to non-collagen protein nitrogen (i.e. elastic tissue plus cell contents). However, results expressed on this basis would indicate the level of enzyme activity within surviving cells, without revealing to what extent the cellular elements have atrophied or necrosed. Referring results to total protein nitrogen would be substantially biased by the considerable quantity of collagen that is formed in the atherosclerotic or hyperplastic intima and atrophied degenerating media (see refs. 5,6,3o). In one experimental series reported here, lipase activity of aortic homogenates was referred both to wet weight and protein nitrogen. In only 3 out of 20 cases were results expressed on these two bases grossly inconsistent with each other. In 2 of these lipid deposition had presumably diluted enzyme activity so that a low "wet weigh t result" was obtained. Enzyme results based on wet weight can take no account of the progressively increasing weight of the ageing aorta, due to lipid deposition and sclerosis. However, this difficulty was partly overcome in this study by comparing decreased wet weight activity with the reciprocal of the age increase in aortic weight from 20-80 years. This is equivalent to relating results to the original weight of the aorta at 20 years. (This age was chosen because extensional somatic growth has virtually stopped and relatively little atherosclerosis is found at this early period of adulthood.) This method of interpreting the results is essentially similar to using intimal surface area as the reference basis. The initial design of this present study did not provide for estimation of both enzyme activity and weight in the same aortic disc. If this has been done, both parameters could have been directly referred to surface area. In retrospect, it is only too clear that such a technique is simpler, more direct and more elegant than comparing the decline in enzyme activity (referred to wet weight) with the reciprocal of the age increase in aortic weight (inferred surface area or inferred weight at age 20 years). In contrast to the above methods of expressing results, quantitative histochemical results--as reported in this paper--reveal the level of enzyme activity in each sectioned zone of the flattened aortic wall. As these estimations were made on zones of approximately equal volume, they present an accurate picture of enzyme activity in each successive layer of the aortic wall from inside outwards.
j. Atheroscler. Res., 1969, 9:87-102
L I P A S E ESTERASE AND T R I G L Y C E R I D E IN T H E A G E I N G HUMAN AORTA
99
We believe that such a combination of methods for preparing tissues and expressing results is considerably more informative than any one method in isolation.
Location of enzymes
When results are expressed on a wet weight basis, overall aortic lipolytic and esterolytic activities tend to decline with advancing age. However, when such reduced overall esterolysis and lipolysis are compared with the reciprocal of the age i~crease in aortic weight (see Figs. 1 and 2), the decline in these enzyme activities largely disappears (see Figs. 7-10). In fact on this basis aortic lipase in women "increases" up to the age of 60 years (Fig. 8). A priori, it could be concluded that these enzymes remain intact in the ageing human aorta. However, the quantitative hi.,;tochemical results disclose a moderate midmedial loss of esterolytic activity in the ageing human aorta. This medial loss is presumably counterbalanced by esterolysis in the thickened atherosclerotic or hypeiplastic tunica intima, as shown here by quantitative histochemistry and previously by "slide" histochemistry zs,29. This balance between midmedial enzyme loss and intimal gain may well explain how total esterolytic activity remains constant when compared with the reciprocal of aortic weight. LEVONEN el al.'s 29 "slide" histochemical studies showed increased esterolytic activity ill the tunica media in the severely atherosclerotic human aorta. Nevertheless, our quantitative histochemical results do not confirm this reported augmentation and, moreover, human aortic muscle fibres usually only display weak esterase activity in slide preparations2, ~~ The position concerning aortic lipolytic activity is more complex. Quantitative histochemical results reveal no specific decline in this enzyme in the midzone of the ageing human aortic media even though activity falls in each layer of the male aorta with advancing age. In "slide" histochemical studies Tween lipase activity has been reported to increase in the smooth muscle cells underlying human atherosclerotic plaques 31 but, using GOMORI'S Tween method, we have been unable to confirm this observation ~0. Tween lipase activity has also been reported to be present in the cells of the human atherosclerotic intima32, 33. However, it is not at all clear what relationship this Tween lipase bears to the arterial wall lipolytic enzyme studied here. Moreover, not more than 4 ~o of arterial lipase activity is retained in aortic tissue- sections after subjecting them to " d u m m y " histochemical incubation or to any other conventional fixative, except formalin (see Table 1). When overall lipolytie activity in whole aortic homogenates is compared with the reciprocal of aortic weight, lipase remains essentially unaffected in the male and actually "increases" in women up to the end of the 6th decade. As with esterase, these results must represent the sum of intimal and medial lipolytic activities. In men, total medial lipase activity falls with advancing age (Fig. 3), but activity gained from the thickened atherosclerotic intima keeps the overall activity constant when results are compared with the reciprocal of aortic weight. In women, however, medial lipolytic J. Atheroscler. Res., 1969, 9 : 8 7 - 1 0 2
100
c.W.M.
ADAMS, O. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER, M. A. ROOT
activity is unaffected up to the age of 60 years (Fig. 4), so that overall activity increases when compared with the reciprocal of aortic weight.
Triglyceride content of arterial wall In general accord with SMITH'S7 observations, we found that the amount of triglyceride in the aortic wall only increases by a trivial amount with advancing age (see Fig. 11) : this contrasts with the greater increment in phospholipid and the striking increase in cholesterol. The above-mentioned results are expressed on a wet weight basis. When these estimations are compared with the reciprocal of aortic weight (interrupted lines in Fig. 11), triglyceride accumulation remains trivial, but the increments of cholesterol and phospholipid become even more substantial. These findings suggest either that triglycerides do not penetrate the arterial wall or that they are rapidly catabolized and do not accumulate to any great extent therein. The first explanation is not plausible, because radioactivity has been detected in the aortic wall after feeding rabbits with 125I-labelled triolein. It follows that triglycerides do enter the arterial wall and then must be actively catabolised by vascular lipolytic enzymes (see refs. 5, 6). Any triglyceride synthesized within the arterial wall35, 36 is presumably also rapidly catabolised. CONCLUSIONS
Cholesterol cannot apparently be significantly catabolised by the arterial wall. Apart from oxidation of the side chain and other minor biochemical but physiologically important modifications of the nucleus, the bodily economy is unable to catabolise sterols in the same sense that triglycerides can be broken down to their constitutent parts. Hence, it has been suggested that the arterial wall can only dispose of cholesterol (and perhaps cholestenones and cholestanol) by processes that promote dispersion and transport of sterols out of the tissue (see refs. 3o,37). The failure and relative inefficiency of such mechanisms may explain the peculiar rather selective accumulation of cholesterol in atherosclerotie lesions (see ref. a0). By contrast triglycerides can be degraded to relatively simple components by the action Of various tissue lipases. However, it must be admitted that it is not clear how the arterial wall disposes of the fatty acids thus released from triglycerides, unless they take part in acylation and transacylation reactions (see ref. 3s) or are conjugated with albumin. The relative absence of triglycerides from human atherosclerotic lesions can be attributed both to the efficiency of arterial lipolytic activityS, 6 and the relative persistence of lipase and esterase in the ageing aortic wall. The midmedial decline in esterolytic activity in both sexes and the overall medial decline in lipase activity in the male aorta appear to be counterbalanced by the gain in enzymic activity from cells in the thickened atherosclerotic intima. Thus, it is inferred that the overall lipolytic and esterolytic activity of the ageing aortic wall is not significantly lower than that which would have pertained in a similar segment of the vessel in early adulthood.
j. Atheroscler. Res., 1969, 9:87-102
LIPASE ESTERASE AND TRIGLYCERIDE IN THE AGEING HUMAN AORTA
101
ACKNOWLEDGEMENTS The authors are indebted to the Department
of M e d i c a l I l l u s t r a t i o n , G u y ' s
H o s p i t a l M e d i c a l S c h o o l for p r e p a r i n g t h e figures. T h e a u t h o r s also w i s h t o a c k n o w l e d g e R e s e a r c h G r a n t s f r o m t h e B r i t i s h H e a r t Foundation
(Y.H.A.), t h e T o b a c c o R e s e a r c h Council (Y.H.A.), Chas. Pfizer L t d .
(C.W.M.A.)a n d
the U.S. Public H e a l t h Service (Grant H E - 0 9 4 5 7 (R.F.M.); Fellow-
s h i p A w a r d 1 - F 3 - A M - 2 9 (M.R.).)
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histochemical observations on certain oxidative and lipolytic enzymes in human aortic wall, Progr. biochem. Pharmaeol., 1967, 4: 218. s ZZ~PL~NYI, T., Enzymes of the arterial wall, J. Atheroscler. Res., 1962, 2: 2. 8 ZEMvLgNYI, T., Enzyme Biochemistry of the Arterial Wall as related to Atherosclerosis, LloydLuke, London, 1968. 7 SMITH, E. B., The influence of age and atherosclerosis on.the chemistry of aortic intima, Part (The iipids), J. Atheroseler. Res., 1965, 5: 224. 8 ADAMS, C. W. M., Y. a . ABDULLA, O. B. BAYLISS AND R. 0 . WELLER, Histoehemieal detection of triglyceride esters with specific lipases and a calcium-lead sulphide technique, J. Histochem. Cytochem., 1966, 14: 385. 9 WORLD HEALTH ORGANIZATION, Classification of atherosclerotic lesions, Wld, Hlth. Org. techn. Rep. Ser., No. 143, 1958. 10 DAVIS, J. N., C. W. M. ADAMSAND O. B. BAYLISS, Gradient in cholesterol concentration across human aortic wall, Lancet, 1963, if: 1254. 11 ABDULLA, Y. H. AND C. W. IV[. ADAMS, The distribution and nature of phospholipids in the human aortic wall, J. Atheroscler. Res., 1965, 5: 504. 12 MAHLER, i . F., TO be published. la KORN, E. D., Clearing factor, a heparin activated lipoprotein Iipase, Part 1 (Isolation and characterization of the enzyme from normal rat heart), J. biol. Chem., 1955, 215: 1. 14 MAIER, N. AND H. HAIMOVICI, Metabolism of arterial tissue with special refelence to esterase and Iipase, Proe. Soe. exp. Biol. ( N . Y . ) , 1965, 118: 258. 15 SELIGMAN, A. M. AND N. M. NACHLAG, The coiorimetric determination of iipase and esterase in human serum, J. clin. Invest., 1950, 2 9 : 3 1 16 FoLey, J., M. LEES AND G. H. SLO.~cE-SrA~LEV, A simple method for the isolation and purification of total lipides from animal tissues, J. biol. Chem., 1987, 226: 497. 17 FREEMAN, C. P. AND D. WEST, Complete separation of lipid classes on a single thin layer plate, J. Lipid Res., 1966, 7: 324. 18 MARINETTI, G. V., Chromatographic separation, identification and analysis of phosphatides, J. Lipid Res., 1962, 3: 1. 19 LEFLER, H. H. AND C. I2[. McDOUGALD, Estimation of cholesterol in serum, Amer. J. clin. Path., 1963, 39: 311. 20 SI
j . Atheroscler. Res., 1969, 9:87-102
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23 ZSOLDOS, S. J. AND H. O. HEINEMANN, Lipolytic a c t i v i t y of r a b b i t a o r t a
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Physiol., 1964, 206: 615. 24 KIRK~ J. E., Aliesterase activities of n o r m a l a n d atherosclerotic h u m a n v a s c u l a r tissue, Lab. Invest., 1965, 14: 573. 25 GEORGE, J. C. AND P. M. AMBADKAR, H i s t o c h e m i c a l d e m o n s t r a t i o n of lipids a n d lipase a c t i v i t y in r a t testis, J. Histochem. Cytochem., 1963, 11: 420. 26 ABE, M., S. P. •RAMER, L. D. ARONSON, M. D. SULKIN, M. G. ROSENFELD AND A. M. SELIGMAN, A n e w m e t h o d for t h e d e m o n s t r a t i o n of p a n c r e a t i c lipase, J. Histochem. Cytochem., 1964, 12: 26. 27 MUNEAKI, M., S. P. KRAMER AND A. M. SELIGMAN, T h e h i s t o e h e m i c a l d e m o n s t r a t i o n of p a n c r e a t i c - l i k e lipase a n d c o m p a r i s o n w i t h t h e d i s t r i b u t i o n of esterase, J. Histochem. Cytochem., 1964, 12: 364. 28 DERIBAS, V. I., B. B. FUKS AND G. S. SCHISCHKIN, E s t e r a s e a c t i v i t y in h u m a n atheroselerotic lesions, Dokl. Akad. Nauk. SSSR, Otd. Biol., 1960, 134: 443. 29 LEVONErL E., J. RAEKALLIO AND U. UOTILA, H i s t o c h e m i e a l d e m o n s t r a t i o n of s o m e h y d r o l y t i c e n z y m e s in a t h e r o m a t o u s a o r t a s , Nature (Lond.), 1960, 188: 677. 3o ADAMS, C. W. M., Vascular Histochemistry, L l o y d - L u k e , L o n d o n , 1967, p. 19. sl LEITES, F. L., A c t i v i t y of lipolytic e n z y m e s in atherosclerosis, Fed. Proc., 1964, 23: T565. 82 GOMORI, G., D i s t r i b u t i o n of lipase in t h e t i s s u e s u n d e r n o r m a l a n d p a t h o l o g i c c o n d i t i o n s , Arch. Path., 1964, 41: 121. 33 TISCHENDORF, F. AND S. B. CURRI, ]:)as V e r h a l t e n der L i p a s e in a t h e r o s k l e r o t i s c h v e r a n d e r t e n A r t e r i e n v o m muskul~iren T y p , Acta histochem. (Jena), 1959, 8: 158. 84 FRIEDMAN, M., S. O. BY~RS, L. FENTON AND P. CADV, Localization a n d r e t e n t i o n of I TM f r o m fed triolein in t h e atherosclerotie infiltration of r a b b i t aortas, J. clin. Invest., 1959, 38: 539. a5 STEIN, O., Z. SELINGER AND Y. STEIN, I n c o r p o r a t i o n of [1-14C]liuoleic acid into lipids of h u m a n u m b i l i c a l arteries, J. Atheroscler. Res., 1963, 3: 189. 36 STEIN, Y., O. ST~IN AND B. SHAPIRO, E n z y m i c p a t h w a y s of glyceride a n d p h o s p h o l i p i d s y n t h e s i s in aortic h o m o g e n a t e s , Biochim. biophys. Acta (Amst.), 1963, 70: 33. 87 ADAMS, C. W . M., S. VIRAG, R. S. MORGAN AND C. C. ORTON, Dissociation of [SH]cholesterol a n d 125I-labelled p l a s m a p r o t e i n influx in n o r m a l a n d a t h e r o m a t o u s r a b b i t aorta. A q u a n t i t a t i v e h i s t o e h e m i c a l s t u d y , J. Atheroscler. Res., 1968, 8: 679-696. 38 ABDULLA, Y. H . , C. C. ORTON AND C. W . M. ADAMS, Cholesterol esterification b y t r a n s a c y l a tion in h u m a n a n d e x p e r i m e n t a l 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 7 - 1 0 2