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The effects of vitamin E deficiency and/or ionizing radiation on uterine ceroid pigment development and… Marchant, Ruth Yu Yoke 1974

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THE EFFECTS OF VITAMIN E DEFICIENCY AND/OR IONIZING RADIATION ON UTERINE CEROID PIGMENT DEVELOPMENT AND SEVERAL OTHER PARAMETERS IN THE RAT by RUTH YU YOKE MARCHANT B.Sc, University of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the D i v i s i o n of HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA June, 1974 In p r e s e n t i n g 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 r e q u i r e m e n t s 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 Co lumb ia , I a g ree 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 r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g 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 g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r 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 g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8. Canada Date ABSTRACT The purpose of t h i s study was to examine the ef f e c t s of Vitamin E defi c i e n c y and/or i o n i z i n g r a d i a t i o n on uterine ceroidogenesis, i n c i s o r depigmentation, serum Vitamin E, haemoglobin concentration, haematocrit, 2,3-DPG and adipose tissue f a t t y acid composition i n r a t s . An attempt was also made to define the nature of ceroid and to examine i t s r e l a t i o n s h i p to l i p o f u s c i n . Rats fed a Vitamin E d e f i c i e n t d i e t deposited increasing amounts of ceroid i n the uterine musculature between 90 to 261 days a f t e r the imposition of the experimental d i e t s . This pigment was found to be e s s e n t i a l l y s i m i l a r to l i p o f u s c i n . Ionizing r a d i a t i o n did not have any apparent e f f e c t on the rate and degree of ceroidogenesis i n Vitamin E d e f i c i e n t r a t s , althought i t did cause a s i g n i f i c a n t decrease i n the serum Vitamin E l e v e l s of both Vitamin E d e f i c i e n t and supplemented animals. The serum Vitamin E of the d e f i c i e n t animals were s i g n i f i c a n t l y lower than those of the supplemented animals, i r r e s p e c t i v e of t h e i r r a d i a t i o n status. Vitamin E deficiency also caused a depigmentation of the maxillary i n c i s o r s of the animals and had no e f f e c t on the haemoglobin concentration, haematocrit and 2,3-DPG l e v e l s of these r a t s . F i n a l l y , Vitamin E de f i c i e n c y and i o n i z i n g r a d i a t i o n resulted i n a decrease i n the polyunsaturated f a t t y acid content of adipose ti s s u e s . - l -ACKNOWLEDGEMENTS Acknowledgement i s g r a t e f u l l y extended to the following persons i Dr. I. D. Desai - D i v i s i o n of Human N u t r i t i o n , School of Home Economics, who served as graduate advisor and provided the f a c i l i t i e s f o r t h i s study Dr. M. Lee - School of Home Economics, f o r the hours he spent on t h i s manuscript Dr. B. March - Department of Poultry Science, f o r her time spent i n c a r e f u l reading of t h i s manuscript Mr. C. C u l l i n g and Mrs. B. Barkosczy - Department of Pathology, f o r invaluable advice and te c h n i c a l assistance on h i s t o l o g i c a l and histochemical matters Dr. Robert Schutz - School of. Physical Education, f o r h i s b r i l l i a n c e , s t a t i s t i c a l and otherwise Wayne Marchant - f o r h i s a r t i s t r y Len Marchant - for h i s guidance and assistance during a l l phases of t h i s study F i n a n c i a l assistance was provided by the UBC Graduate Fellowship (1972-19731 1973-197*0 and NRC Research Grant # 67-^686 (awarded to Dr. I. D. Desai). - i i -TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i i LIST OF TABLES v i LIST OF FIGURES i x LIST OF PLATES x i STATEMENT OF THE PROBLEM Introduction 1 Purpose and scope of the study 2 Significance of the study 3 REVIEW OF THE LITERATURE Introduction 5 The b i o l o g i c a l antioxidant theory of Vitamin E function 6 L i p i d peroxidation and ceroid pigment formation.... 12 The nature of ceroid pigment 16 The r e l a t i o n s h i p between ceroid, Vitamin E and l i p i d s 18 Lipofuscin/ceroid and theories of senescence 21 Ionizing r a d i a t i o n and aging 22 The lysosomal theory of aging 23 Incisor depigmentation i n r a t s 26 Vitamin E de f i c i e n c y and red c e l l metabolism 26 The e f f e c t on heme biosynthesis.. 26 S t a b i l i t y of the red c e l l membrane 27 - i i i -TABLE OF CONTENTS (contd) Vitamin E and 2,3-diphosphoglycerate 29 MATERIALS AND METHODS Animal care 3 1 Experimental d i e t s 31 Experimental groups 35 Experimental procedure. 35 Biochemical determinations 37 Serum Vitamin E l e v e l s 37 Haemoglobin concentrations and haematocrit determinations 38 2,3-DPG analysis M Determination of f a t t y acid composition. 4 l H i s t o l o g i c a l and histochemical techniques Experimental design. S t a t i s t i c a l a nalysis.. ^5 Serum Vitamin E analysis 52 Haematological parameters 53 Adipose tissue f a t t y acid composition 55 RESULTS Raw scores .57 Homogeneity of variance. 57 Body weight and uterus weight gains 57 Ceroid pigment development and accumulation 58 Morphological observations 58 Histochemical observations 67 Incisor depigmentation 82 - i v -TABLE OF CONTENTS (contd) Serum Vitamin E 82 Descriptive s t a t i s t i c s . . . . 82 S t a t i s t i c a l analysis of data. 88 Haematological parameters... 98 Descriptive s t a t i s t i c s . . . . . . . . . . 98 S t a t i s t i c a l analysis of data 99 Fatty acid composition of adipose tissues 102 Descriptive s t a t i s t i c s 102 S t a t i s t i c a l analysis of data 102 DISCUSSION Limitations and delimitations 114 The e f f e c t of Vitamin E d e f i c i e n c y and/or i o n i z i n g r a d i a t i o n on body and uterus weight changes 115 Ceroid pigment development and accumulation 119 The nature of ceroid pigment 121 Incisor depigmentation i n Vitamin E d e f i c i e n t r a t s . 126 The e f f e c t of Vitamin E d e f i c i e n c y and/or i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s 127 Haematological parameters 131 The e f f e c t of Vitamin E d e f i c i e n c y and/pr i o n i z i n g r a d i a t i o n on adipose tissue f a t t y acid composition 132 SUMMARY AND CONCLUSIONS 135 BIBLIOGRAPHY .. 137 APPENDIX 155 v-LIST OF TABLES Table Page I E f f e c t of antioxidants other than Vitamin E on some Vitamin E deficiency-diseases 13 II Composition of Basal Vitamin E-free d i e t 32 III Composition of mineral mix used i n the basal d i e t 33 IV Composition of vitamin mix used i n the basal d i e t V Experimental periods 36 VI Table of hypotheses k9 VII ANOVA table of serum Vitamin E l e v e l s f o r Day 0 to Day 30 of the experiment 52 VIII ANOVA table of serum Vitamin E l e v e l s f o r Day 30 to Day 261 of the experiment 5^ IX Histochemical r e s u l t s 68 X Serum Vitamin E l e v e l s of Group I and Group II rats f o r the f i r s t four sampling periods (Day 0 to Day 30) 85 XI Serum Vitamin E l e v e l s of rats from a l l groups f o r ten sampling periods (Day 30 to Day 261) 88 XII Orthogonal comparison of average Group I and average Group II serum Vitamin E l e v e l s for Days 0 to 30 89 XIII Orthogonal comparison of pooled (Group I and Group II) serum Vitamin E l e v e l s over time 89 XIV Orthogonal comparison of i n t e r a c t i o n e f f e c t between Group I and Group II serum Vitamin E l e v e l s from Day 0 to Day 30 91 XV Orthogonal comparison of average serum Vitamin E l e v e l s of a l l groups f o r Days 30 to 261 91 - v i -Table Page XVI XVII XVIII XIX XX XXI XXII XXIII XXIV XXV XXVI XXVII XXVIII Orthogonal comparison of pooled serum Vitamin E l e v e l s from Day 30 to Day 261 (Groups I to IV) 92 Orthogonal comparison of group e f f e c t of serum Vitamin E l e v e l s over time 93 Orthogonal comparison of the e f f e c t of die t a r y Vitamin E status on serum Vitamin E l e v e l s of nonirradiated rat s from Day 30 to Day 261 9^ Orthogonal comparison of the e f f e c t of di e t a r y Vitamin E status on serum Vitamin E l e v e l s of i r r a d i a t e d r a t s from Day 30 to Day 26l 95 Orthogonal comparison of the e f f e c t of of d i e t a r y Vitamin E status on serum Vitamin E l e v e l s of nonirradiated and i r r a d i a t e d r a t s from Day 30 to Day 261 96 Orthogonal comparison of the e f f e c t of i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s of Vitamin E supplemented rats from Day 30 to Day 26l 97 Orthogonal comparison of the e f f e c t of i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s of Vitamin E d e f i c i e n t r a t s from Day 30 to Day 26l 98 Orthogonal comparison of the e f f e c t of i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s of supplemented and d e f i c i e n t r a t s from Day 30 to Day 261 99 Means and standard deviations f o r haematological parameters 100 Orthogonal comparisons of haematological parameters i n Group I and Group II animals 102 Percent f a t t y acid composition of adipose tissues 103 Multivariate analysis of group e f f e c t on adipose tissue f a t t y acid composition 106 Orthogonal comparison f o r the e f f e c t of di e t a r y Vitamin E status on adipose tissu e f a t t y acid composition of nonirradiated r a t s (Groups I and II) 107 -V11-Table XXIX Page XXX XXXI XXXII XXXIII Orthogonal comparison f o r the e f f e c t of dieta r y Vitamin E status on the adipose tissue f a t t y acid composition of ir r a d i a t e d rats (Groups III and IV) Orthogonal comparison f o r the e f f e c t of dieta r y Vitamin E status on the adipose tissue f a t t y acid composition of supplemented (Groups I and III) and d e f i c i e n t (Groups II and IV) rats Orthogonal comparison t e s t i n g the e f f e c t of i o n i z i n g r a d i a t i o n on the adipose tissue f a t t y acid composition of Vitamin E supplemented rats (Groups I and III) Orthogonal comparison t e s t i n g the e f f e c t of i o n i z i n g r a d i a t i o n on the adipose tissue f a t t y acid composition of Vitamin E d e f i c i e n t r a t s (Groups II and IV) Orthogonal comparison t e s t i n g the e f f e c t of i o n i z i n g r a d i a t i o n on the adipose tissue f a t t y acid composition of nonirradiated and i r r a d i a t e d r a t s 108 109 110 111 112 XXXIV Orthogonal comparisons t e s t i n g the e f f e c t of treatment (Vitamin E d e f i c i e n c y and/ or i o n i z i n g r a d i a t i o n ) on the adipose tissue f a t t y acid composition of rats (Group I vs Groups I I , III and IV) 113 - v i i i -LIST OF FIGURES Figure Page 1 Proposed mechanism fo r the antioxidant function of Vitamin E 7 2 Oxidation products of alpha-tocopherol 11 3 A pathway f o r the formation of fluorescent pigment 20 4 2,3-DPG formation i n the mammalian erythrocyte 30 5 C a l i b r a t i o n curve f o r the determination of serum Vitamin E 39 6 C a l i b r a t i o n curve f o r beta-carotene 40 7.1 Experimental design f o r trend analysis of dependant variables 1,2 and 3 f o r a l l four groups during the entire experimental period 46 7»2 Experimental design f o r trend analysis of serum Vitamin E i n Groups I and II from Day 0 to Day 30 of the experiment 46 7*3 Experimental design f o r trend analysis of serum Vitamin E i n a l l groups f o r Day 30 to Day 26l of the experiment 47 7»4 Experimental design f o r the analysis of dependant variables 5,6 and 7 i n Groups I and II f o r one sampling period 47 7.5 Experimental design f o r the analysis of adipose tissue f a t t y acid composition i n a l l four groups f o r one sampling period 48 8*1 Cumulative body weight gain of animals i n a l l groups from Day 0 to Day 26l 59 8.2 Cumulative weight gain of animals i n a l l four groups from Day 0 to Day 30 60 9 Cumulative uterus weight gain of animals i n a l l groups from Day 0 to Day 261 6l 10 Changes i n serum Vitamin E l e v e l s of Group I and Group II animals from Days 0 to 30 85 - i x -Changes i n serum Vitamin E l e v e l s of animals i n a l l groups from Day 60 to Day 261 The e f f e c t of d i e t a r y Vitamin E d e f i c i e n c y on various haematological parameters Percent f a t t y acid composition of adipose tissues LIST OF PLATES Plate Page I E a r l i e s t morphological evidence f o r ceroid formation i n Vitamin E de f i c i e n c y (3 months) 60 II E f f e c t of Vitamin E d e f i c i e n c y and/or i o n i z i n g r a d i a t i o n on the u t e r i of r a t s 6 l III Animals with u t e r i and abdominal depot f a t tissue i n s i t u 63 IV The appearance of ceroid pigment accumulated i n the uterine tissue at the end of eight months of di e t a r y Vitamin E de f i c i e n c y (Haematoxylin and Eosin) 67 V Pigmentogenesis and accumulation i n muscle c e l l s of u t e r i from Vitamin E def i c i e n c y (Sudan Black B) 68 VI Responses of co n t r o l and Vitamin E d e f i c i e n t uterine tissues to Gomori's Methenamine S i l v e r Reaction 69 VII Accumulation of pigment i n uterine connective tissues of Vitamin E d e f i c i e n t r a t s (Sudan Black B) 70 VIII Ceroid pigment development and accumulation i n Vitamin E d e f i c i e n t r a t s (Schmorl's Reaction) 71 IX Ceroid pigment development and accumulation i n Vitamin E d e f i c i e n t r a t s (Periodic A c i d - S c h i f f Reaction) 7^ X Comparison of ceroid pigment properties a f t e r s i x months of diet a r y Vitamin E def i c i e n c y 77 XI E f f e c t of i o n i z i n g r a d i a t i o n on ceroidogenesis 79 XII Incisor depigmentation i n Vitamin E d e f i c i e n t r a t s 81 - x i -For my husband - x i i -CHAPTER I STATEMENT OF THE PROBLEM Introduction Vitamin E deficiency i s known to cause s t e r i l i t y i n rats (Evans and Bishop,1922? Evans,1925> Evans and Burr,1927? Evans, 1928). In the female t h i s i s manifested by f o e t a l resorption and i s associated with the accumulation of a l i p i d - d e r i v e d pigment, ceroid, i n the uterine myometrium (Martin and Moore,1936} Barrie, 1938$ Martin and Moore,1939). Ceroid pigment was h i s t o l o g i c a l l y and histochemically characterized t h i r t y years ago and these data have neither been confirmed nor challenged. In addition, the ceroid pigment of Vitamin E d e f i c i e n c y i s currently thought to be relat e d to, i f not the same as, the l i p o f u s c i n pigment associated with senescence (Porta and Hartroft,1969» Pearse,1972). No study has been done to v e r i f y t h i s i n Vitamin E d e f i c i e n t r a t s . According to the B i o l o g i c a l Antioxidant Theory of Vitamin E function, Vitamin E def i c i e n c y leads to the generation of excess free r a d i c a l s i n the system so that extensive l i p i d peroxidation occurs, causing widespread damage of c e l l u l a r membranes and l a b i l e constituents (Tappel,1953). Further, Casarett(i960), Upton (I960) and Lindop and Rotblat (196l) have shown that the process of aging i n animals i s at l e a s t p a r t l y the r e s u l t of free r a d i c a l damage of the organism with time. I f i t can be demonstrated that the physical manifestations of the two processes (Vitamin E def i c i e n c y and i o n i z i n g r a d i a t i o n ) are the same - that l i p o f u s c i n -2-and ceroid pigment share e s s e n t i a l s i m i l a r i t i e s - then the v a l i d i t y of the antioxidant theory of Vitamin E function and the free r a d i c a l theory of aging (Maynard-Smith, 1963) may be assessed on the basis of the rate and degree of pigment formation i n appropriately treated animals. Moreover, any potentiation e f f e c t of Vitamin E on the aging process may also be demonstrated. I. Purpose and Scope of the Study. The purpose of t h i s i n v e s t i g a t i o n was to study the development and accumulation of ceroid pigment i n the u t e r i of r a t s i n response to Vitamin E d e f i c i e n c y and/or i o n i z i n g r a d i a t i o n . According to Lindop and Rotblat (1961), i o n i z i n g r a d i a t i o n provides a means whereby free r a d i c a l s are a r t i f i c i a l l y generated within the organism so as to induce premature aging e f f e c t s . Attempts were made to determine the time of onset of ceroidogenesis as well as to characterize the histochemical nature of ceroid pigment during i t s development and accumulation i n the uterus over the entire experimental period of 8i months i n order to assess the r e l a t i o n s h i p of ceroid, i f any, to l i p o f u s c i n . Several other parameters were examined. Serum Vitamin E l e v e l s were measured so as to provide a biochemical yardstick f o r the h i s t o l o g i c a l observations. An attempt was also made to determine the r e l a t i o n s h i p , i f any, between serum Vitamin E l e v e l s and the onset of pigmentogenesis. In addition, the k i n e t i c s of serum Vitamin E depletion i n Vitamin E d e f i c i e n t animals and that of serum Vitamin E increases i n Vitamin E supplemented r a t s , as well as the changes i n t h i s parameter that occurred over the entire -3-experimental period i n i r r a d i a t e d and nonirradiated animals of d i f f e r i n g Vitamin E status, were assessed. Then there was an attempt to delineate differences i n erythrocyte 2,3-diphosphoglycerate l e v e l s , haemoglobin l e v e l s and haematocrits between animals of d i f f e r i n g d i e t a r y Vitamin E status. The e f f e c t of Vitamin E d e f i c i e n c y on the above haematological parameters was examined because previous studies have demonstrated a causal r e l a t i o n s h i p between these factors (Kann,1967; Kurokawa e_t al,1970j Murty et al,1970j Melhorn,1971 $ Kurokawa et al,1972» Menge1,1972). F i n a l l y , the percent f a t t y acid composition of adipose t i s s u e s from i r r a d i a t e d and nonirradiated rat s of d i f f e r i n g d i e t a r y tocopherol status were compared to each other because depot f a t composition i s believed to r e f l e c t d i e t a r y l i p i d composition and, since Vitamin E prevents l i p i d peroxidation (Dam and Granados,1945l Dam,1962} Dam and Sondergaard,1970> Witting,1970j 1972), i t was presumed that lack of antioxidant i n the d i e t as well as i n vivo would a l t e r depot f a t composition. Furthermore, no data are avail a b l e on the adipose tissue composition of i r r a d i a t e d animals. I I . S i g nificance of the Study. Much of the d e f i n i t i v e work on Vitamin E d e f i c i e n c y -induced ceroidogenesis and s t e r i l i t y i n female r a t s was done about t h i r t y years ago. In view of the current resurgence of i n t e r e s t i n Vitamin E, i t i s f e l t that a reevaluation of some of these data i s needed at t h i s time. Furthermore, the question of Vitamin E and the aging process has to be more f u l l y explored since no actual data have ever been gathered to e i t h e r support or dismiss the hypothesis that senescence i s caused by free r a d i c a l damage of the organism due to a lack of the antioxidant, Vitamin E. I t has been demonstrated that ceroid pigment (diet-linked) and l i p o f u s c i n pigment (age-linked) have a common o r i g i n (both due to free r a d i c a l reactions)(Porta and Hartroft,1969 j Lindlar,1970; Pearse,1972) so that a basis f o r the comparison of Vitamin E and/or aging e f f e c t s on pigment development can be formed. I t remains to be demonstrated whether the e s s e n t i a l c h a r a c t e r i s t i c s of ceroid pigment are s i m i l a r to those of l i p o f u s c i n . This study also attempts to c l a r i f y the e f f e c t s of Vitamin E d e f i c i e n c y on the elements of the haematopoeitic system. To date, no such study has been done i n r a t s , only i n mice (Melhorn,19711 Mengel,1972), nor have previous studies u t i l i z e d more than h a l f a dozen animals. F i n a l l y , no data are avai l a b l e on the f a t t y acid composition of adipose tissues i n i r r a d i a t e d r a t s , i r r e s p e c t i v e of t h e i r d i e t a r y Vitamin E status. CHAPTER II REVIEW OF THE LITERATURE Introduction During investigations into the r e l a t i o n s h i p between f e r t i l i t y and n u t r i t i o n , Evans and Bishop (1922) discovered that wheat germ o i l had a remarkably curative e f f e c t on the f o e t a l resorption syndrome observed i n rat s on a rancid l a r d d i e t . The *X-Factor* i n wheat germ responsible f o r t h i s outcome was named Vitamin E by Sure i n 192^ and l a t e r designated as tocopherol (tokos,Gr., c h i l d b i r t h ; pherein,Gr., to bear; - o l , suf., hydroxyl group) (Evans,19^2). The long early h i s t o r y of i n q u i r i e s r e l a t i n g to Vitamin E occupied almost two decades - from 1922 to 19^0 - and was distinguished by many d i s c r e t e , often unrelated, but b i o l o g i c a l l y sound observations as well as a resolute e f f o r t to chemically delineate the new substance. The period from 19^0 up to the present, 197^» has been witness to many more de t a i l e d and extensive studies on t h i s subject. The purpose of t h i s chapter i s to b r i e f l y review the findings i n the area of Vitamin E def i c i e n c y i n rat s and i t s r e l a t i o n s h i p to l i p i d peroxidation (ceroid pigment formation), aging, di e t a r y f a t and various haematological parameters such as 2,3-diphosphoglycerate and haemoglobin l e v e l s . - 6 -I. The B i o l o g i c a l Antioxidant Theory of Vitamin E Function. The b i o l o g i c a l r o l e of Vitamin E i n animals has been a matter of conjecture for many years and remains l a r g e l y unresolved today. Although many workers f e e l that the actions of Vitamin E involve s p e c i f i c biochemical mechanisms yet to be discovered, one major theory has been put forward to explain and apparently correlate most of the known n u t r i t i o n a l and biochemical f a c t s about the vitamin. This i s the antioxidant theory of Tappel ( 1 9 5 3 ; 1 9 6 2 ? 1 9 6 5 ; 1 9 7 2 ) . According to the theory, Vitamin E functions s o l e l y as a p h y s i o l o g i c a l antioxidant. Tappel ( 1 9 5 3 * 1 9 6 2 j 1 9 6 5 ) suggested that tissue unsaturated l i p i d s are c o n t i n u a l l y under attack by free r a d i c a l s (which may e i t h e r be of random o r i g i n or produced i n metabolic reactions) and that, i n the presence of oxygen, these l i p i d s become peroxidized. Alpha-tocopherol functions i n vivo by reacting with the free r a d i c a l s , thus i n h i b i t i n g the tissue peroxidation process (Figure l ) . I f s u f f i c i e n t Vitamin E i s not present, the peroxidation of l i p i d s becomes extensive and uncontrolled, thereby leading to widespread damage to i n t r a c e l l u l a r membranes, enzymes and c e r t a i n l a b i l e metabolites. In general, there are f i v e types of evidence that provide the t h e o r e t i c a l and p r a c t i c a l basis for the antioxidant theory. F i r s t , alpha-tocopherol i s known to have antioxidant a c t i v i t y i n v i t r o (Green,1969)1 a function which i s related to the oxidation--7-FIGURE 1 PROPOSED MECHANISM FOR THE ANTIOXIDANT FUNCTION OF VITAMIN E I n i t i a t i o n Reaction i LIPID (LH) ) FREE RADICAL (L* ) Propagation of Chain Reaction L* + 0 2 ) L 0 2 * L 0 2 * + LH —• » L* + LOOH Termination Reaction LOg* + ocTH (reduced tocopherol) > LOOH + otTQ (oxidized) * l i g h t / i o n i z i n g radiation/metabolic reactions (Tappel,l973) LEAVES 8-9 OMITTED IN PAGE NUMBERING reduction properties inherent i n i t s structure (Drummond,1939» Harrison et a l . 11956| Vasington e_t a l , I960; Tappel, 1962). (Figure 2). Second, there i s a f a i r l y well-substantiated r e l a t i o n s h i p between the amount of dietary unsaturated f a t and the onset and incidence of Vitamin E d e f i c i e n c y diseases i n animals (Harris and Embree,1956j Sondergaard,1967). M a t i l l (1927) f i r s t showed that an increase i n the r a n c i d i t y of the d i e t accelerated the onset of s t e r i l i t y i n r a t s . Many other workers have since demonstrated that unsaturated f a t and long chain polyunsaturated f a t t y acids exacerbate the symptoms of Vitamin E d e f i c i e n c y (Harris and Mason,1956j Harris and Embree,1963> Sondergaard,19671 Lee and Barnes,1969; Witting,1970). This r e l a t i o n s h i p i s a very complex one and cannot be interpreted s o l e l y i n terms of the p e r o x i d i z a b i l i t y of unsaturated f a t and the antioxidant function of Vitamin E (Green and Bunyan,1969» Green,1972). The t h i r d l i n e of evidence, one most relevant to t h i s t h e s i s , i s that the tissues of Vitamin E d e f i c i e n t animals contain more l i p i d peroxides and are more 'peroxidizable' than those of supplemented animals (Dam and Granados,19^5f Dam,1957). One of the best examples of l i p i d peroxidation i n vivo due to Vitamin E d e f i c i e n c y i s the formation of ceroid pigment i n various tissues of a f f l i c t e d animals (Tappel,1962} Lindlar,1970> Horowitz and Hartroft,1971J Raychaudhuri and Desai,1971} Reddy et al.,1973» Tappel,1973). This topic w i l l be discussed i n greater d e t a i l below. In addition to the above evidence, there have also been suggestions that l a b i l e substances such as sulphydryl (SH)-containing enzymes, ascorbic acid and Vitamin A are protected by FIGURE 2 OXIDATION PRODUCTS OF ^ -TOCOPHEROL \ / H 3 C I 6 H 3 3 MAIN PRODUCT FROM LIPID PEROXIDATION MAIN PRODUCT FROM RADIATION oC-TOCOPHEROL H * CH. CH, CH. C I 6 H 3 3 H 3 C CH, H 3 C C . i H 3 . +2H ]*> <h--2H H ' HO OH V CH, C16 H33 , CH, CH, *C-TOCOPHERYLQUINONE oC-TOCOPHERYLHYDROQUINONE CH, CH 3 H,C C I 6 H 3 3 - H -CH. C I 6 H 3 3 CH, 5-EX0METHYLENET0C0PHER - 6 - O N E (Tappel,1962) -.12-Vitamin E from peroxidative destruction i n the tissues (Tappel, 1969). However, the experimental data do not appear to be conclusive i n t h i s regard. F i n a l l y , c e r t a i n other substances such as methylene blue (Dam,1957)1 selenium (Scott et al.,1967; Scott,1969) and synthetic antioxidants such as DDPD (N,N*-diphenylphenylenediamine) and ethoxyquin (6-ethoxy-l,2-dihydro-2,2,4-trimethylquinoline) can f u l f i l l part of the function of Vitamin E i n some species (Table I) (Bieri,1961 i1964; Tappel,1965; Chen,1973? Levander et al. ,1973) . This i s thought to be the strongest argument f o r the antioxidant theory. However, i t i s not cl e a r whether synthetic antioxidants function i n place of Vitamin E, whether they merely spare Vitamin E or whether they have some other functions i n the animal. II. L i p i d Peroxidation and Ceroid Pigment Formation. The generalized antioxidant theory i s an exclusive theory i n the sense that i f Vitamin E functions s o l e l y as a phy s i o l o g i c a l antioxidant, then a l l the diverse secondary manifestations of Vitamin E de f i c i e n c y must be consequences of one primary cause - l i p i d peroxidation. The e a r l i e s t observations relevant to the problem i n vivo were concerned with ceroid pigment development i n Vitamin E d e f i c i e n t r a t s . TABLE I E f f e c t of antioxidants other than Vitamin E on some Vitamin E deficiency diseases Experimental animal Prevented by Disease Vitamin E Selenium Synthetic Antioxidants Foetal resorption female r a t poultry - * S t e r i l i t y male r a t hamster * - -L i v e r necrosis r a t P i g * * -Erythrocyte haemolysis r a t , chick, human * - * Encephalo-malacia chick # Exudative d i a t h e s i s chick turkey * * -Depigmentation r a t * * (Scott,1969) I l a . Uterine Pigmentation i n Vitamin E D e f i c i e n t Rats. Since i t s discovery Vitamin E has been known to be necessary for reproduction i n r a t s . The consumption of a Vitamin E - free d i e t causes t e s t i c u l a r degeneration and permanent s t e r i l i t y i n male animals (Evans,1925J Evans and Burr,1927) i and renders female r a t s incapable of carrying t h e i r pregnancies to term, although a l l other aspects of reproductive function such as ovulation, f e r t i l i z a t i o n and implantation, remains normal (Evans,1928). In 1936, Martin and Moore reported that " . . . . i n r a t s maintained f o r prolonged periods on d i e t s d e f i c i e n t i n Vitamin E a brown di s c o l o u r a t i o n of the uterus occurred...." This d i s c o l o u r a t i o n was subsequently found to be associated with the accumulation of coarse pigment granules i n the smooth muscle c e l l s of the uterine myometrium (Barrie,1938j Martin and Moore,1939). According to Mason and Emmel (1944), the u t e r i of weanling r a t s deprived of Vitamin E were normal i n a l l respects at f o r t y days of age. However, by the s i x t i e t h day, small pigment granules were seen i n the sarcoplasm of many smooth muscle c e l l s . These granules were i n i t i a l l y located predominantly at the poles of the n u c l e i and became more numerous throughout the c e l l s by the e i g h t i e t h day when the u t e r i were f a i n t l y but d e f i n i t e l y yellow i n colour. At t h i s time, the f i r s t resorptions i n pregnant females occurred. Continued d e f i c i e n c y further increased pigment accumulation i n the muscle c e l l s and also caused t h e i r appearance i n many macrophages of the muscle layers and the intermuscular connective t i s s u e . The u t e r i became dark chocolate -15-brovm i n colour and rather f i b r o t i c due to the increasing number, size and pigment content of macrophages and other connective tissue elements i n the muscularis. Marked uterine f i b r o s i s i n c h r o n i c a l l y Vitamin E d e f i c i e n t r a t s was also noted by Barrie (1938). During the course of pigraentogenesis, the smooth muscle c e l l s r e t a i n t h e i r c h a r a c t e r i s t i c h i s t o l o g i c a l features (long, spindle-shaped c e l l s with c e n t r a l l y - l o c a t e d prominent nuclei ) except that they appear abnormally distended due to the enormous accumulation of pigment granules. Often, the n u c l e i are not v i s i b l e f o r the same reason. In the advanced stages of de f i c i e n c y , degenerative nuclear changes such as pyknosis, hyperchromatism and karyolysis have been observed (Lindlar,1970). In r a t s r e s t r i c t e d to the Vitamin E d e f i c i e n t d i e t s f o r periods calculated to cause marked dis c o l o u r a t i o n of the uterus and then given l i b e r a l amounts of wheat germ o i l concentrate or alpha-tocopherol for period up to eleven months, the u t e r i at autopsy showed no evidence of recovery (Martin and Moore, 1939? Raychaudhuri and Desai,1971), although, i f tocopherol was resupplied e a r l y enough (before two months on d e f i c i e n t d i e t s ) , e s s e n t i a l l y normal c e l l functions were restored and the animals could bear l i v e pups (Martin and Moore,1939; Mason and Emmel, 19^5). On the basis of t h e i r experimental observations, Hartroft and Horowitz (1971) recently suggested that ceroidogenesis i s the c a r d i n a l pathological event i n the Vitamin E d e f i c i e n c y -induced f o e t a l resorption syndrome. They found that ceroid pigment formation was concommitant with f o e t a l r e sorption (as did Mason and Emmel, 19^1 19^5) and that ceroid was -16-found i n various s i t e s associated with placentation i n Vitamin E d e f i c i e n t r a t s . l i b . The Nature of Ceroid Pigment. The term 'ceroid' was f i r s t used by L i l l i e et a l (19M) to describe a coarsely globular, yellow, wax-like pigment found i n the c i r r h o t i c l i v e r s of rats maintained on a low protein, low f a t d i e t s . I t s occurrence was attributed to ei t h e r the breakdown products of the damaged l i v e r c e l l s or to some deri v a t i v e of cod l i v e r o i l present i n the experimental d i e t s (Endicott et al,1944). The pigment was systematically characterized by h i s t o l o g i c a l techniques and found to be i n e r t , insoluble i n l i p i d solvents, a c i d - f a s t , b a s o p h i l i c , f a t - p o s i t i v e , iron-negative and strongly fluorescent i n u l t r a - v i o l e t l i g h t (Endicott, 19441 Endicott and L i l l i e , 19W. At the same time, Mason and Emmel (194411945), i n t h e i r studies on the pigmentation of muscle tissue and sex glands i n Vitamin E d e f i c i e n t r a t s , found that the uterine pigment of these animals was e s s e n t i a l l y 'ceroid' i n character. In 1946, Pappenheimer and V i c t o r confirmed these observations and pointed out that, with one or two exceptions, the various experimental d i e t s used i n the production of hepatic c i r r h o s i s had been d e f i c i e n t i n Vitamin E. The only source of tocopherol i n the d i e t used by L i l l i e et a l (194l) was i n the three percent of cottonseed o i l contained i n the mix. This was calculated to mean a d a i l y intake of les s than 0.194 milligrams of tocopherol - much l e s s than the requirement. Further, Blumberg and Orady (1942) had found le s s ceroid when the basal r a t d i e t was supplemented with wheat germ o i l than - 1 7 -was shown by the rats of Blumberg and McCollum (19M) which had received no Vitamin E. Because the pigment, i n vivo, showed l i t t l e or no evidence of breakdown a f t e r i t s retention f o r prolonged periods by the tissue macrophages, Mason and Emmel (1944) suggested that i t was an abnormal metabolite which resulted from some biochemical l e s i o n i n the system. The exact nature of t h i s l e s i o n was not c l e a r at the time. Much e a r l i e r , Pinkerton (1938), i n his work on the pathogenesis of l i p i d pneumonia, a c c i d e n t a l l y discovered that a granular, a c i d - f a s t material accumulated i n the pulmonary parenchyma a f t e r the i n t r a t r a c h e a l i n j e c t i o n of cod l i v e r o i l into r a b b i t s . He bubbled a i r through cod l i v e r o i l and obtained the same, thi c k , gummy substance which was insoluble i n ether and chloroform. He suggested that these properties may be due to hydrolysis and oxidation of the o i l . This, and subsequent experiments by others i n vivo (Hass,1938aj 1939a1 Graef ,1939i Hartroft,19511 Hartroft and Porta,1965) and i n v i t r o (Hass,1938bj 1939bj E n d i c o t t , 1 9 ^ i Hartroft,1951>19531 Casselman,1951» Tappel, 1953» Porta,1963) have indicated the primary importance of unsaturated l i p i d s i n the genesis of ceroid pigment. Hass (1938a,b»1939a,b) investigated the subject most extensively. He injected a large number of crude and p u r i f i e d f a t s , o i l s , f a t t y acids and esters of f a t t y acids into guinea pigs and concluded that long chain f a t t y acids having several double bonds or esters of such acids were necessary f o r the production of the ac i d - f a s t material. Endicott (19^4) showed that parenteral administration of cod l i v e r o i l and linseed o i l -18-produced the c e r o i d - l i k e substance whereas hydrogenated cod l i v e r o i l , hydrogenated cottonseed o i l and beef lard f a i l e d to do so. l i e . The Relationship between Ceroid, Vitamin E and L i p i d s . The exact nature of the r e l a t i o n s h i p among ceroid, d i e t a r y f a t and Vitamin E was not known u n t i l the c l a s s i c a l experiments of Dam and Granados ( 1 9 ^ 5 ) demonstrated that ceroid pigment formation i n animals was caused by the peroxidation of polyunsaturated f a t t y acids i n tissues i n the absence of tocopherol. Using a modified chemical method f o r determining the peroxide values of t i s s u e s , Dam and Granados found that r a t s fed a high l e v e l of cod l i v e r o i l (20%) without tocopherol manifested a brown di s c o l o u r a t i o n of the adipose tissue which was preceded and accompanied by a very marked peroxidation of the body f a t . Exclusion of cod l i v e r o i l or the presence of Vitamin E i n the d i e t prevented t h i s . Ceroid deposition i n r a t u t e r i took much longer than development of d i s c o l o u r a t i o n i n adipose tiss u e (Dam and Granados,19^5? Dam,1962i Dam and Sondergaard,1970) and did not depend on the presence of cod l i v e r o i l . Vitamin E-stripped l a r d has been found to produce the same e f f e c t s (Dam,1962t Dam ans Sondergaard,197Of Raychaudhuri and Desai, 1 9 7 1 ) . Since 1 9 ^ 5 t a great deal of evidence f o r the l i p i d peroxidation theory of ceroid pigment formation i n Vitamin E d e f i c i e n c y has been accumulated. Most of these studies were based on the d i r e c t measurement of the peroxidation products by the t h i o b a r b i t u r i c acid r e a c t i o n (Placer ejt a l , 1 9 6 6 ) or by -19-the estimation of the c h a r a c t e r i s t i c fluorescence of the pigment (Moore and Wang,19^31 Endicott and Lillie,19^4» Moore and Wang,19^71 Oppenheimer and Andrews,19591 Hartroft and Porta, 1965; Chio and Tappel,1969; Porta and Hartroft,1969 ; D i l l a r d and Tappel,1971; Tappel,1972; Reddy et al,1973; Fletcher et al,1973). Most recently, the experiments of Reddy e_t a l (1973) have shown that ra t s fed high l e v e l s of polyunsaturated f a t s and low Vitamin E c h a r a c t e r i s t i c a l l y have fluorescent pigments deposited i n a number of tissues and that there was a larger amount of pigment i n the tissues of ra t s fed cod l i v e r o i l without Vitamin E than from r a t s supplemented with Vitamin E. The spectra of fluorescent pigments from these animals are s i m i l a r to those produced from d e f i n i t i v e i n v i t r o biochemical systems described by D i l l a r d and Tappel (1971) and Fletcher et a l (1973). Further, Bidlack and Tappel (1973) have shown that fluorescent pigments with almost i d e n t i c a l s p e c t r a l c h a r a c t e r i s t i c s are r e a d i l y formed when malonaldehyde, produced during peroxidation of the polyunsaturated f a t t y acids, reacts with aminophospholipids ( e s p e c i a l l y phosphatidyl ethanolamine) to form conjugated S c h i f f base products with the fluorescent chromophoric system (Figure 3). Since l i p i d peroxidation i s a random free r a d i c a l r e action, i t i s apparent that the deposited l i p o f u s c i n pigments represent chromophoric molecular damage s i t e s and t h e i r presence i n tissues can be taken as evidence of la r g e r numbers of concurrent and unconfined free r a d i c a l reactions (Tappel,1972). -20-FIGURE 3 A PATHWAY FOR THE FORMATION OF FLUORESCENT PIGMENT POLYUNSATURATED FATTY ACIDS V 0, (free r a d i c a l peroxidation) Vitamin E i n h i b i t i o n PEROXIDES V I (peroxide decomposition) MALONALDEHYDE PHOSPHATIDYL ETHANOLAMINE (R) R-NH-CH=CH-CH=N-R (FLUORESCENT PIGMENT) (Reddy et al,1973) -21-I I I . Lipofuscin/Ceroid and Theories of Senescence. L i p o f u s c i n age pigments have been recognized as d i s t i n c t i n t r a c e l l u l a r structures f o r well over a century (Hannover,1843jKoneff,1886). Research studies of l i p o f u s c i n have emphasized p h y l e t i c d i s t r i b u t i o n , occurence i n human t i s s u e s , bio p h y s i c a l and biochemical properties, histochemical a f f i n i t i e s and ultrastrueture (Toth,1968). Although various investigations have attempted to elucidate the or i g i n s and function(s) of l i p o f u s c i n , the r e s u l t s obtained have not been conclusive. However, i t i s well established that the accumulation of l i p o f u s c i n i n non-replaceable, f i x e d , postmitotic c e l l s i s an age-correlated process (Jayne,1950> Strehler,1959» Brody,1960). I t has also been pointed out that the pigment of Vitamin E def i c i e n c y , c e r o i d , and the age pigment d i s p l a y the same phy s i c a l and biochemical c h a r a c t e r i s t i c s at one moment or other of t h e i r evolution (Porta and Hartroft,1969)» and that the l o c a l or environmental factors which determine or influence t h e i r formation are v a r i a b l e . In the case of l i p o f u s c i n , the aging pigment apparently unrelated to die t a r y f a c t o r s , l i t t l e i s known of the l o c a l conditions that might induce i t s formation i n c e r t a i n c e l l s and even le s s about the changes of t h e i r surrounding medium. Indeed, i t i s questionable i f l i p o f u s c i n i s a r e s u l t of a genetic programme or the r e s u l t of ' b i o l o g i c a l noise'. Senescence i s a slow process that cannot be reproduced r e a d i l y i n the experimental animal unless one p a t i e n t l y awaits i t f therefore, i t i s at present d i f f i c u l t to study experimentally -22-the mechanism involved i n the formation of the age pigment. However, s a t i s f a c t o r y i s o l a t i o n of l i p o f u s c i n has been achieved (Heidenreich and Siebert,1955» Lang and Siebert,19551 Bjorkerud and Zelander,I960 j Mildvan and Strehler,i960) and i t s physico-chemical properties studied. Resulting data indicate, or suggest at l e a s t , that the b u i l d i n g stones of age pigments are s i m i l a r to those of the pigments that can be produced i n the experimental animal, n u t r i t i o n a l l y or otherwise, and even that produced i n v i t r o . A l l the separate information gathered to date from investigations on the pigments r e l a t e d to aging and to d i e t a r y factors favour t h e i r u n i f i c a t i o n under l i p o f u s c i n pigment. To divide these c l o s e l y r e l a t e d products into subentities seems u n r e a l i s t i c and only an accident of h i s t o r i c a l discoveries (Porta and Hartroft,1969» Pearse,1972). I l i a . Ionizing Radiation and Aging. One of the most widely held theories of senescence i s that i t i s a consequence of somatic mutation occurring i n non-d i v i d i n g c e l l s (eg. Szilard , 1 9 5 9 > Failla,1960» Curtis,1963). By somatic mutations are meant changes i n the chromosomal DNA of somatic c e l l s of a kind which, i f they occurred i n the germ l i n e , would be recognized as genetic mutations. Such a theory i s based on the assumption that even i f no other senescent >. changes took place, animals - some of whose e s s e n t i a l tissues consist of non-dividing c e l l s - would ultimately die from the e f f e c t s of somatic mutation. Thus the issue i s not whether somatic mutations occur, but whether they are common enough to contribute s i g n i f i c a n t l y to aging. -23-Further evidence f o r the theory l i e s i n the observation that i o n i z i n g r a d i a t i o n , i n doses too small to cause r a d i a t i o n sickness, are known to shorten l i f e . Lindop and Rotblat ( 1 9 6 1 ) found that single doses of X-rays given to young mice caused a reduction i n mean l i f e span roughly proportional to dose. Radiation appears to exert i t s i n i t i a l damaging e f f e c t s upon b i o l o g i c a l tissues primarily through the formation of free r a d i c a l s (Bacq and Alexander,1961 j Harman,1962j Tappel,19^5J Slater,1966) which are highly reactive and can be expected to accelerate the peroxidation of polyunsaturated l i p i d s and to damage membranes, proteins and nucleic acids. Haissinsky (1958), Tappel (1965), Roubal and Tappel (1966) and Barber and Bernheim (1967) also c i t e evidence that there are decided s i m i l a r i t i e s between l i p i d peroxidation damage and r a d i a t i o n damage and that the underlying mechanisms of these e f f e c t s may be s i m i l a r at the molecular l e v e l . In t h i s connection, the widely accepted and supported observation that many of the consequences of chronic i r r a d i a t i o n of b i o l o g i c a l organisms are indistinguishable from the aging e f f e c t s of time i s i n t e r e s t i n g . Since such r a d i a t i o n i s known to cause somatic mutations, i t has been suggested that they shorten l i f e span by causing somatic mutations and that i n doing so, they are mimicking the normal aging process which i s also due to somatic mutations (Maynard-Smith,19&3)• I l l b . The Lysosomal Theory of Aging. A number of drugs, p a r t i c u l a r l y c o r t i c o s t e r o i d s , s a l i c y l a t e s and antihistamines, have been found to prolong the -24-l i f e span of the f r u i t f l y Drosophila melanogaster as well as the i n v i t r o s u r v i v a l time of several c e l l types (Hochschild, 1970). These groups of compounds have l i t t l e chemical s i m i l a r i t y but i t i s generally thought that such drugs are s t a b i l i z e r s or protectors of c e l l and organelle membranes. There have also been observations that a number of antioxidants extend the l i f e span of mice, r a t s , guinea pigs, f r u i t f l i e s and macrophages (Bun-Hoi and Ratsimananga,19591 Oeriu and Vochitu,1965i Harman, 1968a,b1 Hochschild,1970). Because antioxidants, l i k e membrane s t a b i l i z e r s , protect c e l l u l a r membranes from free r a d i c a l s and l i p i d peroxidation - induced damage, both l i n e s of evidence have been taken to indicate membrane breakdown as a mechanism which may time other expressions of the aging process. Further, i t has been observed that lysosomes are extremely s e n s i t i v e to peroxides and free r a d i c a l s ( i o n i z i n g r a d i a t i o n ) and that lysosomal membrane damage can r e a d i l y release h y d r o l y t i c enzymes, leading to varying degrees of nonspecific l y s i s of c e l l u l a r components (Desai et al,1964j De Duve and Wattiaux ,1966). The lysosomal theory of aging states that lysosomes may pa r t i c i p a t e i n the aging process i n several ways. Leakage of lysosomal enzymes through damaged lysosomal membranes into the cytoplasm, the nucleus and into the e x t r a c e l l u l a r spaces has been c i t e d by Comfort(1966), Tappel (1965,1968), Packer et a l (1967) and others to produce exactly the kind of damage to DNA, to the c e l l u l a r machinery and to e x t r a c e l l u l a r proteins (eg. collagen and e l a s t i n ) which i s var i o u s l y postulated to underly aging. Such damage includes somatic mutations, DNA deletions, strand breakage and cross linkage, damage to the -25 -machinery of t r a n s c r i p t i o n and t r a n s l a t i o n , catabolism of c e l l proteins, damage to other organelles, stimulation of collagen formation and degradation of other e x t r a c e l l u l a r structures. These kinds of damage, i n turn, have been postulated to lead to other manifestations of the aging process i n organisms. During aging, lysosomes may also become thoroughly congested with i n d i g e s t i b l e material so that they are unable to carry out t h e i r l y t i c a c t i v i t i e s . Much of t h i s i n d i g e s t i b l e matter which clogs lysosomes seems to be membrane fragments of other organelles. Tappel (1968) notes that membranes damaged by l i p i d peroxidation are not well hydrolyzed by lysosomal enzymes, suggesting a second, though i n d i r e c t mechanism whereby l i p i d peroxidation can damage lysosomes - namely, by damaging the membranes of other organelles, thereby contributing to lysosomal engorgement. One of the theories of l i p o f u s c i n formation suggests bthat i t may be due to the progressive a l t e r a t i o n , merging and cross-linkage of l i p i d residues l e f t over i n lysosomes a f t e r repeated bouts of i n d i g e s t i b i l i t y (De Duve and Wattiaux,19661 Tappel,19681 Toth,1968). De Duve and Wattiaux (1966) propose that the i n a b i l i t y of most c e l l s of higher organisms to r i d themselves of engorged lysosomes may be responsible f o r the dis r u p t i o n of c e l l u l a r organization, interference with normal metabolic and l y t i c processes within c e l l s , and for l y t i c i n j u r i e s r e s u l t i n g from.the eventual rupture of congested lysosomes. Such deleterious events may well contribute to c e l l u l a r aging and death. I f so, agents which damage membranes might be expected to accelerate the aging process while membrane protective agents might have the opposite e f f e c t . Vitamin E d e f i c i e n c y r e s u l t s i n -26-the premature accumulation of lipofuscin-type pigment i n young animals where they do not normally occur, and, as pointed out by Tappel (1968), Vitamin E def i c i e n c y , i r r a d i a t i o n and aging a l l lead to much the same kind of damage to c e l l proteins, membranes and other structures. IV. Incisor Depigmentation i n Rats. An i n t e r e s t i n g aspect i n the consideration of tocopherol d e f i c i e n c y and l i p i d peroxidation i s the phenomenon of i n c i s o r depigmentation i n r a t s subject to chronic Vitamin E def i c i e n c y (Dam and Granados, 19^5; Granados and Dam, 19^ -5) Dam, 19621 Dam and Sondergaard,1970). The normal r a t i n c i s o r exhibits an orange-brown colour due to a f e r r i c ion-containing pigment on i t s f r o n t a l surface. Production of t h i s pigment i s i n h i b i t e d when unsaturated f a t such as cod l i v e r o i l or corn o i l i s used i n the basal Vitamin E d e f i c i e n t d i e t . The exact mechanism .:• which causes t h i s condition i s not known at present. V. Vitamin E Deficiency and Red C e l l Metabolism. Va. The E f f e c t on Heme Biosynthesis. One of the symptoms of Vitamin E d e f i c i e n c y which i s only p a r t i a l l y cured by another antioxidant (N,N'-diphenyl-p-phenylenediamine) i s the anemia which occurs i n the Rhesus monkey (Nutr.Revs.,1963). I t has been suggested that the anemia i s the r e s u l t of a defect i n the biosynthesis of heme and heme proteins (Whittaker et al,1967» Darby,1968). Murty et a l (1970) -27-showed that i n the Vitamin E d e f i c i e n t r a t , even i n the absence of a demonstrable anemia, there was a depression i n the biosynthesis of heme. Although i t was not stated c a t e g o r i c a l l y i n the paper, i t was assumed that haemoglobin l e v e l s i n Vitamin E d e f i c i e n t animals were lower than those of the controls. In the bone marrow, the defect resided at the l e v e l of the f i r s t enzyme i n heme synthesis, S-amino l e v u l i n i c acid synthetase, involved i n the formation of 5-amino l e v u l i n i c acid; while i n the l i v e r , i t seemed to be at the l e v e l of the second enzyme, 5-amino l e v u l i n i c acid dehydratase which synthesizes porphobilinogen. The requirement f o r Vitamin E i n erythropoeisis i n the Rhesus monkey i s s p e c i f i c : tocopherol treatment caused a complete remission of the anemia whereas other haematopoeitic agents such as i r o n , f o l a t e and Vitamin B 1 2 were without s i g n i f i c a n t b e n e f i c i a l r e s u l t s (Fitch,1968)1972). This anemia i s s i m i l a r to the one found i n premature infants who are Vitamin E d e f i c i e n t . Vb. S t a b i l i t y of the Red C e l l Membrane. Accompanying the ultimate decrease i n erythrocyte s u r v i v a l of Vitamin E deficiency-induced anemia i n animals i s an increased s u s c e p t i b i l i t y of the red blood c e l l s . t o oxidant haemolysis (Gyorgy and Rose,1948; Gordon et a l , 1965» Melhorn et al,197l). This s u s c e p t i b i l i t y i s e a s i l y demonstrated In v i t r o by adding hydrogen peroxide to erythrocytes from Vitamin E d e f i c i e n t animals or man. Dilute solutions of hydrogen peroxide cause extensive haemolysis i n such cases. Under ordinary conditions, Vitamin E d e f i c i e n t animals do -28-not develop haemolytic anemia despite the increased s u s c e p t i b i l i t y of t h e i r erythrocytes to oxidant stress ( F i t c h , 1972). Nevertheless, severe and even f a t a l haemolytic anemia may be provoked i f the Vitamin E d e f i c i e n t r a t or mouse i s challenged by an unusual oxidant stress such as hyperbaric oxygen (Kann,1967)« ^ w a s noted that tocopherol-supplemented animals showed no change a f t e r hyperbaric oxygen exposure and t h e i r c e l l s were also r e s i s t a n t to l y s i s by hydrogen peroxide. Incontrast, the red c e l l s of Vitamin E d e f i c i e n t mice showed an increased i n v i t r o l y t i c s e n s i t i v i t y to hydrogen peroxide and also a marked haemolytic s u s c e p t i b i l i t y to hyperoxia. The haematocrit l e v e l s of these animals f e l l by about 50 percent. Because of the antioxidant function of Vitamin E, i t was thought that lack of tocopherol probably resulted i n accelerated red c e l l l y s i s due to an increase i n the rate of red blood c e l l membrane l i p i d peroxidation (Mengel,1972). Immediately a f t e r hyperoxic exposure, and before haemolysis began, the red c e l l s of Vitamin E d e f i c i e n t mice contained large quantities of l i p i d peroxides while a l l other parameters of the red c e l l s were normal. No l i p i d peroxides were found i n the plasma. As haemolysis occurred, there was a s i g n i f i c a n t decrease i n haematocrit l e v e l s and a marked depression i n l i p i d peroxide content of the remaining red c e l l s and a subsequent increase of l i p i d peroxides i n the plasma (Mengel,1972). It was also demonstrated that l i p i d peroxides formed before there was any evidence of erythrocyte damage or l y s i s . Once l i p i d peroxidation had occurred, there appeared to be progressive membrane danage i n the red blood c e l l s , with s w e l l i n and osmotic -29-l y s i s . Further evidence supporting the theory that Vitamin E i s required f o r erythrocyte membrane s t a b i l i t y was reported by Kurokawa et a l . (1970), who found that i n v i t r o administration of Vitamin E to blood samples increased the osmotic resistance of the red c e l l s . In addition, Mengel (1972) noted that unsaturated f a t t y acids were l o s t from the l i p i d membranes of Vitamin E d e f i c i e n t erythrocytes whereas there was no change i n the saturated components. Vc. Vitamin E and 2,3-Diphosphoglycerate. 2,3-DPG i s a metabolic intermediate i n the Embden-Myerhoff Pathway i n the mammalian erythrocyte (Figure 4). I t binds p r e f e r e n t i a l l y to deoxyhaemoglobin and i n doing so e f f e c t s the a f f i n i t y of haemoglobin f o r oxygen so that an increased amount of oxygen w i l l be released at a constant p a r t i a l pressure of oxygen (Marchant,1973)* Mengel (1972) showed that tocopherol d e f i c i e n t animals had a s i g n i f i c a n t l y lower 2,3-DPG l e v e l than did the c o n t r o l animals. This e f f e c t became more pronounced when the animals were exposed to hyperoxia. Kurokawa et a l . (1972) subsequently demonstrated that the addition of Vitamin E to haemolysates increased 2,3-DPG l e v e l s within four hours of incubation. It was suggested that these e f f e c t s might be due to the antioxidant action of Vitamin E. -30-FIGURE 4 2,3-DPG FORMATION IN THE MAMMALIAN ERYTHROCYTE 1,3-DIPHOSPHOGLYCERATE 3-PHOSPHOGLYCERATE Embden-Myerhoff Pathway Rapoport-Leubering Shunt GLYCOGEN GLUCOSE } GLUCOSE-6-PHOSPHATE CHAPTER III MATERIALS AND METHODS I. Animal Care. Female weanling Wistar r a t s were purchased from Biobreeding Laboratories of Canada Limited, Ottawa, Ontario. Upon a r r i v a l they were divided into four groups with the use of a table of random numbers and then housed i n pairs i n screen-bottomed s t a i n l e s s s t e e l cages i n an air-conditioned room;; Lig h t i n g was regulated automatically to provide a twelve hour ligh t / d a r k cycle. Food and water were given ad l i b i t u m throughout the experimental period of eight months (October 1st 1972 to June 2*fth 1973). I I . Experimental Diets. Diet materials were obtained from General Biochemicals Incorporation, Ohio, USA. Only two experimental d i e t s were used, tocopherol-supplemented and tocopherol-free. These were based on a modified Draper's Standard Vitamin E-Free d i e t (Draper et al,196 * 0 . The composition of the d i e t s and that of the mineral and vitamin mixes used are shown i n Tables I I , I I I and IV. I t i s noted that although selenium was not included i n the mineral mixture (Table I I I ) , the vitamin-free casein used f o r the d i e t s contained aproximately 0.k$ parts per m i l l i o n selenium. (GBI data, Appendix B). According to Dr.M.L.Scott (personal •32-TABLE II Composition of Basal Vitamin E-Free Diet* Ingredients g/Kg d i e t Dextrose 650 Vitamin-free casein 200 Tocopherol-stripped corn o i l 100 S a l t mix 40 ** Vitamin mix (-Vitamin E) 6 Choline Chloride 5 * modified from Draper e_t a l , 1964 ** Controls supplemented with 2.5 g DL-o(-tocopherol acetate per Kg basal d i e t ** GBI-TD # 72342 -33-TABLE III Composition of Mineral (Salt) Mix used i n the Basal Diet Compound * Percent Composition * *rag/kg Diet Calcium carbonate (CaCO^) 16.3555 6542.200 Dicalcium phosphate (CaHPO^. 2H"20) 35-5555 14-222.200 Cupric sulphate (CuSO^.5H20) 0.0177 7.080 F e r r i c c i t r a t e ( F e f C ^ O ^ O H g O ) 1.6000 640.000 Magnesium carbonate (MgCO^) 4-.0888 1635.520 Manganese sulphate (MnSO^.HgO) 0.1377 55.080 Potassium c i t r a t e (K^C^H^Cv,).H"20) 23.6530 9461.200 Potassium iodide (KI) 0.0044 1.760 Potassium phosphate dibasic (KgHPO^) 7.7333 3093.320 Sodium chloride (NaCl) 10.8088 4-323.520 Zinc carbonate (ZnCO^) 0.0444 I7.76O * GBI data ••calculated f o r use at 4-Og g a i t mix per kg basal d i e t -34-TABLE IV Composition of Vitamin Mix used i n the Basal Diet Vitamin * g A g Vitamin mix **mg/kg Diet B i o t i n 0.0166 0.0996 Vitamin B ^ (0*1% with mannitol) 16.6667 100.0002 Calcium pantothenate 1.6667 10.0002 F o l i c acid 0.1666 0.9996 Menadione (Vitamin K) 0.1666 0.9996 N i c o t i n i c a c i d (Niacin) 4.1666 24.9996 Pyridoxine HC1 0.8333 4.9998 R i b o f l a v i n 0.8333 4.9998 Thiamine HC1 I.6667 10.0002 Dry Vitamin A palmitate (500,000 IU/g) 8.333^  50.0004 Dry Vitamin D 2 (500,000 IU/g) 0.6666 3.9996 Dextrose 964.8166 5788.8996 * GBI TD# 72342 **calculated to be used at 6g Vitamin mix per kg basal d i e t -35-communication,1973)» i f 20% casein was used as i n the present experiment, then the amount of selenium contributed by casein was close to 0.1 parts per m i l l i o n , which i s twice the l e v e l required to prevent l i v e r necrosis i n r a t s . I I I . Experimental Groups. The four experimental groups were designated as follows: Group I : Vitamin E supplemented nonirradiated (controls) +E Group II : Vitamin E d e f i c i e n t nonirradiated -E Group III : Vitamin E supplemented i r r a d i a t e d X+E Group IV : Vitamin E d e f i c i e n t i r r a d i a t e d X-E Animals from Group I acted as the controls f o r the experiment. Animals from Groups III and IV were given a dose of 200 rads of gamma r a d i a t i o n from a Cobalt-60 Bomb i n the Department of Chemistry at the University of B r i t i s h Columbia. IV. Experimental Procedures. The experiment continued over an eight month period during which time animals were randomly chosen from each group and k i l l e d at predetermined time i n t e r v a l s (Table V). The animals were weighed once every three days f o r the f i r s t month and once every two weeks f o r the r e s t of the experimental period i n order to record body weight changes during the entire experiment. Uterus weights were obtained only when the animals had been s a c r i f i c e d as per the preset time i n t e r v a l s . At each of these i n t e r v a l s , the following protocol was car r i e d out: -36-TABLE V Experimental Periods p . - Days a f t e r onset Age of rats rerioas o f e x p e r i m e n t at time of s a c r i f i c e 1 0 21 2 7 28 3 15 36 4 30 51 5 60 81 6 90 111 7 105 126 8 122 143 9 136 157 10 151 172 11 166 187 12 181 202 133 196 217 14 261 282 -37-The animal was f i r s t weighed and then anaesthetized with anhydrous d i e t h y l ether (Fisher S c i e n t i f i c ) . Blood was drawn by cardiac puncture and the blood placed i n heparinized t e s t tubes. Aliquots were removed f o r the determination of haemoglobin concentrations, haematocrit and 2,3-diphosphoglycerate l e v e l s . The remainder was centrifuged at 3000 rpm f o r ten minutes to separate the c e l l u l a r components of the blood from the serum. The serum samples were immediately frozen f o r subsequent analysis of Vitamin E content. Immediately a f t e r exsanguination, the uterus of the animal was removed and fixed i n 2% calcium acetate-buffered formaldehyde. I t was trimmed of f a t and weighed and l a t e r embedded i n p a r a f f i n for subsequent h i s t o l o g i c a l examination. Samples of abdominal adipose tissue (1-2 grams) were removed from the animals and each fixed i n 20 m i l l i l i t r e s of a chloroform/methanol ( 2 t l ) sol u t i o n . Two drops of alpha-tocopherol were added to each v i a l to prevent oxidation of the f a t t y acids. The v i a l s were capped and shaken vigorously to extract the l i p i d moeities. They were then stored i n the r e f r i g e r a t o r u n t i l they could be analyzed for f a t t y acid composition. V. Biochemical Determinations. Va. Serum Vitamin E Levels. Serum Vitamin E l e v e l s were determined according to the method described by B i e r i et al.(1964), which i s a modification of the c l a s s i c Emmerie-Engel technique. The analysis i s based on a reduction of f e r r i c ion (0.2^ F e C l 0 i n -38-95% ethanol) to the ferrous form by tocopherol, with the resultant formation of a red complex of ferrous ion with alpha, alpha'-dipyridyl {0.2% i n 95% ethanol). The formation of t h i s complex was measured i n a Beckman DU-2 spectrophotometer at 520 nm and gave an i n d i c a t i o n of the amount of tocopherol present i n the serum. Because the method entailed a mixed tocopherol/|S-carotene extraction, the readings at 520 nm were corrected f o r |9-carotene content. C a l i b r a t i o n curves for dl-alpha-tocopherol and beta-carotene were prepared (Figures 5 and 6) and the tocopherol content of serum was calculated according to the following formula i Tocopherol concentration (C) i n mg/lOOml = ( A 0 D520nm - <<°-2°5 * A 0 D 4 5 ( J n m ) ) ) x Z where 4°D520nm = A 0 D 5 2 0 n m (corrected) = A0D c o n ^ m - Blank - 0.011 ( 0 D due to reagent 5 2 0 n m impurities) O.205 = percentage of the reading at 520 n m due to ^-carotene i n a mixed tocopherol/ p-carotene extraction. DL-alpha-tocopherol and beta-carotene standards were obtained from Sigma Chemical Company, St.Louis, USA. Vb. Haemoglobin Concentrations and Haematocrit Determinations. Haemoglobin was determined by the spectrophotometric method described by Van Assenfeldt (1970). 0.02 ml of blood was d i l u t e d with 5 nil of Drabkin's Reagent (Fisher S c i e n t i f i c ) and then read at 5^ 0nm using the DU-2 spectrophotometer. Haemoglobin concentration was calculated by multiplying 0°^onm FIGURE 5 CALIBRATION CURVE FOR THE DETERMINATION OF SERUM VITAMIN E 0.300 0.050H CALIBRATION FACTOR = 10.7 0.200 0.400 0.600 0.800 1.000 1.200 1.400 TOCOPHEROL IN mg PER 100 ml ETHANOL 1.600 l .800 2.000 -41-with the fact o r determined on the haemoglobin c a l i b r a t i o n curve which had been previously prepared. Haematocrit was read from a microhaematocrit tube (Becton, Dickinson) a f t e r c e n t r i f u g a t i o n at 11,500 x G f o r f i v e minutes (Albert,1965). Vc. 2 ,3-diphosphoglycerate Analysis. 2,3-DPG was determined using the enzymatic method described i n the Sigma Chemical Company te c h n i c a l b u l l e t i n 35 UV (1971). The assay i s based on the decrease i n o p t i c a l density at 340nm when NADH i s oxidized to NAD i n the rea c t i o n converting 1,3-DPG to glyceraldehyde -3-phosphate (Figure 3). The Beckman DU-2 spectrophotometer was also used i n the determination of 2,3-DPG. 2,3-DPG concentrations were expressed three ways: i n micromoles per ml of whole blood, micromoles per ml of packed blood c e l l s and micromoles per gram of haemoglobin. Vd. Determination of Fatty Acid Composition. The method used was based on a technique described by Hammarstrand (1966). Modifications of the method to s u i t laboratory circumstances were suggested by Dr.J.AngeU t School of Home Economics , UBC. The l i p i d s from the adipose tissue were extracted by the chloroform/methanol (2»1) buffer which also p r e c i p i t a t e d a l l the protein moieties present. The extracts were f i l t e r e d through f a t - f r e e f i l t e r paper into t e s t tubes with v i n y l screw-top caps. 0.9$ NaCl was added to each tube and the tubes shaken -42-vigorously and l e f t to stand overnight. Two phases were separated. The upper (HgO/MeOH/NaCl) phase was discarded and the lower (CHCl^/lipid) phase was washed with a so l u t i o n of CHCl^/MeOH/NaCl (3i47«48) to remove a l l traces of the water phase. The chloroform phase was evaporated to dryness with nitrogen gas at 70'C. Ten ml of a KOH sol u t i o n (lOgm K0H$ 5ml HgOj MeoH up to 100 ml) was added to each sample and the tubes l e f t to saponify at 100°C f o r three to four hours. The saponified f r a c t i o n (containing the free f a t t y acids) was then s o l u b i l i z e d i n 10 ml HgO and the unsaponified f r a c t i o n extracted with petroleum ether three times. Three ml of concentrated HC1 were added to the water phase to release free f a t t y acids into s o l u t i o n and these were then extracted with ether. Methylation of free f a t t y acids was achieved by the addition of BCl^/MeOH (Applied Science Laboratories, C a l i f o r n i a , USA) to the samples and b o i l i n g f o r two minutes i n a water bath. The methylated esters of f a t t y acids were subsequently extracted with petroleum ether and stored i n the r e f r i g e r a t o r with foil-wrapped cork stoppers. The samples were then analyzed using a g a s - l i q u i d chromatograph (Hewlett-Packard Model 5750) with a 10% EGSS-X column. VI. H i s t o l o g i c a l and Histochemical Techniques. U t e r i from the experimental animals were fix e d i n buffered formaldehyde and embedded i n p a r a f f i n blocks which were then sectioned i n a Porter-Blum microtome. The sections were between s i x to eight microns i n thickness. In a l l , s i x d i f f e r e n t types of stains were used on each uterus. A l l the procedures which were carried out can be found outlined i n the Handbook of Histopathological Techniques, (Culling,1968). The stains used were 1. Har r i s ' Haematoxylin and Eosin 2. McManus' Sudan Black B 3. Schraorl's Reaction 4. Periodic A c i d - S c h i f f (PAS) Reaction 5. Gomori's Methenamine S i l v e r Reaction 6. Z i e h l Neelsen Method (prolonged). A l l chemicals and reagents used i n t h i s phase of the experiment were obtained from Fisher S c i e n t i f i c Company, Vancouver, B.C. Ha r r i s ' Haematoxylin and Eosin s t a i n was used as a routine preliminary s t a i n and served to orient the investigator to the main h i s t o l o g i c a l features of the sections. McManus' Sudan Black B was then used to demonstrate the presence of compound l i p i d s i n the uterine sections. Compound l i p i d s may or may not s t a i n black, depending on the complexity of t h e i r composition. Schmorl's Reaction i s s p e c i f i c f o r the demonstration of l i p o f u s c i n and was used f o r t h i s purpose i n t h i s experiment. A p o s i t i v e r e a c t i o n i s caused by the reduction of a f e r r i c c h l o r i d e / f e r r i c cyanide solu t i o n by l i p o f u s c i n . L i p o f u s c i n granules s t a i n blue. The next s t a i n used was the Periodic A c i d - S c h i f f Reaction which demonstrates the presence of adjacent hydroxyl groups and amino alcohols. PAS-positive substances become bright red when stained. Gomori's Methenamine S i l v e r Reaction was employed s p e c i f i c a l l y for detecting the presence of oxidized amines. - 44 -The reaction i s based on the reduction of ammoniacal s i l v e r solutions to m e t a l l i c s i l v e r which appears black i n the sections. L a s t l y , the prolonged Z i e h l Neelsen method was used to demonstrate the 'acid-fastness* of l i p o f u s c i n . I t was thought to s i g n i f y the presence of unsaturated f a t t y acids of high molecular weight. Substances p o s i t i v e f o r t h i s t e s t s t a i n red. However, t h i s method was unsuccessful i n y i e l d i n g conclusive r e s u l t s . The above stains were used f o r sections from every uterus obtained during the course of the experiment. The stains were employed f o r the purpose of showing the properties of c e r o i d / l i p o f u s c i n pigment during i t s development i n Vitamin E deficiency. VII. Experimental Design. Because the study comprises of several d d i s t i n c t sections, various experimental designs were used. The four experimental groups, once again, werei-Group I i Vitamin E supplemented nonirradiated (controls) +E Group II i Vitamin E d e f i c i e n t nonirradiated -E Group III i Vitamin E supplemented i r r a d i a t e d X+E Group IV i Vitamin E d e f i c i e n t i r r a d i a t e d X-E The eight experimental variables werei-1. Cumulative body weight gain 2. Uterus weight gain 3. Ceroid pigment development ( q u a l i t a t i v e assessment) 4-. Serum Vitamin E l e v e l s -^ 5-5. Haemoglobin concentrations 6. Haematocrits 7. Erythrocyte 2,3-diphosphoglycerate l e v e l s 8. Adipose tissue f a t t y acid composition. The experiments were set up as follows»-1. A one by four randomized group design with trend analysis f o r v a riables 1,2 and 3 (Figure 7.1). 2. A one by two randomized group design with trend analysis f o r variable 4 f o r four periods, from Day 0 to Day 30* of the experiment (Figure 7.2). 3. A one by four randomized group design with trend analysis f o r v a r i a ble 4 f o r ten periods, from Day 30 to Day 26l, of the experiment (Figure 7.3). 4. A one by two randomized group design f o r the comparison of dependant v a r i a b l e s 5i6 and 7 (Figure 7.4). 5. A one by four randomized group design f o r the analysis of variable 8 (Figure 7.5). VIII. S t a t i s t i c a l Analysis. Raw scores f o r each r a t f o r each of dependant variables 5,6,7 and 8 and mean scores f o r each r a t f o r variable 4 were key punched onto computer data cards f o r analysis by UBC's IBM 360/67 computer. The Fortran IV programme 'Multivariance -Univariate and Multivariate Analysis of Variance and Covariance' (Finn,1968) was used to analyze the data. The programme - 4 6 -FIGURE 7.1 EXPERIMENTAL DESIGN FOR TREND ANALYSIS OF DEPENDANT VARIABLES 1,2 AND 3 FOR ALL FOUR GROUPS DURING THE ENTIRE EXPERIMENTAL PERIOD Time Body Weight Uterus Weight Ceroid Development Day 0 i i i Day 261 *N = 5 *N = (2-5) *N = (2-5) * f o r each group during each time period FIGURE 7.2 EXPERIMENTAL DESIGN FOR TREND ANALYSIS OF SERUM VITAMIN E IN GROUPS I AND II FROM DAY 0 TO DAY 30 OF THE EXPERIMENT Time Group I Group II Day 0 7 15 30 **N = 2 **N = 4-** f o r each of the time periods -47-FIGURE 7.3 EXPERIMENTAL DESIGN FOR TREND ANALYSIS OF SERUM VITAMIN E IN ALL GROUPS FROM DAY 30 TO DAY 261 OF THE EXPERIMENT Time Group I Group II Group III Group IV Day 60 1 1 l Day 261 *N = (2-4) *N = (4-5) *N = 3 *N = (4-5) * f o r each of the time periods FIGURE 7,4 EXPERIMENTAL DESIGN FOR THE ANALYSIS OF VARIABLES 5, 6 and 7 IN GROUPS I AND II FOR ONE SAMPLING PERIOD Variables Group I Group II #5 (haemoglobin) #6 (haematocrit) #7 (2,3-DPG) N = 12 N = 22 -48-FIGURE 7.5 EXPERIMENTAL DESIGN FOR THE ANALYSIS OF ADIPOSE TISSUE FATTY ACID COMPOSITION IN ALL FOUR GROUPS FOR ONE SAMPLING PERIOD Group I Group II Group III Group IV N = 9 N = 16 N = 5 N = 5 -RO-TABLE VI Table of Hypotheses 1. Vitamin E d e f i c i e n t animals accumulate much larger amounts of uterine ceroid pigment than Vitamin E supplemented animals. 2. Irradiated Vitamin E d e f i c i e n t animals have r e l a t i v e l y more pigment than nonirradiated Vitamin E d e f i c i e n t animals. 3. Irradiated Vitamin E supplemented animals have r e l a t i v e l y more pigment than nonirradiated Vitamin E supplemented animals. 4. A l l Vitamin E d e f i c i e n t animals have more pigment at the end of eight months than at the beginning of the experiment. 5. Morphological and h i s t o l o g i c a l evidence of pigment development i s seen between two to three months a f t e r the onset of Vitamin E deficiency. 6. The histochemical c h a r a c t e r i s t i c s of ceroid pigment are e s s e n t i a l l y the same as those of l i p o f u s c i n pigment ?. Vitamin E d e f i c i e n t animals have lowe serum Vitamin E l e v e l s than Vitamin E supplemented animals. -50-TABLE VI (contd) 8. Irradiated and nonirradiated Vitamin E d e f i c i e n t animals have d i s s i m i l a r serum Vitamin E l e v e l s . 9. Irradiated and nonirradiated Vitamin E supplemented animals have d i s s i m i l a r serum Vitamin E l e v e l s . 10. Irradiated rats of d i f f e r i n g Vitamin E status and nonirradiated r a t s of d i f f e r i n g Vitamin E status have d i s s i m i l a r serum Vitamin E l e v e l s . 11. Vitamin E d e f i c i e n t animals have the same haematocrit values as Vitamin E supplemented animals. 12. Vitamin E d e f i c i e n t animals have lower haemoglobin and 2,3-diphosphoglycerate l e v e l s than Vitamin E supplemented animals. 13. The adipose tissues of Vitamin E d e f i c i e n t animals have a d i f f e r e n t f a t t y acid composition from the adipose tissues of Vitamin E supplemented animals. 14. The f a t t y acid composition of adipose tissues from i r r a d i a t e d and nonirradiated Vitamin E d e f i c i e n t animals are no the same. TABLE VI (contd) 15« The f a t t y acid composition of adipose tissues from i r r a d i a t e d and nonirradiated Vitamin E supplemented animals are not the same. 16. The adipose tissues from i r r a d i a t e d r a t s of d i f f e r i n g Vitamin E status have a d i f f e r e n t f a t t y acid composition from the adipose tissues of nonirradiated rat s of d i f f e r i n g Vitamin E status. -52-generated means and standard deviations where applicable and computed analysis of variance (ANOVA), preplanned orthogonal comparisons and trend analyses. V i l l a . Serum Vitamin E Analysis. The analysis of serum Vitamin E l e v e l s was divided into two phases. Phase I compared Vitamin E supplemented nonirradiated (Group I) and Vitamin E d e f i c i e n t nonirradiated (Group II) animals during the f i r s t t h i r t y days of the experiment. Analysis of variance produced the comparisons outlined i n Table VI. TABLE VII ANOVA table of serum Vitamin E l e v e l s f o r Day 0 to Day 30 of the Experiment Source df Groups 1 Time 3 l i n e a r quadratic cubic 1 1 1 Groups x Time 3 l i n e a r quadratic cubic 1 1 1 T o t a l 7 -53-The second phase of the serum Vitamin E analysis compared the four experimental groups over ten periods from Day 30 to the conclusion of the experiment (Day 261). Analysis of variance produced the comparisons outlined i n Table VII. The multivariance programme f o r t h i s phase of the analysis was computed twice to generate two d i s t i n c t sets of orthogonal comparisons as follows: Computation I Vitamin E supplemented nonirradiated vs Vitamin E supplemented i r r a d i a t e d Vitamin E d e f i c i e n t nonirradiated vs Vitamin E d e f i c i e n t i r r a d i a t e d Vitamin E supplemented nonirradiated plus Vitamin E supplemented i r r a d i a t e d vs Vitamin E d e f i c i e n t nonirradiated plus Vitamin E d e f i c i e n t i r r a d i a t e d Computation II Comparison #1 Comparison #2 -Comparison #3--Comparison #1 Comparison #2 Comparison #3 - Vitamin E supplemented nonirradiated vs Vitamin E d e f i c i e n t nonirradiated - Vitamin E supplemented i r r a d i a t e d vs Vitamin E d e f i c i e n t i r r a d i a t e d  Vitamin E supplemented nonirradiated plus Vitamin E d e f i c i e n t nonirradiated vs Vitamin E supplemented i r r a d i a t e d plus Vitamin E d e f i c i e n t i r r a d i a t e d . V l l l b . Haematological Parameters. Multivariance computed orthogonal comparisons between control and Vitamin E d e f i c i e n t groups (I and II) f o r each of the - 5 4 -TABLE VIII ANOVA table of serum Vitamin E l e v e l s f o r Day 30 to Day 261 of the Experiment Source df Groups 3 Orthogonal Comparisons #1 1 #2 1 #3 1 Time l i n e a r 1 quadratic 1 cubic 1 r e s i d u a l 6 Groups x Time 27 (Interaction) Orthogonal Comparison #1 l i n e a r 1 quadratic 1 r e s i d u a l Orthogonal Comparison #2 7 l i n e a r 1 quadratic 1 r e s i d u a l 7 Orthogonal Comparison #3 l i n e a r 1 quadratic 1 re s i d u a l 7 Total 39 -55-three dependant variables haemoglobin, haematocrit and 2,3-DPG. Data were obtained f o r one sampling period only. VIIIc. Adipose Tissue Fatty Acid Composition. Fatty acid composition data were obtained f o r one sampling period only. Multivariance compared orthogonally the four experimental groups f o r each of the eight f a t t y acids assayed. In addition, an o v e r a l l group e f f e c t was determined also. Results a were obtained by computing the data three times to obtain three d i s t i n c t sets of orthogonal comparisons: Computation I Comparison #1 - Vitamin E supplemented nonirradiated vs Vitamin E d e f i c i e n t nonirradiated Comparison #2 - Vitamin E supplemented i r r a d i a t e d vs Vitamin E d e f i c i e n t i r r a d i a t e d Comparison #3 — Vitamin E supplemented nonirradiated plus Vitamin E d e f i c i e n t nonirradiated vs Vitamin E supplemented i r r a d i a t e d plus Vitamin E d e f i c i e n t i r r a d i a t e d . Computation II Comparison #1 - Vitamin E supplemented nonirradiated vs Vitamin E supplemented i r r a d i a t e d Comparison #2 - Vitamin E d e f i c i e n t nonirradiated vs Vitamin E d e f i c i e n t i r r a d i a t e d Comparison #3 - Vitamin E supplemented nonirradiated plus Vitamin E supplemented i r r a d i a t e d vs Vitamin E d e f i c i e n t nonirradiated plus Vitamin E d e f i c i e n t i r r a d i a t e d . - 5 6 -Computation III Comparison #1 - Vitamin E supplemented nonirradiated (controls) vs a l l other groups (treated) Comparison #2 - Vitamin E d e f i c i e n t nonirradiated vs "both i r r a d i a t e d groups II and IV. CHAPTER IV RESULTS I. Raw Scores. Individual scores f o r a l l rats f o r a l l dependant va r i a b l e s , except ceroid pigment development, are presented i n the tables i n Appendix A. From these r e s u l t s , means and standard deviations of the variables were calculated and presented i n tables and figures herein. I I . Homogeneity of Variance. One of the assumptions underlying s t a t i s t i c a l comparisons of samples i s homogeneity of variance. Therefore, an F „ - t e s t was performed on the f i v e v a r i a b l e s , serum Vitamin max E, haemoglobin, haematocrit, 2,3-DPG and f a t t y acid composition. Body and uterus weight measurements were not tested s t a t i s t i c a l l y . The variance r a t i o s were not s i g n i f i c a n t at at p<0.05, thus homogeneity of variance was assumed f o r the f i v e v a r i a b l e s . I I I . Body Weight and Uterus Weight Changes. The body weight changes of rats i n a l l four groups f o r the entire period of 2 6 l days are shown i n Figure 8. Only the data -58-obtained during the preset time i n t e r v a l s are plotted i n Figure 8.1. No s t a t i s t i c a l comparisons of these data were made. There was no difference i n the growth rates of the four groups during the f i r s t month a f t e r the s t a r t of the experiment. However, by the end of the experimental period, Vitamin E d e f i c i e n t animals were found to be s l i g h t l y smaller than Vitamin E supplemented animals and i r r a d i a t e d animals tended to be smaller than nonirradiated animals. The changes i n the size of the uterus i n animals from a l l four groups f o r the entire experimental period are shown i n Figure 9. Vitamin E d e f i c i e n t animals had smaller u t e r i than Vitamin E supplemented animals and Vitamin E d e f i c i e n t i r r a d i a t e d animals had the smallest u t e r i of a l l four groups. IV. Ceroid Pigment Development and Accumulation. IVa. Morphological Observations. Morphological evidence f o r ceroid pigment development and accumulation i n the u t e r i of Vitamin E d e f i c i e n t animals of d i f f e r i n g r a d i a t i o n status i s presented i n Plates I to I I I . The normal uterus appeared f a i n t l y pink i n colour and was somewhat translucent. I t possessed considerable e x t e n s i b i l i t y and p l i a b i l i t y (Plates Ia, H a , I l i a ) . The u t e r i of Vitamin E supplemented nonirradiated animals (untreated controls) remained normal throughout the experimental period. Animals on a Vitamin E d e f i c i e n t d i e t had normal-appearing u t e r i up to approximately three months a f t e r the onset of deficiency. There was some i n d i v i d u a l v a r i a b i l i t y i n t h i s case. 320.0 300.0 280.0H 20.0 - 5 9 -F I G U R E 8.1 C U M U L A T I V E BODY WEIGHT G A I N OF A N I M A L S FROM DAY 0 TO DAY 2 6 1 GROUP I ( + £ ) x GROUP n (_E) • GROUP m ( X + E ) - i — i — GROUP m ( X - E ) FOR DETAILS OF DAYS '0' to '30 ' SEE FIGURE 8. 2 I I 1 1 1 1 1 1 I 1 1 1 1 O 20 4 0 60 80 100 120 140 160 180 200 220 240 260 280 NUMBER OF DAYS ON EXPERIMENTAL DIET I20.GH 110.0 -60-F IGURE 8.2 CUMULATIVE BODY WEIGHT GAIN OF ANIMALS FROM DAY 0 TO DAY 30 GROUP I (4E) GROUP H („E) GROUP m (X+E) GROUP rz (X-E) 20 — i — 3 0 NUMBER OF DAYS ON EXPERIMENTAL DIET - 6 i -FIGURE 9 CUMULATIVE UTERUS WEIGHT GAIN OF ANIMALS FROM DAY 0 TO DAY 261 0 . 9 0 0 -0 . 8 0 0 -0.700H ~ 0.600-1 < o =) 0 . 5 0 0 -cc UJ t-3 O X o LU 0.400-^ 0.300H 0 .200 -(+E) (-E) (X+E) * — ' — ' — G R O U P I E : (X-E) GROUP I GROUP H GROUP JK 0.100-I I I I I * i i i i i i i i 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 NUMBER OF DAYS ON EXPERIMENTAL DIETS -62-PLATE I Vitamin E supplemented nonirradiated controls. Vitamin E d e f i c i e n t Vitamin E d e f i c i e n t i r r a d i a t e d . nonirradiated. EARLIEST MORPHOLOGICAL EVIDENCE FOR CEROID FORMATION IN VITAMIN E DEFICIENCY (three months). -63-PLATE II a. Vitamin E supplemented nonirradiated controls. b. Vitamin E d e f i c i e n t nonirradiated. EFFECT OF VITAMIN E DEFICIENCY AND/OR IONIZING RADIATION ON THE UTERI OF RATS (six months). - 6 4 -PLATE II (contd) c. Vitamin E supplemented i r r a d i a t e d . Vitamin E d e f i c i e n t i r r a d i a t e d . EFFECT OF VITAMIN E DEFICIENCY AND/OR IONIZING RADIATION ON THE UTERI OF RATS (six months). - 6 5 -PLATE III Vitamin E supplemented nonirradiated control. Vitamin E d e f i c i e n t i r r a d i a t e d . ANIMALS WITH UTERI AND ABDOMINAL DEPOT FAT TISSUE IN SITU. -66-At the time of i n i t i a l pigment deposition, the u t e r i were l i g h t brown i n colour (Plate l b , Ic) and were les s e l a s t i c than those of control animals. There was no d i s c e r n i b l e difference i n the degree of pigmentation between the u t e r i of i r r a d i a t e d and nonirradiated Vitamin E d e f i c i e n t animalsj nor was there any d i s s i m i l a r i t y i n the rate of onset of ceroid development and accumulation between these animals that was not accountable f o r by i n d i v i d u a l v a r i a b i l i t y . In general, i r r a d i a t e d animals had smaller u t e r i than nonirradiated animals (Figure 9 ) , although i t was more marked i n some cases than i n others (Plates l b , I l l b ) . The u t e r i of Vitamin E supplemented i r r a d i a t e d animals were not d i f f e r e n t from those of Vitamin E supplemented nonirradiated animals throughout the enti r e experimental period (Plate I l a , l i e ) . S ix months a f t e r the beginning of the study, the u t e r i of the Vitamin E d e f i c i e n t animals (both i r r a d i a t e d and nonirradiated) had turned a dark chocolate brown colour (Plate l i b , l i d ) , and, i n many cases had become f i b r o t i c i n nature, as evidenced by the appearance of the uterus i n Plate l i b . At t h i s time, there was also no difference i n the amount of ceroid pigment accumulated i n the u t e r i of i r r a d i a t e d and nonirradiated Vitamin E d e f i c i e n t animals. Pigmented u t e r i were inextensible and generally smaller than nonpigmented u t e r i . Ceroid was also evident i n the ovaries of Vitamin E d e f i c i e n t animals at the end of the s i x months (Plate l i b , l i d ) . Furthermore, Vitamin E d e f i c i e n t animals, whether they be i r r a d i a t e d or not, without exception accumulated considerably larger amounts of abdominal depot f a t than did Vitamin E -67-supplemented animals (Plate I I I ) . IVb. Histochemical Observations. The functions of the stains used i n the c h a r a c t e r i z a t i o n of the Vitamin E d e f i c i e n c y pigment have been summarized i n Chapter I I I . A l l stains were su c c e s s f u l l y applied except f o r the Ziehl-Neelsen Method which c o n s i s t e n t l y produced ambiguous r e s u l t s . The reactions of the u t e r i from Vitamin E supplemented and Vitamin E d e f i c i e n t animals (both i r r a d i a t e d and nonirradiated) throughout the e n t i r e experimental period are shown i n Table IX. No attempt was made to quantify the data since the pigment was not deposited i n a uniform fashion i n the t i s s u e . However, i t was observed that the pigment was increasing i n quantity throughout the duration of the study. Ceroid was found to be yellowish-brown i n colour i n the unstained state (Plates IV, Xa) and was deposited i n droplets of varying s i z e s i n the sections of tissue (Plates IX, X). The pigment stained p o s i t i v e l y with Schmorl's Reaction, the Periodic A c i d - S c h i f f (PAS) Reaction and Gomori's Methenamine S i l v e r Method (Plates VI, VIII, IX, X) and stained negatively with Sudan Black B (Plates V, VII, X). In a l l cases, i r r a d i a t e d and nonirradiated animals yielded s i m i l a r r e s u l t s (Plate XI). The most s t r i k i n g r e s u l t s were obtained with Schmorl's Reaction and the PAS Reaction. Thus the s l i d e s were selected to i l l u s t r a t e the development and accumulation of ceroid pigment i n the u t e r i of Vitamin E d e f i c i e n t animals (Plates VIII and IX). There was no evidence of ceroid i n any of the animals u n t i l approximately three months a f t e r the onset of d i e t a r y Vitamin E TABLE IX HISTOCHEMICAL RESULTS Days Haematoxylin and Eosin Schmorl's Reaction PAS Reaction Sudan Black B Methenamine S i l v e r Z i e h l -Neelsen +E -E +E -E +E -E +E -E +E -E +E -E 0 - - - - ? 90 - + + - ? 181 - + + + + + + 261 + + + + + + -+ + + ? +E i Vitamin E supplemented animals ( i r r a d i a t e d and nonirradiated) -E « Vitamin E d e f i c i e n t animals ( i r r a d i a t e d and nonirradiated). -69-PLATE IV Vitamin E supplemented nonir radia ted c o n t r o l s . (l60x) Vitamin E d e f i c i e n t n o n i r r a d i a t e d . (kOOx) THE APPEARANCE OF CEROID PIGMENT ACCUMULATED IN THE UTERINE TISSUE AT THE END OF EIGHT MONTHS OF DIETARY VITAMIN E DEFICIENCY (Haematoxylin and E o s i n ) . -70-PIGMENTOGENESIS AND ACCUMULATION IN MUSCLE CELLS OF UTERI FROM VITAMIN E DEFICIENT RATS (Sudan Black B ) . ( l 6 0 x ) -71-PLATE VI Vitamin E supplemented nonirradiated controls. Vitamin E d e f i c i e n t nonirradiated. RESPONSES OP CONTROL AND VITAMIN E DEFICIENT UTERINE TISSUES TO GOMORI'S METHENAMINE SILVER REACTION. (six months, l60x) - 7 2 -PLATE VII Eight months. ACCUMULATION OF PIGMENT IN UTERINE CONNECTIVE TISSUES OF VITAMIN E DEFICIENT RATS (Sudan Black B). (400x) - 7 3 -CEROID PIGMENT DEVELOPMENT AND ACCUMULATION IN VITAMIN E DEFICIENT RATS (Schmorl's Reaction). (l60x) -74 -PLATE VIII (contd) Four months. d. Six months. CEROID PIGMENT DEVELOPMENT AND ACCUMULATION IN VITAMIN E DEFICIENT RATS (Schmorl's Reaction). (l60x) - 7 5 -PLATE VIII (contd) CEROID PIGMENT DEVELOPMENT AND ACCUMULATION IN VITAMIN E DEFICIENT RATS (Schmorl's Reaction). (I60x) -76-PLATE IX Three months. CEROID PIGMENT DEVELOPMENT AND ACCUMULATION IN VITAMIN E DEFICIENT RATS (Periodic Acid-Schiff Reaction). ( l 6 0 x ) - 7 7 -PLATE IX (contd) Five months. CEROID PIGMENT DEVELOPMENT AND ACCUMULATION IN VITAMIN E DEFICIENT RATS (Periodic Acid-Schiff Reaction). ( l 6 0 x ) -78-PLATE IX (contd) CEROID PIGMENT DEVELOPMENT AND ACCUMULATION IN VITAMIN E DEFICIENT RATS (Periodic Acid-Schiff Reaction). (l60x) -79-PLATE X COMPARISION OF CEROID PIGMENT PROPERTIES AFTER SIX MONTHS OF DIETARY VITAMIN E DEFICIENCY. (400x) - 8 0 -PLATE X (contd) COMPARISON OF CEROID PIGMENT PROPERTIES AFTER SIX MONTHS OF DIETARY VITAMIN E DEFICIENCY. (400x) - 8 1 -PLATE XI c. Vitamin E d e f i c i e n t i r r a d i a t e d . Vitamin E d e f i c i e n t nonirradiated. EFFECT OF IONIZING RADIATION ON CEROIDOGENESIS (Schmorl's Reaction). (400x) deficiency. At t h i s time the f i r s t Schmorl-positive and PAS-p o s i t i v e areas were noted. Some of these were more di s c r e t e e n t i t i e s (Plate V l l l b ) , while others appeared more d i f f u s e throughout the cytoplasm of the muscle c e l l s (Plate IXb). Between three to s i x months of Vitamin E deficiency, there was a progressive accumulation of pigment granules i n the muscle c e l l s (Plate IXc, IXd) as well as i n the connective tissue macrophages (Plate VIIIc, VHId) of the myometrium. In the most advanced stage of Vitamin E d e f i c i e n c y looked at i n t h i s study (261 days), ceroid pigment granules had invaded well into the endometrial layer (Plates V l l l e , IXe). V. Incisor Depigmentation. The e f f e c t of chronic Vitamin E d e f i c i e n c y on i n c i s o r pigmentation i n r a t s i s shown i n Plate XII. These animals l o s t the b r i g h t orange pigment c h a r a c t e r i s t i c of t h e i r top front teeth when subjected to such d i e t a r y Vitamin E s t r e s s . Rats of the same age on a Vitamin E adequate d i e t do not show such a depigmentation of t h e i r i n c i s o r s . VI. Serum Vitamin E. Via. Descriptive S t a t i s t i c s . The c a l i b r a t i o n curve f o r the determination of serum tocopherol by the method described i n Chapter III i s shown i n Figure 5* The means and standard deviations of the values obtained from animals i n Groups I and II f o r the f i r s t four time periods -83-PLATE XII Vitamin E supplemented Vitamin E d e f i c i e n t nonirradiated control. nonirradiated. INCISOR DEPIGMENTATION IN VITAMIN E DEFICIENT RATS. -84-TABLE X •Serum Vitamin E l e v e l s of Group I and Group II r a t s f o r the f i r s t four sampling periods (Day 0 to Day 30) Day Group I Group II 0 1.66 + 0.24 1.55 + 0.20 7 3.36 +0.17 0.54 + 0.07 15 ^.94 + 0.29 0.32 • + 0.05 30 5.90 + 0.06 0.32 + 0.07 * values i n mg/lOOml - 8 5 -FIGURE 10 CHANGES IN SERUM VITAMIN E LEVELS OF GROUP I AND II ANIMALS FROM DAY 0 TO DAY 60 GROUP {+ E) GROUP EC ( - E ) - r -10 -~7— 20 -Tf— 30 4 0 5 0 — i — 6 0 NUMBER OF OAYS ON EXPERIMENTAL DIETS TABLE XI •Serum Vitamin E l e v e l s of r a t s f o r ten sampling periods (Day 60 to Day 26l) Day Group I Group II Group III Group IV 60 8.99 + 1.38 0.31: + 0.01 ••/ 6.37 + 0.24 O.38 + 0.07 90 11.17 + 0.32 0.29 + 0. 08 7.86 + O.36 0.27: + 0.03 105 11.39 + 0.08 0.39 + 0.05 6.64 + O.76 0.32 + 0.10 122 10.40 + 0.04 0.39 + 0.37 4.31 + 0.42 0.34 + 0.02 136 9.29 + o . i5 0.57 + 0.19 9.57 + 0.61 0.30 + 0.07 151 8.55 + 1.35 0.49 + 0.15 8.88 + 0.57 0.21 + 0.07 166 10.27 + 0.63 0.65 + 0.12 9.63 + 0.15 0.26 + 0.06 181 . 9.98 + 0.88 0.33 + 0.22 6.69 + 0.33 0.22 + 0.20 196 9.98 + 0.15 0.30 + 0.07 10.14 + 0.23 0.27 + 0.12 261 9.01 + 1.50 0.29 + 0.14 9.96 + 0.19. 0.25 + 0.05 * values i n mg/lOOml -87-FIGURE 11 CHANGES IN SERUM VITAMIN E LEVELS OF ANIMALS FROM DAY 60 TO DAY 261 - 9.0CH aooH o GROUP r (+E) GROUP n (-E) •• GROUP HE (X+E) — GROUP EX (X-E) o CO o 0) in o CM CM to If) CO CO CM 60 80 100 120 140 160 180 200 220 240 260 NUMBER OF DAYS ON EXPERIMENTAL DIETS -88-(Day 0 to Day 30) are shown i n Table X. This information i s plotted i n Figure 10 to show the rate of depletion of serum Vitamin E l e v e l s i n animals given a d e f i c i e n t d i e t and the concurrent increase i n these l e v e l s i n those animals provided with supplements of Vitamin E. The means and standard deviations of the values obtained from animals i n a l l four groups f o r the l a s t ten time periods (Day 30 to Day 26l) are shown i n Table XI. This same information i s plotted i n the graph i n Figure 11, VIb. S t a t i s t i c a l Analysis of Data - Test of Hypotheses, ( i ) Day 0 to Day 30i The r e s u l t s of the a p r i o r i orthogonal comparison of the average serum Vitamin E l e v e l s between Group I (control) and Group II (Vitamin E d e f i c i e n t nonirradiated) r a t s during the f i r s t four sampling periods (Day 0 to Day 30) are shown i n Table XII. Average serum Vitamin E values of Vitamin E d e f i c i e n t nonirradiated (Group II) r a t s were s i g n i f i c a n t l y lower than the average values of the c o n t r o l (Group I) r a t s during t h i s time. The second orthogonal comparison determined whether the average serum Vitamin E l e v e l s (Groups I and II combined) were s i g n i f i c a n t l y d i f f e r e n t over time. S t a t i s t i c a l analysis indicated that there was a s i g n i f i c a n t difference i n serum Vitamin E l e v e l s over the f i r s t t h i r t y days of the experiment. In order to discover the nature of t h i s change, the pooled values for Group I and Group II were subjected to a trend analysis (Table XIII). The r e s u l t s show that the s i g n i f i c a n t change which occurred over the -89-TABLE XII Orthogonal comparison of average Group I and average Group II serum Vitamin E l e v e l s f o r Days 0 to 30 Comparison df MS P P Group I vs Group II 1,16 57.404 1882.422 <0.0001 TABLE XIII Orthogonal comparison of pooled (Group I and Group II) serum Vitamin E l e v e l s over time (Day 0 to Day 30) Comparison df MS Group I + Group II over time Trend l i n e a r quadratic cubic 3,16 1,16 1,16 1,16 0.5826 1.392 0.268 0.089 19.106 45.630 8.774 2.915 <0.0001 <0. 0001 <0.01 >o.05 -90-f i r s t 30 days was primarily a l i n e a r change with a s i g n i f i c a n t quadratic component (Figure 10). Figure 10 also shows that the time trend of Group I serum Vitamin E l e v e l changes was not i n the same d i r e c t i o n as that of Group II values since Group I l e v e l s increased while Group II l e v e l s decreased during the f i r s t 30 days of the experiment. In order that a more meaningful i n t e r p r e t a t i o n be made of t h i s data, a t h i r d orthogonal comparison was performed. The t h i r d orthogonal comparison determined whether there was a s i g n i f i c a n t i n t e r a c t i o n e f f e c t during the f i r s t four sampling periods. S t a t i s t i c a l analysis indicates that Group I animals had s i g n i f i c a n t l y higher l e v e l s of serum Vitamin E than Group II animals over the f i r s t 30 days (Table XIV). Further, i n order to determine the nature of t h i s i n t e r a c t i o n e f f e c t over time, a trend analysis was performed which showed that the s i g n i f i c a n t difference between the two groups had large l i n e a r and quadratic components (Table XIVj Figure 10). ( i i ) . Day 30 to Day 26l< An a p r i o r i orthogonal comparison was performed i n order to determine i f there was a s i g n i f i c a n t group e f f e c t over the l a s t ten time periods of the experiment. That i s , i f serum Vitamin E l e v e l s were s i g n i f i c a n t l y d i f f e r e n t among the four groups over the entire period between Day 30 and Day 26l. The r e s u l t s , as shown i n Table XV, indicate that there was a. highly - 9 1 -TABLE XIV Orthogonal comparison of i n t e r a c t i o n e f f e c t between Group I and Group II serum Vitamin E l e v e l s from Day 0 to Day 30 Comparison df MS F Group I vs Group II over time Trend l i n e a r quadratic cubic 3,16 1,16 1,16 1,16 7.687 22.o6l 1 . 0 0 1 0 . 0 0 0 4 252.089 723.425 3 2 . 8 2 8 0.013 <0.0001 <0.0001 <0.0001 >o.05 s i g n i f i c a n t general group e f f e c t . TABLE XV Orthogonal comparison of average serum Vitamin E l e v e l s of a l l groups f o r Days 30 to 26l Comparison df MS Groups I + II + III + IV 3,120 9 5 0 . 7 2 0 3 9 9 9 . 7 8 1 <0.0001 -92-A second orthogonal comparison was made i n order to determine i f there was a s i g n i f i c a n t time e f f e c t . That i s , i f serum Vitamin E l e v e l s of a l l four groups, averaged together, was s i g n i f i c a n t l y d i f f e r e n t over time. S t a t i s t i c a l analysis showed that there was a s i g n i f i c a n t time e f f e c t (Table XVI). Next, the time e f f e c t was broken down to determine the trend. I t was found that the differences which occurred over time had a high l i n e a r component. However, t h i s component alone did not account f o r a l l the variance since there was also a highly s i g n i f i c a n t r e s i d u a l e f f e c t . TABLE XVI Orthogonal comparison of pooled (Groups I, I I , III and IV) serum Vitamin E l e v e l s from Day 30 to 26l Comparison df MS P Groups I + II + III + IV over time 9,120 2.045 8.602 < 0.0001 Trend l i n e a r quadratic cubic r e s i d u a l 1,120 1,120 1,120 6,120 2.247 0.205 0.003 2.658 9.455 0.862 0.001 11.184 < 0.01 > 0.05 >0.05 <0.0001 A t h i r d orthogonal comparison was made to determine i f - 9 3 -there was a s i g n i f i c a n t difference betv/een the serum Vitamin E values of Groups I, I I , III and IV over time. Results indicate that there was a highly s i g n i f i c a n t i n t e r a c t i o n a f f e c t (Table XVII). TABLE XVII Orthogonal comparison of group e f f e c t of serum Vitamin E l e v e l s over time Comparison df MS F P Group e f f e c t 4.101 17.254 over time 27,120 <0.0001 ( i n t e r a c t i o n ) The above data provided only general information about group, time and i n t e r a c t i o n e f f e c t s . Further comparisons were made i n order to break down these general e f f e c t s into more meaningful components so that the e f f e c t s of the two experimental treatments (Vitamin E defi c i e n c y and i r r a d i a t i o n ) may be delineated. The r e s u l t s of orthogonal comparisons f o r the determination of the e f f e c t of dietary Vitamin E status on the serum Vitamin E l e v e l s of animals i n the four groups are shown i n Tables XVIII, XIX and XX. Vitamin E d e f i c i e n t nonirradiated r a t s (Group II) had s i g n i f i c a n t l y lower serum Vitamin E l e v e l s than Vitamin E -94-supplemented nonirradiated (Group I) rats over the entire time period. This difference over time had a strong l i n e a r component although t h i s did not account e n t i r e l y f o r a l l the variance as indicated by the s i g n i f i c a n t r e s i d u a l e f f e c t (Table XVIII? Figure 1 1 ) . TABLE XVIII Orthogonal comparison of the e f f e c t of die t a r y Vitamin E status on serum Vitamin E l e v e l s of nonirrad iated r a t s from Day 30 to 261 Comparison df MS F P Group I vs Group II (Group e f f e c t ) 1,120 1093-571 4600.770 <0.0001 Interaction e f f e c t l i n e a r quadratic r e s i d u a l 1,120 1,120 7,120 18.272 0.052 4.622 76.831 0.217 19.446 <0.0001 70.05 <0.0001 Vitamin E d e f i c i e n t i r r a d i a t e d r a t s (Group IV) had s i g n i f i c a n t l y lower serum Vitamin E l e v e l s than Vitamin E supplemented i r r a d i a t e d (Group III) rats i n the period between Day 30 and Day 2 6 l . This difference over time was p r i m a r i l y due to a s i g n i f i c a n t linearchange as well as a s i g n i f i c a n t r e s i d u a l e f f e c t (Table XIX? Figure 1 1 ) . - 9 5 -TABLE XIX Orthogonal comparison of the e f f e c t of di e t a r y Vitamin E status on serum Vitamin E l e v e l s of i r r a d i a t e d r a t s from Day 30 to Day 261 Comparison df MS F P Group III vs Group IV (Group e f f e c t ) 1,120 2.620 11.024 <0.01 Interaction e f f e c t l i n e a r quadratic r e s i d u a l 1,120 1,120 7,120 17.276 0.519 3.763 72.682 2.183 15.832 <0. 0001 >0 .05 <0.0001 When the serum Vitamin E data f o r a l l Vitamin E supplemented animals, regardless of r a d i a t i o n status, was pooled (Group I plus Group III) and compared orthogonally with the pooled data f o r a l l Vitamin E d e f i c i e n t animals of d i f f e r i n g r a d i a t i o n status, i t was found that there was a s i g n i f i c a n t d i e t e f f e c t condensed over time, i r r e s p e c t i v e of the r a d i a t i o n status. That i s , Group II and Group IV considered together as Vitamin E d e f i c i e n t animals had s i g n i f i c a n t l y lower serum Vitamin E l e v e l s than Group I and I I I , the Vitamin E supplemented animals. The i n t e r a c t i o n e f f e c t was also determined and found to have a highly s i g n i f i c a n t l i n e a r trend although a s i g n i f i c a n t amount of variance remains unaccounted f o r (residual component s i g n i f i c a n t ) (Table XX). -96-TABLE XX Orthogonal comparison of the e f f e c t of d i e t a r y Vitamin E status on serum Vitamin E l e v e l s of nonirradiated and i r r a d i a t e d r a t s from Day 30 to Day 261 Comparison df MS F P Groups I & III vs Groups II & IV (Group e f f e c t ) 1,120 5 2 . 5 0 6 220.898 <0.0001 Interaction e f f e c t l i n e a r quadratic r e s i d u a l 1,120 1,120 7,120 31.483 0 .559 7 .307 132.452 2 .350 30.740 <0.0001 >o,05 <0.0001 The r e s u l t s of orthogonal comparisons f o r determining the e f f e c t of i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s of animals i n the four groups are shown i n Tables XXI, XXII and XXIII. S t a t i s t i c a l analysis indicates that the serum Vitamin E l e v e l s of Vitamin E supplemented nonirradiated animals (Group I) were s i g n i f i c a n t l y higher than those of Vitamin E supplemented i r r a d i a t e d animals (Group I I I ) . However, the r e s u l t s also showed that there was no s i g n i f i c a n t i n t e r a c t i o n e f f e c t so that the changes i n serum Vitamin E l e v e l s which occurred over time were s i m i l a r f o r both groups (Table XXI). -97-TABLE XXI Orthogonal comparison of the e f f e c t of i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s of Vitamin E supplemented r a t s from Day 30 to Day 261 Comparison df MS F p Group I vs Group I I I 1,120 3-178 13-371 <0.001 Interaction e f f e c t l i n e a r quadratic r e s i d u a l 1,120 1,120 7,120 0.0003 0.406 0.022 0.001 1.709 0.091 >0.05 >o.05 >o.o5 Another set of orthogonal comparisons indicated that the serum Vitamin E l e v e l s of Vitamin E d e f i c i e n t nonirradiated r a t s (Group II) were s i g n i f i c a n t l y higher than those of Vitamin E d e f i c i e n t i r r a d i a t e d r a t s (Group IV) condensed over time. There was also a s i g n i f i c a n t i n t e r a c t i o n e f f e c t between the two groups during t h i s period which was characterized by a strong l i n e a r component. Again, there was a s i g n i f i c a n t r e s i d u a l e f f e c t which means that a s i g n i f i c a n t amount of variance could not be accounted f o r by the l i n e a r i t y (Table XXII). L a s t l y , when the serum Vitamin E data f o r a l l nonirradiated animals (Group I and Group II) were pooled and compared orthogonally with the pooled data f o r a l l i r r a d i a t e d r a t s (Group III and Group IV), i t was found that there was a highly -98-TABLE XXII Orthogonal comparison of the e f f e c t of i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s of Vitamin E d e f i c i e n t r a t s from Day 30 to Day 26l Comparison df MS F P Group II vs Group IV 1,120 2706.476 11765.074 <0.0001 Interaction e f f e c t l i n e a r quadratic r e s i d u a l 1,120 1,120 7,120 5.577 0.016 3.056 23.464 0.067 12.858 <0.0001 >o.o5 <0.0001 s i g n i f i c a n t e f f e c t due to i r r a d i a t i o n i r r e s p e c t i v e of d i e t a r y Vitamin E status when condensed over time. That i s , nonirradiated animals had s i g n i f i c a n t l y higher serum Vitamin E l e v e l s than i r r a d i a t e d animals (Table XXIII). The i n t e r a c t i o n e f f e c t had s i g n i f i c a n t l i n e a r as well as r e s i d u a l components. VII. Haematological Parameters. V i l l a . Descriptive S t a t i s t i c s . The means and standard deviations f o r haemaglobin concentrations, haematocrit and erythrocyte 2,3-DPG l e v e l s i n Vitamin E supplemented nonirradiated (Group I) animals and Vitamin E d e f i c i e n t nonirradiated (Group II) animals f o r one sampling -99-TABLE XXIII Orthogonal comparison of the e f f e c t of i o n i z i n g r a d i a t i o n on serum Vitamin E l e v e l s of supplemented and d e f i c i e n t rats from Day 30 to Day 261 Comparison df MS F P Group I & II vs Group III & IV 1,120 1755.989 7387.637 <0.0001 Interaction e f f e c t l i n e a r quadratic r e s i d u a l 1,120 1,120 7,120 2.629 0.002 1.898 11.062 0.008 7.983 <0.0001 >o.05 <0.0001 period are shown i n Table XXIV. This same information i s graphed i n Figure 12. V l l l b . S t a t i s t i c a l Analysis of Data - Test of Hypotheses. Three sets of orthogonal comparisons were performed i n order to determine the e f f e c t of Vitamin E d e f i c i e n c y on the haemoglobin concentration, haematocrit and erythrocyte 2,3-DPG l e v e l s of animals i n Groups I and I I . Results of the s t a t i s t i c a l analyses are presented i n Table XXV. No s i g n i f i c a n t differences were observed between the two groups i n a l l three parameters. -100-TABLE XXIV Means and standard deviations f o r haematological parameters Variables Group I Group II (controls) ( d e f i c i e n t s ) haemoglobin concentrations 4 4 . 1 5 + 1 . 5 7 4 4 . 0 6 + 2 . 4 8 (gm/lOOml ) "* ' . ATvtlZiT. ^ . 2 7 ± 0.55 1*.69 ± l . f t 7 2|umoles/gm Kb) 17-62+3.18 1 6 . 9 1 + 3 . 3 1 - 1 0 1 -FIGURE 12 THE EFFECT OF DIETARY VITAMIN E DEFICIENCY ON VARIOUS HAEMATOLOGICAL PARAMETERS 45.0n 44.5 44.0 43.5 43.0 42.5 H 42.0 HEMATOCRIT % VOLUME I5.0n I4.5H I4.0H 13.5' I3.0H 12.5 I2.0H 1 HEMOGLOBIN gm / 100 ml. 2 , 3 - DPG iaon ^ MOLES PER gm Hb 17.5H 17.0 H I6.5H I6.0H 15.5 I50H Gr.I (+E) Gr.II (-E) Gr.I (+E) Gr.II (-E) Gr. I (+E) Gr.II (-E) -102-TABLE XXV Orthogonal comparisons of haematological parameters i n Group I and Group II animals Variables df MS F P Haemaglobin concentration 1.32 1.338 0.878 >0.05 Haematocrit 1,32 0.053 O.Oll >0.05 2,3-DPG concentration 1,32 3.928 O.369 >o.05 IX. Fatty acid composition of Adipose Tissues. IXa. Descriptive S t a t i s t i c s . The means and standard deviations of the percent f a t t y acid composition of adipose tissues from rats of a l l four groups for a single sampling period at the end of the experiment are shown i n Table XXVI, and presented g r a p h i c a l l y i n Figure 13. Only eight f a t t y acids were considered: C12:0 ( l a u r i c a c i d ) j Cl4:0 (myristic acid)} Cl6:0 (palmitic acid)» Cl6:l « 6 (palmitoleic acid)> C18:0 ( s t e a r i c a c i d ) j C l 8 i l « 9 ( o l e i c acid)> Cl8:2*>6 ( l i n o l e i c a c i d ) i and Cl8 : 3 « 3 ( l i n o l e n i c a cid). IXb. S t a t i s t i c a l Analysis of Data - Test of Hypotheses. The r e s u l t s of a multivariate analysis of a group e f f e c t TABLE XXVI Percent f a t t y acid composition of adipose tissues Fatty acid Group I Group II Group III Group IV C 1 2 i 0 2 . 1 4 + 0 . 1 8 1.83 + 0 . 9 5 1.2 2 + 0 .33 0 .70 + 0 . 0 3 C l 4 « 0 4 .14 + 1 .18 3 . 7 0 ' + 1 .68 2 .14 + 0 .37 0.83 + O . 8 9 C 1 6 J 0 12 .53 + 2 .25 14 .31 + 3 . 6 6 18.680 + 0 . 9 0 17 .89 1 .18 C l 6 ! i o » 6 9 .72 + 1 .78 8 . 8 3 ± 3 . 6 3 6 .55 + 0 . 3 1 5. 80 + 0 . 3 7 CI81O 2 . 5 9 + 0 .51 3 . 3 7 + 0.79 1.80 + O.36 3.45 + 0 . 5 8 018:1^9 19.00 + 2.97 2 3 . 9 6 + 5 . 7 3 29 .96 + I . 8 7 2 9 . 4 8 + 4 . 8 0 C18 I2K)6 4 2 . 3 5 ± 5 . 3 8 3 7 . 5 3 + ^ ' 3 1 33.^5 + 1 .18 35. 40 + 0.92 C18«3' j 3 4 . 8 5 + 1. 80 4 . 8 1 + 1 . 3 5 3 . 6 9 + 0. 40 3 . 6 5 + 0.72 FIGURE 13.1 PERCENT FATTY ACID COMPOSITION OF ADIPOSE TISSUES 5.0T C 1 2 i 0 Cl4i0 CI81O 0180^3 \ \\S\ x VN v \ > \SS\ T n m rz FIGURE 13.2 PERCENT FATTY ACID COMPOSITION OF ADIPOSE TISSUES C I 6 1 O 40.CH Cl6«l«6 C18«1«9 C 1 8 I 2 « 6 -106-on the f a t t y acid composition of adipose tissues are shown i n Table XXVII. S t a t i s t i c a l analysis indicates that there was a s i g n i f i c a n t difference between a l l groups for a l l f a t t y acids except Gl8 : 3 " 3 ( l i n o l e n i c a c i d ) . TABLE XXVII Multivariate analysis of group e f f e c t on adipose tissue f a t t y acid composition Fatty acid df MS F P C12«0 3,31 2 .701 5.911 <0.01 Cl4i0 3,31 5.278 2.868 =0.05 Cl6»0 3,31 57.288 7.100 <0.001 C l 6 i l » 6 3,31 23.103 3.205 <o.05 CI81O 3,31 3.977 9.201 <0.001 Cl8:lf>9 3,31 182.964 8.477 <0.001 Cl8i2w6 3,31 103.420 6.181 <0.01 Cl8i3« 3 3,31 3.187 1.722 >o.05 Further comparisons were then made i n order to delineate the e f f e c t s of the two experimental treatments (Vitamin E d e f i c i e n c y and i r r a d i a t i o n ) on the percent f a t t y acid composition of adipose tissues from the four groups. The r e s u l t s of orthogonal comparisons f o r the determination of the e f f e c t of d i e t a r y Vitamin -107-E status on the f a t t y acid composition of adipose tissues from animals i n a l l four groups are shown i n Tables XXVIII, XXIX and XXX. The f i r s t set of orthogonal comparisons tested the e f f e c t of d i e t a r y Vitamin E status i n nonirradiated rats (Group I and Group I I ) . Results indicate that the adipose tissues of r a t s from Group II (Vitamin E d e f i c i e n t ) had s i g n i f i c a n t l y higher l e v e l s of CI81O and s i g n i f i c a n t l y lower l e v e l s of C l 8 i 2 w 6 than the adipose tissues of the untreated controls i n Group I (Table XXVIII). A l l other f a t t y acids were present i n s i m i l a r percentages i n the two groups. TABLE XXVIII Orthogonal comparison for the e f f e c t of diet a r y Vitamin E status on the adipose tissue f a t t y acid composition of nonirradiated r a t s (Groups I and II) Fatty acid df MS F p C12l0 1.31 0 . 1 0 3 0 .225 >o.05 Cl4t0 1.31 0.251 0.136 >0 .05 C1610 1,31 0.671 >0.05 Cl6»l*>6 1.31 0.786 0 .109 >o.o5 CI81O 1.31 4.227 9.779 <0.01 Cl8ila)9 1.31 74.642 3.458 =0.07 C18«2««)6 1,31 88.752 5 . 3 0 4 <o.o5 c1813^3 1.31 0.144 0.080 >o.o5 - 1 0 8 -The second set of orthogonal comparisons determined the e f f e c t of di e t a r y Vitamin E status on the adipose tissue f a t t y acid composition of i r r a d i a t e d rats(Groups III and IV). There was a s i g n i f i c a n t difference between the two groups i n a l l f a t t y acids except Cl8«0. Vitamin E d e f i c i e n t i r r a d i a t e d r a t s had adipose tissues which were s i g n i f i c a n t l y higher i n Cl4i0 and 018:2^6 and s i g n i f i c a n t l y lower i n C12»0, Cl6i0, Cl6iiw6, Cl8il«9 and Cl8i3*>3 than the adipose tissues of Vitamin E supplemented i r r a d i a t e d r a t s (Table XXIX). TABLE XXIX Orthogonal comparison f o r the e f f e c t of di e t a r y Vitamin E status on the adipose tissue f a t t y acid composition of ir r a d i a t e d r a t s (Groups III and IV) Fatty acid df MS F p C12i0 1,31 7.331 16.045 <0.001 Cl4i0 1.31 14.358 7.801 <0.01 CI61O 1,31 164.905 20.436 <0.0001 C I 6 1 K 0 6 1,31 67.099 9.309 <0.01 CI81O 1,31 0.883 2.043 > o . o 5 C l 8 t l « 9 1,31 473.661 21.945 <0.0001 Cl8i2«6 1,31 212.051 12.673 <0.001 018:3^3 1,31 9.413 5.233 <0.05 -109-When the data for a l l Vitamin E supplemented animals (Groups I and III) were pooled and compared orthogonally with the pooled data f o r a l l Vitamin E d e f i c i e n t animals (Groups II and IV), i t was found that the adipose tissues of the Vitamin E d e f i c i e n t rats had s i g n i f i c a n t l y higher l e v e l s of C l8»0 than those of Vitamin E supplemented animals (Table XXX). Thus there was a s i g n i f i c a n t d i e t e f f e c t which was independant of the r a d i a t i o n status of the animals. TABLE XXX Orthogonal comparison f o r the e f f e c t of diet a r y Vitamin E status on the adipose tissue f a t t y acid composition of supplemented (Groups I and III) and d e f i c i e n t (Groups II and IV) r a t s Fatty acid df MS F p C12 I0 1.31 1.180 2.582 >o.05 Cl4i0 1.31 0.109 0.059 >o.o5 C l 6 i 0 1.31 1.726 0.214 >o.05 Cl6ilw»6 1,31 4.719 0.655 >o.05 C18J0 1.31 10.361 1.612 4 0. 001 C 1 8 I 1 « » 9 1,31 34.783 1.612 >o.05 Cl8t2«o6 1.31 14.345 0.857 >o.05 C18«3*>3 1,31 0.012 0.006 >o.05 -110-The r e s u l t s of orthogonal comparisons to te s t the e f f e c t of io n i z i n g r a d i a t i o n on the adipose tissue f a t t y acid composition of animals i n a l l four groups are shown i n Tables XXXI, XXXII and XXXIII. The f i r s t set of comparisons indicated that there was a s i g n i f i c a n t difference i n 6 out of the 8 f a t t y acids between nonirradiated and i r r a d i a t e d Vitamin E supplemented r a t s (Groups I and I I I ) . Group III animals had s i g n i f i c a n t l y lower l e v e l s of C12«0, Cl4j0, C l 6 t l w 6 , Cl8i2tf6 and Cl8«3^3» and s i g n i f i c a n t l y higher l e v e l s of C l 6 « 0 and Cl8tlio9 than Group I animals. TABLE XXXI Orthogonal comparison t e s t i n g the e f f e c t of i o n i z i n g r a d i a t i o n on the adipose tissue f a t t y acid composition of Vitamin E supplemented r a t s (Groups I and III) Fatty acid df MS F p C12t0 1.31 3.125 6.839 <0.05 Cl4i0 1,31 12.034 6.538 <o.05 Cl6»0 1,31 121.542 15.062 <0.001 Cl6tl<«>6 1.31 34.237 4.750 <o.05 CI81O 1,31 0.448 1.037 >0.05 Cl8il*>9 1.31 427.821 19.821 <0.001 C18:2"6 1,31 286.779 17.139 <0.001 Cl8i3<o3 1.31 3.865 2.149 ?o.05 -111-The second set of orthogonal comparisons tested the e f f e c t s of i o n i z i n g r a d i a t i o n on the adipose tissue f a t t y acid composition of Vitamin E d e f i c i e n t r a t s (Groups II and IV). The nanlysis showed that there was a s i g n i f i c a n t difference between the two groups i n only three of the f a t t y acids. Group IV r a t s had s i g n i f i c a n t l y lower l e v e l s of C12:0 and Cl6»1^6, and s i g n i f i c a n t l y higher l e v e l s of Cl6*0 than Group II animals (Table XXXII). TABLE XXXII Orthogonal comparisons t e s t i n g the e f f e c t of i o n i z i n g r a d i a t i o n . . on the adipose tissue f a t t y acid composition of Vitamin E d e f i c i e n t r a t s (Groups II and IV) Fatty acid df MS F p C12t0 1.31 3.797 8.311 <0.01 Cl4»0 1.31 3.690 2 .005 >6.05 Cl6t0 1.31 48.597 6 .023 <o.05 Cl6tl*>6 1.31 3 0 . 3 5 2 4.211 <o.05 CI81O 1,31 1.122 2 .595 >o.05 Cl8tl«9 1,31 86.291 3.998 =0.054 Cl8«2«»6 1,31 9.138 0.546 >o.05 Cl8t3a3 1,31 5.685 3.161 >o.o5 - 1 1 2 -When the data f o r a l l nonirradiated animals (Groups I and II) were pooled and compared orthogonally with the pooled data f o r a l l i r r a d i a t e d animals (Groups III and IV), i t was found that there was a s i g n i f i c a n t difference i n 7 out of the 8 f a t t y acids between the groups. Irradiated r a t s had s i g n i f i c a n t l y lower l e v e l s of C 1 2 » 0 , C l 4 i 0 , Cl6»l»>6, Cl8s2*>6 and Cl8t3*)3. and s i g n i f i c a n t l y higher l e v e l s of C l 6 s 0 and C l 8:l * ) 9 than nonirradiated r a t s (Table XXXIII). TABLE XXXIII Orthogonal comparisons t e s t i n g the e f f e c t of i o n i z i n g r a d i a t i o n on the adipose tissue f a t t y acid composition of nonirradiated (Groups I and II) and i r r a d i a t e d (Groups III and IV) r a t s Fatty acid df MS F P C12»0 1 .31 7.331 1 6 . 0 4 5 <0.001 Cl4:0 1.31 14 .358 7.801 <0.01 Cl6:0 1,31 164 .905 20.436 - 0 . 0 0 0 1 C i 6 i l * 6 1.31 6 7 . 0 9 9 9 . 3 0 9 <0.01 C18«0 1.31 0.883 2 . 0 4 3 vo .05 C18»1U)9 1 .31 473 .661 21.945 5 0 . 0 0 0 1 Cl8s2«>6 1 ,31 212 .051 12 .673 < 0 . 0 1 0180 *0 1 .31 9.413 5 . 2 3 3 < 0 . 0 5 -113-One last set of comparisons were made. The results of this set are shown in Table XXXIV. An orthogonal comparison to test the effect of treatment (Vitamin E deficiency and / or ioniz ing radiation) on the adipose tissue composition of such rats indicated that animals in Groups II, III and IV together had s igni f icant ly lower mean levels of C12t0 and 018:2^6, and s igni f icant ly higher mean levels of C161O and Cl8»lfc)9 than the untreated controls in Group I (Table XXXIV). TABLE XXXIV Orthogonal comparisons testing the effect of treatment (Vitamin E deficiency and/or i r radiat ion) on the adipose tissue fatty acid composition of rats (Group I vs Groups II, III & IV) Fatty acid df MS F p C12«0 1,31. 2.751 6.020 <o.05 Cl4«0 1,31 5.596 3.040 >o.05 Cl6i0 1,31 73.233 9.076 <0.01 Cl6iU>6 1,31 24.454 3.393 >o.05 CI81O 1.31 1.658 3.837 =0.059 C18«1*)9 1.31 343.794 15.928 <0.001 Cl8»2*»6 1,31 241.367 14.425 <0.001 Cl8t3^3 0 1.31 1.538 0.855 >o.o5 CHAPTER V Discussion I. Limitations and Delimitations. The present study, and any conclusions which may be drawn from the r e s u l t s , was subject to several l i m i t a t i o n s and deli m i t a t i o n s . F i r s t , the experiment was carr i e d out over a period of just eight months during which pigmentogenesis was mainfested only i n the uterus. Deposition of ceroid i n other tissues such as the myocardium and those of the c e n t r a l nervous system would not be expected to occur u n t i l approximately one and a h a l f to two years a f t e r the onset of the de f i c i e n c y (Olcott, 19371 Martin and Moore, 1939 J Mason and Emmel, 194-5 i Pappenheimer and V i c t o r , 1 9 4 6 ) . In addition, the e f f e c t s of i o n i z i n g r a d i a t i o n are more profound i n old animals (Russ and Scott,19391 Henshaw, 1944| B e r l i n , 1 9 6 0 f Upton,i960* Lindop and R o t b l a t , 1 9 6 l ) . That i s , i r r a d i a t e d animals surviving the i n i t i a l period a f t e r treatment (with or without acute i l l n e s s ) may l i v e f o r a long time, but eventually tend to die prematurely from natural or pathological causes ( B l a i r et a l . , 1 9 5 6 j C a s a r e t t , i 9 6 0 ) . Second, any conclusions about the e f f e c t s of i o n i z i n g r a d i a t i o n on the symptoms of Vitamin E def i c i e n c y i n these animals can only be based on the actual dose of gamma rays administered to the r a t s . The single sublethal dose of 200 rads was calculated to be too low to cause overt r a d i a t i o n sickness or malignant neoplasias, but high enough to have a l i f e - s h o r t e n i n g e f f e c t on -115-the rats (Furth et al . , 1 9 5 4 > B e r l i n , i 9 6 0 ) . According to Casarett ( I 9 6 0 ) , sublethal i r r a d i a t i o n , when not highly l o c a l i z e d , causes premature death p r i m a r i l y by producing a symdrome with the c h a r a c t e r i s t i c s of premature aging, with concommitant or re l a t e d diseases to which the animals are susceptible, rather than by d i r e c t l y inducing s p e c i a l or s p e c i f i c diseases which are associated with premature death. Third, t h i s study was l i m i t e d by the number of r a t s i n the sample population. Because of the nature of the experiment (four groups, trend a n a l y s i s ) , the t o t a l number of r a t s used was large (greater than 1 0 0 ) , but the actual numbers i n each group per time period were r e l a t i v e l y small (x = 4 ) . In the experiments where a trend analysis was not done (blood parameters and f a t t y acid composition), the number of animals per group was between 5 and 22. I I . The E f f e c t of Vitamin E Deficiency and/or Ionizing Radiation on Body and Uterus Weight Changes. The r e s u l t s of the present study showed that weanling rats fed a Vitamin E-free d i e t with 5% corn o i l did not d i f f e r from the controls (supplemented with 2.5 grams dl-alpha-tocopherol acetate per kg d i e t ) i n body weight gain during the f i r s t month of the experiment, i r r e s p e c t i v e of t h e i r r a d i a t i o n status (Figure 8 . 2 ) . Since the l e v e l of Vitamin E supplied to the control animals was more than adequate f o r the maintenance of high serum Vitamin E l e v e l s (Tables X, XIj Figures 10, 11) , i t may be concluded that -116-Vitamin E d e f i c i e n c y did not a f f e c t the early growth rate of r a t s . These findings concur with those of Reddy et a l (1973) who observed that weanling rat s fed a 15'7$ corn o i l , Vitamin E-free d i e t did not have body weights which were s i g n i f i c a n t l y d i f f e r e n t from those of Vitamin E supplemented rats (10.5 mg to 45.0 mg per kg d i e t ) during the f i r s t 19 weeks of feeding. Figure 8.2 also indicates that the growth rates of i r r a d i a t e d r a t s (Groups III and IV) did not d i f f e r from those of nonirradiated rat s (Groups I and II) f o r the f i r s t 30 days of the experiment. This was expected because the i r r a d i a t e d animals did not become i l l as a r e s u l t of the gamma r a d i a t i o n and also because, according to Lindop and Rotblat ( 1 9 6 l ) , an acute dose of 500 rads caused an i n i t i a l weight l o s s of only 5 grams. This loss was maintained throughout the l i f e span of the treated animals. Between Day 30 and Day 2 6 l , the body weights of a l l four experimental groups followed a common general trend although there was a considerable amount of crossing-over of the data points from one time period to the next, notably up to Day 172 (Figure 8 . 1 ) . There were several reasons f o r these f l u c t u a t i o n s , the most important of which was the f a c t that the sample population f o r each group during any time period was unique to that period. This was because animals were being s a c r i f i c e d at the preset time i n t e r v a l s (Table V) so that the sample population as a whole was constantly changing. In a l l l i k e l i h o o d , t h i s also accentuated the i n d i v i d u a l d i f ferences between the animals. As such, t h i s part of the data cannot be considered as a true 'growth curve', only as a record of body weight changes of animals i n the four groups from Day 30 to Day 2 6 l of the experiment. -117-One of the prime sources of v a r i a t i o n i n the body weights between animals (among and between groups) was the degree of f a t deposition i n the abdomen of these animals, which i s believed to be mainly a function of the appetite of the i n d i v i d u a l r a t f o r the highly palatable sucrose-based d i e t s . Contrary to Raychaudhuri and Desai (1971) who implied that f a t accumulation was s o l e l y the r e s u l t of Vitamin E deficiency, the observations i n t h i s study indicate that Vitamin E supplemented animals also accumulated unusually large amounts of adipose t i s s u e , although never to the ame extent as the d e f i c i e n t animals (Plate I I I ) . This tendency to deposit f a t tissue probably masked the e f f e c t s of Vitamin E de f i c i e n c y and/or i r r a d i a t i o n on the body weights of the animals to some extent. As shown i n Figure 8.1 the e f f e c t s of the two experimental treatments became well defined a f t e r Day 172. By Day 2 6 l , Vitamin E d e f i c i e n c y i t s e l f was found to have caused a decrease i n the body weight gains of Group II animals and that i r r a d i a t i o n i t s e l f caused a s i m i l a r decrease i n Group III animals, while Vitamin E d e f i c i e n c y plus i r r a d i a t i o n appeared to have had an additive adverse e f f e c t on Group IV animals. This i s i n agreement with the observations of Reddy e_t a l (1973) as well as those of Casarett (I960) and Lindop and Rotblat (I96l). The e f f e c t s of the two experimental treatments were much more c l e a r l y defined when changes i n uterus weights were measured (Figure 9 ) , Data points were obtained from the same animals whose body weights were recorded i n Figure 8. Vitamin E d e f i c i e n c y and/or i o n i z i n g r a d i a t i o n did cause a decrease i n the size of the uterus i n appropriately treated animals and, i n the case of Vitamin E -US-deficiency, was a r e f l e c t i o n of uterine dysfunction. According to Casarett ( i 9 6 0 ) , the extent of r a d i a t i o n damage to the tissues of i r r a d i a t e d animals depends q u a n t i t a t i v e l y on the magnitude of the administered dose and, to some degree, on the r a d i o s e n s i t i v i t y of the various t i s s u e s . A whole-body dose of 200 rads was thought to be equivalent to approximately one-third of the l e t h a l dose l i m i t f o r rats (Dr.Walker, UBC Dept.of Chemistryi personal communication) although the exact c r i t i c a l l e t h a l l e v e l s of r a d i a t i o n f o r any animal i s not known at present. In addition, there appears to be no s p e c i a l mechanism or primary change i n radiation-accelerated aging which would d i s t i n g u i s h i t from •normal' aging changes (Russ and Scott, 1939? Blair,1959? Casarett, 1956,1960; B e r l i n , I 9 6 0 } Upton,1060} Lindop and R o t b l a t , 1 9 6 l ? Maynard Smith,1963). Reports from the above sources have indicated that a l l of the histopathological aging changes were b a s i c a l l y i d e n t i c a l i n both control and i r r a d i a t e d animals but that these changes began or were i n i t i a l l y detectable e a r l i e r , progressed at a greater rate, and were therefore more pronounced at given ages i n the i r r a d i a t e d animals. In general, there are four phases of h i s t o p a t h o l o g i c a l e f f e c t s i n the r a d i o s e n s i t i v e tissues of i r r a d i a t e d animals ( C a s a r e t t , i 9 6 0 ) . There i s f i r s t a phase of d i r e c t or early r a d i a t i o n e f f e c t s , characterized by temporary i n h i b i t i o n of c e l l d i v i s i o n and production and by degeneration and necrosis of s e n s i t i v e parenchymal c e l l s , with consequent hypoplastic and atrophic changes i n related tissues and organs. As shown i n Figures 8 and 9, r a d i a t i o n did not a f f e c t i n i t i a l body weight gain, but did cause a retardation i n the growth of the u t e r i i n radiated animals. -119-The u t e r i of weanling rat s are extremely immature and under-developed (average weight le s s than 0.100 g) and are thus l i k e l y to be very sens i t i v e to r a d i a t i o n damage. In addition, a whole body dose of 200 rads was probably s u f f i c i e n t to cause subtle changes i n the components of blood vessel walls so that the blood supply to the uterus may have been inadequate f o r rapid c e l l d i v i s i o n (Casarett,1960). The second and t h i r d phases are those of regeneration and replacement of l o s t parenchymal c e l l s , followed by l i t t l e or no change i n the parenchyma of organs. The rate and degreee of completion of recovery and the length of the period of apparent normalcy vary inversely with the size of the dose. In t h i s study, no attempt was made to delineate these phases because the experimental period was not long enough to warrant t h i s . The l a s t phase of the h i s t o p a t h o l o g i c a l e f f e c t s of i r r a d i a t i o n i s that of accelerated aging. This phase i s nonspecific and begins or i s i n i t i a l l y detectable at somewhat d i f f e r e n t times i n d i f f e r e n t organs, the onset time being dependant on the dose of r a d i a t i o n recieved by the animals. This phase was considered f o r c e r t a i n v a r i a b l e s such as ceroidogenesis, serum Vitamin E and f a t t y acid composition of adipose tissues within the context of the free r a d i c a l theories of aging and the b i o l o g i c a l antioxidant thoery of Vitamin E function. I I I . Ceroid Pigment Development and Accumulation. As indicated by the morphological observations recorded i n Plates I and I I , the 'discolouration' of the u t e r i -120-from Vitamin E d e f i c i e n t r a t s described by Martin and Moore (1936, 1 9 3 8 ) , Barrie (1936,1939) and Mason and Emmel ( 1 9 ^ 5 ) have been confirmed i n t h i s study. There was a progressive deposition of pigment i n the u t e r i of Group II and Group IV animals from approxi mately three months a f t e r the onset of Vitamin E d e f i c i e n c y to the end of the experiment ( 2 6 l days). According to Mason and Emmel ( 1 9 4 5 ) a n d Horwitz and Hartroft (1971), the beginning of ceroidogenesis was concomitant with the onset of s t e r i l i t y i n Vitamin E d e f i c i e n t female r a t s . Thus, i n the present study, i t was assumed that the rat s i n Groups II and IV were unable to carry t h e i r p o t e n t i a l pregnancies to term although no attempt was made to prove t h i s by mating the animals since Barrie (1938,1939) and Raychaudhuri and Desai (1971) have already done so previously. Barrie ( 1 9 3 8 ) had also noted that f i b r o s i s of the uterine w a l l often occurred i n connection with the development of ceroid pigmentation and constitutes further i n d i c a t i o n of uterine abnormality and dysfunction i n Vitamin E d e f i c i e n t r a t s . Such masses of hard, white fibrous connective tissues have been observed i n several rat s i n Groups II and IV (Plate l i b ) . There was no evidence to suggest that a single whole-body, 200 rad-dose of gamma r a d i a t i o n had any e f f e c t on the rate and degree of ceroid development i n the r a t uterus during the experimental period of 8£ months. The u t e r i of Vitamin E supplemented i r r a d i a t e d animals (Group III) did not d i f f e r from those of control animals throughout the experiment (Plate I l a , l i e )i and on the basis of only a q u a l i t a t i v e assessment, Vitamin E d e f i c i e n t i r r a d i a t e d rats (Group IV) did not accumulate more pigment than Vitamin E d e f i c i e n t nonirradiated rat s (Group II) -121-(Plate l i b , l i d ) . The above conclusions were supported by r e s u l t s obtained from the microscopic examination of uterine tissues (Plate XI). Therefore, one must assume that e i t h e r the free r a d i c a l s generated by the gamma rays were not s u f f i c i e n t enough to a f f e c t the peroxidation process r e l a t i v e to the e f f e c t s of Vitamin E def i c i e n c y , or that there were differences which were not apparent q u a l i t a t i v e l y . In ei t h e r case, the nature of ceroid pigment and i t s development w i l l be discussed only within the context of Vitamin E deficiency, assuming that i o n i z i n g r a d i a t i o n had no e f f e c t whatsoever. IV. The Nature of Ceroid Pigment. The l i p i d pigments constitute a large class of substances to which many names have been applied. These include l i p o f u s c i n , haemofuscin, cytolipochrome, ceroid, pseudomelanosis pigment and iron-containing l i p i d pigment (Pearse,1972). No single histochemical property characterizes a l l these substances, and, i n many of them, i t i s d i f f i c u l t or impossible to demonstrate l i p i d by means of the usual tests (Culling,19&3* Pearse,1972). In fa c t , at present there i s only vague understanding of the sig n i f i c a n c e of the various tests which are used to i d e n t i f y l i p o f u s c i n s . However, i n t h i s study, several of these tests were used i n order to examine the c h a r a c t e r i s t i c s of ceroid i n r e l a t i o n to those of l i p o f u s c i n . In addition to being yellow-brown (Plates IVbj Xa), of variable size and morphology (Plate X) and autofluorescent l i k e l i p o f u s c i n (Martin and Moore,19391 Endicott and L i l l i e , 1 9 ^ 4 j Mason and Emmel,19^5i Moore and Wang,1947j Jayne,1950j Oppenheimer -122-and Andrews,19595 S t r e h l e r , 1 9 6 2 j G u l l i n g , 1 9 6 3 j Strehler,1964; Hartroft and Porta,1965? Porta and Hartroft,19691 Pearse,1972), several histochemical properties were found to characterize ceroid i n s i t u and gave support to the concept emphasizing the s i m i l a r i t i e s between i t and the age pigment at some, i f not a l l , stages of t h e i r development (Table IX) (Porta and H a r t r o f t , 1 9 6 9 ) . Ceroid responded to a l l but one (Ziehl-Neelsen) of the histochemical tests i n the same manner as l i p o f u s c i n has been observed to do (Gedigk and Fischer,1959; Wolman,196lj Pearse,I960; Strehler,1964; Porta and Har t r o f t , 1 9 6 9 ; Pearse,1972). As shown i n Table IX, the ceroid pigment of Vitamin E def i c i e n c y was v a r i a b l y a c i d - f a s t at a l l stages of development so that the presence of high molecular weight unsaturated f a t t y acids could not be established with c e r t a i n t y . According to Pearse (1972), the qu a l i t y of 'acid-fastness' depends to some extent on the way i n which the t e s t i s performed and on the i n t e r p r e t a t i o n of what i s aci d - f a s t . He recommended the prolonged Ziehl-Neelsen method which was used i n t h i s study since Berg (1953) bad shown that the acid-fastness of ceroid was dye-specific and obtainable only when basic fuchsin was employed. The r e s u l t s of t h i s study, however, were not d e f i n i t i v e . The main source of d i f f i c u l t y was the dark colour of ceroid which would have masked any f a i n t l y p o s i t i v e reactions. According to Gedigk and Fischer (1959), Pearse ( i 9 6 0 ) , Wolman ( l 9 6 l ) , Strehler ( 1 9 6 4 ) , Porta and Hartroft (1969) and Pearse ( 1 9 7 2 ) , l i p o f u s c i n i s v a r i a b l y a c i d - f a s t and i s thus distinguished from the ceroid pigment of Vitamin E def i c i e n c y . Lee (1950) had also been unable to e s t a b l i s h the acid-fastness of ceroid and attributed t h i s to the variable nature and composition of the pigment. He believed that ceroid, as l i p o f u s c i n , was a mixture' of substances. The presence of various amino acids and other compounds have since been demonstrated by several groups of investigators (Gedigk and Fischer,1959J Pearse,1961j Strehler,1964). However, i t should be kept i n mind that the unique features of l i p o f u s c i n / c e r o i d , by d e f i n i t i o n , c onsist of t h e i r r e l a t i v e i n s o l u b i l i t y and a f f i n i t y f o r f a t s t a i n s . I t quite possible to produce in v i t r o an ' a r t i f i c i a l ceroid* from the i n t e r a c t i o n of polyunsaturated f a t s and oxygen alone, which exhibits both of the above properties o r i g i n a l l y proposed to define these pigments (Hass,1938b, 1939bj Endicott,1944j Hartroft, 1951. 1953? Casselman,1951» Tappel,1953; Porta,19531 Porta and Hartroft,1969)• On t h i s basis, i t follows that proteins, amino acids or other constituents found i n n a t u r a l l y - o c c u r r i n g l i p o f u s c i n s / c e r o i d are not therefore responsible f o r the unique features which allow t h e i r ready d i f f e r e n t i a t i o n from a l l other pigments. The sudanophilia of ceroid was f i r s t noted by L i l l i e e_t a l (1941) and has been repeatedly confirmed by other i n v e s t i g a t o r s (Edwards and Dalton,1942j Endicott and Lillie,1944; Elftmann et al,1949» Hartroft,1950j Lee,1950; Oppenheimer and Andrews,1959). The r e s u l t s i n Table IX and Plates V, VII and Xb indicate that ceroid consists of complex l i p i d s of an undefined nature since i t reacted negatively with Sudan Black B at a l l times. This i s consistent with the l i p i d peroxidation theory of ceroidogenesis proposed o r i g i n a l l y by Dam and Granados (1945) and with the observations of workers such as those mentioned above and i n Chapter I I . -124-Ceroid pigment reacted p o s i t i v e l y to the PAS reaction, Schmorl's Reaction and the Methenamine S i l v e r Reaction (Plates VI, VIII and IX). The PAS Reaction demonstrates the presence of adjacent hydroxyl groups and amino alcohols, and a p o s i t i v e response i s usually interpreted to mean that there are carbohydrate moieties i n the substance being tested (Culling,1963). However, according to Pearse (1972), aldehydes can be produced from non-carbohydrate-containing phosphatides by the action of periodic acid as well as from the oxidation of alcohol groups. Ceroid has been previously reported to be PAS-positive (Hass,1938a1 L i l l i e et a l , 194-2} Edwards and Dalton, 194-21 Endicott and L i l l i e , 19^ 4-} Hartroft,1950), but there has been no other evidence f o r the presence of carbohydrate moieties. Schmorl's Reaction i s s p e c i f i c f o r l i p o f u s c i n (Culling,1963} Pearse,1972) and f o r ceroid (Plates VIII, X and XI). This indicates that during the process of l i p i d peroxidation (that i s , ceroidogenesis), reducing groups are formed which give a p o s i t i v e Schmorl's r e a c t i o n by the reduction of a f e r r i c c h l o r i d e / f e r r i c y a n i d e s o l u t i o n . These groups also weakly reduced ammoniacal s i l v e r solutions to me t a l l i c s i l v e r (Plate VI) suggesting that they were probably oxidized amines (Culling,1863). From these r e s u l t s i t may be concluded that the ceroid pigment of Vitamin E d e f i c i e n c y i s s i m i l a r to the age pigment, l i p o f u s c i n . Bensley (19^7) f i r s t suggested that the colour of the l i p i d pigments was due to the polymerization of aldehydes produced by oxidation. Thus the o r i g i n a l , simple, e a s i l y soluble, colourless l i p i d precursors are peroxidized to a very insoluble, highly coloured product (Pearse,1972). The process varies with the nature -125-of the o r i g i n a l l i p i d and with the c e l l i n which i t takes place (Porta and H a r t r o f t , 1 9 6 9 ) . I t also v a r i e s with the extent to hich protein forms part of the parent substance and on whether the constituent amino acids of the protein are subsequently-oxidized (Gedigk and Fischer,1959; Gedigk and Wessel,1964). According to Pearse ( 1 9 7 2 ) , the chemical and physical c h a r a c t e r i s t i c s of the l i p i d precursors a l t e r as soon as oxidation begins. As the process continues i n the absence of Vitamin E (as i n t h i s study), the c h a r a c t e r i s t i c s of f a t are replaced by those of oxidized f a t , and pigmentation c h a r a c t e r i s t i c a l l y a r ises with time. The early products of the oxidation of t r i g l y c e r i d e s and phosphatides are b a s o p h i l i c , often fluorescent, have weak reducing capacity, are PAS and Sudan Black B p o s i t i v e and may or may not be acid f a s t . Ceroid was considered to be an intermediate stage between auto-oxidizing l i p i d and the t y p i c a l , well-developed brown l i p o f u s c i n pigment which i s strongly b a s o p h i l i c , fluorescent, reduces ferricyanide and s i l v e r solutions and always negative with f a t stains. The ceroid pigment examined i n the present study was t y p i c a l l y l i p o f u s c i n i n character and the reason f o r the discrepancy between Pearse's observations and those reported herein i s the f a c t that Pearse defined the point at which the l i p i d propigment becomes l i p o f u s c i n as the point at which a d e f i n i t e yellow-brown colour becomes v i s i b l e . Since the ceroid pigment, which f i r s t became evident at three months a f t e r the onset of Vitamin E deficiency, was yellow-brown i n colour, i t must be assumed that by then, the l i p o f u s c i n character of the pigment had been f u l l y established; and that what Pearse defined as l i p o f u s c i n -126-was the same as the ceroid pigment of Vitamin E de f i c i e n c y . V. Incisor Depigmentation i n Vitamin E D e f i c i e n t Rats. The observations recorded i n Plate XII indicate that ra t s fed a 5?<> corn o i l d i e t free of Vitamin E f o r several . months exhibited i n c i s o r depigmentation. Such a depigmentation of the maxillary i n c i s o r s of the r a t i s recognized as a common manifestation of Vitamin E d e f i c i e n c y (Dam and Granados,1945; Granados and Dam,1945; Irving,1945; Dam,1962; Dam ans Sondergaard, 1970; Mason and Horwitt,1972). The iron-containing, nonfluorescent pigment i s continuously formed and deposited by the enamel organ as the i n c i s o r i s worn away by a t t r i t i o n (Mason and Horwitt,1972). According to Granados et a l (1945; 1946) and I r v i n g (1945)» depigmentation i s secondary to atrophic changes i n the enamel organ - Irving (1945) described a 'premature and abnormal degeneration of the enamel organ* i n the normally pigmented i n c i s o r s of r a t s which had been on a Vitamin E d e f i c i e n t d i e t f o r 167 days. Oedema and disorganization of the p a p i l l a r y layer, probably caused by c a p i l l a r y damage i n t h i s region, followed by e p i t h e l i a l derangement and cyst formation i n the ameloblast layer has been reported by Pindborg ( 1 9 5 0 ) . There was also a progressive deposition of ceroid pigment i n the macrophages of the highly vascular periodontal connective tissues as depigmentation of the i n c i s o r s continued (Granados et a l , 1 9 4 5 ; 1946). A f t e r Vitamin E therapy, the function of the enamel organ was restored and newly deposited enamel acquired the normal colouring (Granados e_t a l , 1 9 4 5 ) . However, except f o r the changes secondary to macrophage -127-migration, Vitamin E therapy did not appreciably influence the pigment deposition i n the adjacent periodontal tissues. Incisor depigmentation and increased pigmentation of the sof t periodontal tissues are considered unrelated except f o r a common r e l a t i o n s h i p to Vitamin E functions (Granados et al.1945} 1946), but both processes require the presence of highly unsaturated f a t t y acids as well as an inadequacy of Vitamin E i n the d i e t . VI. The E f f e c t of Vitamin E d e f i c i e n c y and/or Ionizing Radiation on Serum Vitamin E l e v e l s . As shown i n Table X and Figure 10,there were two phases i n the depletion of serum Vitamin E l e v e l s i n Group II r a t s . There was an i n i t i a l rapid loss of 65 to 70$ within the f i r s t week af t e r the beginning of feeding, followed by a second phase where Vitamin E l e v e l s f e l l more slowly (5 to 10% a week for the next three weeks). These r e s u l t s are i n l i n e with the observations of B i e r i (1972) who also pointed out that the e f f e c t of Vitamin E depletion was emphasized by the rapid growth and hence increasing tissue mass of the weanling r a t s . The i n i t i a l phase of rapid decrease suggests that there i s a pool of l a b i l e alpha tocopherol which i s r a p i d l y mobilized i n the organism since t h i s loss i n the serum p a r a l l e l e d the decrease i n tissues such as the l i v e r (Peake and Bieri,1971 i Bieri,1972). The second slow phase may represent alpha tocopherol which i s bound to s u b c e l l u l a r structures (such as membranes), probably the c r i t i c a l , f u n c t i o n a l f r a c t i o n of the t o t a l tissue tocopherol. This i s supported by the observation that ceroid pigment did not -128-appear i n the uterine myometrium u n t i l approximately three months a f t e r the onset of Vitamin E deficiency or two months a f t e r a f t e r serum Vitamin E l e v e l s had reached a steady minimal concentration. The evidence i n Figure 10 points to the adequacy of the l e v e l of Vitamin E supplemented to the r a t s i n Groups I and I I I . Levels of serum Vitamin E increased f i v e - f o l d above i n i t i a l concentrations i n Group I animals within 30 days. In concurrence with t h i s , s t a t i s t i c a l analysis indicated that Vitamin E supplemented animals had s i g n i f i c a n t l y higher serum Vitamin E l e v e l s than Vitamin E d e f i c i e n t animals (p<0.000l) between Day 0 and Day 30 of the experiment (Table XIII). Between Day 60 and Day 2 6 l , there was considerable f l u c t u a t i o n of the data points f o r serum Vitamin E l e v e l s i n Group I and Group III rats (Table XI, Figure 11) compared to the l e v e l s f o r Group II and Group IV r a t s . Aside from the problems associated with unequal sample siee and the use of a d i f f e r e n t sample population at each time period, i n d i v i d u a l d i fferences i n the rate and degree of tocopherol uptake and a s s i m i l a t i o n between r a t s probably account f o r most of the v a r i a t i o n (Peake and Bieri,1971» B i e r i , 1 9 7 2 j Losowsky et a l , 1 9 7 2 ) . Serum Vitamin E l e v e l s were more constant f o r the Vitamin E d e f i c i e n t animals because of a depletion of serum and tissue Vitamin E to minimal concentrations. S t a t i s t i c a l analyses of the data have indicated that there was a s i g n i f i c a n t d i e t e f f e c t (Tables XVIII,XIX, XX) and a s i g n i f i c a n t r a d i a t i o n e f f e c t (Tables XXI, XXII, XXIII) on the serum Vitamin E l e v e l s of appropriately treated animals. The d i e t e f f e c t LEAF 129 OMITTED IN PAGE NUMBERING -130-was consistent with the use of two d i f f e r e n t l e v e l s of alpha tocopherol acetate i n the basal mixture (0 and 2.5 g per kg d i e t ) , so that Vitamin E d e f i c i e n t animals had s i g n i f i c a n t l y lower serum Vitamin E concentrations than Vitamin E supplemented animals (p<0.000l) throughout the experiment, i r r e s p e c t i v e of t h e i r r a d i a t i o n status (Table XX). I r r a d i a t i o n caused a s i g n i f i c a n t decrease i n the serum Vitamin E l e v e l s of both Vitamin E supplemented (Group III) and Vitamin E d e f i c i e n t (Group IV) animals between Day 60 and Day 2 6 l . Interpretation of these r e s u l t s i s l i m i t e d by the lack of data f o r these two groups between Day 0 and Day 60. However, the data shown i n Table XXI indicates that Group I had s i g n i f i c a n t l y higher serum Vitamin E than Group III at the 0.001 l e v e l and that there was no i n t e r a c t i o n e f f e c t (p>0.05). This i s taken to mean that there was probably an early d i r e c t e f f e c t of r a d i a t i o n on Group I I I animals so that t h e i r tissue Vitamin E concentrations f e l l below those of the Group I animals. According to 3 i e r i ( 1 9 7 2 ) , during the period of rapid growth, there i s a time of i n i t i a l adjustment to tocopherol supplementation so that tissue tocopherol concentrations may increase, s t a b i l i z e or decrease, depending on the animal and the l e v e l of tocopherol given. Since there was no difference i n body weights between Day 0 and Day 3°i one must assume that the acute dose of r a d i a t i o n caused some permanent, though minor, damage to the metabolism of the r a t , as exemplified by the decrease i n serum Vitamin E l e v e l s . As shown i n Table XXII, Group IV animals had s i g n i f i c a n t l y lower serum Vitamin E l e v e l s than Group II animals ( p < 0 . 0 0 0 l ) . This i s l i k e l y due to a greater i n i t i a l rapid loss of the l a b i l e -131-pool of tocopherol in the irradiated animals and perhaps the premature release of bound tocopherol from damaged ce l lu la r structures (Bieri,1972). In addit ion, the differences in serum Vitamin E levels were probably emphasized by the differences in body size of the two groups (Figure 6.1). VII. Haematological Parameters. Recently, numerous studies have been carried out to investigate the effects of Vitamin E deficiency on red blood c e l l metabolism (Gordon et al,1965j Kann et al,1967i Fitch,1968) Kurokawa et a l . l970i Murty et al,1970j Melhorn et a l , l 71J F i tch , 1972i Kurokawa et al#1972j Mengel,1972). These papers have reported that such dietary stress decreased heme biosynthesis, red c e l l membrane s tab i l i t y and 2,3-DPG formation in mice and monkeys. The results of this study, shown in Tables XXIV and XXV and Figure 12, indicate that chronic Vitamin E deficiency ( 2 6 l days) had no s igni f icant effect on the haemoglobin concentration, haematocrit and 2,3-DPG levels of 22 rats . Therefore, i t may be concluded that Vitamin E does not affect heme biosynthesis in the bone marrow as has been reported by Murty et a l (1970), and that the antioxidant function of Vitamin E does not necessarily extend to the red blood c e l l (Kurokawa et al,1972j Mengel,1972). In agreement with the observations of Kann et a l (1967) and Mengel (1972), there was no s ignif icant difference between the haematocrit levels of Vitamin E def ic ient and control rats (Table XXV). Although the haemolytic sens i t iv i ty of the erythrocytes -132-was not tested, i t may be assumed on the basis of previous studies that the red blood c e l l s of these Vitamin E d e f i c i e n t r a t s were more susceptible to haemolysis than those of the Vitamin E supplemented animals (Kami et al ,1967 ; Kurokawa et a_l»1970j Mengel, 1972). No d i r e c t evidence was obtained, however. In addition, the data from t h i s study i s contrary to the observations of Kurokawa et al ( l972) and Mengel (1972) who reported that Vitamin E d e f i c i e n t mice had lower erythrocyte 2,3-DPG l e v e l s than normal. The r e s u l t s i n Table XXV indicate that there was no s i g n i f i c a n t difference at the o.o5 l e v e l between Group I and Group II animals. Therefore, i t must be concluded that i f Vitamin E a f f e c t s red c e l l metabolism, i t does not do so at the l e v e l of the oxygen binding function of haemoglobin. VIII. The E f f e c t s of Vitamin E Deficiency and / or Ionizing Radiation on Adipose Tissue Fatty Acid Composition. According to Mason et a l (19^6), Morhauer and Holman (1963), Derrick and Wishner (1967), Witting (1970) and Reddy et a l (1873), r a t s fed a Vitamin E d e f i c i e n t , 15-20$ corn o i l d i e t had adipose tissues which were lower i n polyunsaturates than normal r a t t i s s u e s . This was due to an increased peroxidation of the polyunsaturated f a t t y acids i n the adipocytes, exemplified by the deposition of an increasing amount of non-extractable, fluorescent ceroid pigment (Morhauer and Holamn,1963» Reddy et al,1973)> In t h i s study, no attempt was made to measure the l e v e l s of peroxidation or the development of ceroid i n the adipose tissues of the animals. However, on the basis of the evidence reviewed by -133-W i t t i n g (1970) and r e c e n t l y reported by Reddy et a l (1973), r a t s fed a 5$ corn o i l d i e t f r e of Vitami n E should show an apparent p r e f e r e n t i a l decrease i n the polyunsaturated f a t t y a c i d s of adipose t i s s u e s , although perhaps not to the same extent as . those animals fed higher l e v e l s of unsaturated f a t i n the d i e t . The e f f e c t of Vitamin E d e f i c i e n c y and/or i o n i z i n g r a d i a t i o n on the f a t t y a c i d composition of adipose t i s s u e s from r a t s i n a l l four experimental groups are shown i n Tables XXVIII t o XXXIV. Although the percent composition of e i g h t f a t t y a c i d s were determined i n each animal, t h i s d i s c u s s i o n w i l l be mainly concerned w i t h C l 6 t 0 , Cl8s0, C l 8 i l « 9 , Cl8:2t06 because V i t a m i n E i s b e l i e v e d t o f u n c t i o n i n the p r e v e n t i o n of polyunsaturated f a t t y a c i d p e r o x i d a t i o n (Dam and Granados,19451 Dam,1962? Dam and Sondergaard,19701 Witting,1970j1972). I n a d d i t i o n , depot f a t composition r e f l e c t s d i e t a r y f a t composition to a l a r g e extent (Witting,1970) and corn o i l has a f a t t y a c i d composition of 13-0% C I 6 1 O , 1.7$ C18 J0, 27.07S Cl8il«9 and 56.7$ Cl8:2ci)6 (Jaeger, 1969). The r e s u l t s of the s t a t i s t i c a l analyses a r e , h e r e i n considered i n l i g h t of the a c t u a l mean values of the f a t t y a c i d s from each group of animals (Table XXIV) because f a c t o r s such as the sample s i z e and standard d e v i a t i o n s do have an u n r e a l i s t i c e f f e c t on the outcome of the analyses (Sokal and Rohlf,1069). For example, the d i f f e r e n c e i n Gl8:0 l e v e l s between Group I (2.588 + 0.512 %) and Group I I (3.374 + 0.794 %) was s i g n i f i c a n t (p<0.0l), but the d i f f e r e n c e between Group I I I (1.800 + O.362 %) and Group IV (3.451 + 0.576 %) was not (Tables XXVIII and XXIX). S i m i l a r l y , the d i f f e r e n c e i n the l e v e l s of C181I109 between Group I (19.002 + 2.971 %) and Group I I (23.956 + 5*729 %) was not s i g n i f i c a n t -134-while the difference between Group III (29.964 + 1.869 %) and Group IV (29.477 + 4 . 8 0 0 %) was s i g n i f i c a n t at the 0.0001 l e v e l (Tables XXVIII and XXIX). In general, t h i s study has provided i n d i r e c t evidence that Vitamin E d e f i c i e n c y and i o n i z i n g r a d i a t i o n both resulted i n an increase i n the normal low rate of l i p i d peroxidation i n adipose tissue due to the generation of excess free r a d i c a l s i n the system which the organism i s unable to cope with to. a s a t i s f a c t o r y degree. Vitamin E d e f i c i e n t rats had adipose tissues which were s i g n i f i c a n t l y higher i n C 1 8 » 0 (p< 0.001) than the tissues of Vitamin E supplemented r a t s (Tables XXVIII and XXX). Furthermore, i r r a d i a t i o n i t s e l f caused an increase i n C l 6 : 0 and C18 » 1 6)9 l e v e l s ( p < 0 . 0 0 l ) and a decrease i n C 1 8 J 2 « 6 l e v e l s ( p < 0 . 0 0 l ) i n Group III animals. F i n a l l y , as the data i n Table XXXIV in d i c a t e s , Vitamin E d e f i c i e n c y and i o n i z i n g r a d i a t i o n resulted i n a decrease i n the higher polyunsaturated f a t t y acids and a concurrent increase i n the more saturated f r a c t i o n s . The s i g n i f i c a n c e of these findings i s emphasized by the p o s s i b i l i t y that as f a t t y acids 'disappear' from the tissue phospholipids according to the k i n e t i c s of l i p i d peroxidation i n v i t r o (Witting,1967), a homeostatic mechanism which increases the conversion of the lower unsaturated f a t t y acids to higher members of the series comes into focus. As pointed out by Witting ( 1 9 7 0 ) , the actual s i t u a t i o n i s 'rather complex' since the apparent decrease i n the higher polyunsaturated f a t t y acids would be contrary to the p h y s i o l o g i c a l requirement for these PUFAs as e s s e n t i a l membrane components. Thus, any net increase or decrease i n these f a t t y acids must be considered r e a l . CHAPTER VI SUMMARY AND CONCLUSIONS The purpose of t h i s study was to examine the e f f e c t s of Vitamin E def i c i e n c y and/or i o n i z i n g r a d i a t i o n on a number of parameters. These parameters included uterine ceroidogenesis, i n c i s o r depigmentation, serum Vitamin E, haemoglobin concentration, haematocrit, 2,3-DPG and adipose tissue f a t t y acid composition. In addition, an attempt was made to define the nature of ceroid pigment and examine i t s histochemical r e l a t i o n s h i p to the age pigment, l i p o f u s c i n . Female weanling Wistar r a t s were divided into four groups. Two of these groups (II and IV) were fed a modified Draper's Vitamin E-free d i e t , and the other two groups were given the same d i e t supplemented with 2.5 grams of alpha-tocopherol per kilogram of mix. Randomly selected animals from each group were s a c r i f i c e d at preset times and the necessary data obtained. The animals fed a Vitamin E d e f i c i e n t d i e t developed ceroid pigmentation at approximately three months a f t e r the beginning of the experiment. Deposition of ceroid i n the uterine musculature continued u n t i l the end of the experimental period of 261 days. The pigment of Vitamin E de f i c i e n c y was observed to be yellow-brown under the microscope and was Schmorl-positive, PAS-positive, Methenamine S i l v e r - p o s i t i v e , Sudan Black B-negative and v a r i a b l y a c i d - f a s t . I t was therefore concluded that i t was e s s e n t i a l l y s i m i l a r to the age pigment, l i p o f u s c i n and that the two terms, ceroid and l i p o f u s c i n , describe the same substance. -136-Ionizing r a d i a t i o n did not have any apparent e f f e c t on the rate and degree of ceroidogenesis i n Vitamin E d e f i c i e n t r a t s . Animals fed a Vitamin E supplemented d i e t at no time during the experiment acquired any pigment i n t h e i r u t e r i . Serum Vitamin E l e v e l s of Group II animals were r a p i d l y depleted to approximately J0% of the i n i t i a l values within one week a f t e r the beginning of feeding. This was followed by a slower rate of decrease of 10$ a week f o r the next three weeks a f t e r which the l e v e l s s t a b i l i z e d at a minimal concentration. Thus, the onset of pigment deposition at three months did not coincide with loss of the l a b i l e pool of tissue tocopherol, but with the slower depletion of the bound f r a c t i o n . I r r a d i a t i o n caused a s i g n i f i c a n t decrease i n the serum Vitamin E l e v e l s of both Vitamin E d e f i c i e n t and Vitamin E supplemented r a t s . Vitamin E d e f i c i e n c y also caused a depigmentation of the maxillary i n c i s o r s of the animals and had no e f f e c t whatsoever on the haemoglobin concentration, haematocrit and 2,3-DPG l e v e l s of these r a t s . F i n a l l y , Vitamin E d e f i c i e n c y and i o n i z i n g r a d i a t i o n resulted i n a decrease i n the polyunsaturated f a t t y acid content of adipose t i s s u e s . In conclusion, the findings of t h i s study have provided d i r e c t and i n d i r e c t evidence to support the B i o l o g i c a l Antioxidant Theory of Vitamin E function and the Free Radical Theory of Aging. 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APPENDIX - 1 5 6 -Table 1 ^Cumulative Weight Gain f o r Groups I, I I , III and IV from Day o to Day 261 Day Group I Group II Group III Group IV 0 3 12.1 11.0 11.8 11.8 6 25-7 24.1 24.5 24.2 9 37.6 35.1 37.2 35-8 12 59.1 55.8 57.9 58.2 15 74.1 71.0 74.6 70.3 18 78.2 78.7 79.9 76.5 21 84.1 82.9 85.2 82.3 24 90.2 99.5 98.1 94.5 30 106.9 108.9 111.2 110.0 40 122.1 115.6 139.2 121.3 60 166.8 131-1 165.9 161.0 90 179.8 204.0 179.2 185.1 105 190.4 214.9 181.2 190.4 122 197-3 216.4 207.5 192.9 136 201.2 224.5 228.9 202.6 151 214.9 224.5 235.9 212.0 166 222.9 230.5 236.7 217.9 181 253-9 243.2 244.5 212.4 196 2 5 6 . 0 251.4 246.3 280.4 261 312.0 2 6 0 . 5 261.2 241.8 * grams -157-TABLE 2 •Cumulative Uterus Weight Gain f o r Groups I, I I , III and IV from Day 0 to Day 261 Day Group I Group II Group III Group IV 0 0.060 0.071 — 7 0.283 0.290 - -15 0.3^9 0.423 - -30 0.570 0.463 0.520 0.260 60 0.620 0.510 0.680 0.430 90 0.710 0.538 0.620 0.480 105 0.720 0.623 0.680 0.510 122 0.740 0.705 0.735 0.580 136 0.775 0.740 0.750 0.590 151 0.815 0.783 0.755 0.600 166 0.820 0.795 0.825 0.610 181 0.828 0.799 0.840 0.710 196 0.853 0.800 0.845 0.780 261 0.900 0.814 mm -•grams - 1 5 3 -TABLE 3 *Serum Vitamin E l e v e l s f o r Groups I, I I , III and IV from Day 0 to Day 261 Day Group I Group II Group III Group IV 0 1.49 1.55 1.83 1.50 7 3-47 0.49 3.24 O.59 -15 4.72 0.29 5.14 0.35 30 5.85 0.21 5.94 0.42 60 9.96 0.23 0.19 0.42 8.01 0.36 0.54 0.32 0.33. 90 11.39 0.28 8.82 0.24 10.94 0.21 7.43 0.29 0.38 -159-TABLE 3 (contd) Day Group I Group II Group III Group IV 105 11.33 O.38 6.68 O.38 11.43 0.40 6.59 0.24' 0.30 0.43 0.44 122 10.42 0.74 4.60' 0.35 IO.37 0.65 4.01 0.32 0.00 0.13 136 9-18 0.73 9.99 0.34 9.39 O.36 9.13 0.24 0.62 151 7.58 0.42 8.47 0.23 7.42 0.34 9.27 0.13 10.04 0.47 - 0.28 9.18 0.70 - 0.18 166 10.73 0.64 9.73 0.72 10.51 0.63 9.52 0.82 -160-TABLE 3 (contd) Day Group I Group II Group III Group IV 166 9.55 0.47 0.68 0.79 0.72 181 10.19 10.73 9.01 0.00 0.54 0.49 0.37 0.22 6.45 6.92. 0.35 0.08 196 9.87 10.08 0.37 0.38 0.22 0.25 0.27 9.97 10.29' 0.38 O.36 0.14 0.18 261 7.22 9.00 10.89 8.90 0.16 0.17 0.51 0.19 0.13 0.47 - C I -T A B L E 3 (contd) Day Group I Group II Group III Group IV 261 - 0.24 0.50 0.23 0.24f 0.37 0.38 0.16 * mg/lOOml serum -162-TABLE 4 Haematological Data f o r Group I % Haematocrit gm % Haemoglobin umoles DPG/gm Hb 45.0 14.535 20.025 41 . 3 13.981 19.552 43.8 1 4 . 4 4 6 12.686 42 . 9 1 4 . 2 0 4 20.979 45.0 14.508 21.017 45.7 13.055 21.705 43.5 13.961 13.927 45.0 14.306 13.644 4 6 . 2 15-185 16.531 45.8 14.918 17.859 43.8 13.789 15.077 41 . 9 1 4 . 4 0 1 18.447 -163-TABLE 5 Haematological Data f o r Group II % Haematocrit gm $ Haemoglobin umoles DPG/gm Hb 43.0 14.076 18.662 46 .9 15.058 19.125 46 .4 15.695 11.259 40.2 13.808 11.850 44.7 14.791 17.908 42 .4 13.808 20.327 40,9 13.961 19.800 40.8 13.082 19-188 48 .2 15.262 13.345 46 .8 14.447 19.6l4 41 .9 13.705 21.603 46 .4 15.453 16.344 42 .7 13.521 14.209 43.9 13.961 13.678 41.6 13.617 16.342 45.1 14.745 13.238 4l.O 13.617 23.156 47.6 14.975 20.465 44.7 15.836 15.025 47.2 20.311 16.813 43.6 14.516 15.967 43.6 14.918 14.091 TABLE 6 Percent Fatty Acid Composition of Adipose Tissues from Group I animals 12i0 l4i0 l6»0 I6ilto6 I81O 18»1<J9 I8:2w6 l8«3«o3 2.299 3.908 11.954 11.264 2.529 16.552 42.529 3.678 2.138 6.709 13.203 11.472 3.030 20.996 37-379 4.113 1.928 3.030 9.917 10.744 2.755 17.355 40.496 6.612 2.138 5.263 11.978 . 10.889 2.178 17.786 38.657 6.897 1.845 3.321 12.546 6.642 2.214 18.635 45.941 4.428 2.178 3.564 10.693 9.109 3.168 17.426 43.564 4.554 2.138 3.360 13.360 9.717 2.024 18.623 47.773 2.429 2.439 3.656 11.382 10.569 2.033 17.479 50.813 3.252 2.138 4.486 17.757 7.103 3-364 26.168 33.458 7.664 TABLE ? Percent Fatty Acid Composition of Adipose Tissues from Group II animals 12:0 14:0 16:0 16: lw6 18:0 18:14>9 l8:2o)6 18136)i 1.832 7.576 17.424 16.667 4.545 18.181 31.818 3.788 2.985 4.478 11.642 11.045 3.582 18.507 39.403 5-672 2.390 3.984 12.154 11.155 3.187 19.124 39.044 4.582 1.832 4.118 17.647 8.824 5.294 22.941 4l.176 6.855 2.419 4.839 10.887 10.484 3.629 17.337 36.694 2.339 0.585 2.047 6.140 9.421 2.339 31.287 38.012 5.159 2.183 3-571 12.103 8.333 3.175 20.437 40.278 4.533 0.533 1.733 17.600 6.000 3.947 33.333 30.533 4.148 2.402 2.838 13-319 8.734 2.402 22.926 39*083 3.800 1.800 3.200 12.000 10.000 2.400 20.200 42.400 ^•575 TABLE 7 (contd) 1 2 1 0 l4«0 16$0 16I1&)6 13 tO I8ilw9 l8«2o)6 18:3^3 3.992 4.902 11.438 9.477 3.595 17.974 38.235 5-895 1.832 1.474 14.947 6.947 3.368 30.526 36.842 7.895 1.832 6.329 16.245 11.814 2.532 23.628 33-755 3.412 0.853 2.345 18.763 5.757 3.625 31.343 33.902 5.328 1.639 3.484 17.213 10.451 3.074 23.956 46.926 4.121 0.275 2.198 19.505 5.632 3.297 31.593 32.418 4.807 TABLE 8 Percent Fatty Acid Composition of Adipose Tissues from Group III animals 1 2 t 0 14:0 I 6 i 0 16J1W6 I 8 i 0 I 8 i l « 9 18«2<06 I8»3a: 1.179 2.505 18.795 6.793 1.999 27.124 34.452 3.472 0.925 2.111 19.083 6.022 2.171 29.678 33.916 3.106 0.928 1.972 17.111 6.625 1.524 31.845 33.326 3.942 1.327 2.477 19.403 6.624 1.988 29.719 31.479 3.814 1.731 1.608 19.006 6.702 1.316 31.455 34.088 4. 094 TABLE 9 Percent Fatty Acid Composition of Adipose Tissues from Group IV animals 12:0 14:0 16:0 16:16)6 18:0 18:1«>9 18:24)6 18:3*0; 0.713 2.487 17.228 5^677 3.433 30.726 35.210 3.452 0.684 3.011 17.149 5.448 3.239 31.150 35.010 3.011 0.697 4.300 17.487 6.283 4.425 21.029 36.947 4.867 0.750 2.125 17.625 5-500 3.250 33.000 34.500 3.250 0.662 2.249 19.974 6.085 2.910 31.481 35.317 3.645 

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