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Histochemical study of an esterase and a "Tween" lipase in arteries and other tissues under the influence… Orchard, Reynold Graham 1970

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i A HISTOCHEMICAL STUDY OF AN ESTERASE AND A "TWEEN" LIPASE IN ARTERIES AND OTHER TISSUES UNDER THE INFLUENCE OF CERTAIN FACTORS RELATED TO ATHEROSCLEROSIS by REYNOLD GRAHAM ORCHARD B.Sc, University of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the department of PATHOLOGY We accept t h i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1970 In p resent ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be al lowed without my w r i t t e n p e r m i s s i o n . Department of Pathology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 3 0 S e ^ - 1 9 7 0 i i ABSTRACT Nonspecific esterase, and in some cases a "Tween" lipase, were investigated histochemically in arteries and several other tissues of rats and rabbits under various conditions known to affect the development of atherosclerosis: age and sex differences, endocrine and metabolic factors (sex steroids, thyroid function, alloxan diabetes, and fasting), arterial injury, and acute and chronic lipemia. A study of the above enzymes was also made in atherosclerotic rabbit and human aortae. Of the several nonvascular tissues studied, only the enzymes of the male gonads and a male accessory sex organ (the prostate) were influenced by some of the experimental conditions tested: maturation greatly increased the activity of both enzymes in the Leydig cells of the testis, and sti l b e s t r o l markedly diminished the esterase activity in the prostate epithelium. Thus, in certain reproductive organs, the enzymes reflected the functional state of the cells under investigation. The following findings were made concerning the enzymes of the arterial wall: 1. Neither of the enzymes was influenced by ageing or sex difference in the rat, rabbit or human. 2 . Neither of the enzymes was influenced by sex hormones, acute or chronic lipemia, or any of the metabolic conditions studied in the rati; 3. Esterase disappeared from the foc i of acute arterial injury induced by Calciferol treatment in the rat and did not reappear six weeks after i n f l i c t i o n of the injury. i i i 4. Strong esterase activity appeared in the cytoplasm of l i p o -phages within both human and experimental rabbit athero-sclerotic lesions, in contrast to the normal arte r i a l wall which exhibited no esterase activity at a l l in either species with the methods used. 5. Esterase was absent from the superficial fibrous cap of the human atherosclerotic lesion. It i s concluded that: In the rat arteries, which normally exhibit appreciable esterolytic activity, this enzyme appears to be quite stable since i t i s not visibly modified by ageing or a series of endocrine and metabolic influences; i t i s , however, drastically diminished by acute vascular injury and this may account, at least in part, for the well known preferential accumulation of l i p i d s in foci of acute arterial damage. In the rabbit arteries, which normally exhibit no histochemi— cally demonstrable esterase, appreciable esterolytic activity appears only within the cytoplasm of cells that have taken up l i p i d after exposure to chromic lipemia (foam cells of atheromata). Similarly, in human atheromata esterase appeared only in foam cells and was absent from the fibrous cap of the atherosclerotic lesions; thus the absence of l i p i d from the cap cannot be a t t r i -buted to increased enzyme activity in this part of the lesion. i v TABLE OF CONTENTS Page Number ABSTRACT i i LIST OF TABLES v LIST OF FIGURES. v i ACKNOWLEDGEMENT . . v i i CHAPTER I: INTRODUCTION . 1 CHAPTER II:REVIEW OF THE LITERATURE ON ESTEROLYTIC VASCULAR ENZYMES 4 A. Enzymes i n Normal Tissues 4 B. E f f e c t s of Sex Difference and Sex Hormones on the Enzymes 5 C. E f f e c t s of Age on the Enzymes 8 D. E f f e c t s of Thyroid Hormones on the Enzymes 10 E. E f f e c t s of Alloxan Diabetes on the Enzymes 11 F. E f f e c t s of Fasting on the Enzymes 12 G. E f f e c t s of Vascular Injury oh the Enzymes 12 H. Ef f e c t s of Acute Lipemia on the Enzymes 13 I. Enzymes i n Atherosclerotic Human A r t e r i e s . . . . 13 J. Enzymes i n Experimental Atherosclerosis i n Rabbits 14 CHAPTER I I I : METHODS 16 A. Experimental Design 1. Reasons for Performing the Experiments 16 2. Description of S p e c i f i c Experiments .18 B. Histochemical Methods .24 CHAPTER IV: RESULTS 29 CHAPTER V: DISCUSSION AND CONCLUSIONS 34 A. Enzymes i n Normal Tissues and Age E f f e c t s 34 B. Ef f e c t s of Endocrine Factors 35 C. Ef f e c t s of Injury, Lipemia, and Fasting 36 D. Enzymes i n Human and Experimental Atherosclerosis 37 BIBLIOGRAPHY 39 FIGURES 44 APPENDIX ON HISTOCHEMICAL METHODS 58 V LIST OF TABLES Page Number TABLE I: NORMAL RAT ESTERASE AND LIPASE 32 TABLE II: NORMAL RABBIT ESTERASE AND LIPASE 33 v i LIST OF FIGURES Page Number 1. Photograph of a normal r a t pancreas. Esterase 44 2. Photograph of a normal r at adrenal. Esterase 44 3. Photograph of a normal exocrine r a t pancreas. Lipase 45 4. Photograph of a normal rat adrenal. Lipase 45 5. Photograph of the aorta of a young, weanling, 26 day old r a t . Esterase 46 6. Photograph of the coronary artery of a young, 26 day old r a t . Esterase 47 7. Photograph of the coronary artery of a mature r a t . Esterase..47 8. Photograph of the t e s t i s from an 8 day old r a t . Esterase....48 9. Photograph of the t e s t i s from a 30 day old r a t . E s t e r a s e . — 4 8 10. Photograph of the t e s t i s from a mature, 18 month old r a t . Esterase 49 11. Photograph of a normal r a t prostate gland. Esterase 50 12. Photograph of the prostate gland of a rat treated with s t i l b e s t r o l . Esterase 50 13. Photograph of the aorta of a mature, 18 month old rat.Ester-ase 51 14. Photograph of an injured aorta from a rat treated with C a l c i f e r o l . Esterase 51 15. Photograph of a normal r at aorta. H. and E 52 16. Photograph of an injured aorta from a rat treated with C a l c i f e r o l . H. and E 52 17. Photograph of a normal human aorta. Esterase 53 18. Photograph of a human aorta with f a t t y streak. Esterase 53 19. Photograph of part of a pearly plaque of a human aorta. Esterase 54 20. Photograph of a normal rabbit aorta. Esterase 55 21. Photograph of a rabbit aorta with raised plaque. Esterase 55 22. Photograph of a rabbit aorta with raised plaque. Esterase....56 23. Photograph of a rabbit aorta with raised plaque, H. and E....57 24. Photograph of a rabbit aorta with raised plaque. H. and E....57 v i i ACKNOWLEDGEMENT I would l i k e to esp e c i a l l y thank Professor Paris Constantinides for h i s guidance and advice during the course of t h i s work. Mrs. Barbara Barkoczy helped with i n s t r u c t i o n i n cryostat section cutting. Mr. F. Beardow, of the Department of Slavonic Studies, was of assistance i n tr a n s l a t i n g some Russian a r t i c l e s . The human ar t e r i e s were obtained through the courtesy of Dr. J . Burton of the Department of Pathology at the Vancouver General Hospital. 1 CHAPTER ONE INTRODUCTION Central to the atherosclerotic l e s i o n i s a dynamic but abnormal serie s of chemical and morphological changes concerned p a r t i c u l a r l y with l i p i d metabolism. L i p i d accumulates i n the a r t e r i a l wall, and i s also tranformed and degraded there to some extent. Since such l i p i d c atabolizing a c t i v i t y i s dependent upon the c a t a l y t i c e f f e c t s of enzymes, i t was considered f r u i t f u l to investigate histochemically some classes of l i p i d — h a n d l i n g enzymes i n some vascular and nonvascular tissues. While an enormous amount of work on atherosclerosis has been concerned with blood and artery l i p i d s , only r e l a t i v e l y l i t t l e work has been done on the a r t e r i a l enzymes, and most of i t has been accomplished with biochemical techniques. The purpose of t h i s thesis was to make a histochemical study of the behaviour of nonspecific esterase and "Tween 60" l i p a s e primarily i n vascular but also i n some non-vascular tissues under a va r i e t y of conditions known to have a d i r e c t bearing on the pathogenesis of atherosclerosis. For purposes of s i m p l i f i c a t i o n , nonspecific esterase w i l l henceforth be c a l l e d "esterase", and "Tween 60" l i p a s e w i l l be r e f e r r e d to as " l i p a s e " . Since rabbits, l i k e humans, are quite susceptible to athero-s c l e r o s i s but rats quite r e s i s t a n t , investigations were ca r r i e d out i n rabbit, human and r a t a r t e r i e s . In r a t s , some non-vascular tissues were studied along with vessels because i t was b i o l o g i c a l l y i n t e r e s t i n g to f i n d out whether esterase and l i p a s e behave i n p a r a l l e l fashion i n a broader variety of tissues. 2 The atherosclerosis-related conditions investigated for the i r e f f e c t s on the enzymes consisted of sex and age factors, some endocrine and metabolic factors, a r t e r i a l injury, and both acute and chronic lipemia. Sex has a bearing on atherosclerosis because women, before menopause, have le s s c l i n i c a l manifestations of the disease than men(l), while age i s well known to be d i r e c t l y correlated to the severity of spontaneous at h e r o s c l e r o t i c lesions i n both man and a n i m a l s ( 1 ) . The endocrine and metabolic factors investigated were t h i o u r a c i l and thyroxin treatment, alloxan diabetes and f a s t i n g . Both hypothyroidism and diabetes mellitus are known to predispose to a t h e r o s c l e r o s i s ( 1 ) , while f a s t i n g was considered of interest because lean people have been reported to develop le s s athero-s c l e r o s i s and have a greater l i f e expectancy ( 2 ) . Injury to the a r t e r i a l wall was induced by C a l c i f e r o l treatment. Since a r t e r i a l injury i s known to greatly promote the deposition of l i p i d s i n animal arteries(3), i t was hoped to f i n d out whether i t operates by diminishing a r t e r i a l e s t e r o l y t i c a c t i v i t y . The e f f e c t s of acute lipemia i n rats and chronic lipemia i n rabbits were examined i n order to see whether ce r t a i n a r t e r i a l e s t e r o l y t i c enzymes can be induced i n the a r t e r i a l wall i n response to exposure of the artery to a high blood l i p i d l e v e l . Next, a study of the two enzymes was made i n the post-mortem human athe r o s c l e r o t i c l e s i o n , with p a r t i c u l a r attention to the fibrous cap of the l e s i o n . The fibrous cap i s a layer of regenera-t i v e tissue that forms a thick coat over the area of l i p i d deposi-t i o n . Since l i p i d does not accumulate within t h i s regenerative 3 tissue layer, i t was hoped to f i n d out whether or not a p a r t i c u -l a r l y active e s t e r o l y t i c enzyme system i n the fibrous cap i s responsible for t h i s i n t r i g u i n g phenomenon. F i n a l l y , to complement the investigation of human athero-s c l e r o t i c lesions, experimental atherosclerotic lesions were generated i n rabbits by feeding them a di e t containing large amounts of cholesterol. Since t h i s d i e t was given for two months i t also constituted a study of chronic lipemia i n t h i s animal. CHAPTER TWO 4 REVIEW OF THE HISTOCHEMICAL LITERATURE OH ESTEROLYTIC VASCULAR ENZYMES A. ENZYMES IN NORMAL TISSUES 1. Rat Before discussing the l i t e r a t u r e on the e f f e c t s of various atherosclerosis-related factors on lipases and esterases of r a t , rabbit and human tissues, i t i s necessary to consider e x i s t i n g data on the normal d i s t r i b u t i o n of esterase and l i p a s e . In the rats esterase a c t i v i t y i n the normal aorta has been found only i n the media, where i t was reported to be quite intense(4). The smooth muscle c e l l s of the media allegedly contain most of the esterase(5). Non-vascular tissues of the rat where esterase was found include the heart, adrenal gland, pancreas, and t e s t i s . The heart was reported p o s i t i v e throughout the myocardium(6). In the adrenal gland, using naphthyl acetate as substrate, strong a c t i v i t y has been found by some i n the cortex mainly(7). However, Nachlas and Seligman, who used f3 naphthyl acetate as substrate instead of <x naphthyl acetate, found the cortex to be weakly p o s i t i v e i n the zona glomerulosa and zona f a s c i c u l a t a , and only occasionally p o s i t i v e i n the zona r e t i c u l a r i s , with a negative reaction i n the medulla(6). The pancreas a c i n i were described as strongly p o s i t i v e and the ducts as weakly p o s i t i v e , but the i s l e t s and i n t e r s t i t i a l tissue were reported negative(7). In the rat t e s t i s , only the Leydig c e l l s and S e r t o l i c e l l s have been found to show a reaction for esterase(6). 5 The lip a s e d i s t r i b u t i o n i n the rat has been reported to have a s l i g h t l y d i f f e r e n t pattern: In the aorta, no histochemically detectable l i p a s e a c t i v i t y has been found(8,9). The adrenal was positi v e only i n the zona glomerulosa and outer zona f a s c i c u l a t a ( 8 ) . In the pancreas, only the acinar c e l l s were reported p o s i t i v e , and here the a c t i v i t y was described mostly i n the zymogen granules(10). In the r a t t e s t i s a p o s i t i v e reaction for l i p a s e was seen only i n i t s Leydig c e l l s . F i n a l l y , i n the rat prostate, a c t i v i t y has been found to be predominant i n the e p i t h e l i a l c e l l s ( 1 1 ) . 2. Rabbit In the rabbit, the esterases of the normal aorta were found only i n the media, where they exhibited a weak to moderate a c t i v i t y , mostly i n the muscle cells(4,12,13). The e l a s t i c lamellae did not show any a c t i v i t y . In contrast to esterase, no l i p a s e has been detected histochemically i n the normal rabbit aorta(9,12,14). 3. Human Information on esterase and l i p a s e of the normal human vascular wall came as a by-product of studies of human atherosclerotic vessels. Biochemical studies indicated that the normal aorta exhibited a l i t t l e esterase but no l i p a s e a c t i v i t y ( 1 4 ) . Histo-chemical techniques, however, could detect neither of these two groups of enzymes(15,16). B. EFFECTS OF SEX DIFFERENCES AND SEX HORMONES ON THE ENZYMES It i s well established that men have more c l i n i c a l ischemic heart disease and peripheral vascular disease than women. The reason might well be r e l a t e d to the sex hormones. Women who have had a b i l a t e r a l oophorectomy have been found to have a greater degree of coronary atherosclerosis than normal women(17,18). 6 On the other hand, males who have received a s t i l b e s t r o l treatment for p r o s t a t i c carcinoma have been reported to develop a lesser degree of coronary atherosclerosis than t h e i r normal male counter-parts (18). There i s an attempt, i n the l i t e r a t u r e , to see i f this difference between males and females has anything to do with the lipid-handling enzymes of the a r t e r i a l wall. Does a heightened a r t e r i a l l i p o l y t i c a c t i v i t y keep the females r e l a t i v e l y free of atherosclerosis? The r e s u l t s of the investigation of e s t e r o l y t i c enzymes i n the normal aortae of male and female rat s have been confusing so f a r . Mrhova and Zemplenyi(19) , e.g. using the substrate F3 naphthyl acetate, found no difference between the e s t e r o l y t i c a c t i v i t i e s of male and female rat aortae. Mallov(20) also found no such difference with coconut o i l emulsion as substrate. Lacuara et a l (21), however, found the male r a t aorta to have a greater es t e r d — • l y t i c a c t i v i t y than the female, using as substrate ©(naphthyl butyrate. Confounding the issue further, Szendzikowski et al(22), who used lipemic human serum as substrate, found the female r a t aorta to have twice the l i p o l y t i c a c t i v i t y of the male aorta. A l l of these investigators used biochemical techniques. The above c o n f l i c t i n g data make i t probable that there i s no substantial sex difference i n the a r t e r i a l e s t e r o l y t i c enzymes. There i s also a lack of agreement on the e f f e c t of orchidectomy on the biochemically determined esterase of r a t aorta. Mrhova and Zemplenyi(19) found esterase to be increased s l i g h t l y i n the aorta ten days after the time of orchidectomy but to show no s i g n i f i c a n t change at seventeen days. Conversely, Lacuara et al(21) noticed a reduction i n esterase a c t i v i t y following orchidectomy. In l i n e 7 with t h i s Lacuara, et a l also reported that oophorectomy increases esterase a c t i v i t y i n the rat aorta, but found that the admini-s t r a t i o n of e s t r a d i o l and testosterone to gonadectomized females and males, respectively, leads to no change i n a o r t i c esterase a c t i v i t y . Both these teams of investigators used the same substrates here as reported above. There i s further information i n the l i t e r a t u r e concerning the e f f e c t s of sex hormones on esterase or l i p a s e of the prostate and the t e s t i s . In the t e s t i s of r a t s , guinea pigs and mice, the histochemically determined <=< naphthyl acetate esterase was decreased by estrogen treatment i n a l l parts where esterase formerly was positive(23). In another study, i n which a biochemical technique involving esters of p-nitrophenol as substrate was used, i t was discovered that cryptorchism i n the r a t reduces the t o t a l t e s t i c u l a r esterase and causes a s t r u c t u r a l atrophy(24). The same study showed that hypophysectomy w i l l reduce the amount and concentration of esterase i n the r a t t e s t i s , and that a subsequent administration of equine gonadotrophin to these animals w i l l increase the esterase i n amount and concentration back to pre-hypophyseetomy l e v e l s . Studies of the o< naphthyl acetate esterase d i s t r i b u t i o n i n the r a t t e s t i s have revealed such enzyme a c t i v i t y i n both Leydig c e l l s and S e r t o l i cells(6,23,25). With fresh frozen tissue, esterase was detectable only i n the Leydig c e l l s ( 2 5 ) . In the f i x e d t e s t i s , however, both Leydig and S e r t o l i c e l l s were esterase p o s i t i v e , and here the staining d i s t r i b u t i o n varied according to the stage of spermatogenesis(25,26). Early i n spermatogenesis there was intense staining only i n the perinuclear cytoplasm of the S e r t o l i c e l l s , 8 whereas l a t e r there was strong a c t i v i t y only i n the cytoplasmic processes within which spermatozoa are embedded p r i o r to t h e i r release. These findings suggest that an esterase i s necessary for the "nursing" r o l e of the S e r t o l i c e l l v i s - a - v i s the spermatozoa. The esterases of r a t S e r t o l i c e l l s apparently have not the same properties as the r a t Leydig c e l l esterases. Compared to Leydig c e l l esterase, S e r t o l i c e l l esterase i s soluble, dependent on f i x a t i o n for histochemical demonstration, more se n s i t i v e to changes i n pH, i n s e n s i t i v e to gonadotrophin, and r e s i s t a n t to the i n h i b i t o r E600 (diethyl p-nitrophenyl phosphate). Conversely, Leydig c e l l esterase i s insoluble, s e n s i t i v e to gonadotrophin and s e n s i t i v e to E600 inhibition(25,27). Tween substrates for l i p a s e have also been employed. Tween 60 l i p a s e was found only i n the Leydig c e l l s of r a t t e s t i s ( 2 5 ) . In the r a t prostate progesterone was found to have a stimulating e f f e c t on the Tween 80 li p a s e of the epithelium and stroma, but the e f f e c t of estrogen proved dose dependent(11). A low dose (0.1 g) increased l i p a s e a c t i v i t y i n e p i t h e l i a and stroma, but a higher: dose (5 g) caused a marked drop i n l i p a s e along with an atrophied epithelium and a hypertrophied and hyperplastic stroma. The response i n rats given t h i r t y days re s t between high dose estrogen treatment and s a c r i f i c i n g i s suggestive of p a r t i a l recovery of l i p a s e a c t i v i t y . C. THE EFFECTS OF AGE ON THE ENZYMES Investigations of the l i p o l y t i c a c t i v i t y of the rat aorta with respect to age have disclosed that older aortae have s l i g h t l y less 9 l i p a s e a c t i v i t y than younger aortae. These studies were bio-chemical i n methodology and used as substrates human lipemic serum(28), coconut o i l emulsion(20), and l i p o p r o t e i n — t r i g l y c e r i d e s (29). One group noticed that l i p a s e activity i n young rats increased with increasing age between two to seven months but declined somewhat thereafter(28). The e f f e c t s of age on the e s t e r o l y t i c a c t i v i t y of t e s t i c u l a r  tissue have been investigated i n rat s and mice. Generally, an increase i n the esterase of the Leydig c e l l s with age was found i n both species, with a p a r t i c u l a r l y dramatic increase following puberty. In the r a t , t h i s information has been obtained with histochemical techniques using naphthyl acetate and naphthol AS-D acetate as substrates(26,30), and with biochemical techniques using p-nitrophenol esters(24) and a variety of naphthyl, naphthy-lamide, and indoxyl esters(31) ;' i n the mouse, the histochemical technique for naphthyl acetate esterase has been used(32,33,34). B a i l l i e actually made a histochemical study of f i v e esterases i n the mouse t e s t i s and expressed his data as the amount of esterase-active i n t e r s t i t i a l tissue per gram t e s t i s mass(33). He found that indoxyl acetate esterase a c t i v i t y decreases drama-t i c a l l y to n i l between b i r t h and three weeks of age, whereas the other four enzymes increased s t e a d i l y with age, beginning to show detectable a c t i v i t y i n the following order: °c naphthyl acetate esterase, naphthol AS acetate esterase, naphthol AS-LC acetate esterase and naphthol AS-D acetate esterase. The behaviour of some other substances may help to evaluate the movements of t e s t i c u l a r esterase: Sudanophilic l i p i d s of the 10 i n t e r s t i t i a l tissue decrease s t e a d i l y or disappear at puberty i n the rat(30). In the mouse t e s t i s i n t e r s t i t i u m , the following enzymes appear i n the f i r s t few weeks of postnatal l i f e and increase with age: cytochrome oxidase, s u c c i n i c dehydrogenase, and 3 :> |}-hydroxysteroid dehydrogenase (33,34). I t i s of i n t e r e s t that i n p a r t i c u l a r 3 :• .|3-~ hydroxysteroid dehydrogenase behaves s i m i l a r l y to esterase i n both mouse(33) and rat(30), because t h i s enzyme i s d i r e c t l y concerned with androgen biosynthesis. D. THE EFFECTS OF THYROID HORMONES ON THE ENZYMES The rel a t i o n s h i p between thyroid hormones and a o r t i c esterase  and l i p a s e a c t i v i t y has been studied i n both dogs(35) and rats(36) by only one investigator. Substrates used were =K naphthyl acetate for esterase and Tween 80 for l i p a s e . In each species, one group was given methylthiouracil i n an atherogenic high cholesterol d i e t , another group received j u s t methylthiouracil treatment, and a t h i r d group received a high cholesterol d i e t without methylthiouracil. In dogs, experimental hypothyroidism was found to decrease esterase and l i p a s e a c t i v i t y i n the aorta, but when combined with an atherogenic d i e t t h i s e f f e c t was not nearly as marked. Experimental hypothyroidism s i m i l a r l y lowered r a t a o r t i c esterase and l i p a s e a c t i v i t i e s , but the addition of a high cholesterol diet did not a l t e r the extent of t h i s diminution. In another study, .a. cholesterol granuloma was induced i n r a t thigh muscle as a model for assaying the response of l i p o l y t i c enzymes to various factors r e l a t e d to atherosclerosis(37). The granuloma was formed i n response to the intramuscular i n j e c t i o n 11 of pulverized cholesterol i n s a l i n e . Normally, l i p o l y t i c a c t i v i t y appeared i n macrophages and giant c e l l s of the granuloma after a mean time of 6.2+0.85 days. Hypothyroidism was claimed to reduce the granuloma l i p a s e a c t i v i t y because the enzyme could not be detected u n t i l a f t e r an average of 15.3+1.13 days had elapsed. Conversely, under hyperthyroidism l i p o l y s i s appeared at 2.5+0.53 days and therefore an increase i n l i p o l y t i c a c t i v i t y was apparent under conditions of increased thyroid a c t i v i t y . E. THE EFFECTS OF ALLOXAN DIABETES ON THE ENZYMES Studies have been made on the e f f e c t of alloxan diabetes on the l i p o l y t i c enzymes of the rat aorta. Using a biochemical technique with substrate Tween 60 i t was found that the duration of alloxan treatment i s an important factor(38). When treatment was given for a short duration (nine to twelve days) eight out of twelve animals maintained a normal a o r t i c l i p a s e a c t i v i t y , but a more prolonged diabetes of eighteen to eighty-eight days caused a s i g n i f i c a n t reduction i n l i p o l y t i c a c t i v i t y . Also of inte r e s t i s the discovery that t h i s enzyme response appeared to be r e v e r s i b l e , since f i v e out of eight treated animals whose blood glucose was allowed to return to a normal l e v e l exhibited a normal amount of a o r t i c l i p a s e a c t i v i t y . The only histochemical information i n t h i s area i s r e l a t e d to the cholesterol granuloma model mentioned above(37). Under alloxan diabetes the l i p o l y t i c a c t i v i t y did not appear u n t i l 17.1*1.62 days after cholesterol i n j e c t i o n , i n d i c a t i n g a decreased l i p o l y t i c a c t i v i t y as a r e s u l t of t h i s disease. 12 F. THE EFFECTS OF FASTING ON THE ENZYMES Nothing i s known of the d i r e c t e f f e c t s of f a s t i n g on the a r t e r i a l enzymes. Biochemical evidence indicated that a moderate fast i n g of the rat causes increases i n both plasma l i p o p r o t e i n lipase(40) and heart l i p a s e a c t i v i t y ( 4 1 ) . The only histochemical study i n t h i s area concerns the e f f e c t of f a s t i n g on l i p a s e of mouse adipose tissue(42), and i t was found that nonfasted mice showed a c t i v i t y only i n c a p i l l a r y endothelial c e l l s , while the response of fasted mice varied according to the duration of the f a s t . If fasted f o r forty-eight hours, mice showed a drop i n endothelial l i p a s e a c t i v i t y but a marked r i s e of the l i p a s e a c t i v i t y i n the adipose c e l l s . After s i x days of f a s t i n g , no l i p a s e a c t i v i t y could be detected i n either endothelium or adipose c e l l s . G. THE EFFECTS OF VASCULAR INJURY ON THE ENZYMES In order to investigate the r e l a t i o n s h i p between vascular injury and l i p i d - h a n d l i n g enzymes, studies were made of rats fed C a l c i f e r o l . It i s known that at appropriate dosages C a l c i f e r o l can cause various degrees of a r t e r i a l injury and medial necrosis with c a l c i f i c a t i o n (3,43). A biochemical experiment using as substrate human lipemic serum gave r e s u l t s which depended on the duration of C a l c i f e r o l treatment and the dosage(44). Rats given 30,000 u. C a l c i f e r o l per day for f i v e days and k i l l e d on the f i f t e e n t h day showed no change i n a o r t i c l i p a s e a c t i v i t y . This l i p o l y t i c a c t i v i t y decreased s i g n i f i c a n t l y , however, i n the aorta of rats given 45,000 u. C a l c i f e r o l per day f o r seven days and k i l l e d on the twenty-first day. 13 H. THE EFFECTS OF ACUTE LIPEMIA ON THE ENZYMES There are no reports in the literature on the relationship between alimentary or intravenously induced acute lipemia and the vascular lipases and esterases. A l l studies on lipemia have been concentrated with chronic, longterm lipemia, involving an atherogenic diet and resulting in atherosclerosis. I. THE ENZYMES IN ATHEROSCLEROTIC HUMAN ARTERIES Probably the most significant outcome of studies on esterase and lipase in human arterial tissue was the demonstration of a higher activity in areas of l i p i d deposition than in adjacent normal areas of the same specimens. This has been documented by both biochemical(45,46,47) and histochemical(15,16,50-53) procedures. The histochemical techniques for lipid-handling enzymes that have been applied include those with the substrates << naphthyl acetate, naphthyl AS acetate, indoxyl acetate, Tween 60, and Tween 80. The activity was a l l intracellular and could be found in foam cells and smooth muscle cells in the periphery of the atherosclerotic lesion and in endothelial cells over the lesion. Esterase was not found in areas of calcium deposition, and the fibrous cap was not investigated. Also of interest are some findings concerning sex variance in the atherosclerotic human artery. Biochemical studies of the atherosclerotic aortic samples of both sexes using the substrates 9^ naphthyl acetate and naphthyl laurate have shown that women have a higher esterolytic activity than men between the ages 18 and 54 years(47). In contrast to animal data, the problem of sex variance in humans has so far not been studied histochemically. 14 Ji THE ENZYMES IN EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS As with humans, rabbits also have a generally increased amount of esterase and lipase activity in the fat laden areas of the atherosclerotic artery. Biochemical investigations have, however, revealed a certain time dependence in the behaviour of the l i p o l y t i c and esterolytic enzymes of the rabbit aorta. According to one investigator a high cholesterol diet of ten weeks is required before esterase begins to increase(54). According to another, during a two to four month high cholesterol diet both lipase(55) and esterase(56,57) were reported to increase gradually in activity. However, during a six to ten month high cholesterol diet period esterase and lipase values were found to drop to normal or below normal values(53,54), evidently because the late lesion has fewer cells than the early lesion, and hence has less cell-bound enzyme. Histochemical studies of moderately advanced experimental atherosclerotic lesions in the rabbit have shown both lipase(12,59,60) and esterase(12,12,59,61,62) to be increased throughout the entire lesion, whereby the substrates used have included Tween 60, Tween 80, and oinaphthyl acetate, naphthol AS acetate, and indoxyl acetate. Small plaques, on the other hand, were reported by one investigator to exhibit i n i t i a l l y an intense esterase reaction only in the endothelial cells over the plaque(13), and data on the enzyme activity of very large plaques have been contradictory(13,59). In conjunction with larger aortic plaques, most of the esterase was found to be located in endothelial cells and particularly in macrophages (foam cells)(12,13,59,61,62). Smooth muscle cells and to a much lesser extent "fibrocytes" have also been reported 15 positive on occasion. Extracellular esterase activity, however, has been found to be weak in areas of fibrous transformation and negative in calcified areas(12,59). Plaques of other vessels, including the coronaries, showed the same esterase changes as the aorta(59). A study of rabbit aortic cells in tissue culture using histo-chemical techniques has supported some of the above histochemical information(63). Of the three types of cells cultured, endothelial, "intermediate", and fibroblastic c e l l s , the endothelial cells had the greatest amounts of both carboxyl esterase and l i p i d . CHAPTER THREE 16 METHODS A. EXPERIMENTAL DESIGN 1. REASONS FOR PERFORMING THE EXPERIMENTS IN THIS THESIS The experiments I outlined in the introduction were undertaken because I wanted to investigate under standard conditions several problems on which histochemical enzyme information was either not available, or conflicting, or in need of confirmation. Thus, experiments examining the effects of both sex difference and gonadectomy on vascular enzyme activity were done because there were no histochemical data in the literature, and the results of biochemical studies of these problems were contradictory. Similarly, the effects of sex hormones on lipid-handling enzymes were investi-gated in the prostate where no esterase work had been done, and also in the testis where confirmation was needed. As regards investigations of the effects of age on esterase and lipase of the aortae, these were performed because no histo-chemical data were available on this subject. The effects of age on testis enzymes, for which considerable information was already available, were studied in order to compare my results with those of the literature consensus and thus check the performance of my enzyme techniques. Concerning my studies of the effects of certain metabolic changes on arterial enzymes, these were undertaken because (a) the information in the literature on the effects of thyroid activity needed confirmation, (b) no histochemical data on the effects of alloxan diabetes were available, and (c) no data were available on the effects of fasting and acute lipemia. 17 The advanced human atherosclerotic lesion was examined because no specific information was available on i t s fibrous cap, whereas the histochemical effects of injury on animal arteries were investigated because they had not been studied before. Finally, rabbit atherosclerotic lesions were screened in order to compare them for the f i r s t time with human lesions under standard conditions. 18 2. DESCRIPTION OF SPECIFIC EXPERIMENTS (a) AGE EFFECTS Two r a t s each of the following age groups were compared for th e i r esterase and li p a s e : very young rats (eight days), weanling rats (twenty-five days), and old r a t s (about eighteen months). Tissues examined were the aorta, heart (including coronaries), adrenals, pancreas and t e s t i s . In addition, the aortae and coronary a r t e r i e s of two young rabbits were compared with those of two older rabbits. The young rabbits were about 600 grams i n weight each, whereas the older ones weighed about 3,000 grams. (b) ENDOCRINE AND METABOLIC CONDITIONS i) Sex Difference and Gonadal E f f e c t s In order to investigate gonadal e f f e c t s on enzyme a c t i v i t y •, the following rat categories were studied: normal male, normal female, pregnant, s t i l b e s t r o l treated male, and orchidectomized r a t . Two to three 300 gm.rats were used i n each category. Tissues sampled included the aorta, heart, adrenals, and pancreas i n a l l r a t s , and the mammary gland, t e s t i s , and prostate gland where applicable according to sex. (For some tissues more than two rats were used. These exceptions were as follows: from the pregnant female, three mammary glands; from the normal female, four aortae; from the normal male, three aortae; from the s t i l b e s t r o l treated r a t s , three adrenals, three prostate glands, and four testes; and from the orchidectomized r a t s only one prostate gland). 19 The stilbestrol treated rats were given once daily for ten days 0.5 ml of a 0.4% solution of stilbestrol in Mazola o i l subeutaneously. This amounted to 2 mg per rat per day. The rats were k i l l e d on the eleventh day. The orchidectomized rats were k i l l e d ten days following operation. A comparison was also made between the aortae and coronary arteries of two male and two female rabbits. i i ) Thyroid Hormones A hyperthyroid condition was produced in two 250 gm. rats by the subcutaneous administration of thyroxin. Each rat received 1 mg per day for seven days. Tissues examined were the pancreas, heart, adrenals, testis, and aorta. Thiouracil was given to another two rats in order to induce a reduction in thyroid activity. Each rat was given 0.3% 4-methyl-2-thiouracil in drinking water for twelve days. Tissues examined were the same as in the case of the thyroxin treated animals. i i i ) Alloxan Diabetes Two 300 gm. rats were given a single intravenous injection each of alloxan monohydrate at a dose known to produce diabetes in rats(69,70) (40 mg per Kg body weight per rat after a fast of forty-eight hours). The rats were sacrificed forty-eight hours after the alloxan administration and the tissues examined were the aorta, heart, pancreas, adrenals, and testis. 20 iv) Fasting Two 300 gm. rats were each fasted for seventy-two hours causing a reduction in body weight of 40 to 50 grams per rat. Tissues examined were the aorta, heart, pancreas, adrenals, and testis. (c) VASCULAR INJURY Four 250 gm. rats were given Calciferol (also called viosterol) in order to damage the walls of their arteries. Each rat was given by gavage 35,000 I.U. per 100 gram body weight per day for a period of five days. Two of these rats were sacrificed on the seventh day, and the other two on the thirty-sixth and forty-eighth day after the commencement of Calciferol treatment. The tissues sampled were the aorta, heart, and adrenals. (d) ACUTE LIPEMIA Lipemia was induced in 300 gm. rats in two different ways. By the one method, two rats were each given 5 ml of corn o i l by gavage. These rats were fasted for ten hours prior to, and were k i l l e d six hours after the gavage treatment. In addition, the aortae of two ungavaged control rats were sampled. The plasma of each oil-fed rat appeared "milky" white in contrast to the transparent normal plasma, and numerous chylomicrons were visible in i t under the phase contrast microscope. Another method of producing lipemic plasma was an indirect one. Lipemia was f i r s t generated in certain rats by gavage of corn o i l . Lipemic plasma was then removed from the abdominal aortae of the gavaged rats and injected intravenously into other rats once or 21 three times through the t a i l vein. The details of this procedure were as follows: i) I n i t i a l l y lipemic plasma was induced by feeding corn o i l to the rats by gavage. The amount of corn o i l given varied according to the weight of the rat, 5 ml of corn o i l given for each 300 grams of rat. About seven hours after the gavage eight to ten ml of blood was collected from the abdominal aorta of each rat by a 10 cc syringe containing 1 ml of anticoagulant (0.1 M sodium citrate). The blood was then centrifuged and the plasma extracted and pooled. i i ) The collected plasma was considered to be lipemic when i t appeared to be "milky" white, i t s optical density at 500 my was at least 0.36 (when normal plasma was set at a zero reading), and i t displayed numerous chylomicrons under the phase contrast microscope. i i i ) .The pooled lipemic plasma was given (a) as a single intravenous injection to two rats, and (b) as a series of three successive intravenous injections to another two rats. The single intravenous injections consisted of 4 ml of pooled lipemic plasma given within half a minute, and half an hour following this the rats were k i l l e d . Multiple intravenous injections entailed three successive injections of 3 ml each, given at hourly intervals and, subsequently, the rats were k i l l e d half an hour after the last injection. The tissues examined in both the rats made lipemic by l i p i d alimentation and those made lipemic by intravenous infusion of lipemic plasma were the aorta, heart, and spleen. 22 (e) ATHEROSCLEROTIC HUMAN ARTERIES The aortae of eleven male autopsy cases ranging i n age from three to s i x t y years were obtained from the Vancouver General Hospital. As soon as the vessels were removed from the cadaver they were frozen s o l i d , and then kept i n the frozen state u n t i l they could be sectioned, (the storage period varying from a few hours to a few weeks). Small blocks of tissue were selected and removed from the large a o r t i c samples j u s t before sectioning with the cryostat. Normal areas of the aortae were compared with areas of early atherosclerotic lesions and pearly plaques. (f) EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS Six New Zealand White rabbits weighing about 3 Kg. each were put on a high cholesterol diet f o r eight weeks, and the aortae of four of these animals were examined with the esterase and l i p a s e techniques. A sp e c i a l 1% cholesterol d i e t was prepared by impregnating rabbit food p e l l e t s with chol e s t e r o l , as follows: a weighed amount of food p e l l e t s was spread over a large sheet of wax paper to a layer of about half an inch thickj a sol u t i o n of the appropriate amount of cholesterol dissolved i n ethyl ether was then sprinkled three times evenly over the p e l l e t s with a watering can, the p e l l e t s being raked and r e d i s t r i b u t e d between successive s p r i n k l i n g sessions; to remove the ether, the food p e l l e t s were subsequently allowed to dry for half an hour i n the open. 23 Each rabbit was allowed 100 grams of food per day for the duration of the eight week treatment. Although the 100 grams pro-vided is slightly less than the optimal caloric intake per day for such rabbits, i t was given to ensure that the rabbits would receive a l l of the daily dose of cholesterol intended for them. This diet was also supplemented twice a week with dry vegetables, as a source of vitamins. Only the four rabbits that developed the greatest amount of lipemia were sacrificed. The degree of lipemia present was established by withdrawing from the earvein of each rabbit about one cc. of blood into a syringe. This blood was then mixed with anticoagulant in the syringe, and centrifuged. The concentration of chylomicrons in the extracted plasma of each sample was assessed by examining the plasma with a phase contrast microscope. A l l of the extracted plasma had a milky white consistency and a high concentration of chylomicrons. At necropsy of the four maximally lipemic rabbits used, a l l of their aortae displayed small, white, raised, atheromata. These lesions were located particularly at the orifices of the intercostal arteries, but were also randomly distributed along the entire aortic surface. Cryostat sections from both the experimental atheromata and the normal areas between the lesions were then examined with both the esterase and lipase techniques. 24 B. HISTOCHEMICAL METHODS The two histochemical techniques used were the < naphthyl acetate method for nonspecific esterase(64) and Gomori's "Tween" method for lipase(64) (see the Appendix). The PC naphthyl acetate method is a simultaneous capture method, whereby incubation of tissues in the ester substrate naphthyl acetate causes release of c< naphthol at sites of esterolytic enzyme activity. When the <naphthol i s subsequently coupled with the azo dye Fast Blue RR, a black precipitate is formed (see figures 1 and 2). The basis of the lipase technique i s the incubation of frozen sections i n the substrate Tween 60 (polyoxyethylene sorbitan monostearate') in the presence of calcium ions and activator. Tissue lipase s p l i t s off stearic acid from the Tween molecules and this i s captured by calcium. Insoluble calcium stearate soaps form at the sites of enzyme activity, and these are subsequently converted to lead soaps, and then to visibl e brown granules of lead sulphide(64) (see figures 3 and 4). The types of lipid-handling enzymes for which these techniques are specific overlap but do not coincide. Generally, lipases act mainly on triglycerides, whereas esterases have as their substrates aliphatic esters of low molecular weight. However lipase i s also thought to react, though less rapidly, with some low molecular weight aliphatic esters, and esterase i s similarly believed to cross-react less rapidly with some triglycerides(65). x This compound is a water soluble l i p i d ; i t consists of an ester between the polyvalent alcohol sorbitol, on the one hand, and stearic acid and polymers of ethylene oxide, on the other. 25 It i s important to remember that detection techniques employing a given substrate can only detect enzymes which can act on the specific substrate employed. Rat tissue, for instance, has a large variety of different esterases which have been grouped into four classes as carboxylesterases, arylesterases, acetylesterases and cholinesterases(31). No particular histochemical technique would detect a l l of these equally well. However, particular enzymes can be quite similar in terms of chemical structure because, for instance, Okuda and Fujii(66) discovered that a molecule of rat l i v e r lipase i s composed of a molecule of li v e r esterase complexed with l i p i d . A l l tissues were frozen in a cryostat immediately after their removal from the cadaver. Animal tissues were sectioned within an hour, whereas the human tissues were sectioned within hours or days, during which time they were stored frozen solid. A l l tissues were sectioned on a cryostat and subsequently fixed in 10% formalin for ten to thirty minutes. The fixed tissues were then immersed into the incubation media of the two enzyme techniques employed within an hour of sectioning. Many aspects of each enzyme technique were thoroughly investi-gated in numerous pilot studies in order to extract optimal performance from them. Attempts were made to insure a maximal sensitivity with minimal diffusion, dissolution, or crystallization of the reaction products. Included among such methodological pilot studies were the questions of the optimal fixation, substrate, incubation time and pH, and time of section exposure to cSunterstain. 26 Also, with reference just to the lipase technique, the optimal exposure time to ammonium sulphide and the question of the best activators were investigated. A serious cover glass mountant problem was also encountered when i t was discovered that ordinary mounting media cause diffusion and crystallisation of the enzymatic reaction products. Fortunately, an intensive search led to the discovery of an aqueous mountant ("Arlex Gelatin of L i l l i e and Greco") that seemed to preserve sections without change for months. 1. The Esterase Technique The procedure followed for the esterase technique i s given in the Appendix. Controls were run for both enzyme and substrate. Enzyme controls consisted of (a) boiling the sections in d i s t i l l e d water for fifteen minutes, (b) heating them over a Bunsen flame for ten seconds, and (c) exposing them to 5% potassium cyanide for one hour. The substrate controls consisted merely of the substitution of d i s t i l l e d water for the substrate. The esterase technique as prescribed by Pearse(64) was adapted and improved as follows: a) Fixation in 10% formalin; this was done because i t was confirmed in pilot studies that the esterase was distinctly more intensive in fixed than in unfixed tissues. b) Use of the azo dye Fast Blue RR instead of Fast Blue B because the latter was found to be insufficiently soluble in the incu-bation medium. c) Since the azo dye Fast Blue RR used forms a heavy precipitate i f l e f t standing for about ten minutes, the incubation media were always f i l t e r e d in the dark and used immediately. 27 d) The pH ol incubation originally used for this technique by Nachlas and Seligman was pH 7.8, while Pearse suggests pH 7.4(64). Since I found no difference in esterase reaction product between pH 7.4 and pH 8.0, I decided a pH of 7.4 would be adequate. e) The incubation times giving the best results were between five and eight minutes. Six minutes was therefore considered to be a safe standard time of incubation. 2. The Lipase Technique The procedure followed for the lipase technique i s given in the Appendix. Controls were run for both enzyme and substrate as described for the esterase technique. The following improvements of the lipase technique were instituted: a) Fixation in 10% formalin, for the same reasons as given above for the esterase technique. b) The exclusive use of a Tween 60 solution which is not contami-nated with free fatty acids as evidenced by the fact that i t remains clear when tested with calcium chloride(67). c) The use of the more soluble activator sodium taurocholate in the incubation media, as opposed to the less soluble sodium glycocholate or sodium desoxycholate. d) An optimal and sufficient incubation time of three hours was used, (Pearse(64) recommended three to twelve hours), because pilot studies distinctly showed that the maximum lipase reaction occurred at two and a half hours of incubation, with no further increment beyond that interval. e) An incubation temperature of 37 degrees Centigrade instead of room temperature, because the former was found to give a more intense lipase reaction. 28 3. Adaptations Applying to both Esterase and Lipase Techniques a) The method of k i l l i n g , (a blow to the head or ether intoxication,) was found without effect on the results of the techniques. Thus, sacrifice by ether intoxication was used throughout a l l of the animal experiments. b) Since the duration of counterstaining with Mayer's Hematoxylin that gave the best results was between three and five minutes, four minutes was adopted as the standard counterstaining time. c) A problem with mounting media developed because the glycerine j e l l y suggested for both enzyme techniques by Pearse caused fading, diffusion and crystallization of the f i n a l reaction products within a few days. An aqueous mountant had to be used because the f i n a l reaction products are soluble in alcohol or xylol. During the search for an adequate mountant, twelve possible mountants were tried and "Arlex Gelatin of L i l l i e and Greco"(68) was found to be the only mountant which did not cause fading, diffusion, crystallization or tissue damage. Sections mounted with this mountant appeared to be preserved adequately for many months, and in some cases even a year after the time of mounting. For those who may wish to use this mountant, i t should be mentioned that the ingredient Arlex D-sorbitol syrup is unavailable in Canada, but that "Atlas 70% Sorbo" is an available equivalent of this syrup. 29 CHAPTER FOUR RESULTS A. NORMAL TISSUES Tables 1 and 2 l i s t the distribution and intensity ol activity of esterase and lipase in tissues of the rat and rabbit as established in this study. It should be noted that no sex difference in the intensity of activity of the above enzymes could be detected in the normal tissues of the rat, the rabbit, and in the aorta of man. B. THE EFFECTS OF AGE ON THE TISSUES There were no apparent differences in the vascular tissue esterase and lipase between old and young in rats, rabbits and humans except for a more homogeneous distribution of esterase in the mature as against the immature aortae of the rat. (Compare figure 6 with figure 7 and figure 5 with figure 13). Similarly no age effects were discernible in any of the non-vascular tissues studied, except for the Leydig c e l l s . In these testicular cells no esterase activity was evident in any of the Leydig cells of the very young eight day old rats, whereas strong activity appeared in some of the Leydig cells of the twenty-five day old weanling r a t s j and in a l l of the Leydig cells of the eighteen month old mature rats (see figures 8, 9, and 10). Lipase was found to behave similarly to esterase in testicular intertubular tissue. C. THE EFFECTS OF ENDOCRINE AND METABOLIC FACTORS ON THE ENZYMES Orchidectomy caused no changes in esterase activity in any of the tissues studied. Stilbestrol treatment similarly did not affect esterase in any of the tissues examined, except the prostate gland. In each of the three treated rats, prostate esterase diminished greatly in activity. 30 Stilbestrol treatment also caused in the prostate an atrophy of the epithelium and a hypertrophy of the stroma (see figures 11 and 12). The other endocrine or metabolic conditions examined for their effect on esterase and lipase were thiouracil and thyroxin treatments, alloxan monohydrate treatment (at a dose known to produce experi-mental diabetes), and fasting. None of these conditions led to any significant changes of esterase or lipase activity in any of the vascular or non-vascular rat tissues examined. D. THE EFFECTS OF VASCULAR INJURY ON THE ENZYMES In the focal areas of injury in the rat aorta esterase activity disappeared and did not reappear as late as a month and a half after the i n f l i c t i o n of vascular damage (see figures 13, 14, 15, and 16). Von Kossa's technique for calcium revealed calcium deposition in these same focal areas. The lipase reaction did not exhibit any diminution, of course, because the normal aorta was negative for lipase to begin with. There were no other significant vascular or non-vascular enzymatic changes resulting from Calciferol treatment. E. THE EFFECTS OF LIPEMIA ON THE ENZYMES Both types of acute lipemia tested (alimentary and intravenous) failed to cause significant changes in rat esterase or lipase activity in the tissues studied. The multiple intravenous injections were as ineffective as single intravenous injections in this respect. Chronic lipemia produced by cholesterol feeding also failed to cause any changes in vascular esterase of the normal, non-athersclerotic areas of the rabbit aorta. 31 F. THE ENZYMES IN ATHEROSCLEROTIC HUMAN ARTERIES In normal, non-diseased portions of the sampled aorta there was no detectable esterase activity (see figure 17). Esterase appeared only in conjunction with areas of fatty deposition, and in such areas i t was a l l intracellular (see figure 18). In fatty streaks, esterase could be found in round and spindle shaped foam cells scattered throughout the lesion, whereas in pearly plaques esterase positive cells appeared only at the plaque margins, where some foam cells survived. The fibrous cap of the pearly plaque was negative for esterase along with the large masses of extracellular l i p i d , the gruel (see figure 19). G. THE ENZYMES IN EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS The pattern of esterase distribution and reaction intensity was similar to that seen in human atherosclerosis. Positive esterase reaction was found throughout the foam c e l l mass of the lesion (see figures 20, 21, 22, 23, and 24). The distribution of lipase was found to be similar to that of esterase. TISSUE A. ESTERASE 1. Vascular Aorta Coronary artery 2 . Non-vascular Myocardium Adrenal cortex Adrenal medulla Pancreas Testis Prostate Mammary gland Spleen Liver B. LIPASE 1. Vascular Aorta Coronary Artery 2 . Non-Vascular Myocardium Adrenal cortex Adrenal medulla Pancreas Testis Prostate Mammary gland Spleen Liver ENZYME DISTRIBUTION ENZYME ACTIVITY Between the elastic fibres of media Throughout myocardial fiber Positive throughout, mostly in zona glomerulosa Clusters of cells only Acini, only Leydig cells and basal parts of spermatogenic tubule Epithelium only Epithelium only Throughout red pulp and in macrophagesPositive of white pulp Evenly distributed throughout lobule Positive Strongly positive Negative Strongly positive Strongly positive Positive Strongly positive Strong in Leydig c e l l s , other positive cells moderate. Positive Positive Scattered granules in the myocardial fibers Positive throughout cortex, least in zona fasciculata Acini only In Leydig ce l l s only Epithelium only Parts of lactiferous duct only Red pulp only Evenly distributed throughout lobule Negative Negative Weakly positive Weakly positive Negative Strongly positive Strongly positive Weak Positive Weak Strongly positive m 8 ISO Is w H 5 w w to TISSUE ENZYME DISTRIBUTION ENZYME ACTIVITY ESTERASE 1. Vascular Aorta Coronary arteries 2. Non-vascular Myocardium Spleen LIPASE 1. Vascular Aorta Coronary artery 2. Non-vascular Myocardium Spleen Negative Negative Throughout myocardial fiber Positive Throughout both red pulp and white Positive pulp Negative Negative Scattered granules in the myocardial Weakly positive fibers Throughout whole spleen; red pulp Strongly positive stronger than white pulp CHAPTER FIVE 34 DISCUSSION AND CONCLUSIONS The results of my study w i l l be evaluated and correlated with the literature in the order in which they were presented in the previous section on "Results". A. Enzymes in Normal Tissues and Age Effects My results for a l l of the normal tissues agreed with the literature, except for the rabbit aorta which I found negative whereas Zemplenyi et al(4,12,13) found i t very slightly positive. Zemplenyi very likely attached too much importance to the same slightly positive patches which I interpreted as being artifact (see figure 21). My finding that age has no effect on the histochemical non-specific esterase or "Tween" lipase of the rat aorta does not concur with biochemical information obtained with other substrates. Investigators employing human lipemic serum, triglycerides, and coconut o i l emulsion as substrates have claimed that older rat aortae have slightly less l i p o l y t i c activity than younger ones (20,28,29). My finding that rat Leydig tissue esterase and lipase increases dramatically with age agrees with biochemical(24,31) and histo-chemical (26,30) information reported by other investigators who used the same substrates as employed in my study. One would suppose that such increased enzyme activity is associated with androgen production, the principal function of Leydig c e l l s . The fact that with my techniques I obtained information that was in complete agreement with the consensus of opinion supports the technical r e l i a b i l i t y of the histochemical techniques used in the present study. 35 B. The Effects of Endocrine Factors The fact that a l l three types of sex study undertaken (sex difference, s t i l b e s t r o l , and orchideetomy) failed to detect any sex dependent variations of arterial esterase or lipase probably accounts for the contradictory biochemical findings of Mrhova and Zemplenyi(19), Mallov(20), Lacuara et al (21), and Szendzikowski et al(22) on this subject. In the absence of a real sex effect, one would expect various investigators at different times and places to report contradictory results in their investigations. However, sex changes did occur i n a tissue where they would be biologically expected, that i s , in the prostate gland. My finding that daily stilbestrol injection causes a greatly diminished esterase activity in the prostate gland, accompanied by some distinc-tive morphological changes there, confirms the lipase results of Takker et a l ( l l ) , who used estejrogen at a similar dosage. These findings are not surprising because i t is well known that female sex hormone causes an atrophy of the male accessory sex organs, including the prostate epithelia, and i t would be expected that such atrophied cells would display a decreased enzymatic activity. The agreement in my results that in rat, rabbit and human vascular tissue there is no sex difference in lipid-handling enzyme activity make i t unlikely that an increased susceptibility of the male to atherosclerosis i s due to a defiency in vascular enzyme activity. With respect to other endocrine factors I found that neither experimental hypothyroidism nor experimental hyperthyroidism 36 produced any changes in enzyme activity i n the tissues studied. These results do not agree with those of Leites, who found a decreased esterase and lipase in rat and dog aorta in response to experimental hypothyroidism(35,36). This discrepancy i s probably a consequence of the fact that Leites administered a huge dose of methylthiouracil in rat food (12%) which was many times greater than the amount that was added to the drinking water of the animals of the present study (0.3%). Furthermore, since he applied this treatment for a much longer period of time than was employed in my studies, (a few months as against a few days), i t i s quite possible that he may have produced vasotoxic effects. Turning from the thyroid to the pancreas, I found that alloxan monohydrate administration caused no changes in esterase or lipase in any of the tissues studied when rats were sacrificed two days after treatment. This i s actually in agreement with Leites' biochemical studies in which he found that a prolonged alloxan diabetes of over eighteen days i s required for a significant reduction in aortic lipase activity to occur(38,39). His results with pro-longed diabetes make i t possible to speculate that the increased susceptibility of diabetics to atherosclerosis may be related^to a diminished l i p o l y t i c activity in their vessels. c« Effects of Injury. Lipemia and Fasting My results show that experimental injury to the rat aorta with Calciferol caused a dramatic and apparently permanent disappearance of esterase activity in focal areas of injury and are thus in agreement with the biochemical findings the Zemplenyi group(44) made using human lipoprotein as a substrate. Since a l l cellular activities are diminished or absent in necrotic f o c i , a reduction 37 of esterase activity would be expected. An additional factor could be the finding of Zemplenyi that the abnormal mucopolysaccharide composition and the calcium salts seen in Calciferol injured vessels are each, independently, known to inhibit esterolytic activity in aortic tissue(71). Be that as i t may, the above findings may contribute to an explanation for the increased l i p i d deposition in fo c i of vascular injury. Reduced local esterolytic activity would be expected to retard the disposal of wall-invading l i p i d s . Turning from injury to metabolic effects, I discovered that neither lipemia nor acute fasting changed the esterase or lipase activity of the animal arterial tissues studied. This was true for both alimentary lipemia and that induced by intravenous infusions of lipemic plasma. These results indicate that-jsaaspecific esterase and "Tween" lipase do not respond to dramatic increments (acute or chronic) or decrements of l i p i d levels in the blood. They also suggest that neither acute nor chronic lipemia "induces" any visible esterolytic enzyme activity in mammalian arterial c e l l s , unlike bacterial systems in which enzymes have been induced by dramatic changes of ambient substrate concentrations, as reported by Jacob and Monod(72). D. Enzymes in Human and Experimental Atherosclerosis In human aortae significant esterase activity only occurred in areas of atheroma in conjunction with the deposition of fat. A l l activity was intracellular. In fatty streaks, the foam cells of the lesion were esterase positive. Pearly plaques exhibited 38 esterase activity only in foam cells that survived at the margins of the gruel mass. These findings agree in general with the extensive literature on this topic(15,16,45-53). The fibrous cap of the human pearly plaque did not, however, show any esterase activity. Thus the well known absence of l i p i d deposits from this protective superficial layer of the pearly plaque cannot be attributed to an increased esterolytic enzyme activity in this region, and remains unexplained. As for the early experimental rabbit atheroma. this was found to exhibit the same enzyme distribution and activity as the human lesion, in agreement with the literature(12,13,55-62). As in the human lesion, activity was found to be intracellular, in the foam ce l l s . I did not have sufficient time to produce pearly plaques in the rabbit because this would require at least one year(3), so I do not know what the enzyme findings would be in the fibrous cap of the experimental lesion. However, since the early experimental lesion revealed a very similar enzymatic pattern to the early human one, one might expect the advanced experimental atheroma to be enzymatically similar to i t s human prototype. BIBLIOGRAPHY 39 1. Schettler, F.G. and Boyd, G.S., ad., Atherosclerosis. Elsevier New York, (1969). 2. Miasnikov, A.L., Atherosclerosis - Occurance. Clinical Forms. and Therapy, pp.70-78, NathionalInstitutes of Health, Bethesda, Md. (1962). 3. Constantinides, P., Experimental Atherosclerosis, p.49, Elsevier, Amsterdam, (1965). 4. Zemplenyi, T.; Lojda, Z.; and Grafnetter, D. "Relationship of Lipolytic and Esterolytic Activity of the Aorta to Susceptibi-l i t y to Experimental Atherosclerosis" Circ Res 7:286—290(1959). 5. Sotonyi, Van P.; Huttner, I; Jellinek, H; Toth, A.; and Makoi, Z. "Enzymhistochemische Untersuchungen an den Gefap wan den bei Versuchstieren" Acta Histochem 21: 213-218 (1965). 6. Nachlas, M.M. and Seligman, A.M. "The Comparative Distribution of Esterase in the Tissues of Five Mammals by a Histochemical Technique" Anat. Rec. 105: 677-695 (1949). 7. Eranko, 0; Kokko, A.; and Soderholm, U. "Histochemical Evidence of Three Types of Esterases in the Adrenal Medulla of the Rat" Histochemie 2: 383-388 (1962). 8. Gomori, G. "Distribution of Lipase in the Tissues under Normal and under Pathologic Conditions", Arch Path 41: 121-129 (1946). 9. Szendzikowski, St.; and Patelski, J. "Aspects of the Histochemistry of Esterases in the Aorta" Ann Histochim 6: 377-8 (1961). 10. Arnesjo, B. and Filipek-Wender, H. "Intracellular Distribution of Lipolytic Enzymes in the Rat Pancreas" Acta Phvsiol Scand 74: 616-628 (1968). 11. Takkar, G.L.; Kamby, V.P. and Kar, A.B. "Effect of Altered Hormonal States on the Histochemical Distribution of Lipase Activity in the Rat Prostatic Complex" Histochemie 20:21-8 (1969). 12. Lojda, Z., and Zemplenyi, T. "Histochemistry of some Enzymes of the Vascular Wall in Experimental Rabbit Atherosclerosis" J. Ather. Res. 1: 101-120 (1961) . 13. Zemplenyi, T.; Grafnetter, D.; and Lojda, Z. "Some Problems Connected with the Lipolytic and Esterolytic Activity of Tissues" I n : International Conference on the Biochemistry of Lipids. 6th Proc., Marseille, pp.203-212, (1960). 14. Maier, N; and Haimovici, H. "Metabolism of Arterial Tissue with Special reference to Esterase and Lipase" Proc Soc E X P Biol (N.Y.) 118: 258-261 (1965). 40 15. Muller, E. and Neumann, W. "Untersuchungen Uber Esterase-Aktivitat der G e f a 0 intima im Bereiche Arteriosklerotischer Herde" Frankfurter Z Pathol 70: 174-186 (1959). 16. Tischendorf, F. and Curri, S.B. "Das Verhalten der Lipase im Atherosklerotisch veranderten Arterien vora muskularen Typ" Acta Histochem (Jena) 8: 158-166 (1959). 17. Wuest, J.H. Jr.; Dry, T.J.; and Edwards, J.E. "The Degree of Coronary Atherosclerosis in Bilaterally Oophorectomized Women" Circulation 7: 801-809 (1953). 18. Rivin, A.V. and Dimitroff, S.P. "The Incidence and Severity of Atherosclerosis in Estrogen-treated Males, and in Females with a Hypoestrogenic or a Hyperestrogenic State" Circulation 9: 533-539 (1954). 19. Mrhova, O. and Zemplenyi, T. "The Effect of Sex Differences and Gonadectomy on some Aortic Enzymes of the Rat" Quart J. E X P Phvsiol 50: 289-299 (1965). 20. Mallov, S. "Aortic Lipoprotein Lipase Activity in Relation to Species, Age, Sex and Blood Pressure" Circulat Res 14: 357-363 (1964) . .21. Lacuara, J.L.; Gerschemson, L. ; Moguilevsky, H.C; and Malinow, M.R. "Sexual Differences in the Esterase Activity of the Aorta in Rats" J Athero Res 2: 496-498 (1962). 22. Szendzikowski, S.; Patelski, J. and Pearse, A.G.E. "The Influence of Cholinesterase Inhibitors on the Lipolytic Activity, of the Rat Aorta" Enzvmol Biol Clin (Basel) 1: 125^137 (1961-62). 23. Baust, P.; Goslar, H.G.; and Tonutti, E. "Das Verhalten der Esterasen im hoden von Ratte, Maus und Meerschweinchen nach Oestrogenbehandlung" Z Zeltforschung 69: 686-698 (1966). 24. Huggins, C. and Moulton, S.A. "Esterases of Testis and other Tissues" J Exp Med 88: 169-179 (1948). 25. Niemi, M.; Harkonen, M. and Kokko, A. "Localization and Identification of Testicular Esterases in the Rat" J Hist Cvto 10: 186-193 (1962). 26. Niemi, M. and Kormano, M. "Histochemical Demonstration of a C-Esterase Activity in the Seminiferous Tubules of the Rat Testis" J Reprod F e r t i l 10: 49-54 (1965). 27. Niemi, M.; Harkonen, M.; and Ikonen, M. "A Chemical and Histo-chemical study on the Significance of the Nonspecific Esterase Activity in the Adult Rat Testis" Endocrinology 79: 294-300 (1966). 28. Zemplenyi, T. and Grafnetter, D. "The Lipolytic Activity of the Aorta, i t s Relation to Ageing and to Atherosclerosis" Geronto-logia 3/1: 55-64 (1959). 41 29. Chlouverakis, G. "Lipoprotein Lipase Activity in Adipose, Muscle and Aortic Tissue from Hats of Different Age and in Human Subcutaneous Adipose Tissue" Proc Soc Exp Biol (N.Y.) 119: 775-778 (1965). 30. Niemi, M. and Ikonen, M. "Histochemistry of the Leydig Cells in Postnatal Prepubertal Testis of the Rat" Endocrinology 72: 443-448 (1963). 31. Holmes, R.S. and Masters, C.J. "The Developmental Multiplicity and Isoenzyme Status of Rat Esterases" Biochim Biophys Acta 146: 138-50 (1967). 32. Hitzemann, J.W. "Development of Enzyme Activity in the Leydig Cells of the Mouse Testis" Anat. Rec 143: 351-362 (1962). 33. B a i l l i e , A.H. "Esterase Substrate Specificity in the Testis Of the Growing Mouse" Quart J Micr Sci 105: 247-55 (1964). 34. B a i l l i e , A.H. "Further Observations on the Growth and Histo-chemistry of Leydig Tissue in the Postnatal Prepubertal Mouse Testis" J Anat (London) 98: 403-419 (1964). 35. Leites, F.L. and Fedoseev, A.N. "Lipolytic Enzyme Activity in Dogs with Experimental Atherosclerosis" Fed Proc (Trans Suppl) 24: 829-32 (1964). 36. Leites, F.L. "Changes in the Nonspecific Esterase Activity in Experimental Disturbances of Lipoid Metabolism" Pat F i z i o l  eksp Ter (Moskva) 7 (2): 50-54 (1963). 37. Leites, F.L. "Induction of Lipolytic Enzymes by the Connective tissue Cells during Changes of the Endocrine Gland in Experi-mental Conditions" Probl Endokr Gormonoter 9(6): 29-33 (1965) . 38. Leites, F.L. and Chzhou-Su "Lipolytic Activity of the Aortic Wall in Certain Experimental Pathologic Conditions" Fed Proc  (Trans Suppl) 22: 466-69 (1962). 39. Leites, F.L. "Histochemical Peculiarities of Lipid Metabolism and Activity of Lipolytic Enzymes in Alloxan Diabetes" Probl  Endokr Gormonoter 11(3): 88-95 (1965) . 40. Cairns, A. and Constantinides, P. "Endocrine Effects on the Heparin-induced Lipemia-clearing Activity of Rat Plasma" Can J Bioch Phys 33: 530-538 (1955). 41. Zemplenyi, T. and Grafnetter, D. "Fatty Acid Release on Incu-bation of Lipemic Serum with Rat Tissues. The Influence of Heparin and Fasting" Arch int Pharmacodyn 122: 57-66 (1959). 42. Moskowitz, M.S. and Moskowitz, A.A. "Lipase: Localization in Adipose Tissue" Science 149: 72-73 (1965). 43. Gillman, T.; Grant, R.A. and Hathorn, M. "Histochemical and Chemical studies of Calciferol-induced Vascular Injuries" "Brit J Exptl Pathol 41: 1-18 (1960). 42 44. Grafnetter, D. and Zemplenyi, T. "Tissue Lipolytic Activity in Calciferol Intoxicated Rats" Experlentia 18: 85-86 (1962). 45. Adams, C.W.M.; Bayliss, O.B.; Abdulla, Y.H.; Mahler, F.R. and Root, M.A. "Lipase, Esterase and Triglyceride in the Ageing Human Aorta" J Atheroscler Res 9: 87-102 (1969). 46. Isachenko, V.B. "The Relationship between the Lipolytic Enzyme Activity and Lipoidosis of the Aortic Wall" Pat. F i z i o l Eksp Ter 11: 32-5 (1965). 47. Kirk, J.E. Enzymes of the Arterial Wall. Academic Press, N.Y. (1969) p.208-222. 48. Anestiadl, V.Kh. and Russu, S.P. "Considerations sur les Caracteres Histoenzymalogiqu.es des Enzymes Hydrolytiques de la Paroi des Arteres au stade i n i t i a l de 1'Atherosclerose" Ann Anat Path (Paris) 14: 267-74 (1969). 49. Deribas, V.I.; Fuks, B.B.; and Shishkin, G.S. "Modified Reactivity in Terminal States" In: Mechanisms of Disease and  Recovery. Novosibirsk, (1960). p.124. 50. Gonzalez, I.E. "Comparative Histochemical Study of Human and Experimental Atherogenesis" Ann Histochem 8: 335-388 (1963). 51. Gonzalez I.E. and Furman, R.H. "Histochemistry of Spontaneous and Experimental Arterial Lesions" In: Comparative Atheroscle- rosis pp.329-341, ed. by J.C. Roberts Jr. and R. Straus. Hoeber, New York. (1965). 52. Levonen, E.; Raelallio, J. and Uotila, U. "Histochemical Demonstration of some Hydrolytic Enzymes in Atheromatous Aortas" Nature (Lond.) 188: 677-678 (1960). 53. Stein A.A. and Harris, J. "Histochemical Enzyme Distribution on Human Arteries" Surgery 56: 413-418 (1964). 54. Zemplenyi, T. and Mrhova, 0. "Activite Enzymatique de la Paroi Arterielle et Atherogenese" Rev Atheroscler 8(3): 145-151 (1966). 55. Patelski, J.; Bowyer, D.E.; Howard, A.N.; and Gresham, G.A. "Changes in Phospholipase A, Lipase, and Cholesterol Esterase Activity in the Aorta in Experimental Atherosclerosis in the Rabbit and Rat" J Atheroscler Res 8: 221-228 (1968). 56. Maier, N. "Metabolism of Arterial Tissue and i t s Relation to Atherosclerosis" Ann N.Y. Acad Sci 149: 655-665 (1968). 57. Maier, N. and Haimovici, H. "Metabolism of Atherosclerotic Tissue of Rabbit and Dog, with Special Reference to Esterase and Lipase" Circ Res 17: 178-85 (1965). 43 58. Zemplenyi, T. and Grafnetter, D. "The Lipolytic Activity of Heart and Aorta in Experimental Atherosclerosis in Rabbits" Brit J Exp Path 40: 312-317 (1959). 59. Zemplenyi, T.; Lojda, Z.; and Mrhova, 0. "Enzymes of the Vascular Wall in Experimental Atherosclerosis in the Rabbit" In: Atherosclerosis and i t s Origin (M. Sandler and G.H. Bourne, eds.) pp. 459-513. Academic Press. New York. (1963). 60. McMillan, G.C; Klatzo, I.; and Duff, G.L. "Histochemical and Staining Reactions of Tissues and Organs of Cholesterol Fed Rabbits" Lab Invest 3: 451-468 (1954). 61. Wegmann, R. and Fouquet, J.P. "Quelques modifications histo-enzymologiques accompagnant le debut du depot de cholesterol dans 1'aorta du lapin" Ann Histochim 6: 61-65 (1961). 62. Kobernick, S.D. and Hashimoto, Y. "Histochemistry of Atherosclerosis-I: Induced Lesions of the Aorta of Cholesterol-fed Exercised and Sedentary Rabbits" Lab Invest 12: 638-647 (1963). 63. Koide, R. and Pollak, O.J. "Cytochemical Studies of Rabbit Aortic Cells in Tissue Culture" J Histochem Cytochem 11: 265-272 (1963). 64. Pearse, A.G.E. Histochemistry, Thanran.t.i*>ai an r i AppH arf J 2nd ed., J. & A. Churchill, London (1960). 65. Zemplenyi, T. "Enzymes of the Arterial Wall" J Atheroscler  Res 2: 2-24 (1962). 66. Okuda, H. and F u j i i , S. "Relationship between Lipase and Esterase" J Biochem (Tokyo) 64: 377-85 (1968). 67. Gomori, G. Microscopic Histochemistry. p. 204 Univ. of Chicago Press, Chicago, 111. (1952). 68. L i l l i e , R.D. Histopathological Technique and Practical Histo— chemistry. 3rd ed., McGraw-Hill, New York. p. 102 (1965). 69. Brown, D.F. "Triglyceride Metabolism in the Alloxan - Diabetic Rat" Diabetes 16: 90-95 (1967). 70. Shichiri, M.; Miller, M.; and Woodward, A. Jr. "Disappearance of DL-Lactate-2-C14 from Blood in Normal and Diabetic Rats" Soc EXP Biol Med - Proc 125: 1238-42 (1967). 71. Zemplenyi, T., Enzvme Biochemistry of the Arterial Wall. Lloyd-Luke, London, p. 219, (1968). 72. Jacob, F. and Monod, J. "Genetic Regulatory Mechanisms in the Synthesis of Proteins" J. Mol. Biol. 3: 318-356 (1961). 44 Figure 1. Normal rat pancreas. Esterase. Note the contrast in a c t i -vity between the positive acini, l e f t , and a negative large i s l e t of Langerhan, right. The faint brown colouring in the i s l e t i s probably diffusion artifact. Magnification 40X. I Figure 2. Normal rat adrenal. Esterase. Note contrast between cortex, l e f t , and medulla, right. Magnification 40X. 45 Figure 4. Normal rat adrenal. Lipase. The medulla, upper right, is negative, while the rest is positively reacting cortex. Magnification 40X. 46 • * Figure 5. The aorta of a young, weanling, 26 day old rat. Esterase. Magnification 40X. F i g u r e 7. T h e c o r o n a r y a r t e r y o f a m a t u r e r a t . E s t e r a s e . T h e e n z y m e d i s t r i b u t i o n i s t h e same a s t h a t o f f i g u r e 6. M a g n i f i c a t i o n 40X. Figure 9. Testis from a 30 day old rat. Esterase. Some Leydig cells are positive. Magnification 40X. 49 4 Figure 10. Testis from a mature, 18 month old rat. Esterase. Note the large group of Leydig cells to the l e f t which are strongly reacting. The basal parts of the sperm-atogenic tubule are also slightly positive. The boundary between the Leydig c e l l cluster and the spermatogenic tubule i s indicated by the two arrows. Magnification 40X. Figure 12. The prostate gland of a rat treated with s t i l b e s t r o l . Esterase. Note the disappearance of the enzyme activity accompanying the epithelial atrophy. Magnification 40X. 51 Figure 13. The aorta of a mature, 18 month old rat. Esterase. • Figure 14. Injured aorta from a rat treated with Calciferol. Esterase. Note lack of enzyme activity at focus of injury in the upper part of the vessel wall. Magnification 40X. 52 Figure 16. Focus of injury in the upper media of the aorta from a rat treated with Calciferol. H. and E. Magnification 40X. 53 Figure 17. Normal human aorta. Esterase. There is no enzyme activity. Magnification 10X. * Figure 18. Human aorta with fatty streak. Esterase. Positively reacting foam cells and spindle cells can be seen. Magnification 40X. 54 I Figure 19. Boundary between l i p i d and n o n — l i p i d containing areas of a pearly plaque of a human aorta. Esterase. Note how a l l enzyme activity is intracellular, within a few spindle-shaped foam cells just below a negatively reacting fibrous cap. Magnification 40X. 55 Figure 20. Normal rabbit aorta. Esterase. There is no enzyme activity. Magnification 10X. Figure 21. Rabbit aorta with raised atherosclerotic plaque. Esterase. Enzyme activity is strong throughout the plaque area. The apparently slightly positive areas in the media beneath the lesion were observed only in a minority of cases, and possibly represent an artifact. Magnification 10X. • • M i 56 Figure 22. Rabbit aorta with raised atherosclerotic plaque. Esterase. Note that a l l cells of the plaque react for enzyme. Magnification 40X. 57 58 APPENDIX A. oC NAPHTHYL ACETATE METHOD FOR ESTERASE (64) 1. Cut frozen sections 12 microns thick. 2. Fix in 10% formalin. 3. Incubate for six minutes at room temperature in the following medium: a) To 20 mg =< naphthyl acetate in 0.50 ml acetone add 40 ml 0.1 M phosphate buffer (pH7.4). b) Shake until most cloudiness disappears. c) Add 80 mg Fast Blue RR salt. d) Shake and f i l t e r i n the dark. (This medium must be used just as soon as i t i s made). 4. Wash in running water two minutes. 5. Counterstain in Mayer's Hematoxylin four minutes. 6. Blue in bicarbonate 2%. 7. Wash. 8. Mount in aqueous mountant. (Arlex Gelatin of L i l l i e and Greco). 59 APPENDIX B. GOMORI TWEEN METHOD FOR LIPASE(64) 1. Cut frozen sections 12 microns thick. 2. Fix in 10% formalin. 3. Incubate for three hours in the following medium, at 37 degrees Centigrade : 5 ml " t r i s " buffer (trishydroxymethylaminomethane) at pH 7.4 2 ml calcium chloride 10% 2 ml Tween 60, 5% 2 1/2 ml sodium taurocholate 2.33 mM. 37 1/2 ml d i s t i l l e d water. 4. Wash well in d i s t i l l e d water. 5. Immerse in 1% lead nitrate for 15 minutes. 6. Wash in running water for 5 minutes and transfer to a clean coplin jar. 7. Immerse in 1% ammonium sulfide for 2 minutes. 8. Wash; counterstain with Mayer's Hematoxylin for 4 minutes. 9. Blue with bicarbonate 2%. 10. Wash; mount in aqueous mountant (Arlex Gelatin of L i l l i e and Greco). 

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