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Effect of dietary manganese and vitamin E deficiencies on tissue antioxidant status in STZ-diabetic rats Thompson, Katherine Hirsch 1991

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EFFECT OF DIETARY MANGANESE AND VITAMIN E DEFICIENCIES ON TISSUE ANTIOXIDANT STATUS IN STZ-DIABETIC RATS by KATHERINE HIRSCH THOMPSON BA., Pomona College, 1968 M.Sc, The University of British Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES SCHOOL OF FAMILY A N D NUTRITIONAL SCIENCES DIVISION OF H U M A N NUTRITION We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA July 1991 (c) K. H. Thompson, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of pet/yi/ki K / ^ c//V7$ ^ i / ^'^1c^-S The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T I n t e r a c t i o n s between manganese (Mn) d e f i c i e n c y and s t r e p t o z o t o c i n ( S T Z ) - d i a b e t e s w i t h r e s p e c t t o t i s s u e a n t i o x i d a n t s t a t u s were i n v e s t i g a t e d i n male, Sprague-Dawley r a t s . A l l r a t s were f e d e i t h e r a M n - d e f i c i e n t (1 ppm) o r a M n - s u f f i c i e n t (45 ppm) d i e t f o r 8 weeks. D i a b e t e s was t h e n i n d u c e d by t a i l - v e i n i n j e c t i o n o f STZ (60 mg/kg body w e i g h t ) , a f t e r w h i c h t h e r a t s were k e p t f o r an a d d i t i o n a l 4 t o 8 weeks. The c o n t r o l groups c o m p r i s e d r a t s not i n j e c t e d w i t h STZ, w h i c h were e i t h e r Mn-d e f i c i e n t o r M n - s u f f i c i e n t . The M n - d e f i c i e n t d i e t d e c r e a s e d t h e a c t i v i t i e s o f manganese s u p e r o x i d e d i s m u t a s e (MnSOD) i n k i d n e y and h e a r t , and o f c o p p e r -z i n c s u p e r o x i d e d i s m u t a s e (CuZnSOD) i n k i d n e y , i n n o n - d i a b e t i c a n i m a l s . I n t h e d i a b e t i c r a t s , t h e M n - d e f i c i e n t d i e t i n d u c e d more pronounced d e c r e a s e s i n a c t i v i t i e s o f t h e s e same enzymes, and a l s o i n c r e a s e d l i v e r MnSOD a c t i v i t y . P a n c r e a s w e i g h t s were s i g n i f i c a n t l y l o w e r i n M n - d e f i c i e n t , compared t o M n - s u f f i c i e n t r a t s . A l s o , M n - d e f i c i e n t , d i a b e t i c r a t s were s i g n i f i c a n t l y more h y p e r g l y c e m i c i n r e s p o n s e t o a g l u c o s e l o a d t h a n M n - s u f f i c i e n t , s u g g e s t i n g t h a t t h e y may have been more s e v e r e l y d i a b e t i c . S u r p r i s i n g l y , plasma and h e p a t i c v i t a m i n E l e v e l s i n c r e a s e d p r o g r e s s i v e l y w i t h t h e d u r a t i o n o f d i a b e t e s . L i p i d p e r o x i d a t i o n , as measured by H202~induced p r o d u c t i o n o f t h i o b a r b i t u r i c a c i d r e a c t i v e s u b s t a n c e s i n e r y t h r o c y t e s , plasma i i i l i p o p e r o x i d e s , and r e n a l a d i p o s e t i s s u e f l u o r e s c e n c e , a l s o i n c r e a s e d c o n c o m i t a n t w i t h d e c r e a s e d l i v e r and k i d n e y g l u t a t h i o n e l e v e l s . The e f f e c t o f v i t a m i n E - d e f i c i e n c y on M n - d e f i c i e n t , d i a b e t i c r a t s was a l s o i n v e s t i g a t e d . P r e d i c t a b l y , v i t a m i n E - d e f i c i e n t r a t s were a l m o s t e n t i r e l y d e p l e t e d o f plasma and l i v e r v i t a m i n E a f t e r 12 weeks on t h e d e f i c i e n t d i e t s (4 weeks a f t e r STZ t r e a t m e n t ) . C o n s i s t e n t w i t h t h i s , t i s s u e l i p i d p e r o x i d e s were e l e v a t e d compared t o v i t a m i n E - s u f f i c i e n t r a t s . S u p e r i m p o s i n g v i t a m i n E - d e f i c i e n c y on manganese d e f i c i e n c y f a i l e d t o add any f u r t h e r d e f i c i t s i n t i s s u e a n t i o x i d a n t s t a t u s . H i g h e r g l y c o s y l a t e d hemoglobin l e v e l s were obse r v e d i n v i t a m i n E-d e f i c i e n t , compared t o v i t a m i n E - s u f f i c i e n t , d i a b e t i c r a t s . These f i n d i n g s demonstrate f o r t h e f i r s t t i m e an i n t e r a c t i v e e f f e c t between manganese d e f i c i e n c y and S T Z - d i a b e t e s r e s u l t i n g i n a m p l i f i c a t i o n o f t i s s u e a n t i o x i d a n t changes seen w i t h e i t h e r manganese d e f i c i e n c y o r S T Z - d i a b e t e s a l o n e . T h i s e f f e c t o f c o f a c t o r d e p r i v a t i o n i n e x p e r i m e n t a l d i a b e t e s r a i s e s t h e q u e s t i o n o f adequacy of t h e n o m i n a l l y M n - s u f f i c i e n t d i e t i n i n s u l i n - d e p e n d e n t d i a b e t e s m e l l i t u s . i v T A B L E O F C O N T E N T S Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES i x LIST OF APPENDICES X L I S T OF ABBREVIATIONS x i ACKNOWLEDGEMENTS x i v DEDICATION XV 1. INTRODUCTION 1 2. LITERATURE REVIEW 5 2.1 T i s s u e A n t i o x i d a n t s 5 2.1.1 S u p e r o x i d e Dismutases 9 2.1.2 G l u t a t h i o n e P e r o x i d a s e 11 2.1.3 G l u t a t h i o n e R eductase 12 2.1.4 C a t a l a s e 13 2.2 Manganese 14 2.2.1 Manganese Homeo s t a s i s 14 2.2.2 Manganese D e f i c i e n c y and MnSOD A c t i v i t y 17 2.2.3 Manganese D e f i c i e n c y and Abnormal C a r b o h y d r a t e M e t a b o l i s m 19 2.3 D i a b e t e s M e l l i t u s 21 2.3.1 E x p e r i m e n t a l D i a b e t e s 22 2.3.2 O x i d a t i v e S t r e s s i n D i a b e t e s 24 2.4 V i t a m i n E 26 2.5 G l u t a t h i o n e 29 2.6 O t h e r Nonenzymatic A n t i o x i d a n t s 29 2.7 A n t i o x i d a n t I n t e r a c t i o n s 31 3. MATERIALS AND METHODS 34 3.1 C h e m i c a l s and Reagents 34 3.2 A n i m a l s and D i e t s 34 3.2.1 I n d u c t i o n o f D i a b e t e s 39 3.2.2 G l u c o s e T o l e r a n c e T e s t i n g 39 3.3 T i s s u e C o l l e c t i o n 40 V Page 3 .4. 1 T h i o b a r b i t u r i c A c i d R e a c t i v e Substances (TBARS) Assay 40 3 .4. 2 Red C e l l H e m o l y s i s Assay 43 3 .4. 3 Gly c o h e m o g l o b i n D e t e r m i n a t i o n 44 3 .4. 4 Hemoglobin D e t e r m i n a t i o n 45 3 .4. 5 Plasma L i p o p e r o x i d e Assay 46 3 .4. 6 Radioimmunoassay o f Plasma I n s u l i n 47 3 .4. 7 T i s s u e G l u t a t h i o n e Assay 47 3 .5 Enzyme As s a y s 48 3 .5. 1 S u p e r o x i d e Dismutase Assays 48 3 .5. 2 G l u t a t h i o n e P e r o x i d a s e and G l u t a t h i o n e Reductase Assays 49 3 .5. 3 C a t a l a s e Assay 51 3 .6 Manganase A n a l y s i s 52 3 .7 V i t a m i n E Assays 52 3 .8 R e n a l A d i p o s e T i s s u e F l u o r e s c e n c e A s s a y 54 3 .9 H e p a t i c F a t Content D e t e r m i n a t i o n 54 3 .10 P r o t e i n D e t e r m i n a t i o n 55 3 . 11 S t a t i s t i c a l A n a l y s i s 56 4 • RESULTS 58 4 . 1 Food Consumption and Food E f f i c i e n c y 58 4 .2 C o n f i r m a t i o n o f Manganese D e f i c i e n c y 58 4 . 3 Rat Growth, T i s s u e Weights, and T i s s u e Weight/ Body Weight R a t i o s 63 4 .4 E f f e c t s o f STZ Treatment on B l o o d G l u c o s e , H bA l c, and Plasma I n s u l i n L e v e l s 67 4 .5 G l u c o s e T o l e r a n c e T e s t s 69 4 . 6 T i s s u e A n t i o x i d a n t A l t e r a t i o n s 71 4 .6. 1 E f f e c t s o f Manganese D e f i c i e n c y on T i s s u e A n t i o x i d a n t Enzymes 71 4 .6. 2 E f f e c t s o f STZ-d i a b e t e s on T i s s u e A n t i o x i d a n t s 71 4 .6. 3 D i s t r i b u t i o n o f T i s s u e A n t i o x i d a n t s 75 4.7 E f f e c t s o f V i t a m i n E D e f i c i e n c y on T i s s u e A n t i o x i d a n t s 75 v i Page 4.8 E f f e c t s o f S T Z - d i a b e t e s and V i t a m i n E D e f i c i e n c y on Plasma and H e p a t i c V i t a m i n E 83 4.9 E r y t h r o c y t e and T i s s u e S u s c e p t i b i l i t y t o L i p i d P e r o x i d a t i o n i n S T Z - d i a b e t i c r a t s 83 4.10 C o r r e l a t i o n C o e f f i c i e n t s 89 5. DISCUSSION 94 5.1 Manganese D e f i c i e n c y 94 5.2 I n d u c t i o n o f D i a b e t e s 96 5.3 S e v e r i t y o f D i a b e t e s 99 5.4 T i s s u e A n t i o x i d a n t Enzyme A l t e r a t i o n s 100 5.4.1 E f f e c t s o f Manganese D e f i c i e n c y and STZ-d i a b e t e s on S u p e r o x i d e Dismutase A c t i v i t i e s 100 5.4.2 E f f e c t s o f D i a b e t e s on O t h e r T i s s u e A n t i o x i d a n t Enzymes 103 5.5 E f f e c t s o f D u r a t i o n o f D i a b e t e s on N onenzymatic A n t i o x i d a n t A l t e r a t i o n s 104 5.5.1 E f f e c t o f S T Z - d i a b e t e s on Plasma and H e p a t i c V i t a m i n E 105 5.5.2 E f f e c t o f D u r a t i o n o f D i a b e t e s on Mn S t a t u s 107 5.6 O x i d a t i v e S t r e s s i n Manganese D e f i c i e n c y and S T Z - d i a b e t e s 108 5.6.1 E f f e c t o f Manganese D e f i c i e n c y on L i p i d P e r o x i d a t i o n 109 5.6.2 E f f e c t o f S T Z - d i a b e t e s on L i p i d P e r o x i d a t i o n 110 5.7 E f f e c t s o f V i t a m i n E D e f i c i e n c y and STZ-D i a b e t e s on L i p i d P e r o x i d a t i o n and T i s s u e A n t i o x i d a n t s 112 5.8 Compensatory Changes i n T i s s u e A n t i o x i d a n t s 114 5.9 P o s s i b l e E f f e c t s o f Weight L o s s on D i a b e t i c A n i m a l s 115 5.10 Adequacy o f t h e M n - s u f f i c i e n t D i e t 116 5.11 Summary and C o n c l u s i o n s 117 6. REFERENCES 119 v i i L I S T OF TABLES TABLE 1. D i e t f o r d i a b e t i c and n o n - d i a b e t i c Sprague-Dawley r a t s 35 TABLE 2. S t a t i s t i c a l d e s i g n f o r a n a l y s i s o f e x p e r i m e n t s 1 and 2 57 TABLE 3. E f f e c t o f d i e t a r y manganese on r a t body w e i g h t and f o o d i n t a k e 59 TABLE 4. Manganese c o n c e n t r a t i o n i n r a t t i s s u e s 61 TABLE 5. Manganese c o n t e n t o f r a t t i s s u e s e x p r e s s e d as /ig/organ 62 TABLE 6. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on body w e i g h t , t i s s u e w e i g h t s , and t i s s u e / b o d y w e i g h t r a t i o s 65 TABLE 7. E f f e c t s o f Mn d e f i c i e n c y and v i t a m i n E d e f i c i e n c y on body and t i s s u e w e i g h t s , t i s s u e body w e i g h t r a t i o s , b l o o d g l u c o s e , H b A i c , and p l a s m a i n s u l i n i n S T Z - d i a b e t i c r a t s 66 TABLE 8. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on b l o o d g l u c o s e , H b A i c , and plasma i n s u l i n l e v e l s i n r a t s TABLE 9. E f f e c t o f d i a b e t e s and d i e t a r y Mn on MnSOD, CuZnSOD, and GSHPx a c t i v i t i e s i n S p r a g u e -Dawley r a t s 68 72 TABLE 10. E f f e c t o f d i a b e t e s and d i e t a r y Mn on GSH, GSSGRd, and CAT a c t i v i t i e s i n S p r a g u e -Dawley r a t s 73 TABLE 11. E f f e c t o f S T Z - d i a b e t e s on GSH, GSHPx and CAT. One-way ANOVA on p o o l e d M n - s u f f i c i e n t and M n - d e f i c i e n t d a t a 74 TABLE 12. A n t i o x i d a n t s t a t u s o f k i d n e y i n v i t a m i n E-s u f f i c i e n t and v i t a m i n E - d e f i c i e n t , M n - s u f f i c i e n t and M n - d e f i c i e n t , f o u r week S T Z - d i a b e t i c r a t s 79 TABLE 13. A n t i o x i d a n t s t a t u s o f l i v e r i n v i t a m i n E-s u f f i c i e n t and v i t a m i n E - d e f i c i e n t , M n - s u f f i c i e n t and M n - d e f i c i e n t , f o u r week S T Z - d i a b e t i c r a t s 80 v i i i TABLE 14. A n t i o x i d a n t s t a t u s o f h e a r t i n v i t a m i n E-s u f f i c i e n t and v i t a m i n E - d e f i c i e n t , M n - s u f f i c i e n t and M n - d e f i c i e n t , f o u r week S T Z - d i a b e t i c r a t s 81 TABLE 15. A n t i o x i d a n t s t a t u s o f p a n c r e a s i n v i t a m i n E-s u f f i c i e n t and v i t a m i n E - d e f i c i e n t , M n - s u f f i c i e n t and M n - d e f i c i e n t , f o u r week S T Z - d i a b e t i c r a t s 82 TABLE 16. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on p l a s m a and h e p a t i c v i t a m i n E 84 TABLE 17. E f f e c t o f v i t a m i n E d e f i c i e n c y on p l a s m a and h e p a t i c v i t a m i n E i n 4-week d i a b e t i c r a t s 85 TABLE 18. E f f e c t o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on t h i o b a r b i t u r i c a c i d r e a c t i v e s u b s t a n c e s (TBARS) 87 TABLE 19. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on r e d c e l l h e m o l y s i s , plasma l i p i d p e r o x i d e s and r e n a l a d i p o s e t i s s u e f l u o r e s c e n c e 88 TABLE 20. E f f e c t s o f Mn d e f i c i e n c y and v i t a m i n E d e f i c i e n c y on measures o f l i p i d p e r o x i d a t i o n i n 4-week d i a b e t i c r a t s 90 TABLE 21. L i n e a r r e g r e s s i o n c o r r e l a t i o n c o e f f i c i e n t s f o r t i s s u e a n t i o x i d a n t s i n d i a b e t i c r a t s 91 TABLE 22. L i n e a r r e g r e s s i o n c o r r e l a t i o n c o e f f i c i e n t s and s t a t i s t i c a l s i g n i f i c a n c e f o r t i s s u e a n t i o x i d a n t s i n i n d i v i d u a l t i s s u e s i n r a t s 92 i x LIST OF FIGURES FIGURE 1. R e d u c t i o n o f m o l e c u l a r O2 by a s e r i e s o f 1 - e l e c t r o n r e d u c t i o n s 6 FIGURE 2. O v e r a l l scheme o f t i s s u e a n t i o x i d a n t b a l a n c e 8 FIGURE 3. S t r e p t o z o t o c i n s t r u c t u r e 23 FIGURE 4. V i t a m i n E s t r u c t u r e 28 FIGURE 5. G l u t a t h i o n e s t r u c t u r e 3 0 FIGURE 6. E x p e r i m e n t a l d e s i g n 3 6 FIGURE 7. E x p e r i m e n t a l r a t i o n a l e 38 FIGURE 8. Sample p r e p a r a t i o n ( b l o o d ) 41 FIGURE 9. Sample p r e p a r a t i o n ( t i s s u e s ) 42 FIGURE 10. Weekly f o o d i n t a k e o f r a t s 60 FIGURE 11. E f f e c t s o f d i e t a r y manganese (Mn) and S T Z - d i a b e t e s on r a t g r o w t h 64 FIGURE 12. E f f e c t o f d i e t a r y manganese (Mn) on g l u c o s e t o l e r a n c e t e s t r e s p o n s e 70 FIGURE 13. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on MnSOD a c t i v i t y 76 FIGURE 14. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on CuZnSOD a c t i v i t y 77 FIGURE 15. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on GSH c o n c e n t r a t i o n 78 FIGURE 16. E f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on p l a s m a and h e p a t i c v i t a m i n E 86 L I S T O F A P P E N D I C E S A P P E N D I X A . T w o - w a y A N O V A s t a t i s t i c a l e f f e c t s : M a n g a n e s e v s . s t r e p t o z o t o c i n , P v a l u e s 1 3 8 A P P E N D I X B . T w o - w a y A N O V A s t a t i s t i c a l e f f e c t s : M a n g a n e s e v s . v i t a m i n E , P v a l u e s 1 3 9 L I S T OF ABBREVIATIONS Mn manganese STZ s t r e p t o z o t o c i n MnSOD manganese s u p e r o x i d e d i s m u t a s e CuZnSOD c o p p e r - z i n c s u p e r o x i d e d i s m u t a s e GSSGRd g l u t a t h i o n e r e d u c t a s e GSHPx g l u t a t h i o n e p e r o x i d a s e CAT c a t a l a s e GSH r e d u c e d g l u t a t h i o n e GSSG o x i d i z e d g l u t a t h i o n e H 2 0 2 h y d r o g e n p e r o x i d e IDDM i n s u l i n - d e p e n d e n t d i a b e t e s m e l l i t u s TBARS t h i o b a r b i t u r i c a c i d r e a c t i v e s u b s t a n c e s ANOVA a n a l y s i s o f v a r i a n c e H b A l c g l y c o s y l a t e d h e m o g l o b i n HPLC h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y G-6-PD g l u c o s e - 6 - p h o s p h a t e d e h y d r o g r e n a s e PEPCK p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e PC p y r u v a t e c a r b o x y l a s e LDL low d e n s i t y l i p o p r o t e i n VLDL v e r y l o w d e n s i t y l i p o p r o t e i n P G I 2 p r o s t a c y c l i n T X A 2 thromboxane A 2 TCA t r i c h l o r o a c e t i c a c i d TBA t h i o b a r b i t u r i c a c i d MDA m a l o n d i a l d e h y d e x i i DETAPAC d i e t h y l e n e t r i a m i n e p e n t a a c e t i c a c i d EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d SDS sodium d o d e c y l s u l f a t e BHT b e t a - h y d r o x y t o l u e n e O 2 - s u p e r o x i d e OH* h y d r o x y l r a d i c a l LOOH l i p i d h y d r o p e r o x i d e L* f a t t y a c i d r a d i c a l LH p o l y u n s a t u r a t e d f a t t y a c i d L O 2 " p e r o x y l r a d i c a l H + hydrogen i o n M e + n m e t a l i o n Cu copper Se s e l e n i u m Zn z i n c Mg magnesium K e g e q u i l i b r i u m c o n s t a n t K f i r s t o r d e r r a t e c o n s t a n t OD o p t i c a l d e n s i t y U u n i t s nm nanometers ppm p a r t s p e r m i l l i o n pM p i c o m o l a r rpm r e v o l u t i o n s p e r minute DTNB 5 , 5 - d i t h i o b i s - ( 2 - n i t r o b e n z o i c ) a c i d H C l h y d r o c h l o r i c a c i d H 2 S O 4 s u l f u r i c a c i d x i i i H N 0 3 n i t r i c a c i d K C l p o t a s s i u m c h l o r i d e M n C l 2 manganese c h l o r i d e CCI4 c a r b o n t e t r a c h l o r i d e -MnND manganese-def i c i e n t , n o n - d i a b e t i c +MnND m a n g a n e s e - s u f f i c i e n t , n o n - d i a b e t i c -MnD manganese-def i c i e n t , d i a b e t i c +MnD m a n g a n e s e - s u f f i c i e n t , d i a b e t i c -MnD4 manganese-def i c i e n t , 4 w e e k s - d i a b e t i c +MnD4 m a n g a n e s e - s u f f i c i e n t , 4 w e e k s - d i a b e t i c -MnD8 manganese-def i c i e n t , 8 w e e k s - d i a b e t i c +MnD8 manganese-suf f i c i e n t , 8 w e e k s - d i a b e t i c -MnDE- manganese-def i c i e n t , d i a b e t i c , v i t a m i n E - d e f i c i e n t +MnDE- m a n g a n e s e - s u f f i c i e n t , d i a b e t i c , v i t a m i n E - d e f i c i e n t I x i v ACKNOWLEDGEMENTS I w i s h t o t h a n k my r e s e a r c h s u p e r v i s o r , Dr. M e l v i n Lee, f o r h i s k i n d h e l p and k n o w l e d g e a b l e g u i d a n c e t h r o u g h o u t my t h e s i s r e s e a r c h p r o j e c t . I am a l s o d e e p l y g r a t e f u l t o Dr. D a v i d G o d i n and Dr. A l l a n D a v i s o n f o r t h e i r c o n s t r u c t i v e c r i t i c i s m s and i n s p i r i n g d i s c u s s i o n s o f work i n p r o g r e s s . My t h a n k s as w e l l t o Dr. I n d r a j i t D e s a i f o r s e r v i n g on my committee and f o r g u i d i n g me i n t h e v i t a m i n E a n a l y s e s i n t h i s s t u d y . I w o u l d a l s o l i k e t o acknowledge t h e a s s i s t a n c e o f Ms. C o n n i e C h i s h o l m , f o r m e r l y i n t h e Department o f P h y s i o l o g y , f o r p l a s m a i n s u l i n r a d i o i m m u n o a s s a y o f our samples; and o f Mr. Mark Hudon, i n t h e Department o f Pharmacology and T h e r a p e u t i c s , f o r h i s e x c e l l e n t i n s t r u c t i o n i n o p e r a t i n g t h e h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y s p e c t r o p h o t o m e t e r . My t h a n k s a l s o t o Dr. R.A. W a l l f o r u se o f t h i s f a c i l i t y . S p e c i a l t h a n k s t o Mr. Lance Coombe f o r h e l p i n d r a w i n g t h e f i g u r e s f o r t h i s t h e s i s , and t o Mrs. L i a n n e Nunn f o r h e l p i n g me d e s i g n t h e t a b l e s . I would a l s o l i k e t o t h a n k Ms. V i c k i R o t h and Ms. Sandra Tannant f o r t h e i r t e c h n i c a l s u p p o r t i n t h e a n i m a l room. A s i n c e r e t h a n k you a l s o t o t h e t e c h n i c i a n s o f t h e A n i m a l C a r e U n i t who a s s i s t e d me i n l e a r n i n g how t o i n j e c t r a t s , and t o Ms. Mary B a t t e l l , i n t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s , f o r h e l p f u l d i s c u s s i o n s on s t r e p t o z o t o c i n i n d u c t i o n o f d i a b e t e s and t h e c a r e o f S T Z - d i a b e t i c r a t s . I am g r a t e f u l t o my husband, Dr. W i l l i a m Thompson, f o r h e l p w i t h t h e s t a t i s t i c a l a n a l y s e s o f t h e d a t a , and a l s o f o r h i s c o n t i n u e d m o r a l s u p p o r t t h r o u g h a l l t h e t r i a l s o f my g r a d u a t e c a r e e r . T h i s work was s u p p o r t e d i n p a r t by a N a t i o n a l S c i e n c e and E n g i n e e r i n g R e s e a r c h C o u n c i l G r a n t , #A-4692, awarded t o Dr. M e l v i n Lee. DEDICATION To my husband, B i l l , our daughter, J e s s i c a , and our sons, D a n i e l , Zev, and Max 1 1. I N T R O D U C T I O N Manganese (Mn) d e f i c i e n c y and s t r e p t o z o t o c i n ( S T Z ) - d i a b e t e s have complex and p a r a d o x i c a l e f f e c t s on t i s s u e a n t i o x i d a n t enzymes (deRosa e t a l . , 1980; P a y n t e r , 1980; Wohaieb and G o d i n , 1987; G o d i n e t a l . , 1988; Loven e t a l . , 1983). The main t i s s u e a n t i o x i d a n t enzymes a f f e c t e d a r e manganese s u p e r o x i d e d i s m u t a s e (MnSOD), c o p p e r - z i n c s u p e r o x i d e d i s m u t a s e (CuZnSOD), g l u t a t h i o n e p e r o x i d a s e (GSHPx), g l u t a t h i o n e r e d u c t a s e (GSSGRd), and c a t a l a s e (CAT). These enzymes c o m p r i s e a major p a r t o f t h e mammalian i n t r a c e l l u l a r d e f e n s e system a g a i n s t b o t h e n d o g e n o u s l y and e x o g e n o u s l y p r o d u c e d f r e e r a d i c a l s (Chow, 1988; H a l l i w e l l and G u t t e r i d g e , 1986). Nonenzymatic a n t i o x i d a n t s i n c l u d e v i t a m i n E, g l u t a t h i o n e (GSH), a s c o r b i c a c i d , u r i c a c i d , p h e n o l s , and a v a r i e t y o f m e t a l c h e l a t i n g a g e n t s ( H a l l i w e l l , 1987). C a p a c i t y t o scavenge e n d o g e n o u s l y produced r e a c t i v e oxygen s p e c i e s i s i n g e n e r a l i m p a i r e d i n i n s u l i n - d e p e n d e n t d i a b e t e s m e l l i t u s (IDDM) (Godin and Wohaieb, 1988; Metz, 1987; Hunt e t a l . , 1990). Some t i s s u e GSH l e v e l s a r e d e c r e a s e d i n e x p e r i m e n t a l d i a b e t e s (Loven e t a l . , 1983; S l o n i m e t a l . , 1976), and v a r i o u s a n t i o x i d a n t enzyme a c t i v i t i e s a r e e i t h e r e l e v a t e d o r d i m i n i s h e d (Wohaieb and G o d i n , 1987). Plasma and t i s s u e v i t a m i n E l e v e l s r e p o r t e d f o r i n s u l i n - d e p e n d e n t d i a b e t e s a r e n o t c o n s i s t e n t : b o t h i n c r e a s e s and d e c r e a s e s have been o b s e r v e d (Karpen e t a l . , 1984, 1985; Behrens e t a l . , 1 9 8 4 ) . V i t a m i n E s u p p l e m e n t a t i o n m i n i m i z e s , and v i t a m i n E d e f i c i e n c y enhances, 2 t h e d i a b e t o g e n i c e f f e c t s o f STZ ( S l o n i m e t a l . , 1983). I n c r e a s e d l i p i d p e r o x i d a t i o n has been o b s e r v e d i n some t i s s u e s o f d i a b e t i c a n i m a l s ( M o r e l and C h i s h o l m , 1989) . E r y t h r o c y t e t h i o b a r b i t u r i c a c i d r e a c t i v e s u b s t a n c e s (TBARS) (an i n d i r e c t measure o f l i p i d p e r o x i d a t i o n ) a r e p r o d u c e d f o l l o w i n g in vitro p e r o x i d a t i v e c h a l l e n g e i n g r e a t e r q u a n t i t i e s i n d i a b e t i c compared t o n o n - d i a b e t i c a n i m a l s (Wohaieb and G o d i n , 1987). Known p a t h o p h y s i o l o g i c a l r e s p o n s e s accompanying S T Z - d i a b e t e s a l s o i n c l u d e changes i n t i s s u e c o n c e n t r a t i o n s o f s e v e r a l t r a c e m e t a l s , i n c l u d i n g Mn ( F a i l l a and K i s e r , 1981; Bond e t a l . , 1 9 8 3), z i n c (Zn) and copper (Cu) ( F a i l l a e t a l . , 1980). Manganese a c c u m u l a t e d i n m a n g a n e s e - s u f f i c i e n t r a t l i v e r and k i d n e y between 3 and 10 days f o l l o w i n g STZ a d m i n i s t r a t i o n ( F a i l l a and K i s e r , 1981). P a i r - f e e d i n g o f d i a b e t i c and c o n t r o l a n i m a l s showed t h a t t h e e f f e c t was n o t due t o i n c r e a s e d f o o d i n t a k e r e s u l t i n g f r o m t h e i n d u c t i o n o f d i a b e t e s . N i s h i d a e t a l . (1989) i n v e s t i g a t e d t i s s u e Mn c o n t e n t o f d i a b e t i c r a t s 2 weeks a f t e r STZ i n j e c t i o n , w i t h and w i t h o u t Mn s u p p l e m e n t a t i o n . L i v e r Mn was n o t e l e v a t e d i n t h e i r S T Z - d i a b e t i c r a t s ; however, w i t h Mn s u p p l e m e n t a t i o n , Mn i n c r e a s e d i n b r a i n and thymus, d e c r e a s e d i n s p l e e n , p a n c r e a s and k i d n e y , and r emained unchanged i n l i v e r . P r i o r a d m i n i s t r a t i o n o f M n C l 2 l e s s e n e d t h e h y p e r g l y c e m i c e f f e c t s o f STZ. R u b e n s t e i n e t a l . ( 1 9 6 2 ) was t h e f i r s t t o s u g g e s t t h a t Mn s t a t u s 3 may be an i m p o r t a n t c o n s i d e r a t i o n i n t h e management o f IDDM. They n o t e d t h a t l u c e r n e , an he r b h i g h i n Mn, has been u s e d f o r decades by i n d i g e n o u s m e d i c a l p r a c t i t i o n e r s i n So u t h A f r i c a as a t r e a t m e n t f o r d i a b e t e s m e l l i t u s . They a l s o f o u n d t h a t Mn s u p p l e m e n t a t i o n r e d u c e d t h e h y p e r g l y c e m i a o f IDDM. The e f f e c t s o f Mn s u p p l e m e n t a t i o n and d e f i c i e n c y on g l u c o s e h o m e o s t a s i s have been i n v e s t i g a t e d i n c o n s i d e r a b l e d e t a i l ( B a l y e t a l . , 1984, 1985a,b, 1987). Mn d e f i c i e n c y i s accompanied by abnormal g l u c o s e t o l e r a n c e , d e p r e s s e d p a n c r e a t i c i n s u l i n s y n t h e s i s , and enhanced i n s u l i n d e g r a d a t i o n . Mn i s now known t o p l a y a c e n t r a l r o l e i n c a r b o h y d r a t e m e t a b o l i s m , b o t h as an enzyme a c t i v a t o r ( K o r c and Brannon, 1988), and as a p r o m o t o r o f p a n c r e a t i c i n s u l i n mRNA and amylase mRNA s y n t h e s i s ( B a l y e t a l . , 1988a, Chang e t a l . , 1990). A d e f i c i e n c y o f Mn r e p o r t e d l y c o n t r i b u t e s t o t h e e t i o l o g y o f IDDM ( K o r c , 1988). The e f f e c t s o f Mn d e f i c i e n c y on t i s s u e a n t i o x i d a n t s , p a r t i c u l a r l y MnSOD, have a l s o been i n v e s t i g a t e d . I n p a r t i c u l a r , i n v e s t i g a t o r s have found d e c r e a s e d a c t i v i t y o f MnSOD i n k i d n e y and h e a r t o f M n - d e f i c i e n t r a t s ( P a y n t e r , 1980), i n l i v e r o f Mn-d e f i c i e n t c h i c k e n s , i n l i v e r and b r a i n o f M n - d e f i c i e n t m i c e (deRosa e t a l . , 1 9 8 0), and i n h e a r t o f M n - d e f i c i e n t sheep ( P a y n t e r , 1988). A r e d u c t i o n i n t i s s u e s e l e n i u m (Se) was f o u n d t o accompany M n - d e f i c i e n c y i n p i g s (Burch e t a l . , 1 9 7 6), s u g g e s t i n g a p r o b a b l e ( but unmeasured) d e c r e a s e i n GSHPx a c t i v i t y . 4 The o b j e c t i v e o f t h i s s t u d y was t o i n v e s t i g a t e t h e combined e f f e c t s o f Mn d e f i c i e n c y and S T Z - d i a b e t e s on t i s s u e a n t i o x i d a n t s t a t u s i n t h e r a t . Our h y p o t h e s i s was t h a t t h e r e w o u l d be an i n t e r a c t i v e e f f e c t on t i s s u e a n t i o x i d a n t s t a t u s between Mn d e f i c i e n c y and S T Z - d i a b e t e s . We a s s e s s e d t h e f u n c t i o n a l c o n s equences o f t h e s e combined t r e a t m e n t s by m e a s u r i n g GSH, TBARS, MnSOD, CuZnSOD, GSHPx, GSSGRd and CAT i n l i v e r , k i d n e y , h e a r t and p a n c r e a s . We a l s o e v a l u a t e d e r y t h r o c y t e TBARS, plasma and h e p a t i c v i t a m i n E, b l o o d g l u c o s e l e v e l s , p e r c e n t g l y c o s y l a t i o n o f h e m o g l o b i n ( % H b A i c ) , and r e p o n s e t o g l u c o s e t o l e r a n c e t e s t i n g . The p o s s i b l e "masking" e f f e c t o f d i e t a r y v i t a m i n E was t e s t e d by i m p o s i n g a v i t a m i n E d e f i c i e n c y i n a d d i t i o n t o Mn d e f i c i e n c y i n d i a b e t i c r a t s . The same c r i t e r i a were u s e d t o a s s e s s t i s s u e a n t i o x i d a n t s t a t u s i n t h e v i t a m i n E d e f i c i e n t r a t s . S p e c i f i c q u e s t i o n s w h i c h we s e t out t o answer were: 1) Does Mn d e f i c i e n c y e x a c e r b a t e S T Z - d i a b e t e s i n r a t ? 2) Does t h e p r e s e n c e o f v i t a m i n E i n t h e d i e t "mask" changes due t o r e d u c e d Mn i n t a k e ? 3) I s S T Z - d i a b e t e s combined w i t h Mn d e f i c i e n c y a c o n d i t i o n o f h e i g h t e n e d o x i d a n t s t r e s s ? 4 ) A r e a l t e r a t i o n s i n t i s s u e a n t i o x i d a n t s t a t u s r e f l e c t e d i n changes i n l i p i d p e r o x i d a t i o n l e v e l s ? 5 2 . LITERATURE REVIEW 2 . 1 . T i s s u e A n t i o x i d a n t s T h e g r a d u a l e v o l u t i o n o f . a t e r r e s t r i a l a t m o s p h e r e c o n t a i n i n g i n c r e a s i n g l e v e l s o f o x y g e n a p p e a r s t o h a v e b e e n p a r a l l e l e d b y a n e v o l u t i o n o f b i o c h e m i c a l d e f e n s e m e c h a n i s m s t o c o p e w i t h t h e i n e v i t a b l e t o x i c i n t e r m e d i a t e s o f o x y g e n m e t a b o l i s m ( B o v e r i s , 1 9 7 7 ) . T h e r e d u c t i o n o f m o l e c u l a r o x y g e n , O2, c a n p r o c e e d t o H2O b y a s e r i e s o f o n e - e l e c t r o n r e d u c t i o n s ( F i g u r e 1) ( T a u b e , 1 9 6 5 ) , t h u s g e n e r a t i n g t h e t o x i c m e t a b o l i t e s s u p e r o x i d e (O2"") , H2O2, a n d h y d r o x y l r a d i c a l ( O H * ) , w h i c h a r e c o l l e c t i v e l y k n o w n a s r e a c t i v e o x y g e n s p e c i e s . B o t h s u p e r o x i d e a n d h y d r o x y l r a d i c a l a r e a m o n g t h e h i g h l y r e a c t i v e c h e m i c a l s p e c i e s c l a s s i f i e d a s f r e e r a d i c a l s . A f r e e r a d i c a l i s d e f i n e d b y H a l l i w e l l ( 1 9 8 7 ) a s " a n y s p e c i e s c a p a b l e o f i n d e p e n d e n t e x i s t e n c e t h a t c o n t a i n s o n e o r m o r e u n p a i r e d e l e c t r o n s , i . e . , e l e c t r o n s p r e s e n t s i n g l y i n a t o m i c o r m o l e c u l a r o r b i t a l s " . U n d e r n o r m a l m e t a b o l i c c o n d i t i o n s , m o s t 0 2 r e d u c t i o n t a k e s p l a c e v i a e n z y m a t i c p a t h w a y s s u c h a s t h e t e t r a v a l e n t c y t o c h r o m e o x i d a s e p a t h w a y ( A n t o n i o n i e t a l . , 1 9 7 0 ) , t h u s a v o i d i n g t h e r e l e a s e o f f r e e r a d i c a l s . H o w e v e r , e v e n u n d e r n o r m a l c o n d i t i o n s , s o m e o x y g e n r e d u c t i o n ( a b o u t 2%) t a k e s p l a c e v i a o n e - e l e c t r o n r e d u c t i o n p a t h w a y s , a l l o w i n g f o r m a t i o n o f p a r t i a l l y r e d u c e d f o r m s o f o x y g e n . O x i d a t i v e s t r e s s i n a e r o b i c o r g a n i s m s c o m m o n l y r e f e r s t o i n c r e a s e d p r o d u c t i o n o r d e c r e a s e d i n a c t i v a t i o n o f t h e r e a c t i v e o x y g e n s p e c i e s 02" a n d H2O2 ( H a l l i w e l l , 1 9 8 7 ) . e- +2H+ e- + H + 2^2 OH e" + H* H 20 H FIGURE 1. Reduction of molecular O2 by a series of one-electron reductions. Loss of one electron (e") leads to the production of superoxide (O2*); loss of a second electron, in the presence of hydrogen ions (H + ), to the production of hydrogen peroxide (H2O2); of a third, to the evolution of hydroxyl radical (OH") and water (H20); and of a fourth, to the production of water. 7 F r e e r a d i c a l s were c o n s i d e r e d t o be o f v e r y m i n o r i m p o r t a n c e u n t i l t h e d i s c o v e r y o f an enzyme whose s p e c i f i c s u b s t r a t e i s a f r e e r a d i c a l ( O 2 - ) , namely s u p e r o x i d e d i s m u t a s e (McCord and F r i d o v i c h , 1969). The d i s c o v e r y o f c o p p e r - z i n c s u p e r o x i d e d i s m u t a s e (CuZnSOD) was f o l l o w e d by t h e e l u c i d a t i o n o f a complex web o f e n z y m a t i c and n o n e n z y m a t i c s c a v e n g e r s (Chow, 1979). A l t h o u g h n o r m a l l y p r o d u c e d i n minute q u a n t i t i e s , t h e c h e m i c a l l y h i g h l y r e a c t i v e f r e e r a d i c a l s can i n i t i a t e c h a i n r e a c t i o n s and p r o d u c e o t h e r c h e m i c a l l y r e a c t i v e s p e c i e s such as l i p i d h y d r o p e r o x i d e s w h i c h a r e now known t o be a s s o c i a t e d w i t h t i s s u e damage and c y t o t o x i c i t y i n a wide v a r i e t y o f d i s e a s e s t a t e s as w e l l as e n v i r o n m e n t a l c o n d i t i o n s o f i n c r e a s e d o x i d a n t s t r e s s ( e . g . , h y p e r b a r i c 0*2 and ozone) ( B r i t t o n e t a l . , 1978; F r i d o v i c h 1978a, 1978b). S u p e r o x i d e r a d i c a l s have been d e t e c t e d in vivo and in v i t r o (Chance e t a l . , 1979, S o h a l e t a l . , 1 9 8 9 ) . T h e i r g e n e r a t i o n has been d e m o n s t r a t e d as a b y - p r o d u c t o f t h e a u t o x i d a t i o n o f t h i o l s and heme ( M i s r a , 1974) and as a consequence o f p r o s t a g l a n d i n s y n t h e s i s . The p h a g o c y t i c and b a c t e r i c i d a l a c t i v i t y o f p o l y m o r p h o n u c l e a r l e u k o c y t e s and a l v e o l a r macrophages has been shown t o r e q u i r e s u p e r o x i d e r a d i c a l p r o d u c t i o n . X a n t h i n e o x i d a s e - c a t a l y z e d r e a c t i o n s and m i t o c h o n d r i a l r e s p i r a t i o n a l s o p r o d u c e o x y g e n - d e r i v e d f r e e r a d i c a l s (Godin and Wohaieb, 1988b). FIGURE 2. Overall scheme of tissue antioxidant balance. L H , polyunsaturated fatty acid or membrane lipids; L", free radical of polyunsatured fatty acid or membrane lipids; L O O H , lipid hydroperoxide; L O H , hydroxy acid; LOO*, peroxyl radical; OH*, hydroxyl radical; O2-, superoxide radical; *02*, singlet oxygen; GSH, reduced glutathione; GSSG, oxidized glutathione; G-6-PD, glucose-6-phosphate dehydrogenase; Vit E, vitamin E; MnSOD, manganese superoxide dismutase; CuZnSOD, copper-zinc superoxide^dismutase; H2O2, hydrogen peroxide; GSHPx, glutathione peroxidase; GSSGRase, glutathione reductase; MDA, malondialdchydc, Fe , divalent iron ions; Cu , monovalent copper ions. 9 The o v e r a l l scheme o f t i s s u e a n t i o x i d a n t / o x i d a n t b a l a n c e as i t i s now u n d e r s t o o d i s summarized i n F i g u r e 2. The main a n t i o x i d a n t enzymes a r e : MnSOD and CuZnSOD, w h i c h c a t a l y z e t h e d i s m u t a t i o n o f s u p e r o x i d e r a d i c a l t o H2O2; g l u t a t h i o n e p e r o x i d a s e (GSHPx) (Se-dependent), w h i c h d e t o x i f i e s H2O2 and l i p i d h y d r o p e r o x i d e s (LOOH); g l u t a t h i o n e r e d u c t a s e (GSSGRd), w h i c h c a t a l y z e s t h e r e g e n e r a t i o n o f r e d u c e d g l u t a t h i o n e (GSH); and c a t a l a s e (CAT), w h i c h c a t a l y z e s H2O2 r e d u c t i o n (Chow, 1988). The s u b c e l l u l a r l o c a l i z a t i o n o f t h e s e enzymes d i f f e r s : CuZnSOD and c a t a l a s e a r e p r i m a r i l y c y t o s o l i c enzymes; MnSOD i s p r i n c i p a l l y m i t o c h o n d r i a l ; and GSHPx and GSSGRd a r e f o u n d i n b o t h c y t o s o l and m i t o c h o n d r i a l m a t r i x ( W e i s i g e r and F r i d o v i c h , 1973; O s h i n o and Chance, 1977; Lammi-Keefe e t a l . , 1984). 2 . 1 . 1 Superoxide Dismutases T h e r e a r e t h r e e i n t r a c e l l u l a r s u p e r o x i d e d i s m u t a s e s : MnSOD, CuZnSOD and FeSOD, and one e x t r a c e l l u l a r CuZnSOD, a l l o f w h i c h a r e b i o c h e m i c a l l y d i s t i n c t ( D e l M a e s t r o , 1980; H a l l i w e l l and G u t t e r i d g e , 1990, F r i d o v i c h , 1975). MnSOD and FeSOD seem t o s h a r e a common e v o l u t i o n a r y b a c k g r o u n d (a s d e t e r m i n e d by sequence h o m o l o g i e s ) w h i l e t h e CuZnSOD's seem t o have e v o l v e d i n d e p e n d e n t l y ( M i c h e l s o n , 1978). FeSOD i s f o u n d o n l y i n p r o k a r y o t e s , MnSOD i s fo u n d i n p r o k a r y o t e s and i n t r a m i t o c h o n d r i a l l y i n e u k a r y o t e s , and CuZnSOD i s f o u n d o n l y i n e u k a r y o t e s , m a i n l y i n t h e c y t o s o l f o r t h e i n t r a c e l l u l a r f o r m o f t h e enzyme ( F r i d o v i c h , 1978b). 10 MnSOD 1s have a s u b u n i t m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 23,000 d a l t o n s , w i t h one t r i v a l e n t Mn p e r s u b u n i t . Most MnSOD 1s a r e d i m e r i c , t h o u g h a few ( i n c l u d i n g v e r t e b r a t e forms) a r e t e t r a m e r i c (Ludwig e t a l . , 1986). I n t r a c e l l u l a r CuZnSOD has a m o l e c u l a r w e i g h t o f about 31,000, w i t h 1 g-atom o f Cu and 1 g-atom o f Zn p e r mole o f CuZnSOD. E x t r a c e l l u l a r CuZnSOD has a much h i g h e r m o l e c u l a r w e i g h t (about 135,000) due t o a t t a c h e d c a r b o h y d r a t e m o i e t i e s . The r e a c t i o n s c a t a l y z e d by s u p e r o x i d e d i s m u t a s e s i n v o l v e c o n v e r s i o n o f s u p e r o x i d e t o H 2 O 2 and m o l e c u l a r oxygen. The SOD's m e d i a t e e l e c t r o n t r a n s f e r between t h e r e a c t a n t s i n c y c l e s o f r e d u c t i o n f o l l o w e d by o x i d a t i o n ( F r i d o v i c h , 1 9 8 3 ) , as f o l l o w s : M e + n + 0 2 " > M e + ( n - 1 > + 0 2 (1) M e + ( n - l ) + o 2 ~ > M e + n + H2O2 (2), where Me i s t h e m e t a l i o n i n v o l v e d . The r a t e c o n s t a n t f o r d i s m u t a t i o n w i t h e n z y m a t i c c a t a l y s i s i s 2 x 1 0 9 M - 1 s e c - 1 a t 25°C, w h i c h i s s e v e r a l o r d e r s o f m a g n i t u d e g r e a t e r t h a n t h e r a t e c o n s t a n t f o r spontaneous d i s m u t a t i o n ( F r i d o v i c h , 1983) . 11 As n o t e d above, M n - d e f i c i e n t d i e t s r e s u l t i n d e c r e a s e d MnSOD a c t i v i t i e s i n some t i s s u e s . C u - d e f i c i e n t d i e t s l i k e w i s e l e a d t o d e c r e a s e d a c t i v i t i e s o f CuZnSOD, b u t Z n - d e f i c i e n t d i e t s do n o t have s u c h an e f f e c t . The Cu atom i n CuZnSOD i s a p p a r e n t l y c a t a l y t i c a l l y a c t i v e , c y c l i n g between +2 and +1 o x i d a t i o n s t a t e s , whereas t h e Zn, a t t a c h e d t o a c e n t r a l h i s t i d i n e l i g a n d , s e r v e s m o s t l y a s t r u c t u r a l r o l e . The c a t a l y t i c mechanism o f MnSOD, as i t i s c u r r e n t l y u n d e r s t o o d , i n v o l v e s a c y c l i n g o f t h e Mn r e d o x s t a t e between +2 and +3 (Fee and B u l l , 1 9 8 6 ) . 2.1.2 Glutathione Peroxidase G l u t a t h i o n e p e r o x i d a s e (GSHPx) (EC 1.11.1.9) has a m o l e c u l a r w e i g h t o f about 80,000 d a l t o n s , i s t e t r a m e r i c , and c o n t a i n s 4 gram-atoms o f Se p e r mole (Sunde and H o e k s t r a , 1 9 80). I t was f i r s t d i s c o v e r e d i n e r y t h r o c y t e s by M i l l s ( 1 9 5 7 ) , who s u g g e s t e d t h a t t h e major r o l e o f t h e enzyme i s t o p r o t e c t e r y t h r o c y t e membranes and h e m o g o l o b i n a g a i n s t o x i d a t i v e damage. Once t h e f a c t o f i t s b e i n g a selenoenzyme was e s t a b l i s h e d ( R o t r u c k e t a l . , 1973), i n v e s t i g a t o r s d e t e r m i n e d t h a t a s s a y o f t h e enzyme c o u l d be us e d t o r e l i a b l y i n d i c a t e t h e Se s t a t u s o f an a n i m a l o r human (Hafeman e t a l . , 1974; Chavez, 1979). G l u t a t h i o n e p e r o x i d a s e c a t a l y z e s t h e r e d u c t i o n o f H 2 O 2 and a v a r i e t y o f l i p i d h y d r o p e r o x i d e s ( G u n z l e r and F l o h e , 1 9 85), 12 a c c o r d i n g t o t h e f o l l o w i n g e q u a t i o n : ROOH + 2GSH > ROH + GSSG + H 20 D e f i c i e n c y o f GSHPx r e s u l t s f r om Se d e f i c i e n c y ( X i a e t a l . , 1985, Ge e t a l . , 1983, Chen e t a l . , 1980) and i s t h e o n l y known t i s s u e a n t i o x i d a n t d e f i c i e n c y t h a t has been c l e a r l y l i n k e d t o a d i s e a s e s t a t e i n humans w h i c h i s known t o i n v o l v e p e r o x i d a t i v e i n j u r y . GSHPx i s f o u n d i n g r e a t l y r e d u c e d l e v e l s i n a r e a s o f C h i n a t h a t have s e l e n i u m - p o o r s o i l s . I n t h e s e a r e a s , K e s h a n d i s e a s e , a c a r d i o m y o p a t h y c u r a b l e by Se s u p p l e m e n t a t i o n , i s endemic (Chen e t a l . , 1 9 8 0). I n r a t s , S e - d e f i c i e n c y h a s been c l e a r l y l i n k e d t o d e c r e a s e d a c t i v i t y o f GSHPx i n most t i s s u e s . V i t a m i n E s u p p l e m e n t a t i o n c a n p a r t l y a l l e v i a t e t h i s d e f i c i e n c y e f f e c t (Lawrence and B u r k e , 1976). 2.1.3 G l u t a t h i o n e R e d u c t a s e G l u t a t h i o n e r e d u c t a s e (GSSGRd) (EC 1.6.4.2) i s t h e enzyme w h i c h c a t a l y z e s t h e r e d u c t i o n o f GSSG ( o x i d i z e d g l u t a t h i o n e ) t o GSH, a c c o r d i n g t o t h e f o l l o w i n g e q u a t i o n : NADPH + H + + GSSG =======* NADP + + 2GSH The r e a c t i o n i s e s s e n t i a l l y r e v e r s i b l e , w i t h a p i n g - p o n g k i n e t i c mechanism ( V a n o n i e t a l . , 1990). The enzyme has a m o l e c u l a r w e i g h t o f 104,800 and c o n s i s t s o f 2 i d e n t i c a l s u b u n i t s , w i t h b i n d i n g s i t e s f o r r e d u c e d p y r i d i n e n u c l e o t i d e 13 (NADPH) and GSSG a t o p p o s i t e ends o f one s u b u n i t ( M e i s t e r and A n d e r s o n , 1983) . E f f i c i e n t c a t a l y t i c r e m o v a l o f o r g a n i c h y d r o p e r o x i d e s r e q u i r e s maintenance o f a h i g h GSH:GSSG r a t i o (K eg= 1100, pH 7; Mapson.and Isherwood, 1963). S i n c e t h e a c t i v i t y o f GSSGRd i s dependent on t h e a v a i l a b i l i t y o f NADPH (S i m o n a r s o n , 1985), t h e r e i s a t e r t i a r y dependence o f GSHPx a c t i v i t y on t h e p e n t o s e s h u n t enzymes, g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (G-6-PD) and 6-phosphogluconate d e h y d r o g e n a s e , w h i c h g e n e r a t e NADPH from NADP. Under c e r t a i n c o n d i t i o n s , e.g. e x p o s u r e t o ozone (Chow and T a p p e l , 1972), i n c r e a s e d d i e t a r y l i p i d h y d r o p e r o x i d e s (Reddy and T a p p e l , 1974), o r v i t a m i n E d e f i c i e n c y (Chow e t a l . , 1973), a n i m a l s have been shown t o have i n c r e a s e d a c t i v i t i e s o f GSHPx, GSSGRd and G-6-PD. The a b i l i t y o f some a n i m a l s t o adapt t o o x i d a t i v e s t r e s s by i n c r e a s i n g a c t i v i t e s o f t i s s u e a n t i o x i d a n t enzymes may u l t i m a t e l y d e t e r m i n e t h e i r r e l a t i v e s u s c e p t i b i l i t y t o o x i d a t i v e damage (Chow, 1979). 2.1 .4 C a t a l a s e C a t a l a s e (CAT) (EC 1.11.1.6) i s f o u n d i n a l m o s t a l l t i s s u e s , w i t h t h e h i g h e s t a c t i v i t i e s i n e r y t h r o c y t e s and l i v e r c y t o s o l ( A e b i , 1974) . I t s e n z y m a t i c r o l e i s t o c a t a l y z e t h e d e c o m p o s i t i o n o f H 2 O 2 . The r e a c t i o n mechanism i s a 2 - p a r t p r o c e s s i n v o l v i n g a common i n t e r m e d i a t e (Complex I ) (Wheeler e t a l . , 1 9 9 0), as f o l l o w s : 14 CAT + H 2 0 2 > CAT-H 20 2 (Complex I) (1) CAT-H 20 2 \ + H 2 0 2 > CAT + 2H 20 + 0 2 (2) The r a t e o f t h e r e a c t i o n depends on t h e c o n c e n t r a t i o n o f H 2 0 2 . C a t a l a s e i s a t e t r a m e r i c enzyme c o n t a i n i n g 1 m o l / s u b u n i t o f a t i g h t l y bound heme p r o s t h e t i c group ( C l a i b o r n e , 1 9 8 5). A v a r i a n t o f t h e enzyme has been i s o l a t e d (Kono and F r i d o v i c h , 1983) w h i c h i s h e x a m e r i c and c o n t a i n s 1 atom o f Mn ( I I I ) p e r s u b u n i t i n s t e a d o f heme. I t has so f a r been f o u n d o n l y i n m i c r o o r g a n i s m s , most n o t a b l y L a c t o b a c i l l u s plantarum ( B e y e r and F r i d o v i c h , 1986). 2.2 Manganese 2.2.1 Manganese H o m e o s t a s i s The e s s e n t i a l i t y o f manganese ( a t o m i c number 25) was e s t a b l i s h e d by O r e n t and McCollum (1931), who f o u n d t h a t d i e t a r y d e f i c i e n c y o f Mn r e s u l t e d i n r e d u c e d r e p r o d u c t i v e c a p a c i t y i n t h e r a t . Kemmerer e t a l . (1931) d i s c o v e r e d t h a t t h i s t r a n s i t i o n m e t a l was n e c e s s a r y f o r n o r m al g r o w t h and r e p r o d u c t i o n i n t h e mouse. S u b s e q u e n t l y , a number o f i n v e s t i g a t o r s , most n o t a b l y B e l l and H u r l e y ( 1 9 7 3 ) , have e s t a b l i s h e d t h e e s s e n t i a l i t y o f manganese i n a v a r i e t y o f o r g a n i s m s . D e f i c i e n c y i s a s s o c i a t e d w i t h s k e l e t a l a b n o r m a l i t i e s , i n c l u d i n g g r o w t h f a i l u r e , p e r o s i s and c h o n d r o d y s t r o p h y , and v a r i o u s p e r i n a t a l a b n o r m a l i t i e s i n c l u d i n g n e o n a t a l d e a t h , c o n g e n i t a l a t a x i a , d e f e c t i v e o v u l a t i o n and t e s t i c u l a r d e g e n e r a t i o n ( H u r l e y e t a l . , 1970; H u r l e y , 1981; Keen 15 e t a l . , 1984a). The a t a x i a , o r l o s s o f body r i g h t i n g r e f l e x e s , has been shown t o be t h e r e s u l t o f abnormal d evelopment o f t h e i n n e r e a r , w i t h c h a r a c t e r i s t i c d e f e c t i v e m o r p h o g e n e s i s o f t h e o t o l i t h s ( B e l l and H u r l e y , 1973). I n t r a c e l l u l a r a b n o r m a l i t i e s f o u n d i n M n - d e f i c i e n t mice i n c l u d e d e l o n g a t i o n and r e o r i e n t a t i o n o f c r i s t a e i n t h e m i t o c h o n d r i a . H u r l e y e t a l . ( 1 9 7 0 ) a l s o o b s e r v e d a r e d u c e d oxygen u p t a k e i n l i v e r m i t o c h o n d r i a o f s e c o n d g e n e r a t i o n M n - d e f i c i e n t r a t s . No c l e a r - c u t c a s e s o f Mn d e f i c i e n c y i n humans have as y e t been documented. S e v e r a l i s o l a t e d c a s e s i n w h i c h s e e m i n g l y non-s p e c i f i c symptoms have been c u r e d by a d d i t i o n o f manganese t o t h e d i e t s u g g e s t , b u t do n o t p r o v e , t h e e s s e n t i a l i t y o f t h i s u b i q u i t o u s m e t a l i n humans ( O l s e n , 1988). I n a r e c e n t r e p o r t by F r e e l a n d - G r a v e s ( 1 9 8 8 ) , a p o s s i b l e Mn d e f i c i e n c y was i n d u c e d i n 7 men between t h e ages o f 19 and 22 who had been f e d a s e m i -p u r i f i e d d i e t c o n t a i n i n g 0.11 mg Mn/day f o r 39 d a y s . (The recommended n u t r i e n t i n t a k e f o r Mn i s between 2.0 and 5.0 mg/day.) Toward t h e end o f t h e s t u d y , 5 o u t o f 7 s u b j e c t s d e v e l o p e d a s c a l y , e r y t h e m a t o u s r a s h w h i c h c l e a r e d a f t e r Mn s u p p l e m e n t s were begun. Manganese t o x i c i t y i s a l s o r a r e , and f a i r l y h i g h d i e t a r y i n t a k e s seem t o be w e l l - t o l e r a t e d . T o x i c i t y g e n e r a l l y o n l y o c c u r s as t h e r e s u l t o f i n d u s t r i a l e x p o s u r e , e s p e c i a l l y among m i n e r s ( F r e e l a n d - G r a v e s , 1988). Some of t h e p a t h o l o g i c a l c onsequences o f Mn o v e r l o a d a r e r e l a t e d t o c a r b o h y d r a t e m e t a b o l i s m . C h r o n i c 16 i n t a k e o f e x c e s s Mn r e s u l t e d i n a p r o l o n g e d r e a c t i v e h y p o g l y c e m i a f o l l o w i n g a g l u c o s e l o a d (Keen and L o n n e r d a l , 1 986). By c o n t r a s t , an a c u t e change o f Mn s t a t u s , by i n j e c t i o n o f h i g h d o s e s o f Mn, p r o d u c e d h y p e r g l y c e m i a and h y p o i n s u l i n e m i a i n r a t s (Keen e t a l . , 1984b; B a l y e t a l . , 1985a). B e s i d e s a l t e r a t i o n s i n c a r b o h y d r a t e m e t a b o l i s m , v a r i o u s p s y c h o l o g i c a l and n e u r o l o g i c a l symptoms have been f o u n d i n c a s e s o f c h r o n i c Mn o v e r l o a d ( C o t z i a s e t a l . , 1968; Keen and L o n n e r d a l , 1 9 8 6). T y p i c a l symptoms b e a r a c l o s e r e s e m b l a n c e t o P a r k i n s o n ' s d i s e a s e , and a r e p a r t i a l l y r e l i e v e d by a d m i n i s t r a t i o n o f L-dopa (Mena e t a l . , 1970). E x c r e t i o n , r a t h e r t h a n a b s o r p t i o n , i s b e l i e v e d t o be t h e r e g u l a t o r o f Mn h o m e o s t a s i s . B r i t t o n and C o t z i a s (1966) have shown t h a t b i l i a r y e x c r e t i o n i s l i n e a r l y c o r r e l a t e d w i t h d i e t a r y i n t a k e o f Mn i n r a t s . I n t e s t i n a l r e a b s o r p t i o n may p l a y a r o l e i n r e g u l a t i o n o f body s t o r e s and, under c e r t a i n c i r c u m s t a n c e s , up t o o n e - t h i r d o f t h e Mn s e c r e t e d i n t o t h e i n t e s t i n a l lumen may be r e a b s o r b e d . However, t h e main r e g u l a t o r y o r g a n i s t h e l i v e r , w i t h e x c e s s Mn b e i n g e l i m i n a t e d i n b i l e , and a s m a l l e r amount b e i n g e x c r e t e d t h r o u g h t h e u r i n a r y t r a c t o r r e l e g a t e d t o g r o w t h o f h a i r and n a i l s ( B e r t i n c h a m p s e t a l . , 1966; P a p a v a s i l i o u e t a l . , 1 966). Manganese c a n s e r v e b o t h as an enzyme a c t i v a t o r and as a c o n s t i t u e n t o f m e t a l l o e n z y m e s . M n ( I I ) i s a c o f a c t o r f o r k i n a s e s , d e c a r b o x y l a s e s , h y d r o l a s e s and t r a n s f e r a s e s , b u t i s 17 r e p l a c e a b l e by magnesium (Mg)(II) (Brandt and Schramm, 1981). There a r e 3 known metalloenzymes c o n t a i n i n g t i g h t l y - b o u n d Mn: p y r u v a t e c a r b o x y l a s e , s u p e r o x i d e d i s m u t a s e and a r g i n a s e (Schramm, 1982). The manganese i n t h e s e 3 enzymes a c c o u n t s f o r t h e m a j o r i t y (60 - 90 %) o f h e p a t i c manganese. Magnesium can s u b s t i t u e f o r Mn i n bo t h p y r u v a t e c a r b o x y l a s e and a r g i n a s e w i t h l i t t l e l o s s o f c a t a l y t i c a c t i v i t y ( S c r u t t o n e t a l . , 1972). MnSOD, however, i s i n a c t i v a t e d by s u b s t i t u t i o n o f any o t h e r m e t a l i o n . 2 . 2 . 2 Manganese D e f i c i e n c y and MnSOD A c t i v i t y A t t h e b i o c h e m i c a l l e v e l , one o f t h e most i m p o r t a n t and s p e c i f i c e f f e c t s o f Mn d e f i c i e n c y i s a de c r e a s e i n MnSOD a c t i v i t y . U n l i k e o t h e r M n - a c t i v a t e d enzymes (e.g., p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e and p y r u v a t e c a r b o x y l a s e ) , MnSOD does n o t r e t a i n e n z y m a t i c a c t i v i t y when Mn i s r e p l a c e d by a n o t h e r t r a n s i t i o n m e t a l i o n ( S c r u t t o n e t a l . , 1972). DeRosa e t a l . (1980) found t h a t MnSOD a c t i v i t y i n Mn d e f i c i e n t mouse l i v e r s was 17% t h a t o f c o n t r o l s , and i n b r a i n , was 50% t h a t o f c o n t r o l s . They a l s o f o u n d s i g n i f i c a n t r e d u c t i o n s i n MnSOD i n M n - d e f i c i e n t c h i c k e n l i v e r s compared t o c o n t r o l s . I n l i v e r m i t o c h o n d r i a from o f f s p r i n g o f M n - d e f i c i e n t female Sprague-Dawley r a t s , t h e y found no d i f f e r e n c e i n MnSOD compared t o c o n t r o l s ; however, t h e s e o f f s p r i n g had v e r y poor s u r v i v a l , l e s s t h a n 2% by day f o u r . P a y n t e r (1980a), s t u d y i n g Mn d e f i c i e n c y a t v a r i o u s l e v e l s o f d i e t a r y Mn i n male Sprague-Dawleys, found t h a t k i d n e y and h e a r t MnSOD a c t i v i t y l e v e l s were reduced a f t e r 6 weeks on t h e 18 d e f i c i e n t d i e t s . The l e v e l s o f Mn i n t h e d i e t s r a n g e d f r o m 0.2 t o 29.7 mg Mn/kg d i e t and were c l o s e l y c o r r e l a t e d w i t h k i d n e y and h e a r t MnSOD a c t i v i t i e s . M a s t e r s and P a y n t e r (1988) a l s o f o u n d a r e d u c t i o n i n h e a r t MnSOD w i t h v a r y i n g l e v e l s o f d i e t a r y Mn i n sheep and s u g g e s t e d t h a t t h i s t i s s u e may be more s u s c e p t i b l e t o damage c a u s e d by p e r o x i d a t i o n d u r i n g Mn d e f i c i e n c y . That t h e m i t o c h o n d r i a l a b n o r m a l i t i e s , i n c l u d i n g m o r p h o l o g i c a l damage and r e d u c e d oxygen u p t a k e , accompanying Mn d e f i c i e n c y m i g h t be due t o r e d u c t i o n i n MnSOD a c t i v i t y was p o s t u l a t e d by Z i d e n b e r g - C h e r r e t a l . ( 1 9 8 3 ) . T h e i r s t u d y c o r r e l a t e d l i p i d p e r o x i d a t i o n w i t h t h e MnSOD a c t i v i t y d u r i n g development o f fe m a l e Sprague-Dawley r a t s . A s i x f o l d i n c r e a s e i n l i v e r MnSOD a c t i v i t y was o b s e r v e d i n Mn-s u f f i c i e n t r a t s d u r i n g t h e f i r s t 60 days o f development, b u t o n l y a t h r e e f o l d i n c r e a s e was o b s e r v e d i n M n - d e f i c i e n t r a t s d u r i n g t h i s same p e r i o d . M i t o c h o n d r i a l l i p i d p e r o x i d a t i o n was h i g h e r i n M n - d e f i c i e n t r a t s , as was l i v e r CuZnSOD a c t i v i t y . The i n v e s t i g a t o r s p o s t u l a t e d t h a t i n c r e a s e d CuZnSOD d u r i n g Mn d e f i c i e n c y m i g h t be a g e n e r a l compensatory phenomenon. H e a r t MnSOD a c t i v i t y has been p r o p o s e d as an i n d i c a t o r o f Mn s t a t u s ( D a v i s e t a l . , 1990). T h i s s u g g e s t i o n i s s u p p o r t e d by a number o f s t u d i e s i n w h i c h h e a r t MnSOD a c t i v i t y p a r a l l e l e d d i e t a r y Mn i n t a k e (Lee and Johnson, 1984; P a y n t e r , 1980a,b; Z i n d e r b e r g - C h e r r e t a l . , 1983). However, i n c r e a s e d MnSOD a c t i v i t i e s w i t h o u t c o n c o m i t a n t changes i n d i e t a r y Mn have been 19 o b s e r v e d under a v a r i e t y o f c o n d i t i o n s o f i n c r e a s e d o x i d a n t s t r e s s i n c l u d i n g e t h a n o l f e e d i n g i n monkeys (Keen e t a l . , 1985), e x p o s u r e o f y e a s t c e l l s t o h y p e r b a r i c oxygen o r ozone ( W e s t e r b e e k - M a r r e s e t a l . , 1988), and f e e d i n g o f h i g h p o l y u n s a t u r a t e d f a t d i e t s ( D a v i s e t a l . , 1990). H e a r t MnSOD a c t i v i t y w o u l d t h e r e f o r e o n l y be u s e f u l as an i n d i c a t o r o f Mn s t a t u s u n d e r c o n d i t i o n s o f normal o x i d a t i v e m e t a b o l i s m . 2.2.3 Manganese D e f i c i e n c y and Abnormal C a r b o h y d r a t e M e t a b o l i s m A r e l a t i o n s h i p between manganese i n t a k e and a l t e r e d g l u c o s e m e t a b o l i s m was f i r s t s u g g e s t e d by R u b e n s t e i n e t a l . i n 1962. They o b s e r v e d a marked d e c l i n e i n b l o o d s u g a r l e v e l s i n a s e v e r e l y d i a b e t i c p a t i e n t ( i n s u l i n - d e p e n d e n t ) f o l l o w i n g s u p p l e m e n t a t i o n e i t h e r w i t h an i n f u s i o n o f l u c e r n e ( a l f a l f a , Medicago s a t i v a ) , w h i c h i s known t o be h i g h i n manganese, o r w i t h o r a l manganese c h l o r i d e s u p p l e m e n t a t i o n . Two s u b s e q u e n t i n v e s t i g a t i o n s t e n d e d t o c o r r o b o r a t e t h i s f i n d i n g . E v e r s o n and S h r a d e r (1968) f o u n d abnormal g l u c o s e t o l e r a n c e c u r v e s i n Mn-d e f i c i e n t g u i n e a p i g s b o r n t o M n - d e f i c i e n t dams. The e f f e c t was r e v e r s i b l e by d i e t a r y Mn s u p p l e m e n t a t i o n . H i s t o l o g i c a l e x a m i n a t i o n o f t h e d e f i c i e n t a n i m a l s r e v e a l e d h y p e r t r o p h i e d p a n c r e a t i c i s l e t t i s s u e and d e g r a n u l a t e d B - c e l l s , s u g g e s t i n g t h a t Mn d e f i c i e n c y compromised p a n c r e a t i c f u n c t i o n . I n a n o t h e r s t u d y , S h a n i e t a l . (1972) f e d sand r a t s , whose d i e t i s n a t u r a l l y h i g h i n Mn, a low-Mn c o m m e r c i a l r a t chow and p r o d u c e d a d i a b e t i c - l i k e c o n d i t i o n w h i c h was r e v e r s i b l e by r e i n t r o d u c i n g t h e high-Mn n a t u r a l d i e t . 20 I n t h e f i r s t s y s t e m a t i c i n v e s t i g a t i o n o f t h e e f f e c t o f Mn d e f i c i e n c y on c a r b o h y d r a t e h o m e o s t a s i s , B a l y e t a l . (1984) o b s e r v e d an i m p a i r e d g l u c o s e t o l e r a n c e c u r v e i n M n - d e f i c i e n t o f f s p r i n g o f M n - d e f i c i e n t dams f e d a 1 ppm Mn d i e t , compared t o r a t s f e d a 45 ppm Mn d i e t . The a l t e r e d g l u c o s e t o l e r a n c e was f o u n d t o be t h e r e s u l t o f i m p a i r e d i n s u l i n s e c r e t i o n , w i t h l o w e r p a n c r e a t i c i n s u l i n c o n t e n t i n a l l M n - d e f i c i e n t r a t s . An enhanced d e g r a d a t i o n o f i n s u l i n was a l s o o b s e r v e d ( B a l y e t a l . , 1985a). I n v e s t i g a t i o n o f t h e mechanism o f t h e s e changes r e v e a l e d a d e p r e s s e d i n s u l i n mRNA s y n t h e s i s i n M n - d e f i c i e n t , Sprague-Dawley r a t s ( B a l y e t a l . , 1988). M n - d e f i c i e n t d i e t s a l s o d e c r e a s e d a c t i v i t i e s o f l i v e r g l u c o n e o g e n i c enzymes, e s p e c i a l l y t h e manganoenzyme p y r u v a t e c a r b o x y l a s e (PC) (E.C. 6.4.1.1) and t h e M n - a c t i v a t e d enzyme p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e (PEPCK) (E.C. 4.1.1.32) ( B a l y e t a l . 1985b). Impairment o f t h e a c t i v i t y o f b o t h o f t h e s e enzymes i n Mn-d e f i c i e n t o f f s p r i n g was a s s o c i a t e d w i t h h i g h p e r i n a t a l m o r t a l i t y . R e c e n t l y , Mn d e f i c i e n c y was a l s o f o u n d t o have a p r o n o u n c e d e f f e c t on g l u c o s e t r a n s p o r t i n t h e a d i p o s e c e l l ( B a l y , 1988). A 30% d e c r e a s e i n i n s u l i n r e c e p t o r number was o b s e r v e d , a l o n g w i t h pronounced d e c r e a s e s i n b a s a l and i n s u l i n -s t i m u l a t e d g l u c o s e u p t a k e , s u g g e s t i n g t h a t t h e d e f e c t may be a t t h e l e v e l o f t h e t r a n s p o r t p r o t e i n . 21 2.3 D i a b e t e s M e l l i t u s D i a b e t e s m e l l i t u s i s a g e n e r a l t e r m w h i c h r e f e r s t o a v a r i e t y o f m e t a b o l i c d i s o r d e r s whose s o l e u n i f y i n g c h a r a c t e r i s t i c i s p e r s i s t e n t l y e l e v a t e d b l o o d g l u c o s e . The two most common t y p e s o f c l i n i c a l d i a b e t e s a r e termed Type I , o r j u v e n i l e d i a b e t e s , w h i c h i s i n s u l i n - d e p e n d e n t , and Type I I , o r m a t u r i t y - o n s e t d i a b e t e s , w h i c h i s u s u a l l y n o t i n s u l i n - d e p e n d e n t ( R e n o l d e t a l . , 1974) . The e t i o l o g y o f d i a b e t e s m e l l i t u s o f e i t h e r t y p e i s u n c l e a r , . a l t h o u g h a g e n e t i c component has been p o s t u l a t e d f o r b o t h . S e c o n d a r y c o m p l i c a t i o n s i n c l u d e r e t i n o p a t h y , n e u r o p a t h y , a r t e r i o s c l e r o s i s , n e phropathy and i m p a i r e d immune f u n c t i o n ( E l l e n b e r g and R i f k i n , 1983). The p r o x i m a l c a u s e o f t h e h y p e r g l y c e m i a a s s o c i a t e d w i t h Type I d i a b e t e s (IDDM) i s l a c k o f i n s u l i n , w h i c h f a c i l i t a t e s t h e t r a n s p o r t o f g l u c o s e i n t o t h e c e l l s . I n s u l i n i s p r o d u c e d i n t h e 6 c e l l s o f t h e p a n c r e a s and a f f e c t s g l u c o s e u p t a k e , g l y c o g e n o l y s i s , l i p o p r o t e i n s y n t h e s i s and o t h e r b i o c h e m i c a l p athways i n many t i s s u e s o f t h e body. Treatment m o d a l i t i e s u s u a l l y i n c l u d e i n s u l i n r e p l a c e m e n t a t a dosage and a t i n t e r v a l s s u c h t h a t normoglycemia i s m a i n t a i n e d as much o f t h e t i m e a s p o s s i b l e (Koop, 1988). O t h e r m e t a b o l i c a l t e r a t i o n s known t o accompany d i a b e t e s i n c l u d e 22 d e c r e a s e d plasma v i t a m i n A (Basu e t a l . , 1990) and v i t a m i n C (Som e t a l . , 1 9 8 1), plasma, l i v e r , and p a n c r e a s GSH (Loven e t a l . , 1 9 8 3 ) , and p l a t e l e t PGI2 w i t h i n c r e a s e d T x A 2 (Karpen e t a l . , 1 9 8 2 ), a l l o f w h i c h can be l i n k e d t o an i n c r e a s e d " o x i d a n t t o n e " i n d i a b e t e s m e l l i t u s (Chow, 1988; W o l f f , 1987). 2.3.1 E x p e r i m e n t a l D i a b e t e s Much o f what i s known about t h e p a t h o p h y s i o l o g y o f IDDM comes f r o m e x p e r i m e n t a t i o n w i t h a n i m a l models o f t h e d i s e a s e . None o f t h e c u r r e n t l y a v a i l a b l e a n i m a l models e x a c t l y m imics human IDDM; t h e y a r e d i f f e r e n t from each o t h e r and a r e e a c h more o r l e s s s u i t a b l e d e p e n d i n g on what a s p e c t o f t h e d i s e a s e i s b e i n g i n v e s t i g a t e d ( B e l l and Hye, 1983). The two c h e m i c a l models commonly u s e d a r e a l l o x a n - d i a b e t e s and s t r e p t o z o t o c i n - d i a b e t e s . STZ ( 2 - d e o x y - 2 - ( 3 - m e t h y l - 3 - n i t r o s o u r e a ) 1 - d - g l u c o p y r a n o s e ( F i g u r e 3) i s a m e t a b o l i t e o f Streptomyces achromogenes o r i g i n a l l y e x t r a c t e d f o r i t s a n t i o b i o t i c p r o p e r t i e s ( V a v r a e t a l . , 1 960). T h i s compound, c l o s e l y r e l a t e d t o a commonly used a n t i - c a n c e r t h e r a p e u t i c a g e n t , BCNU ( 1 , 3 - b i s ( 2 - c h l o r o e t h y l ) - 1 -n i t r o s u r e a ) , i s a l s o t u m o r i c i d a l , c a r c i n o g e n i c and d i a b e t o g e n i c (Loven and O b e r l e y , 1982). The l a t t e r p r o p e r t y was f i r s t d i s c o v e r e d by R a k i e t a n e t a l . (1963) and has been u t i l i z e d e x t e n s i v e l y s i n c e t h e n ( B e l l and Hye, 1983). STZ i s s e l e c t i v e l y a c c u m u l a t e d i n t h e B c e l l s o f t h e p a n c r e a s and may f u n c t i o n as an o x i d a n t , b i n d i n g t o s u l f h y d r y l groups v i a t h e g l u c o s e m o i e t y o f STZ (Karunanayake e t a l . , 1975). 23 F IGURE 3. Chemical structure of streptozotocin (STZ) 24 STZ a d m i n i s t r a t i o n l e a d s t o p r e f e r e n t i a l d e s t r u c t i o n o f t h e & c e l l s o f t h e p a n c r e a t i c i s l e t s , t o p a t h o l o g i c a l r e n a l changes i n c l u d i n g i n c r e a s e d k i d n e y s i z e , i n c r e a s e i n t h e g l o m e r u l a r c a p i l l a r y f i l t r a t i o n s u r f a c e and i n c r e a s e d g l o m e r u l a r f i l t r a t i o n r a t e ( R a s c h , 1979; B e l l and Hye, 1983). The r e n a l changes a r e v e r y s i m i l a r t o t h o s e s e e n i n t h e e a r l y s t a g e s o f human IDDM. D i a b e t i c r e t i n o p a t h y , a common se c o n d a r y c o m p l i c a t i o n i n human IDDM, does n o t u s u a l l y d e v e l o p w i t h STZ t r e a t m e n t . 2.3.2 O x i d a t i v e S t r e s s i n D i a b e t e s M e l l i t u s F r e e r a d i c a l m e t a b o l i s m i s i n v o l v e d i n IDDM a t a number o f d i f f e r e n t l e v e l s (Loven and O b e r l e y , 1982), i n c l u d i n g i n s u l i n r e l e a s e , g l u c o s e u p t a k e , autoimmune B - c e l l d e s t r u c t i o n , p r o s t a g l a n d i n s y n t h e s i s and t h e development o f d i a b e t i c c o m p l i c a t i o n s . O x i d a n t s t r e s s i n c r e a s e s i n a system when t h e r a t e o f f r e e r a d i c a l p r o d u c t i o n i n c r e a s e s and/or t h e a n t i o x i d a n t mechanisms a r e i m p a i r e d . I n c r e a s e d s u p e r o x i d e r a d i c a l p r o d u c t i o n has been o b s e r v e d i n i n s u l i n - d e p e n d e n t , n o n - k e t o t i c , d i a b e t i c s u b j e c t s (Nath e t a l . , 1984). P r o d u c t i o n o f i n c r e a s e d H 2 O 2 i n e x p e r i m e n t a l d i a b e t e s has a l s o been documented (Loven e t a l . , 1983) . G l u c o s e i t s e l f c a n be a s o u r c e o f s u p e r o x i d e i o n s ( T h o r n a l l y , 1986). M o n o s a c c h a r i d e s e n o l i z e s l o w l y t o p r o d u c e e n e - d i o l s , w h i c h r e a c t n o n e n z y m a t i c a l l y w i t h oxygen t o pr o d u c e a s e m i d i o n e r a d i c a l and s u p e r o x i d e . The s e m i d i o n e r a d i c a l d e cays 25 t o a 1 - h y d r o x y a l k y l r a d i c a l and hydrogen p e r o x i d e a t p h y s i o l o g i c a l pH. The p r e s e n c e o f e x c e s s t r a c e m e t a l i o n s ( e . g . , C u + , w h i c h a c c u m u l a t e s i n l i v e r and k i d n e y o f d i a b e t i c r a t s ) ( F a i l l a and K i s e r , 1981) c a t a l y z e s t h e s e r e a c t i o n s . I m p a i r e d a n t i o x i d a n t s c a v e n g i n g i n d i a b e t e s c a n be i n f e r r e d f r o m t h e many s t u d i e s d e m o n s t r a t i n g i n c r e a s e d l i p i d p e r o x i d a t i o n i n IDDM and e x p e r i m e n t a l d i a b e t e s . Hunt e t a l . (1990) have d e m o n s t r a t e d t h a t p e r o x i d a t i o n and g l y c o s y l a t i o n o f LDL o c c u r c o n c o m i t a n t l y i n d i a b e t e s m e l l i t u s . L i p i d p e r o x i d a t i o n i n d i a b e t i c VLDL and LDL have a l s o been s u g g e s t e d t o be d i r e c t l y r e l a t e d t o development o f d i a b e t i c c o m p l i c a t i o n s ( M o r e l and C h i s h o l m , 1989). I n c r e a s e d s u s c e p t i b i l i t y o f e r y t h r o c y t e s t o H 2 O 2 - i n d u c e d p e r o x i d a t i o n i n S T Z - d i a b e t e s has been shown (Wohaieb and G o d i n , 1987a). T h e r e may be b o t h p r o - o x i d a t i v e and a n t i - o x i d a t i v e f o r c e s a t work i n d i a b e t e s , s i n c e e l e v a t e d c o n c e n t r a t i o n s o f g l u c o s e i n t h e b l o o d a r e n o t p a r a l l e l l e d by h i g h i n t r a c e l l u l a r c o n c e n t r a t i o n s o f g l u c o s e i n a l l t i s s u e s . I n h e a r t and p a n c r e a s , w h i c h a r e dependent on i n s u l i n f o r g l u c o s e u p t a k e , a r e l a t i v e l a c k o f i n t r a c e l l u l a r g l u c o s e w o u l d e x i s t i n t h e d i a b e t i c s t a t e . L a c k o f g l u c o s e would l e a d t o d e c r e a s e d hexose monophosphate s h u n t a c t i v i t y , w i t h d e c r e a s e d p r o d u c t i o n o f NADPH, and hence i m p a i r e d GSH r e g e n e r a t i o n . T h i s i n i t s e l f m i g h t c o n s t i t u t e an i n c r e a s e d o x i d a t i v e s t r e s s s i t u a t i o n ( A l l e n , 1 9 9 0 ) . On t h e o t h e r hand, Sagone e t a l . (1982) have shown t h a t 26 g l u c o s e i s a weak scavenger o f OH* r a d i c a l s , such t h a t i n t h e p h y s i o l o g i c a l c o n c e n t r a t i o n o f g l u c o s e e n c o u n t e r e d i n u n c o n t r o l l e d d i a b e t i c plasma (>440 mg/dl), t h e m i c r o b i c i d a l f u n c t i o n o f p h a g o c y t i c c e l l s , w hich a r e dependent on OH* p r o d u c t i o n , might be i n t e r f e r e d w i t h , t h u s c a u s i n g i n c r e a s e d s u s c e p t i b i l i t y t o i n f e c t i o n . Rucker e t a l . (1991) found no i n c r e a s e i n spontaneous l i p i d p e r o x i d a t i o n i n plasma o f STZ-d i a b e t i c r a t s . N o n e t h e l e s s , a m a j o r i t y o f r e s e a r c h e r s f a v o u r t h e h y p o t h e s i s t h a t d i a b e t e s r e p r e s e n t s a c o n d i t i o n o f i n c r e a s e d o x i d a n t s t r e s s . 2 . 4 V i t a m i n E Among nonenzymatic a n t i o x i d a n t s , v i t a m i n E i s t h e p r i n c i p a l c h a i n - b r e a k i n g a n t i o x i d a n t w i t h i n mammalian membranes ( G u t t e r i d g e and H a l l i w e l l , 1990) and t h e o n l y l i p o p h i l i c a n t i o x i d a n t i n b l o o d ( N i k i , 1988). Of t h e 4 t o c o l and 4 t o c o t r i e n o l d e r i v a t i v e s p o s s e s s i n g v i t a m i n E a c t i v i t y , t h e most common and p o t e n t i s a - t o c o p h e r o l ( F i g u r e 4) ( D e s a i , 1989). A l l a r e s i m i l a r i n t h a t t h e y have a 6-chromanol r i n g s t r u c t u r e and a s i d e c h a i n . They can be d i f f e r e n t i a t e d by t h e number and l o c a t i o n o f m e t h y l groups and t h e number o f d o u b l e bonds. A l p h a - t o c o p h e r o l o c c u r s f r e e i n b l o o d and i n t h e a c e t a t e e s t e r form i n l i v e r . The major b i o l o g i c a l r o l e o f v i t a m i n E i s as a c h a i n - b r e a k i n g a n t i o x i d a n t i n b i o l o g i c a l membranes ( T a p p e l , 1972, 1973). V i t a m i n E i n t e r f e r e s w i t h l i p i d h y d r o p e r o x i d e f o r m a t i o n w h i c h , 27 u n s cavanged, would p r o c e e d a c c o r d i n g t o t h e f o l l o w i n g s t e p s : L* + 0 2 > L 0 2 + " (1) L 0 2 * + LH ---—> LOOH + L* These s t e p s would t e n d t o i n i t i a t e a c h a i n r e a c t i o n i n t h e membrane. The p r e s e n c e o f a - t o c o p h e r o l (aTH) i n t e r f e r e s w i t h t h i s p r o c e s s by d o n a t i n g a hydrogen ( T a p p e l , 1972), as f o l l o w s : L 0 2 * + aTH > LOOH + aT* R e c e n t e v i d e n c e (Chow, 1988) has shown t h a t t h i s r e a c t i o n p r o c e e d s t h r o u g h f o r m a t i o n o f a quinone i n t e r m e d i a t e , w i t h r e g e n e r a t i o n o f r e d u c e d a - t o c o p h e r o l by a s c o r b i c a c i d ( v i t a m i n C) o r g l u t a t h i o n e (GSH) ( N i k i , 1987) i n a s y n e r g i s t i c a s s o c i a t i o n between t h e two v i t a m i n s . The r o l e o f v i t a m i n E i n t h e p r e v e n t i o n o f l i p i d h y d r o p e r o x i d e f o r m a t i o n i s one o f an intramembrane, n o n e n z y m a t i c s c a v e n g e r . The 1:1 s t o i c h i o m e t r i c r a t i o o f s c a v e n g e r : o x i d a n t i s n e c e s s a r i l y l e s s e f f i c i e n t t h a n e n z y m a t i c c a t a l y s i s ; however, v i t a m i n E p l a y s a v i t a l r o l e as t h e o n l y l i p o p h i l i c backup s c a v e n g e r ( L e w i s and D e l M a e s t r o , 1980). Other f u n c t i o n s o f v i t a m i n E i n c l u d e s c a v e n g i n g o f s i n g l e t oxygen (Grams and E s k i n s , 1972) and i n h i b i t i n g s y n t h e s i s o f x a n t h i n e o x i d a s e , a g e n e r a t o r o f s u p e r o x i d e r a d i c a l s ( C a t i g n a n i e t a l . , 1974). HO. CHj i^CH 2(CH 2CH 2CHCH 2),H Ri a- Tocopherol: r = Tocopherol: R1 = R ? = C H ? ; R2 = 7- Tocopherol: R^  = ^ = CH3> R3 = H H 6- Tocopherol: R1 = CH T ; R 2 = R ? = H FIGURE 4. Vitamin E structure. 29 2.5 G l u t a t h i o n e A n o t h e r o f t h e nonenzymatic a n t i o x i d a n t s c a v e n g e r s p r e s e n t i n most a n i m a l t i s s u e s i s GSH, a t r i p e p t i d e o f c y s t e i n e , g l y c i n e , and g l u t a m i c a c i d ( F i g u r e 5) (Chow, 1988). I t i s a c o - s u b s t r a t e f o r GSHPx, a s t a b i l i z e r o f s u l f h y d r y l - c o n t a i n i n g enzymes, a c e l l u l a r r e d o x b u f f e r , and a h y d r o p h i l i c t i s s u e a n t i o x i d a n t ( M e i s t e r , 1983; M e i s t e r and Anderson, 1983). N e u t r a l i z a t i o n o f t i s s u e o x i d a n t s o c c u r s by r e a c t i o n w i t h t h e s u l f h y d r y l group of t h e c y s t e i n e m o i e t y o f GSH ( A l l e n , 1991). GSH i s t h e most abundant o f c e l l u l a r r e d u c i n g a n t i o x i d a n t s , w i t h c o n c e n t r a t i o n s f r e q u e n t l y e x c e e d i n g 1 mM. Under c o n d i t i o n s o f i n c r e a s e d o x i d a t i v e s t r e s s i n a v a r i e t y o f d i s e a s e s t a t e s , o x i d i z e d g l u t a t h i o n e (GSSG) i n c r e a s e s ( S i e s e t a l . , 1972). T h i s i s e s p e c i a l l y t r u e i n i n s t a n c e s o f d i e t a r y p r o t e i n r e s t r i c t i o n , when r e d u c e d l e v e l s o f d i e t a r y c y s t e i n e may p r e c l u d e i n c r e a s e d GSH s y n t h e s i s (Denecke e t a l . , 1983). I n o t h e r c i r c u m s t a n c e s , r e d u c e d g l u t a t h i o n e (GSH) c o n c e n t r a t i o n i n c r e a s e s i n r e s p o n s e t o o x i d a t i v e c h a l l e n g e s , e.g., i n c r e a s e d ambient 0 2 and e x p o sure t o p a r a q u a t i n i n s e c t s ( A l l e n , 1991). These a r e r e p r e s e n t a t i v e o f i n d u c e d GSH s y n t h e s i s i n response t o o x i d a t i v e c h a l l e n g e i n t h e w e l l - f u n c t i o n i n g organism. 2.6 O t h e r nonenzymatic a n t i o x i d a n t s O t h e r h y d r o p h i l i c c h a i n - b r e a k i n g a n t i o x i d a n t s i n c l u d e u r i c a c i d , c y s t e i n e and a s c o r b i c a c i d . I n t r a c e l l u l a r g l u c o s e can a l s o N H 2 I . H O O C C H C H 2 C H 2 C O — N H H S C H 2 C H C O — N H C H 2 C O O H FIGURE 5. Glutathione structure. w o 31 f u n c t i o n a s a weak o x i d a n t s c a v e n g e r . A t l e a s t p a r t o f t h e a n t i o x i d a n t f u n c t i o n o f a s c o r b i c a c i d ( v i t a m i n C) i s i n r e g e n e r a t i n g r e d u c e d a - t o c o p h e r o l . I t a l s o h e l p s m a i n t a i n GSH i n a r e d u c e d s t a t e . A p r e c u r s o r o f v i t a m i n A, 6 - c a r o t e n e , i s an e f f e c t i v e q uencher o f s i n g l e t oxygen and a r a d i c a l - t r a p p i n g a n t i o x i d a n t . A n o t h e r a s p e c t o f t h e c e l l u l a r a n t i o x i d a n t d e f e n s e s y s t e m w h i c h w i l l n o t be d e a l t w i t h i n any d e t a i l h e r e i s m e t a l i o n s e q u e s t r a t i o n . A l t h o u g h f r e e (uncomplexed) m e t a l i o n s s u c h as Fe ( I I ) and Cu ( I ) a r e r a r e l y f o u n d i n t r a c e l l u l a r l y , t h e y may i n c r e a s e i n c o n c e n t r a t i o n as a r e s u l t o f a v a r i e t y o f d i s e a s e c o n d i t i o n s . T h e i r p r e s e n c e promotes f o r m a t i o n o f f r e e r a d i c a l s s u c h as OH*, L* , and LOO*. M e t a l - c h e l a t i n g compounds s u c h as f e r r i t i n , t r a n s f e r r i n , and c e r u l o p l a s m i n may t h e n p l a y an i m p o r t a n t a n t i o x i d a n t r o l e by removing e x c e s s f r e e m e t a l i o n s . 2.7 A n t i o x i d a n t i n t e r a c t i o n s A l l t i s s u e a n t i o x i d a n t s a r e c l o s e l y i n t e r r e l a t e d i n o v e r a l l c e l l u l a r d e f e n s e a g a i n s t o x i d a t i v e damage. The t y p e s o f i n t e r r e l a t i o n s h i p s v a r y : t h e y may be complementary o r a n t a g o n i s t i c , s y n e r g i s t i c , compensatory o r i n t e r d e p e n d e n t (Chow, 1988) . Examples o f s y n e r g i s t i c e f f e c t s i n c l u d e t h e f u n c t i o n i n g o f v i t a m i n s E and C ( N i k i , 1988), and t h e e f f e c t i v e n e s s o f Se s u p p l e m e n t a t i o n i n p r e v e n t i n g v i t a m i n E- and combined v i t a m i n E-and S e - d e f i c i e n c y symptoms i n r a t s ( D r a p e r , 1980). A s y n e r g i s m between v i t a m i n E and Se a g a i n s t mammary tumors i n d u c e d i n r a t s by 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e has been o b s e r v e d ( I p , 1988). 32 An example o f a compensatory r e s p o n s e i s i n c r e a s e d CuZnSOD a c t i v i t y accompanying d e c r e a s e d a c t i v i t y o f MnSOD i n Mn-d e f i c i e n t c h i c k e n l i v e r s (deRosa e t a l . , 1980). L i k e w i s e , CuZnSOD a c t i v i t y i s e l e v a t e d i n G S S G R d - d e f i c i e n t p a t i e n t s (Chow, 1988). B o t h CAT and GSHPx a c t i v i t y i n c r e a s e d i n c e r t a i n t i s s u e s o f r a t s m a i n t a i n e d on a v i t a m i n E - d e f i c i e n t d i e t (Lee e t a l . , 1981) . GSHPx, GSSGRd,and G-6-Pd a c t i v i t i e s were i n c r e a s e d i n p e r i r e n a l a d i p o s e , p a r a e p i d i d y m a l a d i p o s e and m u s c l e t i s s u e s ( b u t n o t i n l i v e r , l u n g o r k i d n e y ) o f v i t a m i n E - d e f i c i e n t r a t s (Chow e t a l . , 1973). An example o f i n t e r d e p e n d e n c e i s t h e r e l a t i o n s h i p o f v i t a m i n E and a v a r i e t y o f t i s s u e m e t a b o l i t e s c a p a b l e o f r e g e n e r a t i n g a-t o c o p h e r o l , GSH among them (McCay e t a l . , 1986, Reddy e t a l . , 1982) . A n o t h e r example i s t h e i n t e r d e p e n d e n c e o f GSHPx, GSSGRd, GSH, and NADPH ( v i d e supra). C o n t r a d i c t o r y e f f e c t s i n c l u d e t h o s e o f a s c o r b i c a c i d , w h i c h can a c t b o t h a s an a n t i o x i d a n t o r a p r o o x i d a n t ( F r e i e t a l . , 1989) and o f CuZnSOD, w h i c h c a n b o t h i n h i b i t and s t i m u l a t e t h e r e d u c t i o n o f oxygen by h y d r o q u i n o n e s (Bandy e t a l . , 1 9 9 0 ) . These a p p a r e n t c o n t r a d i c t i o n s may r e l a t e t o l e v e l s o f uncomplexed m e t a l i o n s and hence may be more o b v i o u s in vitro t h a n in vivo. Under normal p h y s i o l o g i c a l c o n d i t i o n s , uncomplexed m e t a l i o n s a r e r a r e ; o n l y i n p a t h o l o g i c a l s i t u a t i o n s w o u l d f r e e m e t a l i o n s be r e l e a s e d from b i n d i n g p r o t e i n s ( F r e i e t a l . , 1989; H a l l i w e l l and G u t t e r i d g e , 1986). 33 Complementary e f f e c t s i n c l u d e a v a r i e t y o f c o m b i n a t i o n s o f a n t i o x i d a n t s w h i c h have r e c e n t l y been t r i e d i n v a r i o u s t r e a t m e n t m o d a l i t i e s , f o r e x a m p l e , . r e t i n o i d s and Se a g a i n s t p a n c r e a t i c c a n c e r (Curphey e t a l . , 1988). A n o t h e r example o f c o m p l e m e n t a r i t y i s t h e c h a i n - b r e a k i n g a c t i v i t i e s o f v i t a m i n E and B - c a r o t e n e . The f o r m e r i s most e f f e c t i v e a t h i g h oxygen c o n c e n t r a t i o n s and t h e l a t t e r a t low oxygen c o n c e n t r a t i o n s (Chow, 1988). C l a r i f i c a t i o n o f t h e v a r i o u s i n t e r r e l a t i o n s h i p s among a n t i o x i d a n t s may l e a d t o more e f f e c t i v e t r e a t m e n t o f a v a r i e t y o f d i s e a s e s , d i a b e t e s i n c l u d e d . F o r u n d e r s t a n d i n g o f e n z y m a t i c p r o c e s s e s , c o f a c t o r d e p r i v a t i o n can be a u s e f u l model. The p u r p o s e o f t h i s s t u d y was t o d e t e r m i n e t h e e f f e c t o f manganese d e f i c i e n c y , w i t h and w i t h o u t s u p e r i m p o s i t i o n o f v i t a m i n E d e f i c i e n c y , on t i s s u e ( k i d n e y , h e a r t , p a n c r e a s and l i v e r ) a n t i o x i d a n t s i n S T Z - d i a b e t i c r a t s . 34 3. MATERIALS AND METHODS 3.1 C h e m i c a l s and r e a g e n t s A l l c h e m i c a l s and r e a g e n t s were o b t a i n e d f rom Sigma C h e m i c a l Co. ( S t . L o u i s , MO), u n l e s s o t h e r w i s e s p e c i f i e d . D i e t components were o b t a i n e d f rom ICN B i o c h e m i c a l s ( C l e v e l a n d , OH). 3.2 A n i m a l s and D i e t s W e a n l i n g , male Sprague-Dawley r a t s ( C h a r l e s R i v e r , Quebec, Canada) were i n d i v i d u a l l y housed i n s t a i n l e s s - s t e e l s c r e e n b o t t o m c a g e s i n a t e m p e r a t u r e - and l i g h t - c o n t r o l l e d room (21 ± 1°C, 12-h l i g h t - d a r k c y c l e ) . A n i m a l s were f e d ad l i b i t u m p u r i f i e d d i e t s t h a t were e i t h e r M n - d e f i c i e n t (1 ppm by a n a l y s i s ) o r M n - s u f f i c i e n t (45 ppm by a n a l y s i s ) (See T a b l e 1 ) , as p r e v i o u s l y d e s c r i b e d by deRosa e t a l . ( 1 9 8 0 ) . D i e t s were made up i n 5 k g b a t c h e s a p p r o x i m a t e l y e v e r y 2 weeks and m a i n t a i n e d a t 5°C between f e e d i n g s . D e i o n i z e d w a t e r was p r o v i d e d ad l i b i t u m . D a i l y f o o d c o n s u m p t i o n was r e c o r d e d and r a t s were weighed w e e k l y . The o v e r a l l e x p e r i m e n t a l d e s i g n encompassed two p a r t s ( F i g u r e 6 ) . F o r t h e f i r s t e x p e r i m e n t , 32 r a t s were randomly a l l o c a t e d t o 1 o f 4 g r o u p s : M n - s u f f i c i e n t , n o n - d i a b e t i c (+MnND); Mn-d e f i c i e n t , n o n - d i a b e t i c (-MnND); M n - s u f f i c i e n t , d i a b e t i c (+MnD); o r M n - d e f i c i e n t , d i a b e t i c (-MnD). E x p e r i m e n t 1 was d e s i g n e d t o 35 TABLE 1. Diet for diabetic and non-diabetic Sprague-Dawley rats Diet composition Vitamin Mix Salt Mix %(w/w) (g/kg diet) (g/kg diet) Protein (casein3) 30 myo-inositol 0.37500 CaC03 18.00 Salt mix 6 ascorbic acid 0.07500 K2HPO4 19.26 Vitamin mix 1.5 Ca-pantothenate 0.03750 NaCl 10.08 Alpha-cel 545 Thiamine-HCl 0.02250 MgS04 3.60 Corn oilb 8.0 Pyridoxine-HCl 0.02250 CaHP04 3.60 Nicotinic acid 0.02250 FeSO4'7H20 1.50 Menadione 0.01875 KI 0.048 Riboflavin 0.00750 ZnC03 0.015 PABA 0.00750 CUSO4H2O 0.018 Folic acid 0.00045 CrK(SO4)2l2H20 0.021 Vitamin E c 0.17850 Mn(S04)2H20f 0.138 Vitamin A d 0.04095 Vitamin D e 0.00345 Vitamin B12 0.00004 Choline-Cl 0.75080 ^tamin-free casein (ICN) °tocopherol-free lard substituted for corn oil in vitamin E-deficient diets cDL-alpha tocopherol powder, 250 IU/gram (ICN), included in vitamin E-sufficient diets only ^retinol palmitate (Sigma) eactivated 7-dehydrocholesterol cholecalciferol, D 3 (Sigma) i^ncluded in manganese-sufficient diets only 3 6 Manganese-deficient non-diabetic (-MnND) n = 8 diabetic-4 wks (-MnD4) n = 8 Manganese-sufficient non-diabetic (+ MnND) n = 8 diabetic-4 wks (+ MnD4) n = 8 Experiment 2: Manganese-deficient diabetic-4 wks (MnD4) n =. 4 diabetic-8wks (MnD8) n = 4 diabetic-4 wks vit E-deficient (MnDE-) n = 8 Manganese-sufficient diabetic-4 wks (+ MhD4) n = 4 diabetic-4 wks vit E-deficient (+ MnDE-) n = 8 diabetic-8wks ( + MnD8) n = 4 F IGURE 6. Experimental design. Experiment 1: weanling, male, Sprague-Dawley rats were fed purified diets, either Mn-deficient (1 ppm Mn) or Mn-sufficient (45 ppm Mn) for 8 weeks. Diabetes was then induced in rats allocated to groups -MnD4 (Mn-deficient, 4 week-diabetic) and +MnD4 (Mn-sufficient, 4 week-diabetic) by tail vein injection of STZ (60 mg STZ/kg body weight). Al l rats (including ND, non-diabetic) were fed the purified diets for another 4 weeks. Experiment 2: weanling, male, Spragure-Dawley rats were fed purified diets: Mn-deficient, vitamin E-deficient; Mn-sufficient, vitamin E-sufficient; Mn-sufficient, vitamin E-deficient; or Mn-sufficient, vitamin E-sufficient, for 8 weeks prior to diabetic induction. Vitamin E-sufficient rats were injected with 60 mg STZ/kg body weight; vitamin E-deficient rats, with 45 mg STZ/kg body weight. Rats were then maintained on the purified diets for a further 4 weeks or, in groups -MnD8 (Mn-deficient, 8 week-diabetic) and + MnD8 (Mn-sufficient, 8 week-diabetic), for a further 8 weeks before termination. 37 answer t h e p r i m a r y q u e s t i o n : does M n - d e f i c i e n c y e x a c e r b a t e STZ-d i a b e t e s ? F o r t h e second e x p e r i m e n t , 3 6 r a t s were randomly a l l o c a t e d t o 1 o f 4 gr o u p s : M n - s u f f i c i e n t , d i a b e t i c (+MnD), Mn-d e f i c i e n t , d i a b e t i c (-MnD), M n - s u f f i c i e n t , d i a b e t i c , v i t a m i n E d e f i c i e n t (+MnDE-), o r M n - d e f i c i e n t , d i a b e t i c , v i t a m i n E d e f i c i e n t (-MnDE-). E x p e r i m e n t 2 was i n t e n d e d t o answer a s e c o n d m a j o r q u e s t i o n : does t h e absence o f v i t a m i n E "unmask'1 o r e x a c e r b a t e manganese d e f i c i e n c y e f f e c t s t h a t were a b s e n t o r un m e a s u r a b l y s m a l l i n Expe r i m e n t 1? S i n c e E x p e r i m e n t 2 r e q u i r e d t h a t h a l f o f t h e r a t s be f e d a v i t a m i n E d e f i c i e n t d i e t , a l l d i e t s f o r t h i s e x p e r i m e n t were p r e p a r e d w i t h t o c o p h e r o l - s t r i p p e d l a r d r e p l a c i n g c o r n o i l . F o r +MnDE- and -MnDE- g r o u p s , a-t o c o p h e r o l was a l s o e l i m i n a t e d from t h e v i t a m i n mix. The d i e t s were o t h e r w i s e unchanged from E x p e r i m e n t 1. An o v e r v i e w o f t h e e x p e c t e d c o u r s e o f t h i s i n v e s t i g a t i o n i s p r e s e n t e d i n F i g u r e 7. An addendum t o E x p e r i m e n t 2 was d e v i s e d t o answer a t h i r d q u e s t i o n : does l e n g t h o f t i m e t h a t t h e r a t s a r e d i a b e t i c a l t e r t i s s u e a n t i o x i d a n t b a l a n c e ? ( F i g u r e 6 ) . F o r t h e p u r p o s e s o f t h i s s t u d y , 4 r a t s from each o f t h e 2 g r o u p s : +MnD and -MnD i n e x p e r i m e n t 2 were m a i n t a i n e d f o r an a d d i t i o n a l 4 weeks (8 weeks t o t a l , a f t e r STZ t r e a t m e n t ) and compared w i t h t h e 4 week-d i a b e t i c r a t s . These groups were t h e n renamed +MnD4, -MnD4, +MnD8, and -MnD8 t o d i f f e r e n t i a t e between them. Y E S D o e s v i tamin E de f i c i enc y amp l i f y th is e f f e c t ? Dot v i tan supp lem prevent th 38 tin E entat ion is e f f e c t ? Conc lude : •Mn exacerbates oxidant s t ress Jn S T Z - d i abe tes . Ident i fy c o m p e n s a t o r y c h a n g e s Are c h a n g e s in t i s sue levels of Mn para l le l to M n S O D T,i ? C o n c l u d e : •Mn d o e s not a f fec t S T Z - d i abe tes Conc lude : Mn may be marginally def ic ient for S T Z - diabetes FIGURE 7. Fjcperimental rationale. Mn, manganese; -Mn, manganese-deficiency, STZ, streptozotocin; MnSOD, manganese superoxide dismutase. 03 39 3.2.1 I n d u c t i o n o f D i a b e t e s A l l r a t s were m a i n t a i n e d on t h e p u r i f i e d d i e t s f o r 8 weeks p r i o r t o d i a b e t i c i n d u c t i o n . D i a b e t e s was i n d u c e d by t a i l v e i n i n j e c t i o n o f s t r e p t o z o t o c i n (STZ) (60 mg/kg body w e i g h t , i n s t e r i l e s a l i n e , f r e s h l y p r e p a r e d ) . D i a b e t e s was c o n f i r m e d by b l o o d g l u c o s e measurement (Accu-chek b l o o d g l u c o s e m o n i t o r , Quebec, Canada) 24 hours a f t e r STZ t r e a t m e n t and a g a i n p r i o r t o a u t o p s y . B l o o d g l u c o s e l e v e l s g r e a t e r t h a n 11 mM were a c c e p t e d as d i a b e t i c (Unger, 1991). F o r groups +MnDE- and -MnDE-, i t was n e c e s s a r y t o a d j u s t t h e dosage o f STZ downward t o 45 mg/kg body w e i g h t i n o r d e r t o a c h i e v e t h e same degree o f h y p e r g l y c e m i a as v i t a m i n E - s u f f i c i e n t groups w i t h o u t e x c e s s i v e m o r t a l i t y ( S l o n i m e t a l . , 1983). 3.2.2 G l u c o s e T o l e r a n c e T e s t i n g R a t s i n groups +MnD and -MnD o f experiment 2 were t e s t e d f o r g l u c o s e t o l e r a n c e 1 week p r i o r t o autopsy. A l l r a t s were f a s t e d o v e r n i g h t p r i o r t o t e s t i n g . R a t s i n groups +MnD4 and -MnD4, l i g h t l y a n e s t h e t i z e d under h a l o t h a n e , were g i v e n 3.0 ml o f a 180 mg/100 g body w e i g h t g l u c o s e l o a d ( i n water) a d m i n i s t e r e d o r a l l y from a 5-ml s y r i n g e f i t t e d w i t h a r o u n d - t i p p e d , c u r v e d m e t a l f e e d t u b e . Because o f t h e d i f f i c u l t y o f a d m i n i s t e r i n g 3 ml o f g l u c o s e s o l u t i o n t o r a t s , t h e p r o t o c o l f o r g l u c o s e t o l e r a n c e t e s t i n g was m o d i f i e d s l i g h t l y . A n i m a l s i n groups +MnD8 and -MnD8, u n a n e s t h e t i z e d , had v a r i a b l e amounts (<1 m l , volume a d j u s t e d t o g i v e an o r a l dose o f 180 mg g l u c o s e / l O O g body 40 w e i g h t ) o f a 500 mg/ml g l u c o s e s o l u t i o n a d m i n i s t e r e d by stomach t u b e . B l o o d samples were t a k e n v i a t a i l v e i n , s t a r t i n g a t 1300 h o u r s , i m m e d i a t e l y p r i o r t o t h e g l u c o s e l o a d , and e v e r y 3 0 m i n u t e s t h e r e a f t e r t o 120 m i n u t e s p o s t - g l u c o s e l o a d . 3.3 T i s s u e C o l l e c t i o n A t t h e t e r m i n a t i o n o f each e x p e r i m e n t , r a t s were a n e s t h e t i z e d w i t h h a l o t h a n e (MTC P h a r m a c e u t i c a l s , Cambridge, O n t a r i o , Canada) and b l o o d was o b t a i n e d by c a r d i a c p u n c t u r e i n t o h e p a r i n i z e d s y r i n g e s . H e a r t , l i v e r , k i d n e y s and p a n c r e a s were removed, f l a s h - f r o z e n i n l i q u i d n i t r o g e n and s t o r e d a t -70°C f o r <3 months p e n d i n g a n a l y s i s . I n a d d i t i o n , r e n a l a d i p o s e t i s s u e s a m ples were c o l l e c t e d from r a t s i n e x p e r i m e n t 2 and s t o r e d a t -70°C. The o v e r a l l s c h e d u l e f o r a n a l y s i s o f t i s s u e s and t i s s u e homogenates i s p r e s e n t e d i n F i g u r e s 8 and 9. A l l a n a l y s e s were p e r f o r m e d i n d u p l i c a t e , o r i n t r i p l i c a t e where p r a c t i c a l . 3 . 4 . 1 T h i o b a r b i t u r i c A c i d R e a c t i v e S u b s t a n c e s (TBARS) A s s a y L i p i d p e r o x i d a t i o n was a s s e s s e d by measurement o f t h i o b a r b i t u r i c a c i d - r e a c t i v e s u b s t a n c e s (TBARS) f o l l o w i n g p e r o x i d a t i v e c h a l l e n g e w i t h H 2 O 2 , a c c o r d i n g t o t h e method o f Wohaieb and G o d i n (1987) . B l o o d was c e n t r i f u g e d f o r 10 m i n u t e s @ 3000 rpm i n a t a b l e t o p c l i n i c a l c e n t r i f u g e , plasma and b u f f y c o a t were removed, and r e d b l o o d c e l l s were washed t w i c e w i t h i s o t o n i c s a l i n e c o n t a i n i n g 2.0 mM Na a z i d e . Ten p e r c e n t s u s p e n s i o n s o f r e d b l o o d c e l l s h e p a r i n i z e d t u b e s R B C ' s - c e n t r i f u g e p l a s m a 0 . 0 5 m l f r e e z e i H b A l c 0.1 m l f r e s h I T B A R S 0 .2 m l f r e s h I CAT 0.4 m l f r e e z e I G S H P x a n d C u Z n S O D 0 . 7 5 m l f r e e z e I i n s u l i n by R IA 1.5 m l f r e e z e I V i t E FIGURE 8. Blood sample preparation. Hb, hemoglobin; RCH, red cell hemolysis; RBC's, erythrocytes; HbA l c , glycosylated hemoglobin; TBARS, thiobarbituric acid reactive substances (H2C>2-induced); CAT, catalase; GSHPx, glutathione peroxidase; CuZnSOD, copper-zinc superoxide dismutase; RIA, radioimmunoassay; Vit E, vitamin E. 1 CuZnSOD MnSOD GSHPx GSSGRd (28° C) (28° C) (37° C) (37° C) FIGURE 9. Tissue sample preparation. TBARS, thiobarbituric acid reactive substances (H202-mduced); [Mn], manganese concentration; AAS, atomic absorption spectrophotometry; Vit E, vitamin E; HPLC, high pressure liquid chromatography, CAT, catalase; EtOH, absolute ethanol; GSH, reduced glutathione; CuZnSOD, copper-zinc superoxide dismutase; MnSOD, manganese superoxide dismutase; GSHPx, glutathione peroxidase; GSSGRd, glutathione reductase. 43 were made up i n s a l i n e - a z i d e . A l i q u o t s , 0.5 m l , were p r e -i n c u b a t e d f o r 5 min a t 37°C, t h e n t r e a t e d i n t h e same manner as t h e t i s s u e homogenates f o r t h e r e m a i n d e r o f t h e a s s a y p r o c e d u r e . F o r t i s s u e s , 10% homogenates were p r e p a r e d i n 10 mM p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.4 c o n t a i n i n g 30 mM K C l . Homogenates were a l i q u o t e d , 0.5 m l / a l i q u o t , i n t o 1.5 ml Eppendorf c e n t r i f u g e t u b e s . F o r c e d p e r o x i d a t i o n was i n i t i a t e d by a d d i t i o n o f 0.5 ml t - b u t y l h y d r o p e r o x i d e (100 / i l 30 mM t - b u t y l h y d r o p e r o x i d e i n t o 6.9 ml w a t e r ) . Tubes were i n c u b a t e d a t 37°C f o r 30 m i n u t e s . The r e a c t i o n was s t o p p e d by a d d i t i o n o f 0.5 ml T C A - a r s e n i t e (28% t r i c h l o r o a c e t i c a c i d [TCA] + 0.1M sodium a r s e n i t e ) , f o l l o w e d by c e n t r i f u g a t i o n a t 6000 g f o r 10 m i n u t e s . One ml a l i q u o t s o f t h e s u p e r n a t a n t were removed t o P y r e x t u b e s , 0.5 ml 1.0% t h i o b a r b i t u r i c a c i d (TBA) i n 0.01N NaOH added, and t h e t u b e s p l a c e d i n a b o i l i n g w a t e r b a t h f o r 15 m i n u t e s . S o l u t i o n s were d i l u t e d w i t h 0.5 ml d i s t i l l e d w a ter p r i o r t o a b s o r b a n c e measurement a t 532 nm u s i n g a H i t a c h i - C o l e m a n s p e c t r o p h o t o m e t e r . R e s u l t s were c a l c u l a t e d from a s t a n d a r d c u r v e u s i n g m a l o n d i a l d e h y d e b i s ( d i e t h y l a c e t a l ) , 98% ( A l d r i c h C h e m i c a l Co., M i l w a u k e e , WI) as t h e s t a n d a r d . 3.4.2 Red C e l l H e m o l y s i s A s s a y Red c e l l h e m o l y s i s was measured a c c o r d i n g t o t h e method o f Gyorgy e t a l . (1952). F i f t e e n d r o p s o f b l o o d were c o l l e c t e d i n t o 2.0 ml 0.9% NaCI s o l u t i o n c o n t a i n i n g 1% sodium c i t r a t e . The s u s p e n s i o n was c e n t r i f u g e d a t 2500 rpm f o r 5 m i n u t e s and t h e 44 s u p e r n a t a n t d i s c a r d e d . Red b l o o d c e l l s u s p e n s i o n s (2.5 ml r e d c e l l s i n 10 ml s a l i n e - c i t r a t e ) were i n c u b a t e d f o r 1 hou r a t 3 7°C. The s u s p e n s i o n was r e c e n t r i f u g e d , t h e s u p e r n a t a n t d i s c a r d e d and a 5% s u s p e n s i o n o f c e l l s i n normal s a l i n e was made. H e m o l y t i c c a p a c i t y was t e s t e d by a d d i t i o n o f H 2 O 2 ( f i n a l c o n c e n t r a t i o n , 1.2%). Samples were i n c u b a t e d a t 37°C f o r 15 m i n u t e s , t h e n k e p t a t room t e m p e r a t u r e f o r 2.75 h o u r s . They were t h e n d i l u t e d w i t h s a l i n e - b u f f e r s o l u t i o n (0.25 mM p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.4/0.9% s a l i n e , 1:1, v/v) and c e n t r i f u g e d a t 1500 rpm f o r 5 m i n u t e s . O p t i c a l d e n s i t y o f s u p e r n a t a n t s was measured a t 540 nm u s i n g a S p e c t r o n i c 20 s p e c t r o p h o t o m e t e r . P e r c e n t a g e o f c o m p l e t e h e m o l y s i s (by c o m p a r i s o n w i t h a d i s t i l l e d w a t e r - h e m o l y z e d c o n t r o l sample) i n i n d i v i d u a l samples was used a s a measure o f r e d c e l l h e m o l y s i s . 3 . 4.3 G l y c o h e m o g l o b i n D e t e r m i n a t i o n G l y c o h e m o g l o b i n was d e t e r m i n e d as %HbA l c a c c o r d i n g t o t h e method o f W i n t e r h a l t e r ( 1 9 76). E r y t h r o c y t e s were s e p a r a t e d f r o m h e p a r i n i z e d b l o o d by c e n t r i f u g a t i o n a t 2500 rpm and were r i n s e d t w i c e w i t h s t e r i l e s a l i n e . A l i q u o t s o f r i n s e d e r y t h r o c y t e s , 500 111, were l y s e d by a d d i t i o n o f 1.4 ml d i s t i l l e d w a t e r and 0.4 ml C C I 4, w i t h v i g o r o u s s h a k i n g . The h e m o l y s a t e was c e n t r i f u g e d a t 2500 rpm f o r 5 m i n u t e s , t h e s u p e r n a t a n t d i s c a r d e d , and t h e l y s e d b l o o d c e l l s made up t o 10 ml w i t h d i s t i l l e d w a t e r . To 2.0 ml o f t h e d i l u t e d h e m o l y s a t e was added 1.0 ml 1.0 N o x a l i c a c i d . The m i x t u r e was v o r t e x e d g e n t l y . The s o l u t i o n was i n c u b a t e d a t 100°C f o r 4.5 h o u r s and c o o l e d t o room t e m p e r a t u r e by i m m e r s i o n 45 i n a water bath f o r 10 minutes. One ml of 40% t r i c h l o r o a c e t i c a c i d was added, the mixture vortexed, and the p r e c i p i t a t e removed by c e n t r i f u g a t i o n . Two ml of the c l e a r supernatant were mixed with 500 / i l of saturated t h i o b a r b i t u r i c a c i d (TBA) s o l u t i o n (prepared by d i s s o l v i n g 0.05 mol of TBA per l i t e r i n d i s t i l l e d water at 60°C f o r 1 hour, then cooling to 4°C by p l a c i n g i n an i c e bath, and using only the c l e a r supernatant). The s o l u t i o n was kept at 40°C f o r 30 minutes, cooled to room temperature (15 minutes) and the o p t i c a l density of the s o l u t i o n measured at 443 nm i n a Hitachi-Coleman spectrophotometer. An e x t i n c t i o n c o e f f i c i e n t of pure HbA^c, 1% (1 cm path length), 443 nm, equal to 0.060 was used to c a l c u l a t e %HbAi c f o r each sample. 3 . 4 . 4 Hemoglobin D e t e r m i n a t i o n The hemoglobin content of r a t blood was determined according to a Sigma k i t (procedure No. 525). Ten m i c r o l i t e r s of blood (obtained at autopsy) were added to 2.5 ml Drabkin's reagent (sodium bicarbonate:potassium ferricyanide:potassium cyanide, 20/4/1, [w/w/w] i n s o l u t i o n ) . The o p t i c a l density of the s o l u t i o n at 540 nm was measured using a Hitachi-Coleman spectrophotometer and the r e s u l t calculated according to the equation: %gHb= (f x OD x 1.652)+e where e = molar e x t i n c t i o n c o e f f i c i e n t , f = d i l u t i o n f a c t o r , and OD = o p t i c a l density of the s o l u t i o n at 540 nm. The 46 e x t i n c t i o n c o e f f i c i e n t f o r cyanmethemoglobin = 11 a t 540nm. The hemoglobin content of t i s s u e homogenates was determined u s i n g the same reagent, which was added i n equal volume (750/zl) t o the supernatant from t i s s u e homogenates c e n t r i f u g e d a t 30,000 rpm f o r 15 minutes at 10°C (Beckman u l t r a c e n t r i f u g e , Model T50). O p t i c a l d e n s i t i e s of s o l u t i o n s at 540 nm were measured and hemoglobin c a l c u l a t e d as above, using a d i l u t i o n f a c t o r of 2. 3 . 4 . 5 Plasma L i p o p e r o x i d e A s s a y Plasma l i p i d p e r oxide c o n c e n t r a t i o n was determined f l u o r o m e t r i c a l l y a c c o r d i n g t o the method of Yagi (1976). A l i q u o t s of blood, 50 / i l , were c o l l e c t e d by m i c r o c a p i l l a r y a t autopsy from each of the r a t s i n experiment 2 and were immediately added t o 1.0 ml normal s a l i n e i n c e n t r i f u g e tubes and shaken g e n t l y . Samples were c e n t r i f u g e d a t 4000 rpm f o r 10 minutes and 0.5 ml supernatant t r a n s f e r r e d t o c e n t r i f u g e tubes c o n t a i n i n g 4.0 ml N/12 H2SO4. Mixtures were vortexed, 0.5 ml a l i q u o t s of 10% phosphotungstic a c i d were added, mixtures were ag a i n vortexed, then allowed t o stand a t room temperature f o r 5 minutes. Samples were c e n t r i f u g e d f o r 10 minutes a t 4000 rpm and the supernatants d i s c a r d e d . Two ml of N/12 H 2S0 4 and 0.3 ml of 10% phosphotungstic a c i d were added t o the sediments, and the mixtures c e n t r i f u g e d at 4000 rpm f o r 10 minutes. The supernatants were d i s c a r d e d , the sediments resuspended i n 4.0 ml d i s t i l l e d water and 1.0 ml a l i q u o t s of f r e s h l y prepared TBA 47 r e a g e n t (0.67% TBA aqueous s o l u t i o n + g l a c i a l a c e t i c a c i d , 1:1, v/v) added. The r e a c t i o n m i x t u r e s were h e a t e d a t 95°C f o r 60 m i n u t e s i n a h e a t e r b l o c k . Tubes were i m m e d i a t e l y immersed i n a room t e m p e r a t u r e w a t e r b a t h f o r 5 m i n u t e s , t h e n 5.0 ml a l i q u o t s o f n - b u t a n o l were added, t h e m i x t u r e s v o r t e x e d and t h e n c e n t r i f u g e d a t 4000 rpm f o r 15 m i n u t e s . The f l u o r e s c e n c e o f t h e n - b u t a n o l (upper) l a y e r was measured s p e c t r o f l u o r o m e t r i c a l l y (515 nm e x c i t a t i o n , 553 nm e m i s s i o n ) ( F o c i s p e c t r o p h o t o m e t e r ) . R e l a t i v e f l u o r e s c e n c e was used as a measure o f l i p o p e r o x i d e c o n c e n t r a t i o n . 3 . 4 . 6 Radioimmunoassay o f Plasma I n s u l i n I n s u l i n r adioimmunoassay was p e r f o r m e d by Connie C h i s h o l m o f t h e Department o f P h y s i o l o g y , U.B.C. a c c o r d i n g t o t h e p r o t o c o l o f S t e r n e t a l . (1975) u s i n g r a t i n s u l i n 160 /iU/ml (NOVO I n d u s t r i , Copenhagan, Denmark) as t h e r e f e r e n c e s t a n d a r d . 3 . 4 . 7 T i s s u e G l u t a t h i o n e A s s a y GSH was d e t e r m i n e d as t o t a l n o n - p r o t e i n s u l f h y d r y l c o n t e n t i n t i s s u e homogenates by r e a c t i o n w i t h 5 , 5 1 - d i t h i o b i s - ( 2 -n i t r o b e n z o i c ) a c i d (DTNB) (Moron e t a l . , 1979). T i s s u e homogenates, 10% i n p o t a s s i u m phosphate b u f f e r , pH 7.4, c o n t a i n i n g 30 mM K C l , were a l i q u o t e d , 500 / i l / E p p e n d o r f c e n t r i f u g e t u b e , and 125 / i l c o l d 25% TCA added. The m i x t u r e s were c e n t r i f u g e d a t 6000 rpm f o r 5 m i n u t e s and t h e s u p e r n a t a n t s u s e d f o r a s s a y . To 0.2 ml o f s u p e r n a t a n t were added 1.0 ml i m i d a z o l e b u f f e r (0.15M, pH 7.4), 1.7 ml w a t e r , and 0.1 ml DTNB. 48 The a b s o r b a n c e was measured a f t e r 3 mi n u t e s a t 412 nm on a P e r k i n - E l m e r d u a l beam s p e c t r o p h o t o m e t e r . C o n c e n t r a t i o n s were c a l c u l a t e d by c o m p a r i s o n w i t h a s t a n d a r d c u r v e . 3.5 Enzyme A s s a y s 3.5.1 S u p e r o x i d e D ismutase A s s a y s F o r a s s a y o f MnSOD and CuZnSOD, a m o d i f i c a t i o n o f t h e e p i n e p h r i n e a u t o x i d a t i o n method ( M i s r a and F r i d o v i c h , 1972) as o u t l i n e d by Sun and Zigman (1978) was employed. T i s s u e homogenates (10%) were p r e p a r e d i n 10 mM p o t a s s i u m p h o s p h a t e b u f f e r w i t h added 30 mM K C l and s o n i c a t e d f o r 1 m i n u t e on " H i g h " ( i n i c e b ath) w i t h a B i o s o n i k I V S o n i c a t o r ( B r o n w i l l , R o c h e s t e r , NY). F o l l o w i n g s o n i c a t i o n and c e n t r i f u g a t i o n a t 10,000 x g f o r 15 m i n u t e s , t h e p e l l e t was d i s c a r d e d and MnSOD was measured i n t h e s u p e r n a t a n t f r a c t i o n a t pH 10.2, 28°C, i n t h e p r e s e n c e o f 5 mM NaCN. The f i n a l a s s a y m i x t u r e c o n t a i n e d 100 /nM e p i n e p h r i n e , 100 /^M x a n t h i n e , 3.3 pM x a n t h i n e o x i d a s e and 1 mM d i e t h y l e n e t r i a m i n e p e n t a a c e t i c a c i d (DETAPAC) (Sigma B i o c h e m i c a l s , S t . L o u i s , MO) i n 50 mM sodium c a r b o n a t e / s o d i u m b i c a r b o n a t e b u f f e r . The r e a c t i o n was f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y a t 320 nm ( P e r k i n - E l m e r Model 124 d u a l -beam s p e c t r o p h o t o m e t e r , N orwalk, CT). The s l o p e o f t h e c u r v e between 2 and 4 m i n u t e s was u s e d as a measure o f r e a c t i o n r a t e f o r e a c h sample. T o t a l SOD was d e t e r m i n e d s i m i l a r l y i n a 1:10 d i l u t i o n o f s u p e r n a t a n t , w i t h o u t added c y a n i d e . CuZnSOD was t h e n c a l c u l a t e d by s u b t r a c t i n g MnSOD from t o t a l SOD a c t i v i t y ( i n u n i t s / g wet w e i g h t ) . One u n i t i s e q u i v a l e n t t o t h e amount o f 49 enzyme r e q u i r e d t o i n h i b i t t h e a u t o x i d a t i o n by 50%. F o r d e t e r m i n a t i o n o f CuZnSOD i n b l o o d , 10 / i l o f a 1:40 d i l u t i o n o f f r e s h l y p r e p a r e d h e m o l y s a t e was used i n p l a c e o f 10 / i l o f 10% t i s s u e homogenate. 3.5.2 G l u t a t h i o n e P e r o x i d a s e and G l u t a t h i o n e R e d u c t a s e A s s a y s G l u t a t h i o n e p e r o x i d a s e (GSHPx) was d e t e r m i n e d by an i n d i r e c t , c o u p l e d a s s a y p r o c e d u r e o r i g i n a l l y o u t l i n e d by P a g l i a and V a l e n t i n e (1967) and m o d i f i e d by G i i n z l e r and F l o h e (1985) . G l u t a t h i o n e p e r o x i d a s e r e a c t s w i t h t - b u t y l h y d r o p e r o x i d e f o r m i n g o x i d i z e d g l u t a t h i o n e (GSSG) from GSH i n t h e p r o c e s s . W i t h an e x c e s s o f g l u t a t h i o n e r e d u c t a s e (GSSGRd) p r e s e n t i n t h e r e a c t i o n m i x t u r e , GSSG i s r a p i d l y and c o n t i n u o u s l y r e d u c e d b a c k t o GSH, u s i n g up r e d u c e d p y r i d i n e n u c l e o t i d e (NADPH) i n t h e p r o c e s s . The c o n c e n t r a t i o n o f NADPH can be m o n i t o r e d s p e c t r o p h o t o m e t r i c a l l y as an i n d i r e c t measure o f GSHPx a c t i v i t y . The f o l l o w i n g s o l u t i o n s were p i p e t t e d i n t o a m i c r o c u v e t t e : 595 / i l 0.25 M p o t a s s i u m p hosphate b u f f e r , pH 7.0 c o n t a i n i n g 0.25 mM e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) and 2.5 mM sodium a z i d e ; 100 / i l GSSGRd (25 U/ml i n phosphate b u f f e r ) ; 100 / i l GSH, lOmM; and 5 / i l 1% t i s s u e homogenate f o r l i v e r and k i d n e y t i s s u e s (15 / i l 2% t i s s u e homogenate f o r h e a r t and p a n c r e a s ) . A f t e r 10 m i n u t e s i n c u b a t i o n a t 37°C, 100 / i l NADPH, 2.5 mM i n 0.1% sodium b i c a r b o n a t e , was added. I n c u b a t i o n was c o n t i n u e d f o r 3 more m i n u t e s w h i l e r e c o r d i n g b a s e l i n e NADPH co n s u m p t i o n a t 3 66 nm 50 u s i n g a P e r k i n - E l m e r dual-beam s p e c t r o p h o t o m e t e r . L a s t l y , 100 / i l t - b u t y l h y d r o p e r o x i d e , 12 mM, was added and a b s o r b a n c e a t 366 nm, 3 7°C, r e c o r d e d f o r 5.0 m i n u t e s . GSHPx a c t i v i t y was c a l c u l a t e d a c c o r d i n g t o t h e f o l l o w i n g f o r m u l a : /imoles NADPH/min/g wet w e i g h t = - [ (0D2-0D!) / g / t ] x ( 3 / 6 . 2 2 ) , where t = t i m e , i n m i n u t e s , and OD = o p t i c a l d e n s i t y . GSHPx i n b l o o d was d e t e r m i n e d s i m i l a r l y on h e m o l y s a t e s p r e p a r e d a s f o l l o w s : 0.4 ml packed r e d c e l l s were combined w i t h 1.6 ml w a t e r ; f r o z e n and thawed 3 t i m e s and c e n t r i f u g e d a t 6000 rpm f o r 10 m i n u t e s . From t h e s u p e r n a t a n t , 0.1 ml was combined w i t h 1.9 ml w a t e r and 5 / i l u s e d f o r GSHPx r e a c t i o n m i x t u r e , as above f o r t i s s u e homogenates. G l u t a t h i o n e r e d u c t a s e a c t i v i t y was a l s o measured as t h e r a t e o f d i s a p p e a r a n c e o f NADPH (Racker, 1955; V a n o n i e t a l . , 1990). The f o l l o w i n g s o l u t i o n s were p i p e t t e d i n t o a s e m i m i c r o c u v e t t e : 500 / i l 0.2M p o t a s s i u m phosphate b u f f e r , pH 7.4; 200 / i l EDTA, 5mM; 100 / i l GSSG, 10 mM; 100 / i l NADPH, 3 mM; and 100 / i l t i s s u e homogenate (10% f o r h e a r t and p a n c r e a s , 3% f o r l i v e r and k i d n e y ) . The r e a c t i o n was f o l l o w e d a t 340 nm, 37°C, f o r 6 m i n u t e s . 51 3 . 5 . 3 C a t a l a s e A s s a y C a t a l a s e was d e t e r m i n e d a c c o r d i n g t o t h e method o f A e b i (1974) as m o d i f i e d by D a v i s o n e t a l . (1986). T i s s u e homogenates, 10%, were p r e p a r e d i n 10 mM phosphate b u f f e r w i t h added 30 mM K C l , and s o n i c a t e d on "H i g h " f o r 1 m i n u t e . A l i q u o t s , 400 / i l / E p p e n d o r f c e n t r i f u g e t u b e , were t r e a t e d w i t h 4 jul a b s o l u t e e t h a n o l and f r o z e n f o r up t o 5 days a t -20°C p r i o r t o a n a l y s i s . F o r a n a l y s i s , samples were spun down i n an Eppendorf t a b l e t o p c e n t r i f u g e i n t h e c o l d room (4°C) a t 11000 rpm f o r 15 m i n u t e s and t h e s u p e r n a t a n t used f o r a s s a y . An a l i q u o t (8 /ul f o r l i v e r and k i d n e y homogenates, 40 / i l f o r h e a r t and p a n c r e a s homogenates) was mixed w i t h 3.0 ml 19 mM H 2 O 2 i n a q u a r t z c u v e t t e , mixed r a p i d l y by i n v e r s i o n and t h e t i m e r e q u i r e d f o r a d e c r e a s e i n o p t i c a l d e n s i t y e q u a l t o 0.050 a b s o r p t i o n u n i t s ( e . g . , f r o m O D 2 4 0 n m = 0 • 5 5 0 t o O D 2 4 0 n m = 0 • 5 0 ° ) measured by s t o p w a t c h . T h i s p r o v e d t o be a more p r e c i s e method o f m e a s u r i n g r a t e o f d i s a p p e a r a n c e o f H 2 O 2 t h a n f o l l o w i n g t h e d e c r e a s e i n o p t i c a l d e n s i t y f o r a s e t p e r i o d o f t i m e , s i n c e t h e r e a c t i o n i s l i n e a r o n l y f o r s m a l l changes i n p e r o x i d e c o n c e n t r a t i o n . A Beckman DU s p e c t r o p h o t o m e t e r a t room t e m p e r a t u r e was u s e d f o r o p t i c a l d e n s i t y d e t e r m i n a t i o n . F o r b l o o d c a t a l a s e a s s a y , a 1:5 d i l u t e d h e m o l y s a t e was p r e p a r e d f r o m f r e s h l y c o l l e c t e d and washed r e d b l o o d c e l l s . A l i q u o t s , 20 / i l o f a 1:5 d i l u t i o n o f t h e 1:5 d i l u t e d h e m o l y s a t e (1:25 d i l u t i o n t o t a l ) were used i n t h e a s s a y j u s t d e s c r i b e d i n p l a c e 52 o f t i s s u e homogenates. 3 . 6 Manganese Analysis T i s s u e samples were weighed t o t h e n e a r e s t t e n t h o f a m i l l i g r a m , d r i e d a t 105°C o v e r n i g h t , r e w e i g h e d , and p u l v e r i z e d w i t h a m o r t a r and p e s t l e . They were t h e n ashed a t 450°C i n a m u f f l e f u r n a c e ( w i t h g r a d u a l h e a t i n g t o maximum t e m p e r a t u r e o v e r a p e r i o d o f 1 - 2 h o u r s ) , t a k e n up i n 2.0 ml 0.12 N u l t r a p u r e H C l (BDH C h e m i c a l s ) and a n a l y z e d f o r Mn by f l a m e a t o m i c a b s o r p t i o n s p e c t r o s c o p y ( P e r k i n Elmer 2380 a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r , Norwalk, CT), u s i n g an a i r - a c e t y l e n e f l a m e ( F a i l l a and K i s e r , 1981; C l e g g e t a l . , 1981a; I y e n g a r , 1 9 82). H e a r t and p a n c r e a s samples were n o t l a r g e enough t o be a n a l y z e d i n d i v i d u a l l y ; t h e r e f o r e , t h e s e samples were p o o l e d and a n a l y z e d a s p a i r s f rom t h e same g r o u p s . S t a n d a r d s (0 - 0.01 nq/q) were p r e p a r e d f rom a c e r t i f i e d Mn s t a n d a r d s o l u t i o n ( V e i l l o n and V a l l e e , 1978). D i e t samples were wet ashed w i t h 5.0 ml c o n c e n t r a t e d HNO3 a t 95°C o v e r n i g h t , t h e n d i l u t e d t o 25 ml w i t h w a t e r and a n a l y z e d by f l a m e a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( C l e g g e t a l . , 1981b). 3 . 7 Vitamin E Assays P l a s m a samples were a n a l y s e d s p e c t r o f l u o r o m e t r i c a l l y f o r v i t a m i n E a c c o r d i n g t o t h e method o f Hansen and Warwick (1966) as m o d i f i e d by D e s a i (1989). Plasma p r o t e i n s were p r e c i p i t a t e d w i t h a b s o l u t e e t h a n o l , v o r t e x e d and t h e v i t a m i n E e x t r a c t e d w i t h hexane. The hexane and e t h a n o l l a y e r s were t h e n s e p a r a t e d by 53 c e n t r i f u g a t i o n f o r 5 min a t 2000 rpm. F l u o r e s c e n c e o f t h e hexane (upper) l a y e r was measured s p e c t r o f l u o r o m e t r i c a l l y ( e x c i t a t i o n w a v e l e n g t h 295 nm and e m i s s i o n w a v e l e n g t h 340 nm) w i t h 10 mm s l i t w i d t h s , u s i n g 1 /ig/ml q u i n i n e s u l f a t e t o c a l i b r a t e t h e i n s t r u m e n t ( F o c i f l u o r o m e t e r w i t h a xenon la m p ) . D L - a - t o c o p h e r o l , 2 mg/dL, was used as a s t a n d a r d f o r c a l c u l a t i o n o f v i t a m i n E c o n c e n t r a t i o n s i n t e s t samples. L i v e r v i t a m i n E was d e t e r m i n e d by h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y (HPLC) a c c o r d i n g t o t h e method o f Lang e t a l . ( 1 9 8 6 ) . A f t e r a d d i t i o n o f 50 /xl o f b u t y l a t e d h y d r o x y t o l u e n e (BHT, e t h a n o l i c 2 , 6 - d i - t e r t - b u t y l - p - c r e s o l ) t o p r e v e n t l i p i d a u t o x i d a t i o n , w e i g h e d samples ( a p p r o x i m a t e l y 200 mg/sample) f r o z e n i n l i q u i d n i t r o g e n were homogenized a t t h e h i g h e s t s e t t i n g w i t h 1 ml o f w a t e r . One ml o f 0.1 M aqueous sodium d o d e c y l s u l f a t e (SDS) ( F l u k a B i o c h e m i c a l s , New York) was added and h o m o g e n i z a t i o n was c o n t i n u e d f o r 20-30 s e c o n d s . The v i s c o u s m i x t u r e was t h e n t r a n s f e r r e d by p o u r i n g i n t o a 15-ml c e n t r i f u g e t u b e f i t t e d w i t h a g round g l a s s s t o p p e r . The h omogenizer p e s t l e was r i n s e d w i t h 2 ml r e a g e n t a l c o h o l (BDH u l t r a p u r e HPLC g r a d e ) , e t h a n o l / i s o p r o p a n o l 95/5 ( v / v ) , and t h e r i n s e s o l u t i o n combined w i t h t h e homogenate. The c e n t r i f u g e t u b e was capped and v o r t e x e d f o r 30 s e c o n d s . Two ml hexane (BDH u l t r a p u r e HPLC grade) were added and t h e m i x t u r e was v o r t e x e d v i g o r o u s l y f o r 2 m i n u t e s b e f o r e c e n t r i f u g i n g f o r 5 m i n u t e s a t 1000 x g. The hexane l a y e r was t h e n t r a n s f e r r e d by P a s t e u r p i p e t t e t o a 54 s c i n t i l l a t i o n v i a l , p l a c e d i n an i c e b a t h and d r i e d u nder a s t r e a m o f n i t r o g e n . The r e s i d u e was t a k e n up i n 0.2 ml m e t h a n o l (BDH u l t r a p u r e HPLC g r a d e ) / r e a g e n t a l c o h o l 1/1 ( v / v ) . Samples were a n a l y z e d i m m e d i a t e l y by HPLC (26 m i n u t e r u n t i m e , 35°C, 275 nm d e t e c t o r s e t t i n g , f l o w r a t e 1 ml/min, i n j e c t e d volume 20 /nl sample + 5 / i l m e t h a n ol c a r r i e r , e l u a n t c o m p o s i t i o n : m e t h a n o l / r e a g e n t a l c o h o l 9/1, v / v ) , o r were m a i n t a i n e d t i g h t l y c apped a t -20°C u n t i l a n a l y z e d . D L - a - t o c o p h e r o l s t a n d a r d s were r u n e v e r y 60 - 90 m i n u t e s f o r use i n c a l c u l a t i o n o f sample v i t a m i n E c o n c e n t r a t i o n s . 3 . 8 Renal Adipose Tissue Fluorescence Assay F l u o r e s c e n t p r o d u c t s from r e n a l a d i p o s e t i s s u e were e x t r a c t e d w i t h c h l o r o f o r m / m e t h a n o l 2/1 (v/v) i n a volume t o w e i g h t r a t i o o f 20:1. M i x t u r e s were warmed i n a 45°C w a t e r b a t h , t h e n homogenized b r i e f l y , an e q u a l volume o f w a t e r added, and t h e m i x t u r e v o r t e x e d v i g o r o u s l y f o r 2 m i n u t e s . A f t e r c e n t r i f u g a t i o n a t 3,000 rpm f o r 2 min, a 3 ml a l i q u o t o f t h e c h l o r o f o r m ( l o w e r ) l a y e r was mixed w i t h 0.3 ml o f methanol ( f o r c l a r i f i c a t i o n ) i n a f l u o r e s c e n c e c u v e t t e and t h e r e l a t i v e f l u o r e s c e n c e ( e x c i t a t i o n 370 nm, e m i s s i o n 440 nm) r e c o r d e d . Q u i n i n e s u l f a t e , 1 /tg/ml, was u s e d f o r c a l i b r a t i o n ( F l e t c h e r e t a l . , 1973; D e s a i e t a l . , 1975). 3 . 9 Hepatic Fat Content Determination L i v e r s a m p l e s , a p p r o x i m a t e l y 0.5 g p r e v i o u s l y f r o z e n i n l i q u i d n i t r o g e n , were weighed t o t h e n e a r e s t t h o u s a n d t h o f a gram i n t o 55 h o m o g e n i z e r t u b e s . Three ml wa t e r were added and t h e c o n t e n t s warmed t o 45°C f o r 15 m i n u t e s i n a w a t e r b a t h . C o n t e n t s were t h e n homogenized on h i g h speed, s e t t i n g 4-5, f o r 30 s e e s . , 4.0 ml m e t h a n o l and 2.0 ml c h l o r o f o r m were added, and t h e m i x t u r e homogenized a g a i n f o r 30 seconds. Homogenate was p o u r e d i n t o a 30-ml round-bottom P y r e x c e n t r i f u g e t u b e . The h o m o g e n i z e r t u b e was r i n s e d w i t h 6.0 ml m e t h a n o l / c h l o r o f o r m 2/1 ( v / v ) , r i n s i n g s were homogenized b r i e f l y and t r a n s f e r r e d t o t h e same c e n t r i f u g e t u b e . The combined m i x t u r e s were t h e n c e n t r i f u g e d a t 3000 rpm, f o r 2 m i n u t e s . The c h l o r o f o r m (lower) l a y e r was t h e n t r a n s f e r r e d t o a p r e - w e i g h e d round-bottom n a r r o w t e s t - t u b e f i t t e d w i t h a T e f l o n - l i n e d screw cap. C h l o r o f o r m was e v a p o r a t e d u n d e r n i t r o g e n ( a p p r o x i m a t e l y 20 m i n u t e s a t 35°C). Tubes were d r i e d t o c o n s t a n t w e i g h t i n a 101°C oven, capped and c o o l e d i n a d e s s i c a t o r f o r 30 m i n u t e s b e f o r e w e i g h i n g ( R a d i n , 1969; W i l l i a m s , 1984). 3.10 P r o t e i n D e t e r m i n a t i o n T i s s u e homogenate p r o t e i n c o n t e n t was d e t e r m i n e d u s i n g a Sigma k i t (No. P 5656) based on a Lowry e t a l . (1951) p r o c e d u r e . One ml o f a 1:100 t i s s u e homogenate was mixed w i t h 1.0 ml o f Lowry r e a g e n t (an a l k a l i n e c u p r i c t a r t r a t e r e a g e n t s o l u t i o n ) and a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 20 m i n u t e s . Then 0.5 ml F o l i n - C i o c a l t e a u p h e n o l r e a g e n t was added w i t h i mmediate m i x i n g . C o l o r was a l l o w e d t o d e v e l o p f o r 30 m i n u t e s . O p t i c a l d e n s i t y o f t h e s o l u t i o n was r e a d a t 560 nm and compared t o a 56 s t a n d a r d c u r v e u s i n g b o v i n e serum a l b u m i n , 4 00 ug/ml, as t h e s t a n d a r d . 3.11 S t a t i s t i c a l Analysis A l l r e s u l t s , e x p r e s s e d as means ± SEM, were compared u s i n g 2-way a n a l y s i s o f v a r i a n c e i n i t i a l l y , t o i d e n t i f y manganese d e f i c i e n c y S T Z - d i a b e t e s , and v i t a m i n E d e f i c i e n c y e f f e c t s and i n t e r a c t i o n s . A one-way a n a l y s i s o f v a r i a n c e (ANOVA) f o l l o w e d by Duncan's m u l t i p l e r a n g e t e s t s was used t o i d e n t i f y w i t h i n - g r o u p s i g n i f i c a n t d i f f e r e n c e s ( N o r u s s i s , 1989). Based on t h e r e s u l t s o f t h e f i r s t s t a t i s t i c a l a n a l y s e s , w h i c h showed c o n s i s t e n c y between e x p e r i m e n t s 1 and 2, t h e a n t i o x i d a n t enzyme a c t i v i t y and GSH and MDA c o n c e n t r a t i o n r e s u l t s were r e g r o u p e d as f o l l o w s : 1) a l l o f e x p e r i m e n t 1 w i t h t h e a d d i t i o n o f g r o u p s -MnD4, +MnD4, -MnD8, and +MnD8 from e x p e r i m e n t 2; 2) -MnD4 and +MnD4 from e x p e r i m e n t s 1 and 2, w i t h -MnDE- and +MnDE- fr o m e x p e r i m e n t 2. The r e s u l t a n t 2 d a t a s e t s a r e o u t l i n e d i n T a b l e 2. T h i s r e g r o u p i n g r e s u l t e d i n e f f e c t i v e l y l a r g e r samples s i z e s f o r b o t h e x p e r i m e n t a l d e s i g n s . These two groups were t h e n a n a l y z e d by 2-way ANOVA, f o l l o w e d by 1-way ANOVA and Duncan's m u l t i p l e r a n g e t e s t , as o u t l i n e d above. 57 T A B L E 2. Statistical design for analysis of experiments 1 and 2 (number of rats/groups) • Mn-deficient Mn-sufficient Set l a : Non-diabetic 8 8 4 week-diabetic0 9 11 8 week-diabetic 4 4 Set 2b: Vitamin E-sufficientc 9 11 Vitamin E-deficient 8 8 aRats in set 1 were all vitamin E-sufficient ^Rats in set 2 were all 4 week-diabetic All 4 week-diabetic, vitamin E-sufficient, rats from experiments 1 and 2 were included in both sets 58 4 . R E S U L T S 4 .1 Food c o n s u m p t i o n and f o o d e f f i c i e n c y Manganese d e f i c i e n c y had no e f f e c t on f o o d c o n s u m p t i o n o r f o o d e f f i c i e n c y (body w e i g h t g a i n / f o o d consumed) ( T a b l e 3 ) . I n d u c t i o n o f d i a b e t e s t e m p o r a r i l y d e c r e a s e d f o o d c o n s u m p t i o n , w h i c h r e v e r t e d t o normal by 2 weeks a f t e r STZ t r e a t m e n t ( F i g u r e 1 0 ) . An i n c r e a s e i n f o o d consumption o v e r pre-STZ l e v e l s o c c u r r e d o n l y i n r a t s w h i c h were d i a b e t i c f o r more t h a n 4 weeks. V i t a m i n E d e f i c i e n c y had no e f f e c t on f o o d c o n s u m p t i o n o r f o o d e f f i c i e n c y . A verage d a i l y consumption by v i t a m i n E - d e f i c i e n t r a t s was 29.4 g b e f o r e STZ, 27.1 g i m m e d i a t e l y a f t e r STZ a d m i n i s t r a t i o n , and 31.2 g t h e week b e f o r e a u t o p s y (week 1 1 ) , w i t h no a p p a r e n t d i f f e r e n c e between +MnDE- and -MnDE- g r o u p s . 4.2 C o n f i r m a t i o n o f Manganese D e f i c i e n c y Manganese d e f i c i e n c y was c o n f i r m e d by s i g n i f i c a n t l y l o w e r Mn c o n c e n t r a t i o n s i n l i v e r , k i d n e y , and h e a r t i n -MnND compared t o +MnND a n i m a l s ( T a b l e 4 ) . Comparing n o n - d i a b e t i c , M n - d e f i c i e n t , r a t s t o M n - s u f f i c i e n t a n i m a l s , average l i v e r Mn c o n c e n t r a t i o n was 42% l o w e r , k i d n e y Mn was 53% l o w e r , and h e a r t Mn was 48% l o w e r , b u t p a n c r e a t i c Mn c o n c e n t r a t i o n s were n o t s i g n i f i c a n t l y d i f f e r e n t . S i m i l a r l y , c omparing d i a b e t i c M n - d e f i c i e n t t o Mn-s u f f i c i e n t r a t s , Mn c o n c e n t r a t i o n s i n a l l t i s s u e s measured ( e x c e p t p a n c r e a s ) were s i g n i f i c a n t l y l o w e r ( T a b l e 4 ) . D e c r e a s e s i n Mn c o n t e n t / o r g a n as a r e s u l t o f Mn d e f i c i e n c y were s i g n i f i c a n t f o r a l l f o u r t i s s u e s ( T a b l e 5 ) . Manganese c o n t e n t 59 T A B L E 3. Effect of dietary manganese on Sprague-Dawley rat body weight and food intake Mn (-) Mn(+) 00 (16) (16) Body weight (g) 172.4 + 8.9 169.8 ± 8.6 Body weight gain (g/21d) 88.8 ± 2.8 77.5 ± 3.8 Food intake (g/d) 23.4 ± 2 . 1 23.6 ± 2.1 Food efficiency* (g/g) 0.172 +. .008 0.169 +. 0.013 Values are means +_ SEM, after feeding for 21 days. No means were significantly different between groups. *Body weight gain/food intake Mn(-), manganese-deficient Mn( +), manganese-sufficient CO "D CO XL CO T3 O O LL 32 28 24 20 16 12 8 L- Streptozotocin injection -Mn, non-diabetic - 0 - +Mn, non-diabetic -Mn, diabetic - B - *Mn, diabetic 1 2 3 4 i FIGURE 10. Average weekly food intake of rats. 6 7 8 Time (wk) i 9 10 11 12 32 weanling, male Sprague-Dawley rats were randomly divided into 4 groups: 1) Mn-deficient (-Mn), non-diabetic; 2) Mn-sufficient (+Mn), non-diabetic; 3) Mn-deficient (-Mn), diabetic for 4 weeks; and 4) Mn-sufficient (+Mn), diabetic for 4 weeks. Mn-deficient diets contained 1 ppm Mn; Mn-sufficient diets contained 45 ppm Mn. Diabetes was induced by tail vein injection of 60 mg streptozotocin (STZ)/kg body weight after 8 weeks, as indicated. o 61 TABLE 4. Manganese concentration in rat tissues (ug/g wet weight) Non-diabetic Diabetic Mn(-) Mn( + ) Mn(-) Mn( + ) Liver 1.22a+. 0.10 2.88b+.0.22 1.28ai0.18 2.46bi0.27 Kidney 0 .66 a ±0 .10 1.24b+.0.10 0.78a+.0.11 1.29bjf0.32 Heart 1.38ai0.34 2.89bi0.24 1.04a+.0.16 l ^ + O.W Pancreas 134a + 0.25 1.78a + 0.10 1.91a + 0.35 2.85a + 0.31 Values are means +. SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Mn(-), manganese-deficient; Mn( + ), manganese-sufficient TABLE 5. Manganese content of rat tissues (ug/organ) Non-diabetic Diabetic Mn(-) Mn( + ) Mn(-) Mn( + ) Liver 14.6a+.1.47 30 .0 b ±1 .99 10.1 a ±0.88 2.46b+.0.27 Kidney 1.65a+.0.18 3.15b + 0.23 2.10a + 0.22 2.93b ±055 Heart 1 .27 a ±0.36 2 .45 b ±0.20 0.68a+.0.04 1.03b + 0.14 Pancreas 1.26a +. 0.17 1.55b + 0.07 1.02a + 0.21 1.98b + 0.21 Values are means SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Mn(-), manganese-deficient; Mn( + ), manganese-sufficient 63 o f M n - d e f i c i e n t , v i t a m i n E - d e f i c i e n t d i a b e t i c r a t s was a l s o s i g n i f i c a n t l y l o w e r t h a n M n - s u f f i c i e n t , v i t a m i n E - d e f i c i e n t , d i a b e t i c a n i m a l s ( l i v e r Mn 15.61 ± 1.32 /ng/organ i n Mn-s u f f i c i e n t v s . 7.21 ± 0.84 /ig/organ i n M n - d e f i c i e n t , v i t a m i n E-d e f i c i e n t , d i a b e t i c r a t s ) . 4.3 R a t Growth, T i s s u e W e i g h t s , and T i s s u e Weight/Body Weight R a t i o s D i a b e t i c r a t s had s i g n i f i c a n t l y l o w e r body w e i g h t s a t a u t o p s y compared t o n o n - d i a b e t i c r a t s ( T a b l e 6 ) . No d i f f e r e n c e s i n body w e i g h t were o b s e r v e d between M n - s u f f i c i e n t and M n - d e f i c i e n t g r o u p s a t a u t o p s y , and manganese d e f i c i e n c y d i d n o t a f f e c t r a t g r o w t h ( F i g u r e 1 1 ) . H e a r t and l i v e r w e i g h t s d i d n o t d i f f e r between g r o u p s ; however, h e a r t / b o d y w e i g h t r a t i o s were n e a r l y 3 0% h i g h e r i n -MnD8 and +MnD8 r a t s compared t o n o n - d i a b e t i c a n i m a l s . L i v e r / b o d y w e i g h t r a t i o s were h i g h e r i n +/-MnD4 and +/-MnD8 gr o u p s compared t o +/-MnND. K i d n e y w e i g h t s were s i g n i f i c a n t l y h i g h e r (39% a t 4 weeks a f t e r STZ, 57% a t 8 weeks) i n a l l d i a b e t i c compared t o n o n - d i a b e t i c a n i m a l s . K i d n e y / b o d y w e i g h t r a t i o s were 90-100% h i g h e r i n d i a b e t i c r a t s compared t o n o n - d i a b e t i c r a t s . S T Z - d i a b e t e s d i d n o t a f f e c t p a n c r e a s w e i g h t s ; however, p a n c r e a s w e i g h t s were s i g n i f i c a n t l y l o w e r i n M n - d e f i c i e n t compared t o M n - s u f f i c i e n t d i a b e t i c r a t s . V i t a m i n E d e f i c i e n t , M n - s u f f i c i e n t , d i a b e t i c r a t s were a p p r o x i m a t e l y 27% h e a v i e r t h a n v i t a m i n E s u f f i c i e n t , d i a b e t i c a n i m a l s ( T a b l e 7 ) . H e a r t / b o d y w e i g h t r a t i o s i n v i t a m i n 350 325 300 275 3 250 £ 225 O) CD £ 200 175 150 125 100 Streptozotocin injection A- -Mn, non-diabetic O- +Mn, non-diabetic "0- -Mn, diabetic •9- +Mn, diabetic 8 ~T~ 12 16 Time (wk) FIGURE 11. Effect of dietary manganese (Mn) and STZ-diabetes on rat growth. Mn-deficient (-Mn) diet, 1 ppm Mn; Mn-sufficient ( + Mn) diet, 45 ppm Mn; Streptozotocin, 60 mg STZ/kg body weight in sterile saline, freshly prepared. STZ-diabetic rats were significantly smaller than non-diabetic rats (P<0.001). 65 TABLE 6. Effects of manganese deficiency & STZ-diabetes on body weight, tissue weights, and tissue/body weight ratios in rats Non-diabetic Diabetic 4 weeks after STZ 8 weeks after STZ 00 Mn(-) (8) Mn( + ) (8) Mn(-) (11) Mn( + ) (9) Mn(-) (4) Mn( + ) (4) Body weight (g) 312a +. 8 332a +. 13 226b +. 14 224b ± 17 244bjtl4 243b ±25 Heart weight (g) 0.90a +. 0.03 0.84a +. 0.02 0.84a +. 0.02 0.79a +. 0.06 0.80a +. 0.05 0.893 +. 0.05 Heart/body weight ratio (mg/g) 2.71a +. 0.08 2.71a +. 0.07 3.06b +. 0.08 3.03b +. 0.08 3.48b +. 0.16 3.61b +. 0.24 Kidney weight (g) 2.58a +. 0.08 2.54a +. 0.06 3.46b +. 0.21 3.46b ± 0.19 3.84bc +. 0.15 4.21c +. 0.46 Kidney/body weight ratio (mg/g) 7.81a +. 0.14 8.16a +. 0.17 15.23b +. 1.96 15.66b ± 1.99 14.77b +. 0.65 16.51bjt4.11 Liver weight (g) 11.925^0.51 10.473 +. 0.36 9.22a +. 0.40 9.42a ± 0.61 10.02a +. 0.41 10.16a +. 0.74 Liver/body weight ratio (mg/g) 35.9a +. 0.6 33.6a +. 0.9 38.4b +. 0.2 39.1bci0.1 4 1 . 3 b c ± 1 . 6 42.3C +. 2.1 Pancreas weight (g) 0.87b +. 0.03 0.84bjt0.02 0.73a +. 0.02 0.84b+.0.06 0.70a +. 0.05 0.87b +. 0.05 Pancreas/body weight ratio (mg/g) 2.53a +. 0.08 2.80ab.+0.07 3.03b ± 0.08 3.48cd ±0 .08 2 89abc ± 0 1 6 3.66d +. 0.24 Values are means +_ SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Sample size in parentheses. Mn(-), manganese-deficient; Mn( + ), Mn-sufficient 66 TABLE 7. Effects of manganese deficiency & vitamin E deficiency on body and tissue weights, tissue/body weight ratios, blood glucose, hemoglobin glycosylation, and insulin in four week STZ-diabetic rats Vitamin E ( + ) Vitamin E (-) Mn(-) Mn( + ) Mn(-) Mn( + ) 00 (11) (9) (6) (8) Body weight (g) 243a +. 14 242a ± 17 288ab ± 21 331b +. 17 Heart weight (g) 0.80s ± 0 . 0 6 0.793 ± 0.06 0.84a ± 0.02 0.88s+.0.02 Heart/body • weight ratio (mg/g) 3.06a +. 0.08 3.03a +. 0.08 2.96ab +. 0.16 2.68b +. 0.13 Kidney weight (g) 3.46a ± 0.21 3.46a ± 0.19 3.20s +. 0.13 3.15s +_ 0.07 Kidney/body weight ratio (mg/g) 15.23a +. 1.96 15.663 ± 1.99 U.28b+.038 9.64b ± 0.39 Liver weight (g) 9.22s +. 0.40 9.42a ± 0.61 9Sla ± 0.71 10.79s +. 0.49 Liver/body weight ratio (mg/g) 38.4a +. 0.2 39.1a ± 1.0 33.4a +. 1.3 32.7s +. 0.7 Pancreas weight (g) 0.79s +. 0.06 0.93b +. 0.06 0.73a ± 0.02 0.84b +. 0.02 Pancreas/body weight ratio (mg/g) 3.03a +. 0.08 3.48a+.0.08 2.74a +. 0.16 2.88s +. 0.13 Blood glucose (mM) (24 hours post STZ) 15.8s ± 0 . 7 15.0s ± 0.7 16.4a ± 0.9 16.4s ±0.9 Blood glucose (mM) (pre-autopsy) 19.8a ± 0.5 20.1a +. 0.9 17.0s +. 1.6 17.3s +.0.6 HbAlc (%) 8.3a +. 0.9 7.4a ± 1.0 12.4b +. 2.4 U.4b +. 0.9 Insulin (uU/ml) 20.2a +. 2.2(2) 29.2a +. 8.9(3) 12Sb ± 1.0(4) 8.7b +. 3.0(2) Values are means +. SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Sample size in parentheses (where different from n). Mn(-), manganese-deficient; Mn( + ), Mn-sufficient Vitamin E ( + ), vitamin E-sufficient; Vitamin E (-), vitamin E-deficient 67 E - d e f i c i e n t , M n - s u f f i c i e n t r a t s were s i g n i f i c a n t l y l o w e r t h a n i n v i t a m i n E - s u f f i c i e n t r a t s . K i dney/body w e i g h t r a t i o s i n v i t a m i n E - d e f i c i e n t , d i a b e t i c compared t o v i t a m i n E - s u f f i c i e n t , d i a b e t i c r a t s were s i g n i f i c a n t l y l o w e r , a l t h o u g h t h e y were s t i l l 30% h i g h e r t h a n i n n o n - d i a b e t i c r a t s . V i t a m i n E d e f i c i e n c y r e s u l t e d i n i n c r e a s e d p a n c r e as/body w e i g h t r a t i o s and d i d n o t e l i m i n a t e a s i g n i f i c a n t d e c r e a s e i n p a n c r e a s w e i g h t r e s u l t i n g f r o m manganese d e f i c i e n c y ( T a b l e 7 ) . 4 . 4 E f f e c t s o f STZ Treatment on B l o o d G l u c o s e , H b A l c / and Plasma I n s u l i n L e v e l s B l o o d g l u c o s e l e v e l s 24 h o u r s a f t e r STZ a d m i n i s t r a t i o n were s i g n i f i c a n t l y h i g h e r i n d i a b e t i c compared t o n o n - d i a b e t i c r a t s , a v e r a g i n g 16.5 mM (300 mg%) ( T a b l e s 7 and 8 ) . Two r a t s i n t h e M n - d e f i c i e n t , v i t a m i n E - d e f i c i e n t , d i a b e t i c g r o u p w h i c h were h y p e r g l y c e m i c (12.8 mM) 24 hours a f t e r STZ became n o n - d i a b e t i c (<11 mM) by 4 weeks po s t - S T Z . They were e x c l u d e d f r o m t h e s t u d y . One o f t h e r a t s i n t h e M n - s u f f i c i e n t , 4-week d i a b e t i c g r o u p i n e x p e r i m e n t 2, and 2 i n t h i s same group i n e x p e r i m e n t 1, d i e d f o l l o w i n g STZ a d m i n i s t r a t i o n and b e f o r e t h e end o f t h e e x p e r i m e n t a l p e r i o d . One r a t i n t h e M n - d e f i c i e n t , 4-week d i a b e t i c group i n e x p e r i m e n t 1 a l s o d i e d p r e m a t u r e l y . B l o o d g l u c o s e l e v e l s a t a u t o p s y i n a l l d i a b e t i c g r o u p s were s i g n i f i c a n t l y h i g h e r t h a n n o n - d i a b e t i c b l o o d g l u c o s e l e v e l s (P<0.001), c o n f i r m i n g t h e c o n t i n u e d d i a b e t i c s t a t e o f STZ-t r e a t e d r a t s . T h e r e was no s i g n i f i c a n t e f f e c t o f e i t h e r 68 TABLE 8. Effects of manganese deficiency & STZ-diabetes on blood glucose, HbAlc, and plasma insulin levels in rats Non-diabetic Diabetic 4 weeks after STZ 8 weeks after STZ Mn(-) Mn( + ) Mn(-) Mn( + ) Mn(-) Mn( + ) (n) (8) (8) (11) (9) (4) (4) Blood glucose (mM) 15.8+.0.7 15.0+.0.7 18.7 ± 2 . 4 18.8 +. 1.9 (24 hours after STZ) Blood glucose (mM) 4.44a +. 0.20 4.74a +. 0.09 18.6bj+0.58 19.4b +. 0.89 19.9b +. 0.90 19.6b +. 0.05 (pre-autopsy) HbAlc (%) 4.95a +. 0.05(2) 4.85a+. 0.45(2) 8.28b +.0.89(4) 7.40b ±0.92(3) 8.00b+.1.25(4) 9.77b +. 0.70(3) Plasma insulin (uU/ml) 52.3a(l) 20.2b +. 2.2(2) 29.2b +. 8.9(3) 9.5C ± 1.9(4) 9.0C +. 3.0(2) GTT area (mM'min) 519a +. 20(4) 291b +. 14(3) 762a ± 32(4) 467b +. 25(4) Values are means +. SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Sample size in parentheses (where different from n). Mn(-), manganese-deficient; Mn( + ), Mn-sufficient GTT, glucose tolerance test: 180 mg glucose/kg body weight administered orally at time 0. Area under curve calculated with baseline subtracted. (See FIGURE 12) 69 manganese d e f i c i e n c y o r v i t a m i n E - d e f i c i e n c y on b l o o d g l u c o s e c o n c e n t r a t i o n s ( T a b l e s 7 and 8 ) . G l y c o s y l a t e d h e m o g l o b i n (HbA^ c) l e v e l s i n b l o o d o b t a i n e d a t a u t o p s y were 50 - 150% h i g h e r i n d i a b e t i c compared t o non-d i a b e t i c r a t s . V i t a m i n E - d e f i c i e n t , d i a b e t i c a n i m a l s had s i g n i f i c a n t l y h i g h e r H b A i c l e v e l s t h a n v i t a m i n E - s u f f i c i e n t d i a b e t i c r a t s . T here were no d i f f e r e n c e s between M n - s u f f i c i e n t and M n - d e f i c i e n t g r o u p s . P l a s m a i n s u l i n l e v e l s a t a u t o p s y were l o w e r i n d i a b e t i c r a t s t h a n i n n o n - d i a b e t i c r a t s and were l o w e s t i n +MnD8 and -MnD8, and i n v i t a m i n E - d e f i c i e n t a n i m a l s ( T a b l e s 7 and 8 ) . 4 . 5 G l u c o s e T o l e r a n c e T e s t s M a n g a n e s e - d e f i c i e n t r a t s had s i g n i f i c a n t l y g r e a t e r a r e a s under t h e c u r v e f o r g l u c o s e t o l e r a n c e t e s t r e s p o n s e compared t o m a n g a n e s e - s u f f i c i e n t a n i m a l s , when b a s e l i n e s were s u b t r a c t e d ( T a b l e 8 ) . G l u c o s e t o l e r a n c e t e s t s ( F i g u r e 12) on +/-MnD4 and +/-MnD8 r a t s i n e x p e r i m e n t 2 c o n f i r m e d t h e d i a b e t i c s t a t u s o f t h e s e a n i m a l s . I n n o n - d i a b e t i c r a t s , one-hour p o s t - g l u c o s e l o a d b l o o d g l u c o s e l e v e l s a v e r a g e 6-9 mM, d e c l i n i n g r a p i d l y t o n o r m a l l e v e l s w i t h i n t h e second h o u r . An av e r a g e a r e a under t h e c u r v e f o r a n o r m a l g l u c o s e t o l e r a n c e t e s t i n r a t s was 71 min'mM g l u ( E l i a z e r and S h a f r i r , 1982), compared t o 640 min'mM g l u (Mn-d e f i c i e n t , d i a b e t i c ) and 392 min'mM g l u ( M n - s u f f i c i e n t , d i a b e t i c ) i n t h i s s t u d y . 2 £ o o o D O ~o o o CD 28 24 20 16 12 8 3- -Mn, 4 wk •Mn, 4 wk -Mn, 8 wk v - •Mn, 8 wk 0 30 60 90 Time Post-glucose Load (min) FIGURE 12. Effect of dietary manganese (Mn) on glucose tolerance test response. 120 Male, Sprague-Dawley rats were fed purified diets, either Mn-deficient (-Mn) (1 ppm Mn) or Mn-sufficient ( + Mn) (45 ppm Mn) for 8 weeks prior to diabetic induction (60 mg STZ/kg body weight). Glucose tolerance tests (GTT) were performed on rats fasted overnight, either 3 (for + /-Mn, diabetic 4 wks) or 7 (for + /-Mn, diabetic 8 weeks) weeks after STZ treatment. An oral glucose load (180 mg/lOOg body weight) was administered by gastric *J intubation at time 0. Blood glucose concentrations were determined at half-hourly intervals throughout the test. Areas under the curve, with baseline ° subtracted, were significantly higher for Mn-deficient, compared to Mn-sufficient, diabetic rats (P<0.05). 71 4.6 Tissue Antioxidant A l t e r a t i o n s 4.6.1 E f f e c t s of Manganese Deficiency on Tissue Antioxidant Enzymes The p r i n c i p a l e f f e c t s o f Mn d e f i c i e n c y on t i s s u e a n t i o x i d a n t enzyme s t a t u s were i n k i d n e y , where MnSOD and CuZnSOD a c t i v i t i e s were d e c r e a s e d , i n h e a r t , where MnSOD a c t i v i t y was a l s o d e c r e a s e d , and i n l i v e r , where MnSOD a c t i v i t y was p a r a d o x i c a l l y i n c r e a s e d ( T a b l e 9 ) . These changes were e v i d e n t i n b o t h d i a b e t i c and n o n - d i a b e t i c a n i m a l s . The o u t s t a n d i n g e f f e c t s o f d i a b e t e s on t h e t i s s u e a n t i o x i d a n t s examined were r e d u c e d l e v e l s o f k i d n e y and l i v e r GSH, and d e c r e a s e d a c t i v i t i e s o f h e a r t and k i d n e y GSSGRd, k i d n e y GSHPx, and p a n c r e a s MnSOD and CuZnSOD ( T a b l e s 9 and 10, A p p e n d i x A ) . A c t i v i t i e s o f h e a r t CAT and CuZnSOD, and p a n c r e a s GSHPx were e l e v a t e d i n d i a b e t i c compared t o n o n - d i a b e t i c a n i m a l s . 4.6.2 E f f e c t s of STZ-diabetes on Tissue Antioxidants A one-way ANOVA f o l l o w e d by Duncan's m u l t i p l e range t e s t on t h o s e v a r i a b l e s n o t a f f e c t e d by M n - d e f i c i e n c y r e v e a l e d a complex i n t e r p l a y o f t i s s u e a n t i o x i d a n t changes accompanying i n d u c t i o n o f d i a b e t e s ( T a b l e 1 1 ) . I n h e a r t , CAT was e l e v a t e d a t 4 weeks a f t e r STZ t r e a t m e n t , and f u r t h e r s i g n i f i c a n t l y e l e v a t e d a t 8 weeks compared t o 4 weeks. I n l i v e r , GSH was l o w e r a t 4 weeks a f t e r STZ compared t o n o n - d i a b e t i c s , b u t n o t f u r t h e r r e d u c e d a t 8 weeks, w h i l e i n k i d n e y , GSH was l o w e r 4 weeks a f t e r STZ 72 T A B L E 9. Effect of diabetes and dietary Mn on MnSOD, CuZnSOD and GSHPx activities in Sprague-Dawley rats Non-diabetic Diabetic 4 weeks after STZ 8 weeks after STZ Mn(-) Mn( + ) Mn(-) Mn( + ) Mn(-) Mn( + ) (S) (8) (8) (") (4) (4) MnSOD (U/g wet weight) Heart 342 ± 16' 572 ± 2 8 437 ± 23' 631 ± 4 1 390 ± 65' 443 ± 2 4 Liver 543 ± 69' 498 ± 6 0 726 ± 2 6 * ' 614 ± 27* 677 ± 5 2 * ' 603 ± 6 5 * Kidney 468 ± 2 4 ' 607 ± 4 3 392 ± 15*' 547 ± 2 8 * 390 ± 1 8 * ' 447 ± 2 6 * Pancreas 354 +.31 334 ± 3 1 3 0 9 ± 3 0 288 ± 14 197 ± 1 4 * 217 ± 1 8 * CuZnSOD (U/g wet weight) Heart 3170 + 215 3066 ± 1 8 3 3670 +_ 213* 3803 +_ 152* 3661 _+ 143* 3857 ± 383* Liver 10482 + U44 10902 _+1092 10420 +_ 439 8408 +_ 441 8724 _+ 500 8897 ± 628 Kidney 6958 ± 290' 8343 ± 3 4 6 6208 ± 2 9 1 * 6876 ± 426* 5611 ± 4 8 3 * 6253 ± 607* Pancreas 3215 ± 295 2785 ± 2 1 1 2246 ± 2 0 3 * 2373 ± 146* 2703 ± 2 0 6 2633 ± 2 9 7 GSHPx (umol NADPH/min/g wet weight) Heart 9.31 ± 0.92' 7.70 ± 0.85 8.35 ± 032' 7.87 ± 037 8.40 ± 0.29' 7.88 ± 030 Liver 33.6 ± 5.3 44.4 ± 2.8 2S.8 ± 1.6* 21.8 ± 2.7* 22.8 ± 3.8* 23.6 ± 1.2* Kidney 28.2 ± 1.9 295 ±25 21.2 ± 1 . 1 * 25.4 ± 2.7* 21.4 ± 2.0* 19.2 ± 1.2* Pancreas 9.18 ± 0 . 9 4 957 ± 0.84 9.75 ± 0.63 9.61 ± 034 12.14 ± 1 . 7 0 * 13.93_± 1.01* Values are means ± SEM. Mn SOD, Mn-superoxide dismutase; CuZnSOD, CuZn-superoxide dismutase; GSHPx, glutathione peroxidase * Diabetic significantly different from non-diabetic, P < 0.05 t Manganese-deficient significantly different from manganese-sufficient, P < 0.05 Mn(-), manganese-deficient; Mn( + ), manganese-sufficient 73 T A B L E 10. Effect of diabetes and dietary Mn on GSH, GSSGRd and CAT activities in Sprague-Dawley rats Non-diabetic Diabetic 4 weeks after STZ 8 weeks after STZ Mn(-) Mn( + ) Mn(-) Mn( + ) Mn(-) Mn( + ) (n) (8) (8) (") (9) (4) (4) GSH (umol/g wet weight) Heart 1.99 ± 0.09 2.13 ± 0 . 0 4 2.13 ± 0.07 2.07 ± 0.09 2.26 ± 0.03 2.06 ± 0.07 Liver 6.19 ± 0.19 6.05 ± 0.22 4.49 ± 0 . 2 1 * 4.73± 0.24* 4.89 ± 0.39* 433 ± 0.18* Kidney 3.76 ± 0.09 4.08 ± 0.14 3.39 ± 0.19* 3.36 ± 0 . 1 4 * 233 ± 0 . 2 1 * 2.99 ± 0.42* Pancreas 1.64 ± 0 . 1 6 1.74 ± 0.06 1.39 ± 0.20 136 ± 0 . 1 3 1.27 ± 0 . 1 6 1.43 ± 0.07 GSSGRd (umol NADPH/min/g wet weight) Heart 0.16 ± 0.01 0.16 ± 0.01 0.10 ± 0 . 0 1 0.13 ± 0 . 0 1 0.13 ± 0.02 0.15 ± 0.02 Liver 1.77 ± 0 . 1 3 ' 2.19+.0.23 2.32 ± 0.20 2.33 ± 0.18 1.99 ± 0.09 2.86 ± 0.08 Kidney 438 ± 0.23 435 ± 0.42 434 ± 0.26 4.78 ± 0.43 3.71 ± 0.39* 3.64 ± 036* Pancreas 0.33 ± 0.07 0.45 ± 0.08 0.27 ± 0.03 0.29 ± 0 . 0 5 030 ± 0.05 0.25 ± 0.05 CAT (K/g wet weight) Heart 0.60 ± 0.12 0.62 ± 0.08 1.09+.0.12* 1.24 ± 0.09* 134 ± 0 . 1 9 * 139 ± 0 . 2 0 * Liver 21.74 ± 151 23.06 ± 2.09 19.03 ± 2.20* 1531 ± 1.80* 1830 ± 230* 19.08 ± 2.21* Kidney 4.94 ± 0.84 4.84 ± 0.88 3.86 ± 032 3.74 ± 0.62 3.94 ± 0.21 3.74 ± 0.74 Pancreas 0.39 ± 0.05 0.38 ± 0.07 0.40 ± 0.03 0.46 ± 0.12 037 ±0.08 0.46 ±0.03 Values are means ± SEM. GSH, reduced glutathione; GSSGRd, glutathione reductase; CAT, catalase; K, first-order rate constant Diabetic significantly different from non-diabetic, P< 0.05 1 Manganese-deficient significantly different from manganese-sufficient, P<0.05 Mn(-), manganese-deficient; Mn( + ), manganese-sufficient 74 T A B L E 11. Effect of diabetes on glutathione, glutathione peroxidase & catalase. One-way analysis of variance (ANOVA) on pooled manganese-sufficient, manganese-deficient data. (n) Heart Liver Kidney Pancreas Non-diabetic (16) 2.06 ± 0.05* 6.12 +. 0.1 l a 3.93 +. 0.093 1.69 + 0.07* Diabetic-4 weeks (20) GSH (umol/g wet weight) 2 . 1 1 ± 0 . 0 6 a 4 . 6 0 ± 0 . 1 5 b 3.38 +. 0.12b 1.47 + 0.12a Diabetic-8 weeks (8) 2.16 +. 0.05a 4.71 +_ 0.21b 2.66 +, 0.25c 1.35 + 0.093 Heart Liver Kidney Pancreas 831 +. 033a 39.01 +. 4.003 28.86 +. 1.23* 9.38 + 0303 GSHPx (umol NADPH/min/g wet weight) 8.14 +. 0.38a 24.00 +. 1.09b 23.00 +. 1.40b 9.69 + 0.373 8.14 ± 0.28a 23.22 +. 2.18b 20.28 +. 1.18b 13.04 + 0.98b Heart Liver Kidney Pancreas 0.61 +. 0.06a 22.40 +. 1.293 4.89 +. 0.48* 0.38 + 0.03s CAT (K/g wet weight) 1.16+. 0.08b 17.45 +. 1.47* 3.81 _+ 0.393 0.43 + 0.05a 137 ± 0 . 1 3 c 18.69 ± 1.49* 3.84 ± 0.36a 0.42 + 0.04a Values are means+. SEM. Means not sharing a common superscript are significantly different, P < 0.05. 7 5 t r e a t m e n t t h a n i n n o n - d i a b e t i c s , a n d f u r t h e r d e p l e t e d b y 8 w e e k s f o l l o w i n g d i a b e t e s i n d u c t i o n . I n p a n c r e a s , G S H P x w a s u n c h a n g e d a t 4 w e e k s d i a b e t i c ( c o m p a r e d t o n o n - d i a b e t i c s ) , b u t w a s s i g n i f i c a n t l y i n c r e a s e d b y 8 w e e k s p o s t - S T Z . 4 . 6 . 3 D i s t r i b u t i o n o f T i s s u e A n t i o x i d a n t s C o m p a r i s o n o f t i s s u e a n t i o x i d a n t s i n t h e f o u r t i s s u e s s t u d i e d ( F i g u r e s 1 3 - 1 5 ; T a b l e s 9 a n d 1 0 ) s h o w e d t h e h i g h e s t a c t i v i t i e s o f m o s t a n t i o x i d a n t s m e a s u r e d w e r e i n l i v e r , w i t h k i d n e y n e a r l y a s h i g h . A n o t a b l e e x c e p t i o n w a s k i d n e y G S S G R d a c t i v i t y , w h i c h w a s a p p r o x i m a t e l y t w i c e a s h i g h i n k i d n e y a s i n l i v e r ( T a b l e 1 0 ) . H e a r t a n d p a n c r e a s c o n t a i n e d r e l a t i v e l y l o w e r a m o u n t s o f a n t i o x i d a n t e n z y m e s a n d G S H , e s p e c i a l l y G S S G R d a n d C A T . P a n c r e a t i c C A T a c t i v i t y w a s o n l y 2% t h a t o f l i v e r C A T . M n S O D a c t i v i t i e s w e r e c l o s e s t t o b e i n g c o n s t a n t f r o m o n e t i s s u e t o t h e n e x t . 4.7 E f f e c t s o f V i t a m i n E D e f i c i e n c y on T i s s u e A n t i o x i d a n t s V i t a m i n E d e f i c i e n c y e f f e c t s i n c l u d e d i n c r e a s e d a c t i v i t i e s o f C A T i n k i d n e y , G S S G R d i n h e a r t a n d l i v e r , a n d G S H P x i n p a n c r e a s o f + / - M n D E - r a t s c o m p a r e d t o v i t a m i n E - s u f f i c i e n t d i a b e t i c r a t s ( T a b l e s 1 2 - 1 5 , A p p e n d i x B ) . B y t h e s a m e c o m p a r i s o n , l i v e r a n d p a n c r e a s M n S O D a c t i v i t i e s w e r e d e c r e a s e d , a s w e r e h e a r t G S H P x a n d p a n c r e a t i c C A T . S u r p r i s i n g l y , k i d n e y M n S O D a c t i v i t y w a s d e c r e a s e d i n v i t a m i n E - s u f f i c i e n t , b u t n o t v i t a m i n E - d e f i c i e n t , d i a b e t i c r a t s . B y c o n t r a s t , k i d n e y G S H P x a n d h e a r t M n S O D a c t i v i t i e s w e r e d e c r e a s e d i n b o t h - M n D 4 a n d - M n D E - g r o u p s . Kidney Pancreas Heart Manganese - sufficient Manganese - deficient ND 4 8 ND 4 8 ND 4 8 ND 4 Non-diabetic, 4 or 8 weeks diabetic 8 FIGURE 13. Effects of dietary manganese (Mn) and streptozotocin (STZ)-diabetes on manganese superoxide dismutase (MnSOD) activity in kidney, pancreas, heart and liver of male, Sprague-Dawley rats. Error bars indicate +SEM. Manganese-deficient diets contained 1 ppm Mn; manganese-sufficient diets, 45 ppm Mn. •Diabetic significantly different from non-diabetic (ND) (P<0.05). manganese-deficient significantly different from manganese-sufficient (P<0.05). Kidney Pancreas Heart Liver o o o CD D CO CO O) CO "E 3 12 10 8 6 4 2 Manganese - suf f i c ient Manganese - def ic ient ND 4 8 ND 4 8 ND 4 8 ND 4 8 Non-diabetic, 4 or 8 weeks diabetic FIGURE 14. Effects of dietary manganese (Mn) and streptozotocin (STZ)-diabetes on copper-zinc superoxide dismutase (CuZnSOD) activity in kidney, pancreas, heart and liver of male, Sprague-Dawley rats. Error bars indicate +SEM. Manganese-deficient diets contained 1 ppm Mn; manganese-sufficient diets, 45 ppm Mn. •Diabetic significantly different from non-diabetic (ND) (P<0.05). ** manganese-deficient significantly different from manganese-sufficient (P<0.05). I I h i • it Pancreas Heart M a n g a n e s e - s u f f i c i e n t M a n g a n e s e - d e f i c i e n t ND 4 8 ND 4 8 ND 4 8 ND 4 8 Non-diabetic, 4 or 8 weeks diabetic FIGURE 15. Effects of dietary manganese (Mn) and streptozotocin (STZ)-diabetes on reduced glutathione concentration (GSH) in kidney, pancreas, heart and liver of male, Sprague-Dawley rats. Error bars indicate +SEM. Manganese-deficient diets contained 1 ppm Mn; manganese-sufficient diets, 45 ppm Mn. •Diabetic significantly different from non-diabetic (ND) (P<0.05). ** manganese-deficient significantly different from manganese-sufficient (P<0.05). 79 TABLE 12. Antioxidant status of kidney in vitamin E-sufficient and vitamin E-deficient, manganese-sufficient and manganese-deficient, four week STZ-diabetic rats Vitamin E sufficient Vitamin E-deficient Mn(-) Mn( + ) Mn(-) Mn( + ) 00 (11) (9) (6) (8) GSH(umol/g) 3.39 +.0.19 3.36 ±0.14 3.47 ± 0.22 3.64 ±0 .12 MnSOD 3.92 a ±0.15 5.47 b ±0.28 4 .98 a b ±0 .30 4 . 7 0 a b ± 0 . 3 2 (U/g x IO"2) CuZnSOD 62.1 ± 2 . 9 68.8 ± 4 . 3 65.8 ± 3 . 5 68.0 ± 3 . 4 (U/g x IO'2) GSSGRd 4.54 ± 0.26 4.78 ±0 .43 4.56 ± 0.28 4.60 ±0 .34 (umol NADPH/min/g) GSHPx 2 1 . 2 a ± l . l 25 .4 b ±2.7 19 .8 a ±1.2 23 .0 b ±1 .4 (umol NADPH/min/g) Catalase (K/g) 3.86 ±0 .52 3.74 ±0.62 4.63 ± 0.58 5.84 ±0 .63 Values are means ± SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Mn(-), manganese-deficient; Mn( + ), manganese-sufficient GSH, reduced glutathione; MnSOD, manganese superoxide dismutase; CuZnSOD, copper-zinc superoxide dismutase; GSSGRd, glutathione reductase; GSHPx, glutathione peroxidase U/g, units/g wet weight; K, first order rate constant 80 TABLE 13. Antioxidant status of liver in vitamin E-sufficient and vitamin E-deficient, manganese-sufficient and manganese-deficient, four week STZ-diabetic rats Vitamin E sufficient Vitamin E-deficient Mn(-) Mn( + ) Mn(-) Mn( + ) (n) (11) (9) (6) (8) GSH(umol/g) 3.49 ±0.21 4.73 ± 0.24 4.60 ±0.20 4.20 ±0 .12 MnSOD 7.26 ± 0.26 6.14 ±0 .27 6.03 ±0 .39 5.70 ±0 .28 (U/gxlO-2) CuZnSOD 104.2a ± 4 . 4 84 .1 b ±4.4 9 8 . 5 a b ± 3 . 8 9 7 . 6 s b ± 5 . 5 (U/gxlO"2) GSSGRd 2.32s ±0 .20 2.33 a ±0.18 2.95 b ±0.17 3 .04 b ±0.18 (umol NADPH/min/g) GSHPx 25.8 ± 1 . 6 21.8 ± 2 . 7 21.3 ± 1 . 6 21.7 ± 1 . 2 (umol NADPH/min/g) Catalase (K/g) 19.0 ± 2 . 2 15.5 ± 1.8 19.8 ± 1 . 2 21.2 ± 1 . 6 Values are means ± SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Dietary notations and antioxidant abbreviations are the same as in Table 12. 81 TABLE 14. Antioxidant status of heart in vitamin E-sufficient and vitamin E-deficient, manganese-sufficient and manganese-deficient, four week STZ-diabetic rats Vitamin E sufficient Vitamin E-deficient 00 Mn(-) (11) Mn( + ) (9) Mn(-) (6) Mn( + ) (8) GSH (umol/g) 2 13 ± 0.07 2.07 ±0 .09 2.12 ±0 .09 2.25 ± 0.05 MnSOD (U/g x IO"2) 4.37a ± 0.23 6.31b ± 0.41 4.65 a ±0.21 6.09b ± 0.30 CuZnSOD (U/g x IO"2) 36.7 ± 2.1 38.0 ± 1.5 41.7 ± 2 . 3 39.1 ± 1.1 GSSGRd (umol NADPH/min/g) 0.103a ± 0.012 0.1273 ± 0.011 0.192b ± 0.014 0.210b ± 0.018 GSHPx (umol NADPH/min/g) 8.35a ± 0.52 7.87a ± 0.57 6.96b ± 0.64 6.72b ± 0.29 Catalase (K/g) 1.09+.0.12 1.24 ±0 .09 1.03 ± 0.15 1.11 ±0 .11 Values are means ± SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Dietary notations and antioxidant abbreviations are the same as in Table 12. 8 2 TABLE 15. Antioxidant status of pancreas in vitamin E-sufficient and vitamin E-deficient, manganese-sufficient and manganese-deficient, four week STZ-diabetic rats Vitamin E sufficient Vitamin E-deficient (n) Mn(-) (11) Mn( + ) (9) Mn(-) (6) Mn( + ) (8) GSH (umol/g) 1.39a ± 0.20 1.56a ± 0.13 1.77b ± 0.15 2.18b ± 0.10 MnSOD (U/gxlO-2) 3.09a ± 0.30 2.88 a ±0.14 2.36b ± 0.24 2.35b ± 0.29 CuZnSOD (U/gxlO-2) 22.5 ± 2.0 23.7 ± 1 . 5 25.9 ± 1.5 26.9 ± 1.8 GSSGRd (umol NADPH/min/g) 0.27 ±0 .03 0.29 ± 0.05 0.27 ±0 .04 0.21 ± 0.04 GSHPx (umol NADPH/min/g) 9.75 ± 0.63 9.61 ± 0.34 9.84 ±0.66 11.9 ±1 .16 Catalase (K/g) 0.40 a ±0.03 0.46a ± 0.01 0.24b ± 0.03 0.21b ± 0.03 Values are means ± SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Dietary notations and antioxidant abbreviations are the same as in Table 12. 83 4 . 8 E f f e c t s o f S T Z - d i a b e t e s and V i t a m i n E D e f i c i e n c y on P lasma and H e p a t i c V i t a m i n E An u n e x p e c t e d and p a r t i c u l a r l y i n t e r e s t i n g f i n d i n g i n t h i s s t u d y was e l e v a t i o n i n plasma and h e p a t i c v i t a m i n E as a consequence o f d i a b e t i c i n d u c t i o n ( T a b l e s 16 and 17, F i g u r e 1 6 ) . E l e v a t i o n i n p l a s m a h e p a t i c v i t a m i n E appeared t o be p r o g r e s s i v e , i n c r e a s i n g w i t h t h e d u r a t i o n o f d i a b e t e s . A s i m i l a r p a t t e r n a p p e a r e d i n h e p a t i c v i t a m i n E, though o n l y t h e 8-week d i a b e t i c v i t a m i n E l e v e l s were s i g n i f i c a n t l y h i g h e r t h a n i n non-d i a b e t i c s . H e p a t i c v i t a m i n E/g f a t i n l i v e r r e v e a l e d a p r o g r e s s i v e l y i n c r e a s i n g l e v e l o f v i t a m i n E w i t h d u r a t i o n o f d i a b e t e s ( T a b l e 1 6 ) . No d i f f e r e n c e s between M n - d e f i c i e n t and M n - s u f f i c i e n t v i t a m i n E l e v e l s were o b s e r v e d . 4 . 9 E r y t h r o c y t e and T i s s u e S u s c e p t i b i l i t y t o L i p i d P e r o x i d a t i o n i n S T Z - d i a b e t i c R a t s E r y t h r o c y t e s u s c e p t i b i l i t y t o ^ C ^ - i n d u c e d l i p i d p e r o x i d a t i o n , a s measured by t i s s u e TBARS, was s i g n i f i c a n t l y i n c r e a s e d i n 8-week S T Z - d i a b e t i c r a t s , compared t o b o t h 4-week d i a b e t i c and n o n - d i a b e t i c a n i m a l s ( T a b l e 1 8 ) . O t h e r t i s s u e TBARS d i d n o t d i f f e r s i g n i f i c a n t l y w i t h r e s p e c t t o manganese d e f i c i e n c y o r d u r a t i o n o f d i a b e t e s o v e r t h i s t i m e p e r i o d . P lasma l i p o p e r o x i d e s and r e n a l a d i p o s e t i s s u e f l u o r e s c e n c e were e l e v a t e d i n d i a b e t i c , compared t o n o n - d i a b e t i c r a t s , b u t d i d n o t d i f f e r w i t h r e s p e c t t o manganese d e f i c i e n c y ( T a b l e 1 9 ) . 84 TABLE 16. Effects of manganese deficiency & STZ-diabetes on plasma and hepatic vitamin E Non-diabetic Diabetic 4 weeks after STZ 8 weeks after STZ Mn(-) Mn( + ) Mn(-) Mn( + ) Mn(-) Mn( + ) Plasma vit E (mg/dL) 0.48a±0.06(6) 0.44a ±0.02(5) 0.89b±0.15(6) 0.99b±0.18(5) 1.14b±0.23(4) 1.56b ±0.21(4) Hepatic vit E 46a ±2.6(4) 52a ± 2.4(4) (nmol/g wet weight) 76a ± 6.5(4) 61a ± 11.3(4) 120b ± 16(4) 113b ± 18(4) Hepatic vit E 0.93a ± 0.08 1.17a ± 0.10 1.84 b ±0.14 1.29 b ±0.24 2.95c ± 0.34 2.80c ± 0.38 (umol/g fat) Values are means ± SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Sample size in parentheses. Mn(-), manganese-deficient; Mn( + ), Mn-sufficient; vit E, vitamin E 85 Table 17. Effect of vitamin E deficiency on plasma and hepatic vitamin E in four week diabetic rats Vitamin E-deficient Vitamin E-sufficient (a) (8) (8) Plasma vitamin E (mg/dl) 0.04a±0.02 0.92b±0.11 Hepatic vitamin E (nmol/g) 5.00a±1.82 65.6b ± 7.12 Hepatic vitamin E(ug/g fat) 0.15a±0.02 1.57b ± 0.15 Values are means +. SEM. Means not sharing a common superscript are significantly different, P< 0.001 ND 4 8 ND 4 8 Non-diabetic, 4 or 8 weeks diabetic FIGURE 16. Effects of dietary manganese (Mn) and streptozotocin (STZ)-diabetes on plasma and hepatic vitamin E in male, Sprague-Dawley rats. Error bars indicate +SEM. Manganese-deficient diets contained 1 ppm Mn; manganese-sufficient diets, 45 ppm Mn. * 4 week-diabetic significantly different from non-diabetic (ND) (P<0.05). ** 8 week-diabetic significantly different from 4 week-diabetic (P<0.05). Manganese-deficient were not significantly different from manganese-sufficient rats. co C7\ 8 7 TABLE 18. Effects of manganese deficiency & STZ-diabetes on thiobarbituric acid reactive substances (TBARS) Non-diabetic Diabetic 4 weeks after STZ 8 weeks after STZ Mn(-) Mn( + ) Mn(-) Mn( + ) Mn(-) Mn( + ) TBARS: RBC(nmolMDA/gHb) 295s +. 10(8) 289a +. 11(8) 298a ± 10(11) 331a +. 11(9) 571b +. 33(4) 526bjt42(4) Heart (nmol MDA/g) 49.7a ±6.5 41.2a ± 3.1 30.2b +. 2.4 36.2b +. 3.1 3 9 7ab ± 6 7 53.7a +. 11.8 Kidney (nmol MDA/g) 51.0a ± 5.9 48.1a +. 2.7 44.3a +. 2.1 48.9a +. 3.4 51.3s ± 8.7 63.7a +. 9.3 Liver (nmol MDA/g) 62.6a +. 6.0 59.2a +. 3.4 58.6a +. 3.2 63.9s +.4.0 61.3s +. 9.4 85.2a +. 9.5 Pancreas (nmol MDA/g) 33.9s +. 3.3 34.0a +. 6.9 29.9a +. 1.6 35.5a ± 2.8 38 .0 a ±5 .9 41.8s ±4.4 Values are means +. SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Sample size in parentheses. Mn(-), manganese-deficient; Mn( + ), Mn-sufficient; MDA, malondialdehyde; Hb, hemoglobin 88 TABLE 19. Effects of manganese deficiency & STZ-diabetes on red cell hemolysis, plasma lipoperoxides, and renal adipose tissue fluorescence Non-diabetic Diabetic 4 weeks after STZ 8 weeks after STZ Mn(-) Mn( + ) Mn(-) Mn( + ) Mn(-) Mn( + ) Red cell hemolysis (%) 6.8a ± 0.2(2) 6.0*^ 0.7(6) 3 3 b ± 0.9(5) 3 . 8 b ± 0.4(3) 3.1b +. 1.0(4) 1.4b+. 0.2(4) Plasma lipoperoxides (r.f.*) .205a +. .130(2) .404b ± .030(4) .489b +. .110(4) Renal adipose tissue fluorescence 373a +. 0.85(2) .905b+. .074(4) .773b +. .151(2) .836b +. .202(4) .813b+. .161(2) Values are means +_ SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Sample size in parentheses. •r.f., relative fluorescence, compared to 1 ug/ml quinine sulfate = 21 Mn(-), manganese-deficient; Mn( + ), Mn-sufficient 89 C u r i o u s l y , r e d c e l l h e m o l y s i s appeared t o d e c l i n e i n d i a b e t i c , compared t o n o n - d i a b e t i c , r e d c e l l s . A l l t i s s u e TBARS were h i g h e r i n +/-MnDE- compared t o +/-MnD4 r a t s ( T a b l e 2 0 ) . E r y t h r o c y t e h e m o l y s i s was e l e v a t e d i n +/-MnDE-r a t s compared t o v i t a m i n E - s u f f i c i e n t r a t s . R e n a l a d i p o s e t i s s u e f l u o r e s c e n c e was n o t a f f e c t e d by v i t a m i n E o r manganese d e f i c i e n c y . 4 . 1 0 C o r r e l a t i o n C o e f f i c i e n t s R e s u l t s o f l i n e a r r e g r e s s i o n a n a l y s i s f o r GSH and a l l t i s s u e a n t i o x i d a n t enzymes measured f o r a l l t i s s u e s combined ( i n d i a b e t i c , v i t a m i n E s u f f i c i e n t r a t s , s e t 1) a r e p r e s e n t e d i n T a b l e 21. The r e were s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s between MnSOD, CuZnSOD and GSH; between CuZnSOD, GSHPx, GSH and CAT; between GSHPx, GSH and GSSGRd, and between CAT and GSH. S i m i l a r c o r r e l a t i o n c o e f f i c i e n t s were found f o r n o n - d i a b e t i c r a t s ( n o t shown), w i t h t h e e x c e p t i o n t h a t a l l r v a l u e s f o r MnSOD were <0.47. E x a m i n a t i o n o f c o r r e l a t i o n s between a n t i o x i d a n t s i n i n d i v i d u a l t i s s u e s o f v i t a m i n E - s u f f i c i e n t , d i a b e t i c r a t s ( T a b l e 22) r e v e a l e d t h a t GSH was s i g n i f i c a n t l y c o r r e l a t e d w i t h MnSOD, CuZnSOD, and GSHPx i n k i d n e y ; and w i t h MnSOD and CuZnSOD i n p a n c r e a s . G l u t a t h i o n e was n e g a t i v e l y c o r r e l a t e d w i t h GSHPx i n h e a r t and w i t h MnSOD i n l i v e r . C a t a l a s e was p o s i t i v e l y c o r r e l a t e d w i t h GSHPx i n l i v e r and k i d n e y . 9 0 TABLE 20. Effects of manganese deficiency & vitamin E deficiency on measures of lipid peroxidation in 4-week STZ-diabetic rats Vitamin E-sufficient Vitamin E-deficient Mn(-) Mn( + ) TBARS: RBC (nmol MDA/gHb) 298a ± 10(11) 331a ± 11(9) Heart (nmol/g tissue) Kidney (nmol/g tissue) Liver (nmol/g tissue) Pancreas (nmol/g tissue) 30.2a ± 2.4(11) 36.2a ± 3.1(9) 44.3a ± 2.1(11) 48.9s ± 3.4(9) 58.6a ± 3.2(11) 63.9s ±4.0(4) 29.9s ± 1.6(11) 35.5a ± 2.8(4) Mn(-) Mn( + ) 400 a ±59(6) 286 a ±27(8 ) 55.2b ± 7.0(6) 67.3b ± 7.4(8) 67.7b ± 8.1(6) 80.7b ± 7.4(8) 68.6b ± 6.0(6) 78.1b ± 9.1(8) 4 1 . 6 b ± 4.6(6) 4 5 . 8 b ± 3.2(8) Red cell hemolysis (%) Plasma lipoperoxides (r.f.*) Renal adipose tissue fluorescence 3 . 5 a ± 0.9(5) 3 . 8 a ± 0.4(3) 7 . 8 b ± 2.8(7) 10.04b ± 2.7(7) .344 ± .030(6) .418 ± .039(8) .773a ± .151(2) .836a ± .202(4) .893a ± .199(6) .8993 ± .264(8) Values are means ± SEM. Means not sharing a common superscript in each row are significantly different at P < 0.05. Sample size in parentheses. *r.f., relative fluorescence, compared to 1 ug/ml quinine sulfate = 21 Mn(-), manganese-deficient; Mn( + ), Mn-sufficient 91 TABLE 21. Linear regression correlation coefficients for tissue antioxidants measured in diabetic rats Correlations: MnSOD CuZnSOD GSH GSHPx GSSGRd CAT MnSOD 1.000 0.719* 0.693* 0.403 0.269 0.628* CuZnSOD 1.000 0.864* 0.751* 0.556* 0.827* GSH 1.000 0.700* 0368* 0.805* GSHPx 1.000 0.724* 0.649* GSSGRd 1.000 0.296 CAT 1.000 * Significant correlation coefficient, P< 0.001 MnSOD, manganese superoxide dismutase; CuZnSOD, copper-zinc superoxide dismutase; GSH, glutathione; GSHPx, glutathione peroxidase; GSSGRd, glutathione reductase; CAT, catalase 92 Table 22: Linear regression correlation coefficients and statistical significance for tissue antioxidants in individual tissues in rats Tissue Correlations Vitamin E Heart Kidney Liver Pancreas GSH vs. GSHPx + GSH vs. MnSOD + GSH vs. CuZnSOD + GSH vs. GSHPx + CuZnSOD vs. MnSOD + CATvs. GSHPx + GSH vs. MnSOD + GSH vs. MnSOD + GSH vs. CuZnSOD + -0.357 0317 0.626 0.434 0331 0.403 -0.364 0.450 0349 <0.01 < 0.001 < 0.001 <0.01 < 0.001 <0.01 <0.01 <0.01 < 0.001 Heart Kidney GSHPx vs. CAT GSH vs. CuZnSOD 0.648 0.730 <0.01 < 0.001 GSH, reduced glutathione; GSHPx, glutathione peroxidase; MnSOD, manganese superoxide dismutase; CuZnSOD, copper-zinc superoxide dismutase; CAT, catalase 93 C o r r e l a t i o n s o f n o t e i n v i t a m i n E - d e f i c i e n t g r o u p s compared by t i s s u e ( T a b l e 22) were p o s i t i v e c o r r e l a t i o n s between GSHPx and CAT i n h e a r t and between GSH and CuZnSOD i n k i d n e y . 94 5. DISCUSSION The major o b j e c t i v e s o f t h i s s t u d y were t o answer t h e f o l l o w i n g q u e s t i o n s : 1) does manganese d e f i c i e n c y e x a c e r b a t e STZ-d i a b e t e s ? ; 2) does t h e d u r a t i o n o f d i a b e t e s a f f e c t t i s s u e a n t i o x i d a n t b a l a n c e ? ; 3) i s S T Z - d i a b e t e s combined w i t h manganese d e f i c i e n c y a c o n d i t i o n o f h e i g h t e n e d o x i d a n t s t r e s s ? ; and 4) does v i t a m i n E - d e f i c i e n c y "unmask" changes due t o r e d u c e d manganese i n t a k e ? I n o r d e r t o a d d r e s s t h e f i r s t q u e s t i o n , we needed t o e s t a b l i s h t h a t b o t h manganese d e f i c i e n c y and STZ-d i a b e t e s were i n d u c e d i n t h e a p p r o p r i a t e groups. 5.1 Manganese d e f i c i e n c y The manganese d e f i c i e n t s t a t u s o f r a t s f e d M n - d e f i c i e n t d i e t s was c o n f i r m e d by d e c r e a s e d manganese c o n c e n t r a t i o n s i n h e a r t , l i v e r and k i d n e y ( T a b l e 4 ) . Decreases o f 40 - 50% i n l i v e r , h e a r t and k i d n e y were c o n s i s t e n t w i t h t h e r e s u l t s o f D a v i s e t a l . (1989), P a y n t e r (1980a) and Kawano e t a l . (1989) f o r male Sprague Dawley r a t s on m a n g a n e s e - d e f i c i e n t d i e t s f o r 6 - 8 weeks. Manganese c o n t e n t o f m a n g a n e s e - d e f i c i e n t t i s s u e s (/xg Mn/organ) was a l s o s i g n i f i c a n t l y d e c r e a s e d compared t o manganese-s u f f i c i e n t t i s s u e s ( T a ble 5 ) . I n p a r t i c u l a r , Mn c o n t e n t o f Mn-d e f i c i e n t p a n c r e a s was s i g n i f i c a n t l y d e c r e a s e d compared t o Mn-s u f f i c i e n t p a n c r e a s when compared on a whole organ b a s i s , even though no s i g n i f i c a n t d i f f e r e n c e was found when compared on a 95 /zg/g wet w e i g h t b a s i s . Food e f f i c i e n c y r a t i o s (bodyweight g a i n / f o o d i n t a k e ) were n o t s i g n i f i c a n t l y d i f f e r e n t , a v e r a g i n g 0.171 f o r b o t h manganese-s u f f i c i e n t and m a n g a n e s e - d e f i c i e n t d i e t s ( T a b l e 3 ) . T h e r e were a l s o no d i f f e r e n c e s i n w e i g h t g a i n between t h e 1 /zg Mn/g d i e t and t h e 45 /zg Mn/g d i e t ( F i g u r e 11) . These r e s u l t s a r e c o n s i s t e n t w i t h t h o s e o f B a l y e t a l . (1984), Kawano e t a l . (1987) and D a v i s e t a l . (1989), who a l s o showed no d i f f e r e n c e s i n body w e i g h t a t a u t o p s y between m a n g a n e s e - d e f i c i e n t and m a n g a n e s e - s u f f i c i e n t r a t s . I n t h e s t u d y by P a y n t e r ( 1 9 8 0 a ) , f o o d e f f i c i e n c y v a r i e d from 0.148 f o r a 0.2 /zg Mn/g d i e t t o 0.255 f o r a 9.7 /zg Mn/g d i e t . R a t s f e d d i e t s c o n t a i n i n g 0.2 /zg Mn/g o r 0.7 /zg Mn/g g a i n e d l e s s w e i g h t d u r i n g t h e s t u d y p e r i o d (10 weeks) t h a n r a t s f e d h i g h e r l e v e l s o f Mn. The c o n c l u s i o n t h a t 2 /zg Mn/g d i e t i s n e c e s s a r y f o r maximal g r o w t h o f male, Sprague-Dawley r a t s s h o u l d be t a k e n as an a p p r o x i m a t i o n ; however, s i n c e r a t s f e d 1.7 /zg Mn/g d i e t were n o t s i g n i f i c a n t l y s m a l l e r t h a n a n i m a l s f e d 9.7 /zg Mn/g d i e t . T h i s r e s u l t i s c o n s i s t e n t w i t h t h e r e c e n t r a d i o i s o t o p e s t u d y o f Lee and J o h n s o n (1988) w h i c h showed c o n c l u s i v e l y t h a t t h e r e q u i r e m e n t f o r g r o w t h o f male r a t s i s n o t more t h a n 1.4 /zg Mn/g d i e t when t h e AIN f o r m u l a t i o n ( B i e r i e t a l . , 1977) i s used. R a t g r o w t h , f o o d i n t a k e and f o o d e f f i c i e n c y f o r M n - d e f i c i e n t and M n - s u f f i c i e n t r a t s i n t h e p r e s e n t s t u d y t h u s c o r r o b o r a t e r e s u l t s o f p r e v i o u s i n v e s t i g a t i o n s . 96 5.2 I n d u c t i o n o f D i a b e t e s D i a b e t e s was c o n f i r m e d by b l o o d g l u c o s e l e v e l s >11 mM, b o t h 24 h o u r s a f t e r STZ t r e a t m e n t and i m m e d i a t e l y p r i o r t o a u t o p s y . S u s t a i n e d h y p e r g l y c e m i a a t t h i s l e v e l has been e s t a b l i s h e d as t h e p r i n c i p a l c r i t e r i o n f o r d e t e r m i n i n g d i a b e t e s i n r a t s (Ungar, 1991). Other c r i t e r i a i n c l u d e p o l y d i p s i a , p o l y u r i a , w e i g h t l o s s and h y p e r p h a g i a . I n c r e a s e d t h i r s t (and consequent i n c r e a s e d c onsumption o f water) and i n c r e a s e d u r i n a r y o u t p u t were o b s e r v e d i n a l l d i a b e t i c a n i m a l s , but were not q u a n t i f i e d . S u b s t a n t i a l w e i g h t l o s s ( F i g u r e 11) was obse r v e d i n a l l d i a b e t i c r a t s , w h i c h l o s t up t o 2 0% o f t h e i r body w e i g h t w i t h i n 2 weeks o f STZ a d m i n i s t r a t i o n . Four weeks a f t e r STZ t h e d i a b e t i c a n i m a l s were 30% l i g h t e r t h a n n o n - d i a b e t i c r a t s . R a t s w h i c h were m a i n t a i n e d f o r an a d d i t i o n a l f o u r weeks tended t o s t a b i l i z e t h e i r w e i g h t ; some g a i n e d up t o 15% (compared t o t h e i r l o w e s t w e i g h t a f t e r STZ t r e a t m e n t ) i n t h e l a s t 2 weeks p r i o r t o a u t o p s y . These r e s u l t s compare w i t h r e c o r d e d w e i g h t l o s s e s o f 29% o f pre-STZ w e i g h t i n 12 weeks o f d i a b e t e s (Wohaieb and Godin, 1987a), 21% i n 3 weeks, 31% i n 6 weeks and 32% i n 9 weeks o f d i a b e t e s ( P i l l i o n e t a l . , 1988), and 10% i n 10 days ( F a i l l a and K i s e r , 1981). A f o u r t h symptom o f e x p e r i m e n t a l d i a b e t e s : h y p e r p h a g i a , was n o t c l e a r l y d e monstrated i n t h e s e r a t s . R a t s w h i c h were m a i n t a i n e d f o r 8 weeks f o l l o w i n g STZ a d m i n i s t r a t i o n were o b s e r v e d t o have i n c r e a s e d f o o d consumption (average i n t a k e i n week 7: 37.5g/day), compared t o n o n d i a b e t i c r a t s (27.7 g / d a y ) ; however, t h i s c o m p a r i s o n i s not s t r i c t l y v a l i d , s i n c e t h e 7-week d i a b e t i c 97 r a t s were 3 weeks o l d e r (but n o t h e a v i e r ) t h a n t h e n o n d i a b e t i c r a t s . The degree o f i n c r e a s e d f o o d c o n s u m p t i o n ( o r h y p e r p h a g i a ) accompanying S T Z - i n d u c t i o n o f d i a b e t e s v a r i e s . F a i l l a and K i s e r (1981) o b s e r v e d a 37% i n c r e a s e i n f o o d c o n s u m p t i o n w i t h i n 2 weeks o f STZ a d m i n i s t r a t i o n . A 70% i n c r e a s e was o b s e r v e d i n male W i s t a r r a t s o v e r a 6-week d i a b e t i c p e r i o d (Basu e t a l . , 1 9 9 0 ) , w h i c h i s s i m i l a r t o t h e r e s u l t s o f S i n h a e t a l . (1990) i n n o n - p r e g n a n t , f e m a l e Sprague-Dawley r a t s , and o f Ho e t a l . (1988) i n male Sprague-Dawley r a t s . The s i t u a t i o n i n p r e g n a n t , f e m a l e Sprague-Dawleys seems t o be d i f f e r e n t . B o t h U r i u - H a r e e t a l . (1989) and G i a v i n i e t a l . (1990) r e c o r d e d 2 t o 3 t i m e s i n c r e a s e d f o o d c o n s u m p t i o n a f t e r STZ i n d u c t i o n o f d i a b e t e s . The l a c k o f an immediate h y p e r p h a g i c r e s p o n s e i n t h i s s t u d y may have been due t o t h e h i g h f i b r e c o n t e n t o f t h e p u r i f i e d d i e t . I n c r e a s i n g t h e f i b r e c o n t e n t o f a d i e t g e n e r a l l y r e q u i r e s i n c r e a s e d f e e d c o n s u m p t i o n i n o r d e r t o m a i n t a i n n o r m a l w e i g h t g a i n ( P e t e r s o n e t a l . , 1954; S i b b a l d e t a l . , 1956; P e t e r s o n and Baumgardt, 1971a,b). T h i s has been shown t o o c c u r as l o n g as d i e t a r y p r o t e i n i s adequate ( a t l e a s t 25%) (Delorme and W o j c i k , 1982, S i e g e l e t a l . , 1976). I n t h i s s t u d y , d i e t a r y p r o t e i n was more t h a n adequate (30%) and w e i g h t g a i n a p p e a r e d t o be normal ( B i e r i e t a l . , 1976; UFAW, 1986) and was s i m i l a r t o t h a t d e m o n s t r a t e d by P a y n t e r (1980a). Numerous i n v e s t i g a t o r s ( e.g., S i e g e l e t a l . , 1976, 1977; Trapp e t a l . , 1976; T r a c k e t a l . , 1982; Lee, 1982; Schmidt e t a l . , 1982) have n o t e d i m p r o v e d d i a b e t i c c o n t r o l , a l o n g w i t h i n c r e a s e d w e i g h t and b e t t e r g e n e r a l 98 c o n d i t i o n o f a n i m a l s , w i t h i n c r e a s e d f i b r e c o n t e n t o f d i e t s . T h i s may e x p l a i n t h e d e l a y i n h y p e r p h a g i c r e s p o n s e i n t h e p r e s e n t s t u d y . C o r r o b o r a t i v e e v i d e n c e f o r i n d u c t i o n o f d i a b e t e s i n c l u d e d changes i n t i s s u e / b o d y w e i g h t r a t i o s ( T a b l e 6 ) . K i d n e y h y p e r t r o p h y was e v i d e n t i n a l l d i a b e t i c r a t s . K i d n e y w e i g h t s i n t h i s s t u d y a v e r a g e d 3.46 g (as compared t o 2.56 g f o r n o n d i a b e t i c r a t s ) ; k i d n e y t o body w e i g h t r a t i o s were 15.45 mg/g (compared t o 7.99 mg/g), and h e a r t t o body w e i g h t r a t i o s were 3.05 mg/g (compared t o 2.71 mg/g). A l l were s i g n i f i c a n t l y d i f f e r e n t , P< 0.05, when d i a b e t i c and n o n - d i a b e t i c r a t s were compared. H e a r t w e i g h t s d i d n o t d i f f e r between d i a b e t i c and n o n - d i a b e t i c a n i m a l s . These r e s u l t s c o r r o b o r a t e e a r l i e r o b s e r v a t i o n s ( P i l l i o n e t a l * . , 1988; Rasch, 1979), and have been c o n s i d e r e d d i s t i n g u i s h i n g f e a t u r e s o f S T Z - d i a b e t e s i n r a t s . An i n t e r e s t i n g c o r r o l l a r y o b s e r v a t i o n i n t h i s s t u d y was s i g n i f i c a n t l y d e c r e a s e d p a n c r e a s w e i g h t as an accompaniment t o manganese d e f i c i e n c y ( T a b l e 6 ) . P a n c r e a t i c a t r o p h y as a r e s u l t o f i n a d e q u a t e d i e t a r y i n t a k e o f manganese has n o t been p r e v i o u s l y r e p o r t e d ; however, M y l o r i e e t a l . (1988) o b s e r v e d an even g r e a t e r d e c r e a s e i n p a n c r e a s w e i g h t (76% d e c r e a s e by week 7) i n c o p p e r - d e f i c i e n t male Sprague-Dawley r a t s . They h y p o t h e s i z e d t h a t d e c r e a s e s i n b o t h CuZnSOD and MnSOD a c t i v i t i e s were i n t e g r a l l y i n v o l v e d i n a t r o p h y o f p a n c r e a t i c t i s s u e . 99 5 .3 S e v e r i t y o f D i a b e t e s H a v i n g e s t a b l i s h e d t h a t manganese d e f i c i e n c y and S T Z - d i a b e t e s were s u c c e s s f u l l y i n d u c e d , we can now a d d r e s s d i a b e t i c s e v e r i t y and i t s r e l a t i o n t o manganese d e f i c i e n c y . C r i t e r i a f o r e s t a b l i s h i n g t h e s e v e r i t y o f e x p e r i m e n t a l d i a b e t e s a r e open t o d e b a t e ( R e n o l d e t a l . , 1974; E l l e n b e r g and R i f k i n , 1983) . G l y c o s y l a t e d hemoglobin i s u s e f u l as a measure o f l o n g -t e r m d i a b e t i c c o n t r o l , b o t h i n humans (Tze e t a l . , 1977; Gabbay e t a l . , 1977), and i n e x p e r i m e n t a l a n i m a l s ( K o e n i g and C e r a m i , 1980) . I t s u t i l i t y f o r s h o r t - t e r m a s s e s s m e n t s may be compromised by l a c k o f s e n s i t i v i t y and p r e c i s i o n . I n t h i s s t u d y , H b A i c l e v e l s were s i g n i f i c a n t l y h i g h e r i n d i a b e t i c t h a n n o n - d i a b e t i c r a t s , b u t were n o t s i g n i f i c a n t l y d i f f e r e n t between M n - d e f i c i e n t and M n - s u f f i c i e n t groups ( T a b l e 8 ) . V a l u e s o b t a i n e d were s i m i l a r t o t h o s e r e c o r d e d by Wohaeib and G o d i n (1987a) (8.78 ± 1.20%) f o r d i a b e t i c r a t s 12 weeks a f t e r STZ t r e a t m e n t . P a n c r e a t i c i n s u l i n r e l e a s e (PIR) has been p r o p o s e d as an a c c u r a t e measure o f s e v e r i t y o f l o n g - t e r m e x p e r i m e n t a l d i a b e t e s ( S t u d e r and M u l l e r , 1974). Plasma i n s u l i n l e v e l s ( T a b l e 8) i n d i a b e t i c r a t s were c l e a r l y d e p r e s s e d compared t o n o r m a l l e v e l s (50 - 100 jLtU/ml; S t e r n e t a l . , 1975; Radder e t a l . , 1982; B a l y e t a l . , 1 984), b u t showed no d i f f e r e n c e s between M n - d e f i c i e n t and M n - s u f f i c i e n t r a t s . 100 A n o t h e r commonly u s e d measure o f d i a b e t i c s e v e r i t y i s r e p o n s e t o g l u c o s e t o l e r a n c e t e s t s (Ekoe, 1988; S e i g e l e t a l . , 1 982). T e s t s were p e r f o r m e d 1 week p r i o r t o a u t o p s y i n o r d e r t o a v o i d any c o n f o u n d i n g e f f e c t s o f o v e r n i g h t f a s t i n g on t i s s u e a n t i o x i d a n t s t a t u s (Wohaieb and G o d i n , 1987b). Comparison o f a r e a s u n d e r t h e c u r v e , w i t h b a s e l i n e s u b t r a c t e d ( T a b l e 8, F i g u r e 12) , i n d i c a t e d a s i g n i f i c a n t l y i n c r e a s e d r e s p o n s e t o g l u c o s e t o l e r a n c e t e s t i n g w i t h manganese d e f i c i e n c y . A v e r a g e a r e a s u n d e r t h e c u r v e f o r m a n g a n e s e - d e f i c i e n t , d i a b e t i c r a t s were 640 mM'min compared t o 392 mM'min f o r m a n g a n e s e - s u f f i c i e n t , d i a b e t i c a n i m a l s , an i n c r e a s e o f 63%. E i g h t week d i a b e t i c r a t s t e n d e d t o have h i g h e r a r e a s under t h e c u r v e t h a n 4-week d i a b e t i c r a t s , b u t t h e d i f f e r e n c e s were n o t s t a t i s t i c a l l y s i g n i f i c a n t . These r e s u l t s s u g g e s t an e x a c e r b a t e d g l u c o s e t o l e r a n c e t e s t r e s p o n s e i n m a n g a n e s e - d e f i c i e n t , d i a b e t i c a n i m a l s . 5 . 4 T i s s u e A n t i o x i d a n t Enzyme A l t e r a t i o n s 5 . 4 . 1 E f f e c t s o f Manganese D e f i c i e n c y and S T Z - d i a b e t e s on S u p e r o x i d e D i s m u t a s e A c t i v i t i e s Manganese d e f i c i e n c y d e c r e a s e d a c t i v i t i e s o f k i d n e y and h e a r t MnSOD, and o f k i d n e y CuZnSOD. L i v e r MnSOD was p a r a d o x i c a l l y i n c r e a s e d ( T a b l e 9, Appe n d i x A ) . K i d n e y MnSOD was d e c r e a s e d 10% compared t o M n - s u f f i c i e n t , n o n - d i a b e t i c r a t s and 18% co m p a r i n g M n - d e f i c i e n t t o M n - s u f f i c i e n t r a t s . M n - d e f i c i e n t d i a b e t i c r a t s had 35% l o w e r MnSOD compared t o m a n g a n e s e - s u f f i c i e n t , non-d i a b e t i c a n i m a l s . T h i s shows a g r e a t e r t h a n a d d i t i v e (and 101 h e n ce, p o s s i b l y s y n g e r g i s t i c ) e f f e c t f o r k i d n e y MnSOD a c t i v i t i e s . I n h e a r t , M n - d e f i c i e n c y d e c r e a s e d MnSOD a c t i v i t y by 30%, whereas S T Z - d i a b e t e s r e s u l t e d i n a 10% i n c r e a s e i n a c t i v i t y . The combined t r e a t m e n t s c a used a 24% d e c r e a s e i n a c t i v i t y . I n l i v e r , M n - d e f i c i e n c y i n c r e a s e d MnSOD a c t i v i t y by 8%, d i a b e t e s i n c r e a s e d i t by 23%, and t h e combined t r e a t m e n t s r e s u l t e d i n a 4 6% i n c r e a s e . K i d n e y CuZnSOD was d e c r e a s e d i n b o t h M n - d e f i c i e n t and d i a b e t i c r a t s (17% and 18%, r e s p e c t i v e l y ) , b u t t h e combined e f f e c t s were n o t a d d i t i v e (26% f o r manganese-d e f i c i e n t , d i a b e t i c compared t o m a n g a n e s e - s u f f i c i e n t , non-d i a b e t i c a n i m a l s ) . The i n t e r a c t i v e e f f e c t s o f manganese d e f i c i e n c y and S T Z - d i a b e t e s a r e t h u s p r i n c i p a l l y c o n c e n t r a t e d i n m i t o c h o n d r i a l e n z y m a t i c a c t i v i t y changes. P r e v i o u s i n v e s t i g a t i o n s o f e f f e c t s o f manganese d e f i c i e n c y on t i s s u e a n t i o x i d a n t s t a t u s have d e m o n s t r a t e d d e c r e a s e d k i d n e y and h e a r t MnSOD a c t i v i t y ( P a y n t e r , 1980a; Z i d e n b e r g - C h e r r e t a l . , 1983; D a v i s e t a l . , 1989) i n M n - d e f i c i e n t compared t o Mn-s u f f i c i e n t r a t s . S i m i l a r l y , c o p p e r d e f i c i e n c y i n r a t s r e s u l t e d i n d e c r e a s e d h e a r t CuZnSOD a c t i v i t y , a l t h o u g h z i n c d e f i c i e n c y had no s u c h e f f e c t ( B e t t g e r and B r a y , 1989). E f f e c t s o f an o x i d a t i v e s t r e s s s u c h a s e t h a n o l c o n s u m p t i o n (Keen e t a l . , 1985) o r i n c r e a s e d p o l y u n s a t u r a t e d f a t t y a c i d d i e t ( D a v i s e t a l . , 1990) i n c l u d e i n c r e a s e d MnSOD a c t i v i t y i n l i v e r and h e a r t , r e s p e c t i v e l y . I n a s t u d y by Z i d e n b e r g - C h e r r e t a l . ( 1 9 8 5 ) , manganese d e f i c i e n c y combined w i t h e t h a n o l c o n s u m p t i o n i n r a t s r e s u l t e d i n no s i g n i f i c a n t d i f f e r e n c e s i n l i v e r MnSOD a c t i v i t y 102 compared t o c o n t r o l r a t s (246.8 ± 29.3 U MnSOD/g l i v e r f o r Mn-d e f i c i e n t , compared t o 195.9 ± 39.5 U MnSOD/g l i v e r f o r Mn-s u f f i c i e n t , e t h a n o l - c o n s u m i n g r a t s ) . D i a b e t e s , w i t h o u t added c o f a c t o r d e p r i v a t i o n , has a l s o been shown p r e v i o u s l y t o r e s u l t i n d e c r e a s e d k i d n e y and l i v e r CuZnSOD a c t i v i t y (Wohaieb and Godin, 1987a; Loven e t a l . , 1986; M a t k o v i c s , 1977). T h i s may be t h e r e s u l t o f a d i r e c t i n h i b i t o r y e f f e c t o f t h e i n c r e a s e d p r o d u c t i o n o f H2O2 i n e x p e r i m e n t a l d i a b e t e s . CuZnSOD, but not MnSOD a c t i v i t y i s known t o be i n h i b i t e d by H2O2 (Loven e t a l . , 1983; Kono and F r i d o v i c h , 1982), w i t h no change i n immunoreactive CuZnSOD. On t h e o t h e r hand, i n c r e a s e d p r o d u c t i o n o f s u p e r o x i d e does n o t have an i n h i b i t o r y e f f e c t on s u p e r o x i d e d i s m u t a s e and has been shown t o have a s t i m u l a t o r y e f f e c t on s y n t h e s i s o f MnSOD (Wong e t a l . , 1989). I n c r e a s e s i n t h e p r o d u c t i o n o f s u p e r o x i d e i n d i a b e t i c p a t i e n t s have been measured in vivo (Nath e t a l . , 1984), b u t d i d n o t p a r a l l e l i n c r e a s e d MnSOD a c t i v i t y i n p o l y m o r p h o n u c l e a r l e u k o c y t e s . Copper d e f i c i e n c y i n c o m b i n a t i o n w i t h S T Z - d i a b e t e s p r o d u c e d no i n t e r a c t i o n w i t h r e g a r d t o s u p e r o x i d e d i s m u t a s e s t a t u s (Rucker e t a l . , 1991). Copper d e f i c i e n c y i n c o m b i n a t i o n w i t h a d i f f e r e n t o x i d a t i v e s t r e s s , i n t h i s case i n f l a m m a t i o n , r e s u l t e d i n an e x a c e r b a t i o n o f t h e r e d u c t i o n i n l i v e r and e r y t h r o c y t e 103 CuZnSOD seen w i t h e i t h e r t r e a t m e n t a l o n e ( D i S i l v e s t r o and M a r t e n , 1990). Manganese d e f i c i e n c y i n c o m b i n a t i o n w i t h S T Z - d i a b e t e s r e s u l t e d i n an i n t e r a c t i v e e f f e c t between manganese d e f i c i e n c y and d i a b e t e s w i t h r e s p e c t t o k i d n e y , h e a r t and l i v e r MnSOD, and k i d n e y CuZnSOD i n t h e p r e s e n t s t u d y . 5 . 4 . 2 . E f f e c t s o f D i a b e t e s on Other T i s s u e A n t i o x i d a n t Enzymes L i v e r and k i d n e y g l u t a t h i o n e p e r o x i d a s e a c t i v i t i e s were d e c r e a s e d , and h e a r t c a t a l a s e a c t i v i t y was i n c r e a s e d w i t h STZ-d i a b e t e s , independent o f manganese s t a t u s ( T a b l e 1 1 ) . The d e c r e a s e i n l i v e r GSHPx i n d i a b e t i c r a t s c o r r o b o r a t e s t h e r e s u l t s o f e a r l i e r s t u d i e s by Wohaieb and Godin (1987b) and Rucker e t a l . (1991) showing s i m i l a r d e c r e a s e s . The p r o g r e s s i v e i n c r e a s e i n h e a r t CAT a c t i v i t y (90% i n c r e a s e a t 4 weeks, and 157% i n c r e a s e a t 8 weeks, a f t e r STZ a d m i n i s t r a t i o n , compared t o n o n - d i a b e t i c c o n t r o l s ) may not be e n t i r e l y c o n s i s t e n t w i t h t h e 1 3 - f o l d i n c r e a s e i n h e a r t CAT a c t i v i t y o b s e r v e d by Wohaieb and Go d i n (1987b) 12 weeks a f t e r STZ t r e a t m e n t , a l t h o u g h t h e a c t u a l v a l u e s f o r d i a b e t i c r a t h e a r t a r e s i m i l a r (1.57 ± 0.13 compared t o 0.94 ± 0.20 K/g wet w e i g h t ) . The 25% d e c r e a s e i n k i d n e y GSHPx a c t i v i t y w i t h d i a b e t e s (4 o r 8 weeks) i n t h i s s t u d y was n o t i n a c c o r d a n c e w i t h e a r l i e r o b s e r v a t i o n (Wohaieb and Go d i n , 1987b) o f a 75% i n c r e a s e i n t h i s enzyme a f t e r 12 weeks o f d i a b e t e s , i n female , W i s t a r r a t s . T h i s d i f f e r e n c e c o u l d be due 104 t o l o n g e r d u r a t i o n o f d i a b e t e s , o r t o s t r a i n o r gender d i f f e r e n c e s . 5 . 5 E f f e c t s o f D u r a t i o n o f D i a b e t e s on N onenzymatic A n t i o x i d a n t A l t e r a t i o n s A m a j o r d i f f i c u l t y i n u s i n g any p a r t i c u l a r t i s s u e a n t i o x i d a n t as an i n d i c a t o r o f d i a b e t i c changes i s t h a t t h e v a r i o u s components o f t h e t i s s u e a n t i o x i d a n t scheme appear t o change r e l a t i v e t o one a n o t h e r o v e r t i m e . T i s s u e a n t i o x i d a n t s , b o t h e n z y m a t i c and n o n e n z y m a t i c , changed o v e r a 4 week p e r i o d , as c a n be s e e n by c o m p a r i n g 4 w e e k - d i a b e t i c and 8 w e e k - d i a b e t i c r a t s ( T a b l e s 9, 10, 11, and 1 6 ) . I n p a r t i c u l a r , h e a r t c a t a l a s e a c t i v i t y , p l a s m a and h e p a t i c v i t a m i n E p r o g r e s s i v e l y i n c r e a s e d , w h i l e k i d n e y GSH d e c r e a s e d , w i t h t h e d u r a t i o n o f d i a b e t e s . L i v e r GSH l e v e l s d e c l i n e d by 4 weeks a f t e r STZ a d m i n i s t r a t i o n , b u t r e m a i n e d unchanged between 4 and 8 weeks d i a b e t i c . D e c r e a s e d l i v e r GSH l e v e l s i n 2-week and 12-week d i a b e t i c r a t s compared t o non-d i a b e t i c c o n t r o l s have been o b s e r v e d p r e v i o u s l y and c o u l d be c o r r e c t e d t o n o n - d i a b e t i c l e v e l s by t r e a t m e n t w i t h e i t h e r i n s u l i n o r GSH (100 mg d i s s o l v e d i n 2 ml o f w a t e r ) (Loven e t a l . , 1986; G o d i n and Wohaieb, 1987b). To t h e b e s t o f o u r k nowledge, t h e p r o g r e s s i v e change o v e r t i m e i n t i s s u e a n t i o x i d a n t l e v e l s w i t h S T Z - d i a b e t e s o b s e r v e d i n t h e p r e s e n t s t u d y has n o t been r e p o r t e d p r e v i o u s l y . 105 5.5.1 E f f e c t o f S T Z - d i a b e t e s on Plasma and H e p a t i c V i t a m i n E P l asma and h e p a t i c v i t a m i n E l e v e l s i n c r e a s e d more t h a n t w o f o l d w i t h i n 8 weeks a f t e r STZ t r e a t m e n t i n t h i s s t u d y (0.46 mg/dl t o 1.35 mg/dl, and 15.5 ^iq/q t i s s u e t o 37.3 pq/q wet w e i g h t [116.5 nmoles/g wet w e i g h t ] i n d i a b e t i c a n i m a l s ) ( T a b l e 1 6 ) . T h i s c o n t r a s t s s h a r p l y w i t h an e a r l i e r s t u d y by H i g u c h i (1982) showing a 92% d e c r e a s e i n h e p a t i c v i t a m i n E i n 20-week d i a b e t i c r a t s , w i t h no change i n plasma v i t a m i n E compared t o non-d i a b e t i c c o n t r o l s . The s e v e r e d e p l e t i o n o f l i v e r v i t a m i n E may have o c c u r r e d due t o i n a d e q u a t e d i e t a r y i n t a k e . U n f o r t u n a t e l y , no d i e t a r y i n f o r m a t i o n was g i v e n . P a r a d o x i c a l l y , v i t a m i n E-d e f i c i e n t , d i a b e t i c r a t s were shown t o have t e n t i m e s h i g h e r h e p a t i c v i t a m i n E l e v e l s t h a n t h e i r v i t a m i n E - s u f f i c i e n t c o n t r o l s , w h i c h seems h i g h l y u n l i k e l y c o n s i d e r i n g o t h e r v i t a m i n E - d e f i c i e n c y s t u d i e s ( e . g . , De and Darad, 1988; P r i t c h a r d e t a l . , 1 986). However, d e p l e t i o n o f v i t a m i n E as a c o n c o m i t a n t o f d i a b e t e s i n e x p e r i m e n t a l a n i m a l s has a l s o been p r o p o s e d by K a r p e n e t a l . (1984, 1985), who showed d e p l e t i o n o f p l a t e l e t ( b u t n o t plasma) v i t a m i n E t o n e a r z e r o w i t h i n 8 t o 12 weeks o f a d m i n i s t r a t i o n o f STZ. S u p p l e m e n t a t i o n o f t h e l a b o r a t o r y chow d i e t ( a p p r o x i m a t e l y 44 mg/kg d i e t ) w i t h v i t a m i n E (200 mg/kg d i e t ) r e s t o r e d p l a t e l e t v i t a m i n E l e v e l s t o n o r m a l , d e s p i t e t h e g r e a t e r t h a n 60% w e i g h t l o s s i n t h e i r r a t s . I n c r e a s e d h e p a t i c and plasma v i t a m i n E w i t h d i a b e t e s have a l s o been o b s e r v e d p r e v i o u s l y . A s t u d y by P r i t c h a r d e t a l . (1986) i n 106 male, Sprague-Dawley r a t s showed a more t h a n t w o f o l d i n c r e a s e i n h e p a t i c v i t a m i n E a t 14 weeks a f t e r STZ (80 mg/kg body w e i g h t ) t r e a t m e n t o n l y i f t h e r a t s were supplemented w i t h v i t a m i n E (200 mg/kg d i e t ) f o r most of t h a t t i m e . Plasma v i t a m i n E i n c r e a s e d n e a r l y t h r e e f o l d (compared t o n o n - d i a b e t i c r a t s ) d u r i n g t h e same t i m e p e r i o d , w h e ther t h e r a t s r e c e i v e d d i e t a r y v i t a m i n E s u p p l e m e n t a t i o n o r n o t . I n a s t u d y by Behrens e t a l . (1984) i n s p o n t a n e o u s l y d i a b e t i c BB r a t s , u n s u p p l e m e n t e d w i t h v i t a m i n E, b o t h p l a s m a and h e p a t i c v i t a m i n E i n c r e a s e d a p p r o x i m a t e l y two and a h a l f t i m e s compared t o n o n - d i a b e t i c W i s t a r r a t s w i t h i n 4 days o f t h e o n s e t o f d i a b e t i c symptoms (0.34 mg/dl t o 0.86 mg/dl, and 15.9 n g / g t o 34.4 n g / g t i s s u e , r e s p e c t i v e l y ) . Treatment w i t h i n s u l i n f o r 30 d a y s c a u s e d r e v e r s a l o f plasma and t i s s u e v i t a m i n E l e v e l s t o n o r m a l . Our r e s u l t s a r e c o n s i s t e n t w i t h t h e o b s e r v a t i o n s o f B e h rens e t a l . (1984) o v e r a l o n g e r t i m e p e r i o d , i n S T Z - d i a b e t i c r a t s . I n c r e a s e d plasma and h e p a t i c v i t a m i n E do n o t n e c e s s a r i l y p r e c l u d e d e c r e a s e d p l a t e l e t v i t a m i n E, s i n c e many q u e s t i o n s o f t o c o p h e r o l s t o r a g e and d i s t r i b u t i o n a r e c u r r e n t l y unanswered. S u p p l e m e n t a t i o n w i t h v i t a m i n E has been shown t o r e s t o r e p l a s m a , l i v e r and k i d n e y l e v e l s o f t h i s v i t a m i n t o n o r m a l o r above i n d i a b e t i c a n i m a l s . I n t h e p r e s e n t s t u d y , S T Z - d i a b e t e s o f 4 t o 8 weeks d u r a t i o n w i t h o u t d i e t a r y v i t a m i n E s u p p l e m e n t a t i o n ( i . e . , 44 IU v i t a m i n E/kg d i e t ) r e s u l t e d i n i n c r e a s e d plasma and h e p a t i c v i t a m i n E. The o b s e r v e d v a r i a t i o n s i n t i s s u e e n z y m a t i c and n o n e n z y m a t i c a n t i o x i d a n t s between 4 and 8 weeks o f d i a b e t e s 107 a r e c o n s i s t e n t w i t h an h y p o t h e s i s o f c o n t i n u i n g t i s s u e a n t i o x i d a n t change i n S T Z - d i a b e t e s . 5.5.2 E f f e c t o f D u r a t i o n o f D i a b e t e s on Mn S t a t u s Mn s t a t u s may a l s o change o v e r t i m e i n d i a b e t i c a n i m a l s . I n t h e p r e s e n t s t u d y , t i s s u e Mn c o n t e n t was n o t i n c r e a s e d 4 weeks a f t e r STZ a d m i n i s t r a t i o n . B o t h F a i l l a and K i s e r (1981) and Bond e t a l . ( 1 9 8 3 ) f o u n d an a c c u m u l a t i o n o f Mn i n l i v e r 3 t o 10 days f o l l o w i n g STZ i n j e c t i o n ; however, N i s h i d a e t a l . (1989) f o u n d no i n c r e a s e i n l i v e r Mn a t 14 days. I n t h e l a t t e r s t u d y , STZ-d i a b e t e s a f f e c t e d t h e Mn d i s t r i b u t i o n among t i s s u e s o f r a t s p r e t r e a t e d w i t h MnCl2 s u p p l e m e n t a t i o n . P a n c r e a s , s p l e e n and k i d n e y Mn d e c r e a s e d i n d i a b e t i c compared t o n o n - d i a b e t i c a n i m a l s , w h i l e b r a i n and thymus Mn i n c r e a s e d and l i v e r Mn r e m a i n e d unchanged. F u r t h e r m o r e , STZ t r e a t m e n t r e s u l t e d i n a t r a n s l o c a t i o n o f Mn among s u b c e l l u l a r compartments o f l i v e r w h i c h c o u l d be overcome by s u p p l e m e n t a l manganese. An endogenous r e d i s t r i b u t i o n o f t i s s u e Mn i n S T Z - d i a b e t i c r a t s has been d e m o n s t r a t e d d i r e c t l y u s i n g 5 4 M n as a t r a c e r (Bond e t a l . , 1982) . A t l e a s t 30% o f t h e i n c r e a s e d l i v e r Mn i n d i a b e t i c r a t s was f o u n d t o come from body s t o r e s , w i t h t h e r e m a i n d e r coming f r o m d i e t . I t i s p o s s i b l e t h a t what was o b s e r v e d i n t h e s h o r t -t e r m d i a b e t i c r a t s were t e m p o r a r y i n c r e a s e s i n Mn c o n c e n t r a t i o n . Whether o r not t h e r e i s a c o n s i s t e n t p a t t e r n o f change i n t i s s u e Mn c o n c e n t r a t i o n w i t h d u r a t i o n o f d i a b e t e s d e s e r v e s f u r t h e r s t u d y . 108 I n answer t o t h e s e c o n d q u e s t i o n o f t h e s t a t e d o b j e c t i v e s o f t h i s s t u d y , d u r a t i o n o f d i a b e t e s a f f e c t e d t i s s u e a n t i o x i d a n t s t a t u s , b o t h i n t e r m s o f e n z y m a t i c and n o n e n z y m a t i c t i s s u e a n t i o x i d a n t s . To r e s o l v e t h e q u e s t i o n o f w h e t her t i s s u e Mn d i s t r i b u t i o n changes o v e r t i m e i n S T Z - d i a b e t e s w o u l d r e q u i r e a l o n g i t u d i n a l e v a l u a t i o n o f t i s s u e and s u b c e l l u l a r Mn c o n c e n t r a t i o n s a t w e e k l y i n t e r v a l s f o r 3 o r 4 months f o l l o w i n g STZ a d m i n i s t r a t i o n . Comparison o f changes i n l i v e r , p a n c r e a s , k i d n e y and o t h e r t i s s u e s would be o f p a r t i c u l a r i n t e r e s t , s i n c e s u b c e l l u l a r d i s t r i b u t i o n , r e s p o n s e t o Mn s u p p l e m e n t a t i o n and t o d i a b e t e s seem t o be t i s s u e - s p e c i f i c ( S a k u r a i e t a l . , 1985). 5 . 6 O x i d a t i v e S t r e s s i n Manganese D e f i c i e n c y and S T Z - d i a b e t e s To answer t h e t h i r d q u e s t i o n , o f whether S T Z - d i a b e t e s combined w i t h manganese d e f i c i e n c y i s a c o n d i t i o n o f h e i g h t e n e d o x i d a n t s t r e s s , r e q u i r e d t h a t we f i r s t e s t a b l i s h how t o measure o x i d a n t s t r e s s . O x i d a n t s t r e s s i m p l i e s an i n c r e a s e i n t h e p r o d u c t i o n o f r e a c t i v e oxygen s p e c i e s and/or a d e c r e a s e i n c a p a c i t y t o s c a v a n g e s u c h s p e c i e s . I n c r e a s e d l i p i d p e r o x i d a t i o n i s one i n d i c a t i o n o f h e i g h t e n e d o x i d a t i v e s t r e s s . However, measurement o f l i p i d p e r o x i d a t i o n i n a c o n s i s t e n t and r e p r o d u c i b l e f a s h i o n i s d i f f i c u l t , a t b e s t ( P r y o r e t a l . , 1989; J a n e r o , 1985; Weber, 1990). There i s s t i l l no one p r e f e r r e d and c o m p l e t e l y r e l i a b l e method. We e v a l u a t e d e r y t h r o c y t e and t i s s u e TBARS i n r e s p o n s e t o f o r c e d p e r o x i d a t i o n ( T a b l e 1 8 ) , a r e l a t i v e l y common method, as w e l l as plasma l i p i d p e r o x i d e s , r e d c e l l h e m o l y s i s , and r e n a l a d i p o s e t i s s u e f l u o r e s c e n c e . 109 5 . 6 . 1 E f f e c t o f Mn D e f i c i e n c y on L i p i d P e r o x i d a t i o n Manganese d e f i c i e n c y d i d n o t r e s u l t i n e l e v a t e d l i p i d p e r o x i d a t i o n by any o f t h e measures employed ( T a b l e s 18 and 1 9 ) . I n c r e a s e d l i p i d p e r o x i d a t i o n as an accompaniment t o manganese d e f i c i e n c y has been n o t e d i n s e v e r a l r e c e n t r e v i e w s (Keen e t a l . , 1984a; Z i d e n b e r g - C h e r r and Keen, 1987). On c l o s e r e x a m i n a t i o n , b o t h r e v i e w s r e f e r t o t h e work o f Z i d e n b e r g - C h e r r e t a l . (1983), i n w h i c h h i g h e r l e v e l s o f l i p i d p e r o x i d a t i o n (as measured by i r o n - i n d u c e d t i s s u e TBARS f o r m a t i o n ) were f o u n d i n l i v e r m i t o c h o n d r i a o f s e c o n d - g e n e r a t i o n M n - d e f i c i e n t r a t s between 20 and 60 days o f age, compared t o M n - s u f f i c i e n t c o n t r o l s . The same s t u d y f o u n d no d i f f e r e n c e i n e i t h e r s p o n t a n e o u s o r i n d u c e d t i s s u e TBARS from l i v e r homogenates o f t h e s e a n i m a l s . A second s t u d y ( P a y n t e r , 1980b), i n f i r s t g e n e r a t i o n , M n - d e f i c i e n t r a t s , u s i n g i n d u c e d t i s s u e TBARS i n c r u d e l i v e r m i t o c h o n d r i a l f r a c t i o n s , a l s o c o n c l u d e d t h a t t i s s u e p e r o x i d a t i o n i n c r e a s e d w i t h i n a d e q u a t e d i e t a r y i n t a k e o f manganese. R a t s f e d a v e r y low l e v e l o f d i e t a r y manganese, 0.2 Ug Mn/g d i e t , were compared t o M n - s u f f i c i e n t c o n t r o l s , and t o v i t a m i n E - d e f i c i e n t , M n - d e f i c i e n t r a t s . V i t a m i n E - d e f i c i e n c y f u r t h e r enhanced t h e i n c r e a s e i n l i p i d p e r o x i d a t i o n r e s u l t i n g f r o m Mn d e f i c i e n c y . The use o f m i t o c h o n d r i a l f r a c t i o n s i n t h e s e s t u d i e s may have e f f e c t i v e l y improved t h e s e n s i t i v i t y o f t h e a s s a y as t h e s e o r g a n e l l e s a r e h i g h l y s u s c e p t i b l e t o v i t a m i n E-dependent l i p i d p e r o x i d a t i o n ( T a p p e l , 1972). The l a c k o f an e f f e c t o f manganese d e f i c i e n c y on t i s s u e homogenate TBARS i n t h e 110 p r e s e n t s t u d y i s t h u s c o n s i s t e n t w i t h p r e v i o u s i n v e s t i g a t i o n s . P l asma l i p i d p e r o x i d e s and r e n a l a d i p o s e t i s s u e f l u o r e s c e n c e a s s a y s a l s o f a i l e d t o d e m o n s t r a t e an e f f e c t o f manganese d e f i c i e n c y on l i p i d p e r o x i d a t i o n . The r e n a l a d i p o s e t i s s u e f l u o r e s c e n c e a s s a y may have been compromised by t h e r e l a t i v e l y young age o f t h e r a t s i n t h i s i n v e s t i g a t i o n compared t o e a r l i e r s t u d i e s ( S t r e h l e r and M i l d v a n , 1962). O v e r a l l , t h e s e n s i t i v i t y o f t h e a s s a y s u s e d i n t h i s s t u d y f o r d e t e c t i o n o f l i p i d p e r o x i d a t i o n may n o t have p e r m i t t e d d i f f e r e n t i a t i o n between Mn-d e f i c i e n t and M n - s u f f i c i e n t r a t s . 5.6.2 E f f e c t o f S T Z - d i a b e t e s on L i p i d P e r o x i d a t i o n S T Z - d i a b e t e s r e s u l t e d i n i n c r e a s e d l i p i d p e r o x i d a t i o n as measured by e r y t h r o c y t e TBARS and plasma l i p o p e r o x i d e s 8 weeks a f t e r STZ t r e a t m e n t , and by r e n a l a d i p o s e t i s s u e f l u o r e s c e n c e 4 and 8 weeks a f t e r STZ ( T a b l e s 18 and 1 9 ) . T i s s u e TBARS, as measured by H 2 O 2 - i n d u c e d m a l o n d i a l d e h y d e p r o d u c t i o n , were n o t e l e v a t e d as a r e s u l t o f STZ t r e a t m e n t . The r e l a t i v e i n s e n s i t i v i t y o f t h i s a s s a y under c o n d i t i o n s o f i n c r e a s e d H 2 O 2 -p r o d u c t i o n ( K o s t k a and Kwan, 1989) may have compromised t h e r e s u l t s , o r t h e r e may have been adequate t i s s u e v i t a m i n E t o compensate f o r enzyme d e f i c i e n c i e s i n t h i s s y s t e m . Red c e l l h e m o l y s i s was s i g n i f i c a n t l y r e d u c e d f o l l o w i n g STZ t r e a t m e n t ; however, a l l a s s a y r e s u l t s were c l o s e t o n e g l i g i b l e . The p r o b a b l e c a u s e o f t h i s u n e x p e c t e d r e s u l t i s t h a t t h e r e d I l l c e l l h e m o l y s i s a s s a y i s d r a s t i c a l l y r e d u c e d i n e f f e c t i v e n e s s under h y p e r g l y c e m i c c o n d i t i o n s ( H o r w i t t , 1962). H i g h b l o o d g l u c o s e l e v e l s p r o b a b l y s u p p r e s s e d h e m o l y s i s i n d i a b e t i c r a t s and r e n d e r e d t h i s a s s a y i n c o n c l u s i v e . S T Z - d i a b e t e s i n c r e a s e d plasma l i p i d p e r o x i d e l e v e l s , f l u o r e s c e n t pigment p r o d u c t i o n i n r e n a l a d i p o s e t i s s u e , and s u s c e p t i b i l i t y t o l i p i d p e r o x i d a t i o n i n e r y t h r o c y t e s . These measures o f l i p i d p e r o x i d a t i o n and, i n d i r e c t l y , o f o x i d a t i v e s t r e s s , c o r r o b o r a t e d r e s u l t s o f e a r l i e r i n v e s t i g a t i o n s ( e . g . , K a r p e n e t a l . , 1982; G o d i n e t a l . , 1988a; M o r e l and C h i s h o l m , 1989). Manganese d e f i c i e n c y combined w i t h S T Z - d i a b e t e s d i d n o t r e s u l t i n i n c r e a s e d l i p i d p e r o x i d a t i o n , o r o f e x a c e r b a t e d o x i d a n t s t r e s s . To summarize t h e r e s u l t s o f e x p e r i m e n t 1: We have d e m o n s t r a t e d a n o v e l i n t e r a c t i v e e f f e c t between manganese d e f i c i e n c y and STZ-d i a b e t e s w i t h r e s p e c t t o d e c r e a s e d a c t i v i t i e s o f k i d n e y and h e a r t MnSOD, and k i d n e y CuZnSOD, and i n c r e a s e d l i v e r MnSOD. G l u c o s e t o l e r a n c e t e s t r e s p o n s e s , as a measure o f d i a b e t i c s e v e r i t y , were h i g h e r i n M n - d e f i c i e n t , compared t o Mn-s u f f i c i e n t , d i a b e t i c r a t s . No o t h e r measures o f d i a b e t i c s e v e r i t y , t i s s u e a n t i o x i d a n t s t a t u s , o r l i p i d p e r o x i d a t i o n i n d i c a t e d an e x a c e r b a t i o n o f d i a b e t e s by manganese d e f i c i e n c y . 112 5.7 E f f e c t s o f v i t a m i n E - d e f i c i e n c y and S T Z - d i a b e t e s on l i p i d p e r o x i d a t i o n and t i s s u e a n t i o x i d a n t s The s p e c i f i c o b j e c t i v e o f e x p e r i m e n t 2 was t o answer t h e q u e s t i o n : does adequacy o f d i e t a r y v i t a m i n E "mask" changes due t o manganese d e f i c i e n c y ? Mn d e f i c i e n c y was c o n f i r m e d , as b e f o r e , by d e c r e a s e d t i s s u e Mn c o n t e n t (vide supra). D i a b e t i c i n d u c t i o n was c o n f i r m e d by h y p e r g l y c e m i a , e l e v a t e d H b A i c , d e c r e a s e d RIA plasma i n s u l i n , and i n c r e a s e d k i d n e y / b o d y w e i g h t r a t i o s ( T a b l e 7 ) . V i t a m i n E - d e f i c i e n c y was c o n f i r m e d by s e v e r e r e d u c t i o n s i n plasma and h e p a t i c v i t a m i n E l e v e l s ( T a b l e 1 7 ) . These v a l u e s a r e c o n s i d e r a b l y l o w e r t h a n w o u l d be e x p e c t e d w i t h a v i t a m i n E - d e f i c i e n c y a l o n e f o r 12 weeks (De and Darad, 1988; P r i t c h a r d e t a l . , 1986). T h i s c o u l d be a r e s u l t o f i n d u c t i o n o f d i a b e t e s a f t e r 8 weeks on t h e v i t a m i n E - d e f i c i e n t d i e t s . I n c r e a s e d s u p e r o x i d e p r o d u c t i o n and l i p i d p e r o x i d a t i o n as a r e s u l t o f accompanying d i a b e t e s would be e x p e c t e d t o i n c r e a s e r e q u i r e m e n t s f o r v i t a m i n E as a major f r e e r a d i c a l s c a v e n g e r ( S l o n i m e t a l . , 1983). V i t a m i n E - d e f i c i e n t r a t s had s i g n i f i c a n t l y h i g h e r l e v e l s o f t i s s u e TBARS compared t o v i t a m i n E - s u f f i c i e n t a n i m a l s ( T a b l e 2 0 ) . Red c e l l h e m o l y s i s , plasma l i p i d p e r o x i d e s , and % H b A i c were a l s o s i g n i f i c a n t l y e l e v a t e d i n v i t a m i n E - d e f i c i e n t compared t o v i t a m i n E - s u f f i c i e n t r a t s . T h i s c o r r o b o r a t e s e a r l i e r e v i d e n c e t h a t v i t a m i n E - d e f i c i e n c y enhances S T Z - d i a b e t e s ( S l o n i m e t a l . , 1983) and r e s u l t s i n i n c r e a s e d l i p i d p e r o x i d a t i o n 113 (Karpen e t a l . , 1982; Chow, 1990; L i e b o v i t z e t a l . , 1 9 9 0 ) . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n measures o f l i p i d p e r o x i d a t i o n between Mn s u f f i c i e n t and M n - d e f i c i e n t r a t s among v i t a m i n E - d e f i c i e n t r a t s . Some t i s s u e a n t i o x i d a n t enzyme a c t i v i t i e s were s i g n i f i c a n t l y d i f f e r e n t between v i t a m i n E - d e f i c i e n t and v i t a m i n E - s u f f i c i e n t , d i a b e t i c r a t s ( T a b l e s 12-15, Appendix B ) . I n c r e a s e d a c t i v i t i e s o f k i d n e y c a t a l a s e , l i v e r and h e a r t GSSGRd and p a n c r e a t i c CuZnSOD were f o u n d i n v i t a m i n E - d e f i c i e n t r a t s . D e c r e a s e d a c t i v i t i e s o f l i v e r and p a n c r e a s MnSOD and h e a r t GSHPx were a s s o c i a t e d w i t h v i t a m i n E - d e f i c i e n c y . D e creased a c t i v i t y o f p a n c r e a s MnSOD i n r a t s f e d a v i t a m i n E - d e f i c i e n t d i e t f o r 8 weeks has been o b s e r v e d p r e v i o u s l y (Asayama e t a l . , 1 9 8 6 ) . As w e l l , l i v e r MnSOD a c t i v i t y was s i g n i f i c a n t l y r e d u c e d i n v i t a m i n E - d e f i c i e n t r a t s a f t e r 1 y e a r on t h e d e f i c i e n t d i e t (De and Darad, 1988). Chow e t a l . (1973) found d e c r e a s e d a c t i v i t y o f l i v e r and e r y t h r o c y t e GSHPx i n v i t a m i n E - d e f i c i e n t r a t s . ( H e a r t GSHPX a c t i v i t y was n o t measured.) These r e s u l t s , i n v i t a m i n E-d e f i c i e n t , n o n - d i a b e t i c r a t s , t e n d t o c o r r o b o r a t e t i s s u e a n t i o x i d a n t changes o b s e r v e d by us i n v i t a m i n E - d e f i c i e n t , d i a b e t i c r a t s . Manganese d e f i c i e n c y was a s s o c i a t e d w i t h d e c r e a s e d h e a r t and k i d n e y MnSOD, k i d n e y GSHPx and i n c r e a s e d l i v e r MnSOD and CuZnSOD. V i t a m i n E - s u f f i c i e n t , M n - d e f i c i e n t r a t s e x h i b i t e d d e c r e a s e d k i d n e y MnSOD and i n c r e a s e d l i v e r CuZnSOD b u t v i t a m i n 114 E - d e f i c i e n t , M n - d e f i c i e n t , r a t s d i d n o t (Note s i g n i f i c a n c e o f i n t e r a c t i o n s i n Appendix B ) . For b o t h o f t h e s e a n t i o x i d a n t enzymes, v i t a m i n E d e f i c i e n c y r e s u l t e d i n a c t i v i t y l e v e l s i n t e r m e d i a t e between t h e M n - d e f i c i e n t and M n - s u f f i c i e n t , v i t a m i n E - s u f f i c i e n t , d i a b e t i c l e v e l s , w i t h no s i g n i f i c a n t d i f f e r e n c e s between m a n g a n e s e - s u f f i c i e n t and m a n g a n e s e - d e f i c i e n t , v i t a m i n E-d e f i c i e n t , r a t s ( T a b l e s 12 and 13). Only t h e d e c r e a s e i n k i d n e y GSHPx a c t i v i t y r e p r e s e n t s a s i g n i f i c a n t change from t h e r e s u l t s o f e x p e r i m e n t 1. D i m i n i s h e d k i d n e y GSHPx a c t i v i t y may r e p r e s e n t a d e c r e a s e i n a v a i l a b l e t i s s u e Se, which c o r r o b o r a t e s an e a r l i e r r e p o r t by B u r c h e t a l . (1975) o f d e c r e a s e d t i s s u e Se accompanying Mn d e f i c i e n c y i n p i g s . O v e r a l l , we would have t o c o n c l u d e t h a t v i t a m i n E - d e f i c i e n c y d i d n o t "unmask" changes due t o M n - d e f i c i e n c y . 5 .8 Compensatory Changes i n Tissue Antioxidants H a v i n g c o n s i d e r e d t h e major o b j e c t i v e s o f t h i s s t u d y , we can now h i g h l i g h t some i n t e r e s t i n g c o r r o l l a r y o b s e r v a t i o n s . One o f t h e a s p e c t s o f t i s s u e a n t i o x i d a n t a l t e r a t i o n s o f i n t e r e s t i s whether t h e r e a r e any compensatory mechanisms i n o p e r a t i o n . I n none o f t h e t i s s u e s s t u d i e d d i d we f i n d compensatory i n c r e a s e s i n CuZnSOD as a c o r r e l a t e o f d e c r e a s e d MnSOD ( T a b l e s 9 and 2 2 ) , a l t h o u g h p r e v i o u s i n v e s t i g a t o r s have p r o p o s e d t h a t i n c r e a s e d CuZnSOD a c t i v i t y may be a u s u a l compensatory change i n M n - d e f i c i e n c y s t a t e s (deRosa e t a l . , 1980). The o n l y p o s s i b l y compensatory r e s p o n s e s noted i n t h i s s t u d y were i n h e a r t , where 115 GSH was n e g a t i v e l y c o r r e l a t e d w i t h GSHPx ( r = -0.357, P<0.01) and i n l i v e r , where GSH was n e g a t i v e l y c o r r e l a t e d w i t h MnSOD ( r = -0.364, P<0.01). I n b o t h c a s e s , d e c l i n i n g l e v e l s o f r e d u c e d g l u t a t h i o n e , a nonenzymatic scavenger, may be accompanied by i n c r e a s e s i n r e a c t i v e oxygen m e t a b o l i t e s , r e q u i r i n g a c c e l e r a t e d e n z y m a t i c s c a v e n g i n g . A l l r e m a i n i n g s i g n i f i c a n t c o r r e l a t i o n s by t i s s u e were p o s i t i v e , i n d i c a t i n g t h a t t i s s u e a n t i o x i d a n t s t a t u s i n d i a b e t e s was p r o b a b l y compromised. P a r t i c u l a r l y s t r o n g c o r r e l a t i o n s were n o t e d between GSH and b o t h CuZnSOD and MnSOD i n p a n c r e a s and k i d n e y , s u g g e s t i n g t h a t i n t h e s e two t i s s u e s , d e c l i n i n g nonenzymatic scavenger l e v e l s were n o t compensated by a c c e l e r a t e d s u p e r o x i d e dismutase a c t i v i t y , w i t h consequent i n c r e a s e d s u s c e p t i b i l i t y t o t i s s u e damage from r e a c t i v e oxygen i n t e r m e d i a t e s . T h i s agrees w i t h t h e assessment o f G r a n k v i s t e t a l . (1981) t h a t p a n c r e a s i s p a r t i c u l a r l y s u s c e p t i b l e t o o x i d a t i v e damage compared t o o t h e r t i s s u e s . K i d n e y i s one o f t h e t i s s u e s most f r e q u e n t l y a f f e c t e d by c h r o n i c d i a b e t e s (Rasch, 1979). E v i d e n c e f o r l a c k o f an adequate compensatory r e s p o n s e i s a l s o p r o v i d e d by t h e observed i n c r e a s e s i n measures o f l i p i d p e r o x i d a t i o n . 5.9 Possible E f f e c t s of Weight Loss i n Diabetic Animals I n o r d e r t o a v o i d , as much as p o s s i b l e , t h e c o n f o u n d i n g e f f e c t s o f s t a r v a t i o n on t i s s u e a n t i o x i d a n t change, r a t s i n t h i s s t u d y were n o t f a s t e d p r i o r t o STZ t r e a t m e n t nor p r i o r t o a u t o p s y . They were f e d ad l i b i t u m r a t h e r t h a n b e i n g p a i r - f e d , a l t h o u g h i n 116 f a c t , t h e a v e r a g e w e e k l y f o o d i n t a k e s o f M n - s u f f i c i e n t and Mn-d e f i c i e n t , d i a b e t i c r a t s d i d n o t d i f f e r s i g n i f i c a n t l y f r o m Mn-s u f f i c i e n t and M n - d e f i c i e n t , n o n - d i a b e t i c r a t s e x c e p t f o r a week t o 10 d a y s f o l l o w i n g STZ a d m i n i s t r a t i o n . The e f f e c t s o f s t a r v a t i o n w h i c h have been n o t e d (Godin and Wohaieb, 1988b; Wohaieb and G o d i n , 1987b) i n c l u d e : i n c r e a s e d a c t i v i t i e s o f CAT i n h e a r t and p a n c r e a s , CuZnSOD i n k i d n e y and p a n c r e a s , and GSHPx i n k i d n e y . D e c r e a s e s i n CAT a c t i v i t y i n l i v e r , CuZnSOD a c t i v i t y i n h e a r t and GSH c o n c e n t r a t i o n i n l i v e r were n o t e d i n r a t s s t a r v e d f o r 72 h o u r s compared t o ad l i b i t u m c o n t r o l s . Of t h e s e , o n l y t h e changes i n CAT a c t i v i t e s and l i v e r GSH c o n c e n t r a t i o n o b s e r v e d i n o u r e x p e r i m e n t s c o u l d p o s s i b l y be a t t r i b u t e d t o s t a r v a t i o n ( o r e m a c i a t i o n ) e f f e c t s . Changes i n k i d n e y GSHPx a c t i v i t y , and i n k i d n e y , h e a r t and p a n c r e a s CuZnSOD i n o u r s t u d y were o p p o s i t e f r o m t h o s e a n t i c i p a t e d w i t h s t a r v a t i o n a l o n e . I t i s w o r t h n o t i n g t h a t t h e w e i g h t l o s s o v e r a p e r i o d o f 72 h o u r s i n t h e s t u d y o f G o d i n and Wohaieb (1987b) was g r e a t e r t h a n w e i g h t l o s s d u r i n g a 4 week p e r i o d i n t h i s s t u d y . 5.10 Adequacy o f the M n - s u f f i c i e n t D i e t No e f f e c t o f manganese d e f i c i e n c y was seen i n t i s s u e GSH c o n c e n t r a t i o n s , l i p i d p e r o x i d a t i o n measures, v i t a m i n E l e v e l s , % H b A i c o r h y p e r g l y c e m i a , c o n t r a r y t o e x p e c t a t i o n s . T h i s c o u l d i n d i c a t e an i n s u f f i c i e n t d i f f e r e n c e between t h e M n - d e f i c i e n t and t h e M n - s u f f i c i e n t d i e t s . I f t h e M n - s u f f i c i e n t d i e t were o n l y m a r g i n a l l y a d e q u a t e f o r d i a b e t i c a n i m a l s , we mi g h t see l i t t l e o r no d i f f e r e n c e between t h e s e r a t s and t h e M n - d e f i c i e n t a n i m a l s . 117 A r e c e n t i n v e s t i g a t i o n o f m a r g i n a l z i n c d e f i c i e n c y i n STZ-d i a b e t e s ( U r i u - H a r e e t a l . , 1989) c o n c l u d e d t h a t a n o m i n a l l y a dequate z i n c d i e t was i n e f f e c t z i n c d e f i c i e n t f o r p r e g n a n t d i a b e t i c r a t s . A l s o , i f t h e M n - s u f f i c i e n t d i e t were o n l y m a r g i n a l l y adequate f o r d i a b e t i c a n i m a l s , we m i g h t e x p e c t t o see a m e l i o r a t i o n o f d i a b e t i c symptoms w i t h manganese s u p p l e m e n t a t i o n . No d i r e c t e v i d e n c e i n f a v o u r o f t h i s h y p o t h e s i s i s y e t a v a i l a b l e ; however, Mn s u p p l e m e n t a t i o n o f ob/ob m i c e , a s p o n t a n e o u s l y d i a b e t i c a n i m a l model, l e d t o improvement i n ' t h o s e p a r a m e t e r s o f l i v e r m i t o c h o n d r i a l f u n c t i o n t h a t were measured (Welsh e t a l . , 1985). On t h e o t h e r hand, our M n - d e f i c i e n t d i e t may n o t have been d e f i c i e n t enough. A l t h o u g h d e c r e a s e s i n Mn c o n t e n t o f h e a r t , l i v e r , k i d n e y and p a n c r e a s c o n f i r m e d t h e M n - d e f i c i e n t s t a t u s o f t h e s e r a t s , t h e d e f i c i e n c y was n o t as p r o f o u n d as t h a t e s t a b l i s h e d by deRosa e t a l . (1980) u s i n g s e c o n d - g e n e r a t i o n d e f i c i e n t r a t s . 5.11 Summary and conclusions To summarize, t h e r e s u l t s o f t h i s i n v e s t i g a t i o n i n d i c a t e t h a t t i s s u e a n t i o x i d a n t s t a t u s i n S T Z - d i a b e t e s was a f f e c t e d by manganese d e f i c i e n c y o n l y i n terms o f f u r t h e r d e c r e a s e d a c t i v i t i e s o f k i d n e y and h e a r t MnSOD, and k i d n e y CuZnSOD, and i n c r e a s e d l i v e r MnSOD i n Sprague-Dawley r a t s . H e i g h t e n e d g l u c o s e t o l e r a n c e t e s t r e s p o n s e i n M n - d e f i c i e n t compared t o Mn-s u f f i c i e n t d i a b e t i c r a t s s u g g e s t e d a p o s s i b l e e x a c e r b a t i o n o f S T Z - d i a b e t e s by Mn d e f i c i e n c y . V i t a m i n E d e f i c i e n c y d i d n o t appe a r t o "unmask" any p r e v i o u s l y u n d i s c l o s e d i n t e r a c t i v e 118 e f f e c t s between S T Z - d i a b e t e s and Mn d e f i c i e n c y . L a c k o f adequ a t e d i e t a r y v i t a m i n E r e s u l t e d i n i n c r e a s e d s u s c e p t i b i l i t y o f h e a r t , k i d n e y l i v e r and panc r e a s t o l i p i d p e r o x i d a t i o n as measured by t i s s u e TBARS, and i n c r e a s e d g l y c o s y l a t i o n o f h e m o g l o b i n . I n d u c t i o n o f d i a b e t e s by STZ r e s u l t e d i n a l t e r a t i o n s i n t i s s u e a n t i o x i d a n t s , i n c l u d i n g d e c r e a s e d k i d n e y MnSOD and CuZnSOD, i n c r e a s e d l i v e r MnSOD and h e a r t CuZnSOD, d e c r e a s e d l i v e r and k i d n e y GSHPx, d e c r e a s e d l i v e r CAT, and i n c r e a s e d h e a r t CAT. T i s s u e GSH was d e c r e a s e d i n k i d n e y and l i v e r , and v i t a m i n E was i n c r e a s e d i n l i v e r and p l a s m a . These changes a p p e a r e d t o be p r o g r e s s i v e o v e r t i m e i n d i a b e t i c a n i m a l s , and were r e f l e c t e d i n i n c r e a s e s i n l i p i d p e r o x i d a t i o n l e v e l s by 8 weeks a f t e r STZ t r e a t m e n t ( s o o n e r f o r v i t a m i n E d e f i c i e n t r a t s ) . S t r o n g p o s i t i v e c o r r e l a t i o n s between GSH c o n c e n t r a t i o n s and b o t h CuZnSOD and MnSOD a c t i v i t i e s i n p a n c r e a s and k i d n e y s u g g e s t e d t h a t d e c l i n i n g n o n e n z y m a t i c s c a v e n g e r l e v e l s i n t h e s e t i s s u e s were not compensated by a c c e l e r a t e d s u p e r o x i d e d i s m u t a s e a c t i v i t y , w i t h c o n s e q u e n t i n c r e a s e d s u s c e p t i b i l i t y t o t i s s u e damage from r e a c t i v e oxygen s p e c i e s , e s p e c i a l l y s u p e r o x i d e . 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M n - s u p e r o x i d e d i s m u t a s e , C u Z n - s u p e r o x i d e d i s m u t a s e , Mn, Cu, Fe, and Zn i n mouse t i s s u e s . Biol. Trace Elem. Res. 7 : 209-222 . 138 APPENDIX A. Two-way A N O V A statistical effects: manganese vs. streptozotocin, P values TISSUE GSH MnSOD CuZnSOD GSHPx GSSGRd C A T Heart Mn NS < 0.001 NS NS NS NS STZ NS 0.003 0.003 NS 0.002 <0.001 Interaction NS 0.063 NS NS NS NS Kidney Mn NS < 0.001 0.004 NS NS NS STZ < 0.001 0.001 < 0.001 0.001 0.05 NS Interaction NS NS NS NS NS NS Liver Mn NS 0.028 NS NS 0.041 NS STZ < 0.001 0.002 0.052 < 0.001 0.050 NS Interaction NS NS NS NS NS NS Pancreas Mn NS NS NS NS NS NS STZ NS < 0.001 0.003 < 0.0001 0.059 NS Interaction NS NS NS NS NS NS NS, not significant; Mn, manganese; STZ, streptozotocin; GSH, reduced glutathione; MnSOD, manganese superoxide dismutase; CuZnSOD, copper-zinc superoxide dismutase; GSHPx, glutathione peroxidase; GSSGRd, glutathione reductase; CAT, catalase 139 APPENDIX B. Two-way A N O V A statistical effects: Manganese vs. vitamin E, P values TISSUE GSH MnSOD CuZnSOD GSHPx GSSGRd C A T Heart Mn NS < 0.001 NS NS NS NS V i t E NS NS NS 0.023 < 0.001 NS Interaction NS NS NS NS NS NS Kidney Mn NS - 0.001 NS 0.025 NS NS V i t E NS NS NS NS NS 0.023 Interaction NS 0.028 NS NS NS NS Liver Mn NS 0.016 0.017 NS NS NS V i t E NS 0.008 NS NS 0.001 NS Interaction NS NS 0.045 NS NS NS Pancreas Mn NS NS NS NS NS NS V i t E 0.004 0.021 NS NS NS 0.001 Interaction NS NS NS NS NS NS NS, not significant; Mn, manganese; STZ, streptozotocin; GSH, reduced glutathione; MnSOD, manganese superoxide dismutase; GSHPx, glutathione peroxidase; GSSGRd, glutathione reductase; CAT, catalase 

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