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Effect of thyroid status on plasma silicon levels of rats Louie, Victoria Yvonne 1986

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EFFECT OF THYROID STATUS ON PLASMA SILICON LEVELS OF RATS by VICTORIA YVONNE LOUIE B.H.E., Uni v e r s i t y of B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES -Di v i s i o n of Human Nu t r i t i o n School of Family and N u t r i t i o n a l Sciences We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e g k j s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August 1986 ©Victoria Yvonne Louie, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Departmen The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 )E-6 (3/81) ABSTRACT The p r e s e n t study was undertaken t o i n v e s t i g a t e the n a t u r e of the r e l a t i o n s h i p between t h y r o i d s t a t u s and plasma s i l i c o n c o n c e n t r a t i o n s i n bone m a t u r a t i o n of r a t s from b i r t h u n t i l the time of weaning. V i r g i n female Sprague-Dawley r a t s were b r e d o v e r n i g h t and then a s s i g n e d t o one of t h r e e g r o u p s : c o n t r o l , h y p o t h y r o i d or h y p e r t h y r o i d . O f f s p r i n g s of dams were made h y p o t h y r o i d or h y p e r t h y r o i d r e l a t i v e t o e u t h y r o i d c o n t r o l s d u r i n g the n e o n a t a l p e r i o d . H y p o t h y r o i d pups were g i v e n 0.05% (w/v) p r o p y l t h i o u r a -c i l i n d e i o n i z e d water as the s o l e source of d r i n k i n g f l u i d of the dams from day 12 of g e s t a t i o n u n t i l the young were s a c r i -f i c e d . H y p e r t h y r o i d pups r e c e i v e d d a i l y subcutaneous i n j e c t i o n of L - t h y r o x i n e a t a dose of 0.15-0.30 ug/g of body weight from p o s t n a t a l day 2 u n t i l the day of s a c r i f i c e . A l l a n i m a l s were a l l o w e d ad l i b i t u m a c c e s s t o s t a n d a r d P u r i n a Rat Chow and t o d e i o n i z e d water i n p o l y e t h y l e n e b o t t l e s . No attempt was made to c o n t r o l the d i e t a r y i n t a k e of s i l i c o n i n the s e a n i m a l s . A l l dams were weighed weekly throughout pregnancy and l a c t a t i o n or u n t i l the l i t t e r of young was s a c r i f i c e d . Food consumption was r e c o r d e d f o r c o n t r o l and h y p o t h y r o i d dams d u r i n g pregnancy. A l l o f f s p r i n g s of the dams were weighed d a i l y u n t i l s a c r i f i c e d . S i x pups from the c o n t r o l group were each k i l l e d on p o s t n a t a l days 3, 6, 9, 12, 15, 20, 25 and 30. Twenty f o u r pups each.from hypo- and h y p e r t h y r o i d groups were k i l l e d on each of days 7, 14, 21, and 28. B l o o d samples of i i t h e s e pups were l a t e r a n a l y z e d f o r plasma s i l i c o n by f u r n a c e atomic a b s o r p t i o n s p e c t r o m e t r y and f o r t h y r o x i n e and t r i i o d o -t h y r o n i n e by radioimmunoassay. S i x pups from the l o n g e s t s u r v i v i n g l i t t e r s i n each group were x - r a y e d weekly f o r s e r i a l assessment of bone m a t u r a t i o n by the method of Hughes and Tanner. The study found no d i f f e r e n c e s among the t h r e e groups of dams i n food i n t a k e d u r i n g pregnancy or i n t o t a l weight g a i n d u r i n g pregnancy and l a c t a t i o n . There was a s i g n i f i c a n t e f f e c t of t r e a t m e n t on body weight and bone m a t u r a t i o n of hypo- and h y p e r t h y r o i d pups as compared w i t h c o n t r o l . S i g n i f i c a n t l y reduced body weight was apparent i n hypo- and h y p e r t h y r o i d pups by days 2 and 10 r e s p e c t i v e l y . H y p o t h y r o i d pups e x h i b i t e d r e t a r d e d bone development by day 14, w h i l e h y p e r t h y r o i d pups showed advanced s k e l e t a l m a t u r a t i o n by day 7. These were accompanied by c o r r e s p o n d i n g l y lower and h i g h e r l e v e l s of t h y r o i d hormones r e s p e c t i v e l y . T h y r o i d f u n c t i o n appeared t o i n f l u e n c e the p a t t e r n of plasma s i l i c o n l e v e l s d u r i n g the p o s t -n a t a l p e r i o d . Plasma s i l i c o n of c o n t r o l pups averaged 0.668 ppm on day 6, which rose t o a maximum of 0.970 ppm on day 12 and then d e c l i n e d t o 0.247 ppm a f t e r weaning. S i l i c o n l e v e l s i n t h y r o i d p r i v i a and exce s s were g e n e r a l l y lower and a l t e r e d i n d e v e l o p m e n t a l p a t t e r n s r e l a t i v e t o l e v e l s found i n e u t h y r o i d c o n d i t i o n . Changes i n plasma s i l i c o n c o n c e n t r a t i o n w i t h age were s i g n i f i c a n t i n each group of pups. The h i g h e s t v a l u e s f o r plasma s i l i c o n were 0.404 and 0.365 ppm on days 7 and 14 f o r h y p o t h y r o i d pups and 0.466 ppm on day 14 f o r h y p e r t h y r o i d pups. P e r i o d s of maximal plasma v a l u e s f o r s i l i c o n c o r r e s p o n -ded t o p e r i o d s of the h i g h e s t increment i n bone m a t u r i t y s c o r e s i n each group. However, the d a t a of the p r e s e n t study d i d not p e r m i t s t a t i s t i c a l a n a l y s i s of the r e l a t i o n s h i p between plasma s i l i c o n , c h r o n o l o g i c a l age and bone age of r a t s d u r i n g the n e o n a t a l p e r i o d . I t i s c o n c l u d e d t h a t under the e x p e r i m e n t a l c o n d i t i o n s employed i n t h i s s t u d y , plasma s i l i c o n l e v e l i s not i n f l u e n c e d by t h y r o i d s t a t u s or r e l a t e d t o bone development i n r a t s . i v ACKNOWLEDGEMENT S I am e s p e c i a l l y i n d e b t e d t o my mentor and a d v i s o r , Dr. M e l v i n Lee, f o r h i s d i r e c t i o n and guidance throughout my Ma s t e r ' s Programme. I am a l s o g r a t e f u l t o Dr. James Farmer f o r h i s encouragement, a s s i s t a n c e and thorough i n s t r u c t i o n on the o p e r a t i o n of the s p e c t r o m e t e r and g r a p h i t e f u r n a c e as w e l l as s e r v i n g on my t h e s i s committee. I am t h a n k f u l t o Dr. Joseph L e i c h t e r f o r h i s c o n s t r u c t i v e c r i t i c i s m i n t h e s i s r e v i s i o n and f o r s e r v i n g on my t h e s i s committee. I want t o exte n d s p e c i a l t h a n k s t o V i r g i n i a Green f o r the hours spent on s t a t i s t i c a l a n a l y s e s and i n t e r p r e t a t i o n of my r e s u l t s . Acknowledgement i s a l s o e x p r e s s e d t o the f o l l o w i n g s u p p o r t p e o p l e who have a s s i s t e d me d u r i n g my s t u d y : Amar D h i l l o n f o r g e n e r a l a n i m a l c a r e ; Stanya Horsky of UBC G e o l o g i c a l S c i e n c e s f o r p r a c t i c a l t i p s on plasma a n a l y s i s of s i l i c o n ; Lance Coombe f o r h i s time and s k i l l s i n p r o d u c i n g a l l of my graphs; my b r o t h e r P e t e r f o r h i s a r t i s t i c r e p r o d u c t i o n of the r a t s k e l e t o n ; l a s t but not l e a s t , my son T r a v i s f o r h i s t i m e , d e d i c a t i o n and l o y a l t y i n washing m i c r o t u b e s , c h a n g i n g the a n i m a l s ' beddings and r e f i l l i n g t h e i r d r i n k i n g b o t t l e s . T h i s study was su p p o r t e d by Grant No.4692 to Dr. M e l v i n Lee from the N a t u r a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l of Canada. v TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS V LIST OF TABLES v i i i LIST OF FIGURES i x CHAPTER I. INTRODUCTION 1 I I . REVIEW OF LITERATURE 5 A. P r e v a l e n c e and D i s t r i b u t i o n of S i l i c o n 5 B. Chemical Nature of S i l i c o n 5 C. P h y s i o l o g i c a l Role of S i l i c o n 6 1. E s s e n t i a l i t y of S i l i c o n i n C h i c k s 6 2. E s s e n t i a l i t y of S i l i c o n i n Rat s 9 3. R o l e of S i l i c o n i n G l y c o s a m i n o g l y c a n and C o l l a g e n 10 4. R o l e of S i l i c o n i n Bone C a l c i f i c a t i o n 13 D. E n d o c r i n e C o n t r o l of S i l i c o n 15 E. Role of T h y r o i d Hormones i n Bone Development 15 I I I . EXPERIMENTAL DESIGN AND METHODOLOGY 19 A. Treatment of Animals 19 1. C o n t r o l E u t h y r o i d Group 21 2. H y p o t h y r o i d Group 22 3. H y p e r t h y r o i d Group 22 B. C o l l e c t i o n of B l o o d 23 C. D e t e r m i n a t i o n of Plasma S i l i c o n 24 D. Assessment of S k e l e t a l M a t u r i t y 26 E. Assay of Plasma T h y r o x i n e and T r i i o d o t h y r o n i n e 30 F. S t a t i s t i c a l A n a l y s i s of R e s u l t s 30 IV. RESULTS 32 A. Food I n t a k e of Dams 32 B. Body Weight of Dams 32 1. Pregnancy 32 2. L a c t a t i o n 34 v i C. Body Weight of Pups 37 D. S k e l e t a l M a t u r i t y Scores 39 E. Plasma S i l i c o n C o n c e n t r a t i o n s 39 F. Plasma T h y r o x i n e and T r i i o d o t h y r o n i n e 42 V. DISCUSSION 45 A. Method of Plasma S i l i c o n D e t e r m i n a t i o n 46 B. Plasma S i l i c o n C o n c e n t r a t i o n s i n C o n t r o l Pups 49 C. E f f e c t i v e n e s s of Treatments W i t h P r o p y l t h i o u -r a c i l and L- T h y r o x i n e 53 1. M a t e r n a l N u t r i t i o n a l S t a t u s D u r i n g Pregnancy and L a c t a t i o n 53 2. Body Weight of Pups 55 3. S k e l e t a l M a t u r a t i o n of Pups 58 4. Plasma L e v e l s of T h y r o i d Hormones i n Pups 58 D. Plasma S i l i c o n C o n c e n t r a t i o n s i n Hypo- and H y p e r t h y r o i d Pups 59 E. S u g g e s t i o n s For F u t u r e C o n s i d e r a t i o n 62 BIBLIOGRAPHY 64 v i i LIST OF TABLES Table Page I . O p e r a t i n g Parameters For S i l i c o n D e t e r m i n a t i o n i n Plasma of Rats 27 I I . Food Consumption of C o n t r o l and H y p o t h y r o i d Dams D u r i n g Pregnancy 33 I I I . Plasma L e v e l s of T4 and T3 of S e l e c t e d Pups i n C o n t r o l , Hypo- and H y p e r t h y r o i d Groups a t D i f f e r e n t Ages D u r i n g the P o s t n a t a l P e r i o d 44 v i i i LIST OF FIGURES Figure Page 1. E x p e r i m e n t a l D e sign 20 2. Bone S c o r i n g i n the Rat 29 3. Weekly Body Weight of Dams D u r i n g Pregnancy 35 4. Weekly Body Weight of Dams D u r i n g L a c t a t i o n 36 5. D a i l y Body Weight of Rat Pups From P o s t n a t a l Day 2 t o 28 38 6. Weekly Bone M a t u r i t y S c o r e s of C o n t r o l , Hypo-and H y p e r t h y r o i d Pups D u r i n g the P o s t n a t a l P e r i o d 40 7. Plasma S i l i c o n C o n c e n t r a t i o n s of C o n t r o l , Hypo- and H y p e r t h y r o i d Pups a t D i f f e r e n t Ages D u r i n g the P o s t n a t a l P e r i o d 43 i x CHAPTER I INTRODUCTION S i l i c o n i s u b i q u i t o u s . As the second most abundant element on e a r t h , i t i s w i d e l y d i s t r i b u t e d i n n a t u r e (Weast, 1981). R e l a t i v e l y l a r g e q u a n t i t i e s of s i l i c o n may be i n c o r p o r a t e d by p l a n t s , but o n l y t r a c e amounts a r e found i n the t i s s u e s and body f l u i d s of a n i m a l s i n c l u d i n g man. In a n i m a l s , the h i g h e s t c o n c e n t r a t i o n o c c u r s i n the c o n n e c t i v e t i s s u e s i n g e n e r a l . E s t i m a t e s of s i l i c o n c o n c e n t r a t i o n i n the b l o o d of man and o t h e r mammalian s p e c i e s have averaged 5.0 mg/1 of b l o o d plasma ( C a r l i s l e , 1974b). The e s s e n t i a l i t y of s i l i c o n has been w e l l - e s t a b l i s h e d f o r the u n i c e l l u l a r d iatoms. Among the v e r t e b r a t e s , however, o n l y the r a t and c h i c k have demonstrated a requirement f o r s i l i c o n (Schwarz and M i l n e , 1972; C a r l i s l e , 1972). E v i d e n c e of s i l i c o n e s s e n t i a l i t y i n e x p e r i m e n t a l a n i m a l s has u n d e r s c o r e d the d i f f i c u l t y i n v o l v e d i n p r o d u c i n g c l e a r - c u t d e f i c i e n c y s t a t e s . The u b i q u i t o u s n a t u r e of s i l i c o n i n the environment and i n the l a b o r a t o r y r e q u i r e s e l a b o r a t e p r e c a u t i o n s and s p e c i a l i z e d f a c i l i t i e s i n or d e r t o e l i m i n a t e c o n t a m i n a t i o n by s i l i c o n . T h i s p r e s e n t s major o b s t a c l e s t o i n v e s t i g a t o r s and s t u d i e s i n s i l i c o n m e t a b o l i s m have been l i m i t e d t o a s m a l l number of l a b o r a t o r i e s . In a d d i t i o n , t e c h n i c a l d i f f i c u l t i e s a s s o c i a t e d w i t h the d e t e r m i n a t i o n of s i l i c o n have c o m p l i c a t e d assessment of i t s d i s t r i b u t i o n and c o n c e n t r a t i o n i n b i o l o g i c a l t i s s u e s and 1 f l u i d s . R e s e a r c h i n t o the m e t a b o l i c s i g n i f i c a n c e of s i l i c o n has p r o g r e s s e d s l o w l y . There i s e v i d e n c e f o r a r o l e of s i l i c o n i n the e a r l y s t a g e s of bone c a l c i f i c a t i o n . S i l i c o n i s c o n c e n t r a t e d a t r e l a t i v e l y h i g h l e v e l s w i t h i n the zone of a c t i v e c a l c i f i c a t i o n i n l o n g bones d u r i n g bone f o r m a t i o n ( C a r l i s l e , 1970a). S i l i c o n i s accumulated w i t h i n o r g a n e l l e s i n the c y t o p l a s m of the o s t e o b l a s t s , the a c t i v e bone f o r m i n g c e l l s ( C a r l i s l e , 1975). S t u d i e s of o s t e o b l a s t s from young bone show t h a t s i l i c o n c o n c e n t r a t i o n i n i t i a l l y i n c r e a s e s i n d i r e c t p r o p o r t i o n t o c a l c i u m but d i m i n i s h e s t h e r e a f t e r as the c a l c i u m t o phosphorus r a t i o approaches t h a t of mature bone a p a t i t e when c a l c i f i c a t i o n i s completed ( C a r l i s l e , 1969). I t i s h y p o t h e s i z e d t h a t s i l i c o n p r o v i d e s the n u c l e i f o r b i n d i n g of c a l c i u m t o the bone m a t r i x , and t h i s c o n s t i t u t e s the f i r s t c r i t i c a l s t e p i n the i n i t i a t i o n of m i n e r a l i z a t i o n . In v i v o s t u d i e s have demon-s t r a t e d t h a t s i l i c o n s u p p l e m e n t a t i o n i n c r e a s e s the r a t e of m i n e r a l i z a t i o n ( C a r l i s l e , 1970a) and t h a t the h i g h e r s i l i c o n c o n t e n t of the p e r i o s t e u m of bone i s r e f l e c t e d i n h i g h e r plasma s i l i c o n c o n c e n t r a t i o n s ( C a r l i s l e , 1980b). In a d d i t i o n , s i l i c o n has been shown t o be a component of c o l l a g e n and g l y c o s a m i n o -g l y c a n ( C a r l i s l e , 1974a) and, t h e r e f o r e , i s r e q u i r e d i n the b i o s y n t h e s i s of a r t i c u l a r c a r t i l a g e and c o n n e c t i v e t i s s u e ( C a r l i s l e , 1976). S t u d i e s have shown t h a t the s i l i c o n c o n c e n t r a t i o n of b l o o d i n the mature r a t i s a f u n c t i o n of age, sex and a c t i v i t y of 2 v a r i o u s e n d o c r i n e o r g a n s , p a r t i c u l a r l y the a d r e n a l , gonad and t h y r o i d g l a n d s (Charnot and P e r e s , 1971a; 1971b). T h y r o i d -ectomy-induced h y p o t h y r o i d i s m i n the mature r a t r e s u l t s i n a s t a t i s t i c a l l y n o n s i g n i f i c a n t s h i f t of s i l i c o n c o n t e n t between the bone t i s s u e and b l o o d compartment r e s u l t i n g i n lower and h i g h e r l e v e l s of plasma s i l i c o n i n male and female r a t s r e s p e c t i v e l y . I t i s a l s o w e l l - r e c o g n i z e d t h a t the t h y r o i d hormones i n f l u e n c e the growth and m a t u r a t i o n of the s k e l e t a l system of young a n i m a l s (Greenberg, N a j j a r and B l i z z a r d , 1974). T h y r o i d hormones appear t o have l i t t l e d i r e c t e f f e c t on bone m i n e r a l i z a t i o n (Ogden, 1980). However, they a r e i m p o r t a n t i n the r e g u l a t i o n of e a r l y p h y s e a l growth which i s l a t e r s u p p r e s s e d by i n c r e a s e d c a l c i f i c a t i o n of the m a t r i x p r i o r t o e p i p h y s e a l c l o s u r e a t m a t u r i t y . I t i s p o s s i b l e t h a t , i f hypo-t h y r o i d i s m i s induced i n young r a t s a t a time when s i l i c o n has the most impact on bone development, then t h e e f f e c t s of d e l a y e d m a t u r i t y and r e t a r d e d bone c a l c i f i c a t i o n a s s o c i a t e d w i t h h y p o t h y r o i d i s m may r e s u l t i n s i g n i f i c a n t a l t e r a t i o n s of bone s i l i c o n and plasma s i l i c o n c o n c e n t r a t i o n s . The o b s e r v a t i o n would l e n d support t o the h y p o t h e s i z e d r e l a t i o n of s i l i c o n t o bone f o r m a t i o n . The p r e s e n t study was d e s i g n e d t o i n v e s t i g a t e the e f f e c t s of hypo- and h y p e r t h y r o i d i s m on the plasma l e v e l s of s i l i c o n . H y p o t h y r o i d i s m i s a s s o c i a t e d w i t h reduced s e c r e t i o n s of t h y r o x i n e and t r i i o d o t h y r o n i n e , w h i l e h y p e r t h y r o i d i s m i s c h a r a c t e r i z e d by a s u s t a i n e d r i s e i n the plasma l e v e l of the 3 t h y r o i d hormones. I t was h y p o t h e s i z e d t h a t t h y r o i d hormone d e f i c i e n c y or exce s s i n the young growing r a t s may r e s u l t i n s t a t i s t i c a l l y s i g n i f i c a n t a l t e r a t i o n s of b l o o d s i l i c o n l e v e l s and of bone m a t u r a t i o n . In t h i s s t u d y , r a t pups were made h y p o t h y r o i d or h y p e r t h y r o i d and the plasma s i l i c o n l e v e l s were de t e r m i n e d and compared t o those of normal c o n t r o l pups as a f u n c t i o n of age. No attempt was made t o c o n t r o l s i l i c o n i n -ta k e s or e x p o s u r e s . Bone m a t u r i t y was a s s e s s e d s e r i a l l y by whole body r a d i o g r a p h y . 4 CHAPTER II REVIEW OF LITERATURE A. Prevalence and Dis t r i b u t i o n of S i l i c o n S i l i c o n i s the second most p r e v a l e n t element i n the l i t h o s p h e r e , b e i n g exceeded o n l y by oxygen. I t comprises 25.7% by weight of the e a r t h ' s c r u s t (Weast, 1981). I t o c c u r s i n na t u r e i n v a r i a b l y bound t o oxygen as the d i o x i d e s i l i c a ( SiO,,) and as the monomeric and p o l y m e r i c s i l i c a t e s . S i l i c a forms the major c o n s t i t u e n t of r o c k s and s o i l and i s found i n the human s k e l e t o n and i n the ashes of p l a n t s . S i l i c a t e appears i n numerous m i n e r a l s i n c l u d i n g c l a y , f e l d s p a r , g r a n i t e and a s b e s t o s . The h y d r o l y s i s of S i - O - S i bonds i n s i l i c a and s i l i c a t e s r e l e a s e s s i l i c i c a c i d i n t o s o l u t i o n . I t i s i n t h i s form t h a t s i l i c o n e n t e r s the b i o s p h e r e and i s found i n a l l l i v i n g o rganisms. S i l i c o n appears t o be p r e s e n t i n a l l l i v i n g o rganisms. R e l a t i v e l y l a r g e amounts of s i l i c o n a r e i n c o r p o r a t e d by Diatomae, R a d i o l a r i a n , H e l i o z o a and s i l i c e o u s sponges. Only t r a c e amounts a re found i n almost a l l h i g h e r a n i m a l s . In a n i m a l s , s i l i c o n i s a s s o c i a t e d p r e d o m i n a n t l y w i t h the c o n n e c t i v e t i s s u e s found i n c a r t i l a g e , l i g a m e n t and s k i n . B. Chemical Nature of S i l i c o n The s o l u b i l i t y of s i l i c i c a c i d i n pure water i s of the or d e r of 100 - 140 ppm w i t h i n the pH range of 1 - 9 ( A l e x a n d e r , 5 Heston and I l e r , 1954). I t i s i n c r e a s e d i n the p r e s e n c e of d i s s o l v e d o r g a n i c s u b s t a n c e s and under c o n d i t i o n s of pH g r e a t e r than 9.0 as the weak a c i d b e g i n s t o i o n i z e . C e r t a i n c a t i o n s such as c a l c i u m , aluminum and i r o n reduce the s o l u b i l i t y of s i l i c i c a c i d . At lower c o n c e n t r a t i o n s s i l i c i c a c i d s e x i s t as the monomer [ S i ( O H ) 4 ] . At h i g h e r c o n c e n t r a t i o n s monomeric s i l i c i c a c i d s condense t o form p o l y s i l i c i c a c i d as the h y d r a t e d , amorphous s i l i c a . In a n i m a l t i s s u e s and f l u i d s , the m o l e c u l a r form of endogenous s i l i c o n has been the s u b j e c t of numerous i n v e s t i g a t i o n s . I t i s s p e c u l a t e d t h a t s i l i c o n o c c u r s as f r e e i n o r g a n i c m o i e t i e s or as bound o r g a n i c d e r i v a t i v e s . I t i s p o s t u l a t e d t h a t s i l a n o l a t e , an e t h e r - l i k e d e r i v a t i v e of s i l i c i c a c i d i s bound t o the p o l y s a c c h a r i d e m a t r i x of g l y c o s a m i n o g l y c a n s (Schwarz, 1977). The bound f r a c t i o n presumably does not r e a d i l y h y d r o l y z e t o l i b e r a t e s i l i c i c a c i d . E s t e r d e r i v a t i v e s of o r g a n i c s i l i c a t e s have been v a r i o u s l y i d e n t i f i e d as l i p i d , c a r b o h y d r a t e and p r o t e i n i n n a t u r e . C. Physiological Role of S i l i c o n 1. E s s e n t i a l i t y of S i l i c o n in Chicks The e s s e n t i a l i t y of s i l i c o n f o r normal growth and development was f i r s t d emonstrated i n c h i c k s ( C a r l i s l e , 1972). White Leghorn c o c k e r e l s were g i v e n a c h e m i c a l l y d e f i n e d , amino a c i d d i e t under s i l i c o n - c o n t r o l l e d environment. The d a y - o l d c h i c k s were d e u t e c t o m i z e d a t the time of h a t c h i n g t o remove the 6 y o l k r e s e r v e of s i l i c o n . They were f e d the b a s a l d i e t , which c o n t a i n e d l e s s than 0.8 ppm s i l i c o n , or g i v e n s i l i c o n supplement as sodium m e t a s i l i c a t e a t 100 ppm of the b a s a l d i e t . W i t h i n t h r e e weeks, c h i c k s on the b a s a l d i e t demonstrated s i g n i f i c a n t growth r e t a r d a t i o n and s k e l e t a l a b n o r m a l i t i e s . S u p p l e m e n t a t i o n of the b a s a l d i e t w i t h s i l i c o n improved growth s i g n i f i c a n t l y i n 3 groups of c h i c k s by 30%, 49.8% and 30%. S i l i c o n - d e p r i v e d c h i c k s showed absence of w a t t l e s , d i m i n i s h e d combs, f l e x i b l e beaks and p a l l i d - c o l o u r e d s k i n and mucuous membrane. They dev e l o p e d a s m a l l , a b n o r m a l l y shaped s k u l l . The femur and t i b i a of the l e g were s h o r t e r , and t h i n n e r i n the c o r t e x , and were e a s i l y f r a c t u r e d . The study was c r i t i c i z e d i n t h a t the f o r m u l a t e d b a s a l d i e t s u p p o r t e d l e s s than the optimum growth i n the c o n t r o l group. The o b s e r v e d a b n o r m a l i t i e s , t h e r e f o r e , c o u l d not be a t t r i b u t e d e n t i r e l y t o s i l i c o n def i c i e n c y . The e a r l i e r s t u d i e s by C a r l i s l e (1980a) were s u b s e q u e n t l y r e p e a t e d u s i n g a newly f o r m u l a t e d , s e m i - s y n t h e t i c d i e t based on c a s e i n , which gave o p t i m a l growth i n c o n t r o l a n i m a l s . In t h i s s t u d y , deutectomy was not performed i n the d a y - o l d c o c k e r e l s . They were f e d the b a s a l or supplemented d i e t s which s u p p l i e d s i l i c o n at the l e v e l of 1 ppm and 250 ppm r e s p e c t i v e l y . At the end of f o u r weeks the c h i c k s were e v a l u a t e d f o r growth. Plasma c o n c e n t r a t i o n s of s i l i c o n , c a l c i u m , phosphorus and magnesium were measured as were the i n o r g a n i c and o r g a n i c c o n s t i t u e n t s of the s k u l l and l o n g bones. C o l l a g e n i s the predominant 7 s t r u c t u r a l p r o t e i n w i t h i n the i n t e r c e l l u l a r m a t r i x of bone and c o n n e c t i v e t i s s u e s ( T r i f f i t t , 1980). Other o r g a n i c c o n s t i -t u e n t s of the bone m a t r i x i n c l u d e n o n c o l l a g e n o u s p r o t e i n s such as p r o t e o g l y c a n s , and l i p i d s . P r o t e o g l y c a n s a r e p r o t e i n s con-j u g a t e d t o g l y c o s a m i n o g l y c a n s , which c o n t a i n r e p e a t i n g u n i t s of hexosamine and u r o n i c a c i d r e s i d u e s . These p r o v i d e support t o t h e c o n n e c t i v e t i s s u e s by forming a h i g h l y h y d r a t e d network between the c o l l a g e n f i b r i l s . The g l y c o s a m i n o g l y c a n s found i n compact bone t i s s u e have r e p e a t i n g u n i t s which i n c l u d e k e r a t a n s u l p h a t e , h y a l u r o n i c a c i d and c h o n d r o i t i n 4 - s u l p h a t e . As the c o l l a g e n m a t r i x undergoes c r y s t a l l i z a t i o n , h y d r o x y a p a t i t e c r y s t a l s of c a l c i u m phosphate a r e d e p o s i t e d between the c o l l a g e n f i b r i l s . T h i s i s accompanied by a c o r r e s p o n d i n g r e d u -c t i o n i n water and n o n - c o l l a g e n o u s c o n s t i t u e n t s . In c o n t r a s t t o the p r e v i o u s d i e t , s i l i c o n d e p r i v a t i o n i n t h i s s t u d y produced no s i g n i f i c a n t d i f f e r e n c e i n growth or outward appea-r a n c e s . However, t h e r e were s t r u c t u r a l a b n o r m a l i t i e s of the s k u l l which i n v o l v e d n a r r o w i n g of the f r o n t a l a r e a and s t u n t i n g of the p a r i e t a l , o c c i p i t a l and t e m p o r a l bones. These abnorma-l i t i e s were a s s o c i a t e d w i t h d e p r e s s e d c o l l a g e n c o n t e n t of the f r o n t a l bone. There were no s i g n i f i c a n t d i f f e r e n c e s between the c o n t r o l and s i l i c o n - s u p p l e m e n t e d groups i n the c o n t e n t of hexosamine and n o n - c o l l a g e n o u s p r o t e i n or i n the degree of m i n e r a l i z a t i o n of the s k u l l f r o n t bones. 8 2. E s s e n t i a l i t y of S i l i c o n in Rats S t u d i e s performed i n d e p e n d e n t l y from those of C a r l i s l e d emonstrated t h a t s i l i c o n was a l s o r e q u i r e d by r a t s f o r normal growth (Schwarz and M i l n e , 1972). Male w e a n l i n g F i s c h e r 344 r a t s were i s o l a t e d i n an a l l - p l a s t i c t r a c e e l e m e n t - c o n t r o l l e d system. S i l i c o n c o n t a m i n a t i o n was m i n i m i z e d by p r e v e n t i n g c o n t a c t of the a n i m a l s w i t h m e t a l , g l a s s , dust and c a r e t a k e r s . R o u t i n e a n a l y s e s of the a n i m a l feeds were a l s o performed t o c o n t r o l f o r t r a c e element c o n t a m i n a n t s . The r a t s were f e d p u r i f i e d , b a l a n c e d amino a c i d d i e t s which s u p p l i e d s i l i c o n a t l e s s than 5 ppm. S i l i c o n d e p r i v a t i o n i n growing r a t s was m a n i f e s t e d by growth r e t a r d a t i o n and by a b n o r m a l i t i e s of s k e l e t a l s t r u c t u r e s . There was n o t a b l e impairment i n p i g m e n t a t i o n of the i n c i s o r , r e d u c t i o n i n the l e n g t h of the s k u l l and d i s t o r t i o n i n the bony a r c h i t e c t u r e of the eye s o c k e t . S i l i c o n s u p p l e m e n t a t i o n i n the form of sodium m e t a s i l i c a t e ( N a 2 S i 0 3 . 9 H 2 0 ) at 50 mg/lOOg of the b a s a l d i e t s A and B produced s i g n i f i c a n t growth i n c r e a s e s by 33.8% and 25.2% r e s p e c t i v e l y , compared t o the c o n t r o l groups. However, s i l i c o n was o n l y p a r t i a l l y e f f e c t i v e i n r e s t o r i n g normal p i g m e n t a t i o n of the i n c i s o r w h i l e s i g n i f i c a n t improvements (16% - 44%) were a c h i e v e d w i t h p h y s i o l o g i c a l l e v e l s (500 ppm) of t i n , vanadium and f l u o r i n e . Other a b n o r m a l i t i e s such as l o s s of h a i r , s e b o r r h e a and a t o n i c i t y were observed i n s i l i c o n -d e p r i v e d r a t s , but they were not a m e l i o r a t e d by s i l i c o n s u p p l e -m e n t a t i o n . 9 3. Role of S i l i c o n in Glycosaminoglycan and Collagen S i g n i f i c a n t r e d u c t i o n i n the s i z e of the l o n g bones was l a t e r r e p o r t e d i n s i l i c o n - d e p r i v e d c h i c k s g i v e n the semi-s y n t h e t i c d i e t ( C a r l i s l e , 1980b). D i f f e r e n c e s i n the t i s s u e wet weight of t i b i a between the b a s a l and supplemented groups were not s i g n i f i c a n t and were l e s s pronounced than the p r e v i o u s f i n d i n g w i t h d e u t e c t o m i z e d c h i c k s on p u r i f i e d amino a c i d d i e t . A n a l y s e s of the t i b i a r e v e a l e d s i g n i f i c a n t l y h i g h e r c o n t e n t of water, c o l l a g e n and hexosamine i n the supplemented group than i n s i l i c o n - d e p r i v e d c h i c k s . There were no d i f f e r e n c e s i n non-c o l l a g e n o u s p r o t e i n , c a l c i u m or bone m i n e r a l . S i m i l a r l y , the femur and m e t a t a r s u s of the supplemented group gave h i g h e r v a l u e s f o r water c o n t e n t and presumably hexosamine but were comparable t o the d e p r i v e d group i n t i s s u e wet weight and bone ash. A l t h o u g h bone m i n e r a l as a p e r c e n t a g e of dry weight of t i b i a was e q u i v a l e n t i n both g r o u p s , the m i n e r a l c o m p o s i t i o n d i f f e r e d s i g n i f i c a n t l y f o r s i l i c o n but not f o r c a l c i u m , t o t a l phosphorus, magnesium or z i n c . The h i g h e r s i l i c o n c o n c e n t r a t i o n of t i b i a l p e r i o s t e u m p a r a l l e l e d t h e s i g n i f i c a n t l y h i g h e r plasma s i l i c o n c o n c e n t r a t i o n i n the s i l i c o n - s u p p l e m e n t e d group. In a d d i t i o n , s i l i c o n - d e p r i v e d c h i c k s showed h i s t o l o g i c a l e v i d e n c e of a b n o r m a l i t i e s i n the zone of c a r t i l a g e growth p l a t e s c o n c e r n e d w i t h l o n g i t u d i n a l and l a t i t u d i n a l bone e x p a n s i o n . The e v i d e n c e of s k e l e t a l a b n o r m a l i t i e s s u p p o r t e d the c o n c l u s i o n t h a t s i l i c o n was r e q u i r e d f o r b oth t y p e s of bone 10 f o r m a t i o n , e x e m p l i f i e d by the s k u l l and t i b i a . S i l i c o n d e f i c i e n c y a s s o c i a t e d w i t h reduced g l y c o s a m i n o g l y c a n was i n f e r r e d from lower water and hexosamine c o m p o s i t i o n of t i b i a , femur and m e t a t a r s u s . The e f f e c t of s i l i c o n on g l y c o s a m i n o g l y c a n was g r e a t e r than on c o l l a g e n and g e n e r a l l y more pronounced f o r the f o r m a t i o n of o r g a n i c m a t r i x than f o r bone m i n e r a l i z a t i o n p r o c e s s . S i m i l a r o b s e r v a t i o n s were r e p o r t e d i n p r e v i o u s s t u d i e s ( C a r l i s l e , 1976). S i l i c o n d e f i c i e n c y was c o n s i s t e n t l y a s s o c i a t e d w i t h reduced g l y c o s a m i n o g l y c a n and d i m i n i s h e d c a r t i l a g e s on the a r t i c u l a t i n g s u r f a c e s of the t i b i a and femur. In the s e v e r e l y a t t e n u a t e d combs of the c o c k e r e l s , t h e r e were s u b s t a n t i a l l y lower amounts of c o n n e c t i v e t i s s u e and g l y c o s a m i n o g l y c a n . However, the p u r i f i e d b a s a l amino a c i d m i x t u r e employed i n the s t u d y d i d not support optimum growth i n the c h i c k s and induced more se v e r e d e f i c i e n c y s t a t e s than d i d the s e m i - s y n t h e t i c d i e t s of l a t e r s t u d i e s . The p r e v i o u s f i n d i n g t h a t s i l i c o n was i n v o l v e d w i t h g l y c o s a m i n o g l y c a n s i n a r t i c u l a r c a r t i l a g e and c o n n e c t i v e t i s s u e f o r m a t i o n were c o r r o b o r a t e d by the r e s u l t s of s t u d i e s on bone c o n s t i t u e n t s . These s t u d i e s have d i f f e r e n t i a t e d f r e e s i l i c a t e from a bound form of s i l i c o n (Schwarz, 1973). I t was h y p o t h e s i z e d t h a t the bound s i l i c o n was p r e s e n t as s i l a n o l a t e , an e t h e r or e s t e r d e r i v a t i v e of s i l i c i c a c i d which s e r v e d as a b i o l o g i c a l c r o s s - l i n k i n g agent i n g l y c o s a m i n o g l y c a n s and c o n n e c t i v e t i s s u e t h rough the f o r m a t i o n of R.- 0 - S i - 0 - R, 1 1 l i n k a g e s . T i s s u e a n a l y s e s showed t h a t s i l i c o n o c c u r r e d i n l a r g e q u a n t i t i e s as c o n s t i t u e n t s of c e r t a i n g l y c o s a m i n o g l y c a n and p o l y u r o n i c a c i d s i n c l u d i n g h y a l u r o n i c a c i d , dermatan s u l f a t e and c h o n d r o i t i n 4 - s u l f a t e . S i m i l a r h i g h v a l u e s f o r bound s i l i c o n i n c o l l a g e n were o b t a i n e d from d i f f e r e n t p r e p a r a t i o n s of c o n n e c t i v e t i s s u e s (Schwarz and Chen, 1974). However, the i n v e s t i g a t o r s have c o n c l u d e d t h a t t h e s e v a l u e s f o r bound s i l i c o n were e r r o n e o u s l y h i g h due t o c o n t a m i n a t i o n from c o m m e r c i a l l y p r e p a r e d samples and from the i n a d v e r t e n t use of diatomaceous e a r t h as a f i l t e r i n g agent (Schwarz, 1977). Subsequent r e p e a t a n a l y s e s of thes e samples y i e l d e d v a l u e s f o r t o t a l s i l i c o n i n the ground m a t r i x of c o n n e c t i v e t i s s u e s much lower than those r e p o r t e d p r e v i o u s l y . The i n i t i a l h y p o t h e s i s was q u a l i f i e d t o a r o l e of s i l i c o n i n the c r o s s - l i n k i n g of o n l y s p e c i a l i z e d o r g a n e l l e s , which a c c o u n t e d f o r c o r r e s p o n d i n g l y s m a l l e r p r o p o r t i o n s of the c o n n e c t i v e t i s s u e . These i n c l u d e d the m i t o c h o n d r i a of bone c e l l s i n the c a l c i f y i n g a r e a s of bone t i s s u e , the m i c r o t u b u l a r elements of c e l l s , and the base membrane s t r u c t u r e s of c o n n e c t i v e t i s s u e s . However, the s p e c i f i c m e t a b o l i c f u n c t i o n of s i l i c o n i n h i g h e r a n i m a l s remains open t o s p e c u l a t i o n . S t u d i e s w i t h v i t a m i n D3 have demonstrated t h a t s i l i c o n e x e r t e d a s i g n i f i c a n t e f f e c t on the c o l l a g e n of the c o n n e c t i v e t i s s u e m a t r i x which was independent of v i t a m i n D3 ( C a r l i s l e , 1981). R e g a r d l e s s of the l e v e l of v i t a m i n D3 s u p p l e m e n t a t i o n , a l l the c h i c k s on s i l i c o n - d e p r i v e d d i e t s showed g r o s s 1 2 a b n o r m a l i t i e s of the s k u l l a r c h i t e c t u r e and s u b s t a n t i a l l y reduced c o l l a g e n c o n t e n t . In v i t r o s t u d i e s on the a c t i v i t y of the enzyme p r o l y l h y d r o x y l a s e f u r t h e r s u b s t a n t i a t e d the r e q u i r e m e n t of s i l i c o n i n the c o l l a g e n b i o s y n t h e s i s of bone and c o n n e c t i v e t i s s u e m a t r i c e s . The enzyme, which c a t a l y z e s the h y d r o x y l a t i o n of p r o l i n e t o h y d r o x y p r o l i n e i n c o l l a g e n s y n t h e s i s was s t i m u l a t e d by the a d d i t i o n of s i l i c o n ( C a r l i s l e , Berger and A l p e n f e l s , 1981). 4 . Role of S i l i c o n in Bone C a l c i f i c a t i o n Presumably s i l i c o n i s a s s o c i a t e d w i t h bone c a l c i f i c a t i o n ( C a r l i s l e , 1969). E l e c t r o n probe a n a l y s e s of bones from young r a t s and mice showed t h a t s i l i c o n was l o c a l i z e d i n the a c t i v e growth r e g i o n s . Both s i l i c o n and c a l c i u m c o n t e n t s i n c r e a s e d c o n g r u e n t l y from v e r y low l e v e l s i n the e a r l y s t a g e s of bone c a l c i f i c a t i o n . As c a l c i f i c a t i o n p r o g r e s s e d , the maximal s i l i c o n l e v e l was a c h i e v e d when the c a l c i u m t o phosphorus r a t i o s a p proximated 0.7. In advanced s t a g e s as c a l c i u m approached the p r o p o r t i o n p r e s e n t i n mature bone a p a t i t e , s i l i c o n c o n t e n t d e c l i n e d t o below the d e t e c t i o n l i m i t . S u b s e q u e n t l y , q u a n t i t a t i v e m i c r o a n a l y s e s were performed on specimens of normal t i b i a from young r a t s and mice ( C a r l i s l e , 1970a). The t i b i a was a n a l y z e d f o r s p a t i a l d i s t r i b u t i o n of c a l c i u m , phosphorus and s i l i c o n i n c r o s s - s e c t i o n t r a v e r s e s a c r o s s the p e r i o s t e u m , the o s t e o i d l a y e r and the t r a b e c u l a r 1 3 r e g i o n s of the bone. The o s t e o i d l a y e r c o n t a i n e d 25 times as much s i l i c o n as the a d j a c e n t pre-osseous t i s s u e of p e r i o s t e u m . The s i l i c o n c o n t e n t of f u l l y m i n e r a l i z e d bone w i t h i n the t r a b e c u l a r r e g i o n s was r e p o r t e d l y v e r y low. I t appears t h a t s i l i c c - n i s l o c a l i z e d i n r e g i o n s where t h e r e i s low t o moderate c a l c i u m c o n t e n t and a c t i v e c a l c i f i c a t i o n i s i n p r o g r e s s . S i l i c o n l o c a l i z a t i o n i n the l o n g bones of young r a t s and embryonic c h i c k s has been v e r i f i e d r e c e n t l y by L a n d i s et a l . (1986), u s i n g imaging i o n mi c r o s c o p y based on secondary i o n mass s p e c t r o m e t r y . The element i s found d i s c r e t e l y l o c a l i z e d w i t h i n the e x t r a c e l l u l a r m a t r i c e s of u n c a l c i f i e d o s t e o i d r e g i o n s of the t i b i a . S i l i c o n d i s t r i b u t i o n appears t o f o l l o w c l o s e l y the t i s s u e d i s t r i b u t i o n of c a l c i u m and magnesium. T h i s i s s u p p o r t e d by a subsequent i n v i v o experiment which a s c e r t a i n e d the r e l a t i o n s h i p between the l e v e l of d i e t a r y s i l i c o n and bone m i n e r a l i z a t i o n ( C a r l i s l e , 1970b; 1974b). Weanling r a t s from dams g i v e n a s i l i c o n - l o w d i e t p r i o r t o mating were p l a c e d on d i e t s c o n t a i n i n g 3 l e v e l s of c a l c i u m and 3 l e v e l s of s i l i c o n . I n c r e a s e s of d i e t a r y s i l i c o n on the low c a l c i u m d i e t r e s u l t e d i n a h i g h l y s i g n i f i c a n t i n c r e a s e i n the p e r c e n t a g e ash c o n t e n t of l o n g bones. I t appears t h a t s i l i c o n a c c e l e r a t e s the r a t e of bone m i n e r a l i z a t i o n . F u r t h e r i n v i v o s t u d i e s w i t h c h i c k s demonstrated a r e l a t i o n s h i p between s i l i c o n , magnesium and f l u o r i n e i n growing bones ( C a r l i s l e , 1971). A d d i t i o n of magnesium and f l u o r i n e t o a p u r i f i e d d i e t r e s u l t e d i n severe l e g weaknesses i n young 1 4 c h i c k s . T h i s was accompanied by g r e a t l y reduced bone c a l c i f i c a t i o n and somewhat reduced growth r a t e . The e f f e c t s a r e a s s o c i a t e d w i t h l a r g e changes i n b l o o d s i l i c o n l e v e l as w e l l as changes i n s i l i c o n , magnesium and f l u o r i n e of bone. Adding magnesium or f l u o r i n e s i n g l y t o the d i e t d i d not produce t h e s e r e s u l t . The f i n d i n g s s u p port the concept t h a t s i l i c o n i s i n v o l v e d w i t h c a l c i u m i n the m i n e r a l i z a t i o n p r o c e s s . D. Endocrine C o n t r o l of S i l i c o n S t u d i e s have demonstrated t h a t s i l i c o n m e t a b o l i s m i s under e n d o c r i n e c o n t r o l (Charnot and P e r e s , 1971a). Plasma concen-t r a t i o n of s i l i c o n i s i n c r e a s e d by t h y r o i d e c t o m y i n female r a t s , p o s s i b l y through enhanced i n t e s t i n a l a b s o r p t i o n of the element and a c o r r e s p o n d i n g s h i f t of s i l i c o n c o n t e n t from i n t e s t i n a l t i s s u e t o b l o o d compartment. However, t h y r o i d e c t o m y i n the male r a t has the o p p o s i t e e f f e c t . In a d d i t i o n , plasma l e v e l s of s i l i c o n a r e i n f l u e n c e d by a g i n g , and a d r e n a l and sex hormones (Charnot and P e r e s , 1971b). E. Role of T h y r o i d Hormones in Bone Development I t i s w e l l e s t a b l i s h e d t h a t t h y r o i d hormones p l a y a s i g n i f i c a n t r o l e i n the growth and m a t u r a t i o n of the s k e l e t a l system d u r i n g p r e n a t a l and p o s t n a t a l l i f e ( Greenberg, N a j j a r and B l i z z a r d , 1974). Bone growth i s measured by a l i n e a r i n c r e a s e i n s i z e . Bone c e l l s undergo a c y c l e of p r o l i f e r a t i o n , d e g e n e r a t i o n and r e s o r p t i o n . N o r m a l l y i t proceeds c o n c u r r e n t l y 1 5 w i t h bone m a t u r a t i o n d u r i n g which c e n t r e s of o s s i f i c a t i o n a r e e s t a b l i s h e d i n an o r d e r l y sequence. O s s i f i c a t i o n c e n t r e s a t the e p i p h y s e s of l o n g bones u l t i m a t e l y fuse a t m a t u r i t y and f u r t h e r bone growth i s p r e v e n t e d . The development of t h y r o i d - d e p e n d e n t s k e l e t a l system i n the r a t o c c u r s to a c o n s i d e r a b l e e x t e n t p o s t n a t a l l y . T h y r o i d g l a n d i n the r a t f e t u s i s d i f f e r e n t i a t e d by about day 18 of g e s t a t i o n ( C a r p e n t e r and R o n d o n - T a r c h e t t i , 1957). There i s e v i d e n c e of a f u n c t i o n i n g t h y r o i d - p i t u i t a r y feedback mechanism at t h i s s t a g e of f e t a l development (Hwang and W e l l s , 1959). However, the f e t a l p i t u i t a r y - t h y r o i d a x i s becomes f u l l y i n t e g r a t e d t o the hypothalamus when the t h r e e g l a n d s mature almost s i m u l t a n e o u s l y i n the p o s t n a t a l p e r i o d ( D u s s a u l t and L a b r i e , 1975). I t i s p r o b a b l e t h a t e a r l y i n t r a u t e r i n e f e t a l growth o c c u r s i n d e p e n d e n t l y of t h y r o i d s t a t u s . Moreover, s k e l e t a l and somatic l i n e a r growth i n u t e r o can be c o m p l e t e l y d i s s o c i a t e d from t h y r o i d - d e p e n d e n t s k e l e t a l m a t u r a t i o n (Greenberg, N a j j a r and B l i z z a r d , 1974). S t u d i e s have shown t h a t t h y r o i d e c t o m i z e d f e t a l r a t s a t t a i n normal body s i z e and weight at b i r t h (Hamburgh, Lynn and Weiss, 1964). In f e t a l r a t s d e c a p i t a t e d on day 16 of g e s t a t i o n , t h e femur d i s p l a y e d a t b i r t h a t h i c k e n e d lamina of the p e r i o s t e u m , wider t r a b e c u l a e of the endochondral bone, marked c a r t i l a g e remnants and d e l a y e d c a l c i f i c a t i o n of d i a p h y s i s of the l o n g bone (Geloso e t a l . , 1968). However, the study f a i l e d t o d i f f e r e n t i a t e the e f f e c t s of t h y r o i d hormones 16 from p i t u i t a r y growth hormones on bone growth and m a t u r a t i o n . N e v e r t h e l e s s , t h e r e i s e v i d e n c e t h a t t h y r o i d hormones i n f l u e n c e bone growth by s t i m u l a t i n g the p r o d u c t i o n of growth hormone from the p i t u i t a r y g l a n d (Solomon and Greep, 1959). Both t h y r o i d and growth hormones a c t s y n e r g i s t i c a l l y on bone t i s s u e , each p o t e n t i a t i n g the e f f e c t s of the o t h e r (Thorngren and Kansson, 1973). By c o n t r a s t , p o s t n a t a l growth and development i n the r a t i s c l e a r l y dependent on a e u t h y r o i d s t a t e . Becks et a l . (1950) demonstrated d e l a y e d s k e l e t a l m a t u r a t i o n i n 60-day o l d r a t s t h y r o i d e c t o m i z e d a t b i r t h . The e p i p h y s e a l o s s i f i c a t i o n c e n t r e s i n t h e s e a n i m a l s remained s m a l l , the e p i p h y s e a l c a r t i l a g e were not s e a l e d by bony p l a t e s and the d i f f e r e n t i a t i v e and p r o l i f e -r a t i v e a c t i v i t y p r o g r e s s e d a t a slow pace. T h y r o x i n e d e f i c i e n c y has been a s s o c i a t e d w i t h l o s s of i n t e g r i t y of the a r t i c u l a r c a r t i l a g e , v a s c u l a r i z a t i o n of the h y a l i n e c h o n d r o e p i p h y s i s and i t s e r o s i o n by bone and f i b r o u s t i s s u e , a l t e r e d d i s t r i b u t i o n i n m a t r i c e a l g l y c o s a m i n o g l y c a n and r e d u c t i o n i n p h y s e a l w i d t h p a r t i c u l a r l y i n the zone of h y p e r t r o p h y (Ogden, 1980). In t h y r o p a r a t h y r o i d e c t o m i z e d r a t s , growth and m a t u r a t i o n of the femur, t i b i a , s k u l l and the s i x t h c a u d a l v e r t e b r a were s i m i l a r -l y r e t a r d e d (Ray e t a l . , 1950). C o n v e r s e l y , t h y r o i d hormone exces s enhances the r a t e of l i n e a r growth and bone m a t u r a t i o n . Hamburgh (1968) demonstrated t h a t h y p e r t h y r o i d pups d e v e l o p a d u l t body p r o p o r t i o n s and f a c i a l c o n f i g u r a t i o n w i t h advanced s k e l e t a l m a t u r a t i o n . Growth i s u l t i m a t e l y s t u n t e d because of 1 7 premature e p i p h y s e a l c l o s u r e a s s o c i a t e d w i t h a c c e l e r a t e d s k e l e t a l m a t u r a t i o n i n h y p e r t h y r o i d i s m . 18 C H A P T E R I I I E X P E R I M E N T A L D E S I G N A N D M E T H O D O L O G Y A . T r e a t m e n t o f A n i m a l s T h i r t y v i r g i n f e m a l e S p r a g u e - D a w l e y r a t s (200-250 g) were p u r c h a s e d f r o m B i o b r e e d i n g L a b o r a t o r i e s o f C a n a d a , L t d . ( O t t a w a , C a n a d a ) . They were h o u s e d i n d i v i d u a l l y 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 a t 21 + 1 d e g r e e s C e l s i u s w i t h a 12 h o u r o n - 12 h o u r o f f - l i g h t c y c l e . A l l a n i m a l s were a l l o w e d ad  1 i b i t u m a c c e s s t o s t a n d a r d P u r i n a R a t Chow and t o d e i o n i z e d w a t e r i n p o l y e t h y l e n e b o t t l e s . No a t t e m p t was made t o c o n t r o l c o n t a m i n a t i o n o f a n i m a l s a nd f o o d s by e n v i r o n m e n t a l s i l i c o n . A f t e r a one week a d j u s t m e n t p e r i o d t h e y were b r e d o v e r n i g h t t o m a l e s o f t h e same s t r a i n . The f i n d i n g o f a v a g i n a l p l u g o r t h e a p p e a r a n c e o f s p e r m i n t h e v a g i n a l w a s h i n g on t h e f o l l o w i n g m o r n i n g e s t a b l i s h e d day 1 o f p r e g n a n c y . P r e g n a n t r a t s were t h e n a s s i g n e d t o one o f t h r e e g r o u p s : e u t h y r o i d c o n t r o l , h y p o t h y r o i d o r h y p e r t h y r o i d ( F i g u r e 1 ) . E a c h dam was w e i g h e d on d a y s 1, 7, 14, 21 a n d 22 o f g e s t a t i o n . Two-day f o o d c o n s u m p t i o n r e c o r d s were m a i n t a i n e d f r o m d a y 1 t o day 20 o f g e s t a t i o n f o r f i v e o r s i x a n i m a l s e a c h i n t h e c o n t r o l a n d h y p o t h y r o i d g r o u p s . F o o d i n t a k e o f h y p e r -t h y r o i d dams were n o t e s t i m a t e d a s t h e y were s i m i l a r t o c o n t r o l r a t s s i n c e n e i t h e r g r o u p r e c e i v e d t r e a t m e n t d u r i n g p r e g n a n c y . E a c h dam was t r a n s f e r r e d t o a p l a s t i c c a g e w i t h S a n - I - C e l b e d d i n g s on day 21 o f g e s t a t i o n . 19 30 PREGNANT DAMS 1 P R E N A T A L P E R I O D r 6 CONTROL DAMS 12 H Y P O T H Y R O I D DAMS 12 H Y P E R T H Y R O I D DAMS NO T R E A T M E N T 0.05% P R O P Y L T H I O U -R A C I L FROM DAY 12 OF PREGNANCY IN D R I N K I N G WATER NO T R E A T M E N T r i POSTNATA L PERIOD 48 CONTROL PUPS 96 HYPOTHYROID PUPS 96 HYPERTHYROID PUPS NO TREATMENT 0.05 PROPYLTHIOURACIL IN DRINKING WATER 0.15 - 0.30 ug THYROXINE PER GM BODY WEIGHT FROM DAY 2 Figure 1. Experimental Design 20 A l l dams bore l i t t e r s i n e x c e s s of e i g h t pups. On the day of b i r t h , each l i t t e r was c u l l e d t o f o u r males and f o u r f e m a l e s . Pups of each l i t t e r were then t a t t o o e d f o r permanent i d e n t i f i c a t i o n (Avery and Spyker, 1977). The dorsum of the f o o t j u s t p r o x i m a l t o the t o e s was i n j e c t e d i n t r a d e r m a l l y wi-th I n d i a i n k . No c a n n i b a l i z a t i o n by the mothers was o b s e r v e d from f o o t t a t t o o e s of the pups. Each dam was weighed on days 1, 7, 14, 21 and 28 of l a c t a t i o n or u n t i l the o f f s p r i n g s of these dams were s a c r i f i c e d . 1. Control Euthyroid Group The c o n t r o l group was composed of s i x l i t t e r s (48 pups). S i x pups (2 l i t t e r m a t e s from each of 3 l i t t e r s ) were s a c r i f i c e d on p o s t n a t a l days 3, 6, 9, 12, 15, 20, 25 and 30. Pups c u l l e d i n i t i a l l y from these l i t t e r s p r o v i d e d b l o o d samples f o r day one d e t e r m i n a t i o n s of s i l i c o n and t h y r o i d hormones. The time of s a c r i f i c e was d i f f e r e n t from the sa m p l i n g method a p p l i e d t o t r e a t e d pups i n orde r t o c h a r a c t e r i z e the e a r l y p o s t n a t a l changes i n plasma s i l i c o n l e v e l s of normal e u t h y r o i d pups. Blo o d was c o l l e c t e d and s t o r e d a t -20 degrees C e l s i u s f o r l a t e r d e t e r m i n a t i o n s of s i l i c o n and t h y r o i d hormones. The e f f e c t of p r o g r e s s i v e l y s m a l l e r l i t t e r s i z e i n the c o n t r o l group was a v o i d e d by pr o m p t l y r e p l a c i n g the pups removed f o r s a c r i f i c e . Pups from replacement l i t t e r s were matched w i t h c o n t r o l pups f o r age and sex. Both groups were run c o n c u r r e n t l y and were o f f e r e d the same food and d e i o n i z e d water. However, replacement 21 pups were not s a c r i f i c e d or used f o r any of the t e s t s des-c r i b e d . C o n t r o l pups were i n j e c t e d w i t h the s a l i n e v e h i c l e a l o n e on a d a i l y b a s i s s i m i l a r t o h y p e r t h y r o i d pups. 2. Hypothyroid Group The h y p o t h y r o i d group was composed of t w e l v e l i t t e r s (96 p u p s ) . Pups were made h y p o t h y r o i d by the a d m i n i s t r a t i o n t o the mother of 0.05% (w/v) 6 - p r o p y l - 2 - t h i o u r a c i l (Sigma Chemical Company, Mo., U.S.A.) i n d e i o n i z e d water as the s o l e d r i n k i n g f l u i d ( B u r s t e i n e t a l . , 1979). T h i s was begun on day 12 of g e s t a t i o n t o induce n e o n a t a l h y p o t h y r o i d i s m and c o n t i n u e d u n t i l t he l i t t e r s of pups were s a c r i f i c e d . Three e n t i r e l i t t e r s (24 pups) were s a c r i f i c e d on each of p o s t n a t a l days 7, 14, 21 and 28 f o r s i l i c o n and t h y r o i d hormones d e t e r m i n a t i o n s . 3. Hyperthyroid Group The h y p e r t h y r o i d group was composed of t w e l v e l i t t e r s (96 p u p s ) . The pups r e c e i v e d once d a i l y subcutaneous i n j e c t i o n s of L - t h y r o x i n e (Sigma Chemical Company, Mo. U.S.A., compliments of Dr. J.H. M c N e i l , F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s ) i n s a l i n e . N e o n a t a l h y p e r t h y r o i d i s m of the o f f s p r i n g s was induced by s t a r t i n g i n j e c t i o n s on p o s t n a t a l day 2 and c o n t i n u i n g u n t i l the day of s a c r i f i c e . The pH of the p r e p a r a t i o n was a d j u s t e d t o 7.4 w i t h 0.01N NaOH t o improve i t s s t a b i l i t y and i n c r e a s e i t s d u r a t i o n of hormonal a c t i o n from one t o t h r e e days (Dr_. J.H. M c N e i l , p e r s o n a l c o m m u n i c a t i o n ) . The dose a d m i n i s t e r e d was 22 reduced from 0.4 ug T4/g of body weight ( D u s s a u l t , Coulombe and Walker, 1982) t o 0.3 ug T4/g of body weight due t o the l o n g e r d u r a t i o n of treatment i n the p r e s e n t s t u d y . The m a j o r i t y of pups t o l e r a t e d the p r o c e d u r e except f o r e i g h t pups from one e n t i r e l i t t e r which were found dead on day 10 of t r e a t m e n t . The i n j e c t e d dose f o r the r e m a i n i n g two l i t t e r s i n t h i s group (day 28 of s a c r i f i c e ) was f u r t h e r reduced t o 0.15 ug/g body w e i g h t . Three e n t i r e l i t t e r s (24 pups) were s a c r i f i c e d on p o s t n a t a l days 7, 14, 21 and 28 f o r s i l i c o n and t h y r o i d hormones d e t e r m i -n a t i o n s as i n the h y p o t h y r o i d group. Pups from a l l t h r e e groups were weighed d a i l y and s i x pups (two pups from each of 3 l i t t e r s ) were x - r a y e d weekly on p o s t -n a t a l days 7, 14, 21 and 28 f o r s e r i a l assessment of s k e l e t a l m a t u r i t y . B. Col l e c t i o n of Blood Every p r e c a u t i o n was taken t o m i n i m i z e c o n t a m i n a t i o n of the b l o o d samples by e n v i r o n m e n t a l s i l i c o n ( C a n t l e , 1982a). A l l tubes used f o r b l o o d c o l l e c t i o n were p o l y e t h y l e n e or p o l y -s t y r e n e i n n a t u r e . They were soaked o v e r n i g h t i n 20% reagent grade n i t r i c a c i d , r i n s e d ten t i m e s w i t h d e i o n i z e d water and d r i e d i n a c o v e r e d p l a s t i c c o n t a i n e r a t 65 degrees C e l s i u s . They were c o v e r e d w i t h p l a s t i c s h e e t s f o l l o w i n g a c i d - w a s h i n g and d r y i n g , and d u r i n g the e n t i r e b l o o d c o l l e c t i o n p r o c e d u r e . The i n t e r i o r of the m i c r o - h e m a t o c r i t c e n t r i f u g e was wiped c l e a n of d u s t . Benchtop s u r f a c e s were c o v e r e d w i t h a c l e a n p l a s t i c 23 sheet. Perturbations of a i r flow within the room where work was in progress was minimized. The investigator worked alone behind closed doors. Pups were s a c r i f i c e d between 1300 and 1500 hours, following anaesthesia by ether. Younger pups (from b i r t h u n t i l 12 days of age) were k i l l e d by decapitation. Truncal blood was c o l l e c t e d into heparinized, glass c a p i l l a r y hematocrit tubes. The blood was centrifuged for 5 minutes and the plasma transferred immediately to polyethylene micro tubes for storage. The maximum duration of contact with glass for any sample was 32 minutes.. Blood from older pups was taken by cardiac puncture. The chest cavity was opened and heparinized disposable p l a s t i c syringes were used to withdraw blood as completely as possible. The animals were then k i l l e d by cutting open the heart. Blood samples were transferred to. p l a s t i c tubes and immediately centrifuged in a refrigerated centrifuge at 3000 rpm for 10 minutes. The plasma was aspirated into polystyrene microcentrifuge tubes. A l l plasma samples were capped and stored at -20 degrees Celsius for l a t e r determination of s i l i c o n , thyroxine and triiodothyronine. C. Determination of Plasma S i l i c o n Plasma s i l i c o n was analyzed by flameless atomic absorption spectrometry. The sample was heated e l e c t r i c a l l y through a programmed sequence of drying, charring and atomization in a graphite tube. S i l i c o n atoms thus generated absorbed l i g h t 24 from a h o l l o w - c a t h o d e s i l i c o n lamp. The r e s u l t a n t absorbance s i g n a l had a peak h e i g h t which was p r o p o r t i o n a l t o the q u a n t i t y of s i l i c o n p r e s e n t i n the sample a l i q u o t ( C a n t l e , 1982b). S i l i c o n a n a l y s i s was performed on a P e r k i n - E l m e r Model 2380 Spectrophotometer i n c o n j u n c t i o n w i t h HGA-400 G r a p h i t e Furnace. The sp e c t r o p h o t o m e t e r i s equipped w i t h a d e u t e r i u m ar c background c o r r e c t o r which a u t o m a t i c a l l y c o r r e c t e d f o r n o n s p e c i f i c background a b s o r p t i o n . A s i n g l e element, s i l i c o n h o l l o w - c a t h o d e lamp s e r v e d as the l i g h t s o u r c e . I t was s e t a t the most s e n s i t i v e l i n e a t 251.6 nm and o p e r a t e d a t 40 mA w i t h s p e c t r a l s l i t w i d t h of 0.2 nm. P r e - p u r i f i e d argon gas (Medigas P a c i f i c L t d . , Vancouver, Canada) purged the e x t e r i o r of the g r a p h i t e tube c o n t i n u o u s l y t o p r e v e n t i t s o x i d a t i o n a t h i g h t e m p e r a t u r e s . I t f l o w e d a t a r a t e of about 900 ml/min when the i n l e t p r e s s u r e of the compressed gas c y l i n d e r was s e t a t 41 p s i g . The tube i n t e r i o r was purged s e p a r a t e l y t o e l i m i n a t e sample m a t r i x m o l e c u l a r forms which v o l a t i l i z e d d u r i n g the p r e l i m i n a r y c h a r r i n g stage of the f u r n a c e h e a t i n g program. The i n t e r n a l gas f l o w was reduced t o 50 ml/min d u r i n g sample a t o m i z a t i o n t o improve f u r n a c e s e n s i t i v i t y f o r s i l i c o n . T h i s ensured t h a t the maximum p o p u l a t i o n of v o l a t i l i z e d s i l i c o n atoms remained i n the l i g h t p a t h t o g i v e g r e a t e r a bsorbances. P y r o l y t i c a l l y c o a t e d g r a p h i t e tubes were s e l e c t e d over s t a n d a r d tubes f o r t h e i r l o n g e r u s e f u l l i f e t i m e and g r e a t e r s e n s i t i v i t y i n the d e t e r m i n a t i o n of r e f r a c t o r y elements such as s i l i c o n . The i n e r t argon gas met the recommended minimimm q u a l i t y 25 ( O p e r a t o r ' s Manual, Bodenseewerk, P e r k i n - E l m e r & Co., U b e r l i n g e n ) f o r degree of p u r i t y (99.996%), oxygen c o n t e n t (5 ppm) and m o i s t u r e c o n t e n t (4 ppm) which were 99.998%, 3 ppm and 4 ppm r e s p e c t i v e l y . No v a l u e was a v a i l a b l e f o r the n i t r o g e n c o n t e n t of argon gas. For a l l a n a l y s e s , the plasma sample was d i l u t e d one t o t e n . Due t o the c o m p l e x i t y of the plasma m a t r i x , the method of s t a n d a r d a d d i t i o n s was used (Lo and C h r i s t i a n , 1978). A l i q u o t s of the sample were d i l u t e d w i t h d e i o n i z e d water, 0.5 or 1.0 ppm aqueous s i l i c o n s t a n d a r d s o l u t i o n s (Sodium s i l i c a t e , F i s h e r S c i e n t i f i c Company, N.Y., U.S.A.). Each s t a n d a r d a d d i t i o n was d e t e r m i n e d i n t r i p l i c a t e f o l l o w e d by a b l a n k t o ensure t h a t the b a s e l i n e was r e c o v e r e d . A m i c r o p i p e t t e (Socorex ISBA SA, S w i t z e r l a n d ) w i t h a double a c t i o n p l u n g e r system was used t o d i s p e n s e 20 u l volumes of d i l u t e d sample i n t o the g r a p h i t e f u r n a c e . The sample was a n a l y z e d by the f o l l o w i n g p r e - d e t e r m i n e d a n a l y t i c a l program (Table 1 ) . Temperature and time f o r each s t e p i n the d r y i n g , c h a r r i n g and a t o m i z a t i o n of plasma samples were o p t i m i z e d to g i v e the maximum absorbance. S i l i c o n c o n c e n t r a t i o n i n the sample was then c a l c u l a t e d by l i n e a r r e g r e s s i o n of t h r e e absorbance v a l u e s of added s t a n d a r d s . D. Assessment of S k e l e t a l M a t u r i t y S k e l e t a l growth and development i n r a t s was e s t i m a t e d u s i n g the p r o c e d u r e s of Hughes and Tanner (1970) based on whole 26 T A B L E I O p e r a t i n g P a r a m e t e r s F o r t h e D e t e r m i n a t i o n o f S i l i c o n i n P l a s m a o f R a t s A p p a r a t u s : P e r k i n - E l m e r Model 2380 HGA 400 S o u r c e : P e r k i n - E l m e r I n t e n s i t r o n s i l i c o n h o l l o w - c a t h o d e lamp C u r r e n t : 4 0 mA W a v e l e n g t h : 2 5 1 . 6 nm S l i t W i d t h : 0 . 2 nm M o d e:Peak h e i g h t Atomic a b s o r p t i o n Deuterium a r c background c o r r e c t i o n P u r g e g a s : A r g o n a t 40 p s i g S a m p l e a l i q u o t : 2 0 u l S T E P S 1 2 3 4 5 6 7 T e m p e r a t u r e s ( s ) 100 200 R a m p T i m e ( s ) 20 10 H o l d T i m e ( s ) 50 20 R e a d ( s ) M i n i F l o w ( m l / m i n ) 500 750 1000 1500 2700 10 5 5 5 0 10 10 10 20 11 1 2 50 27 body r a d i o g r a p h y . The a n i m a l s were s e c u r e d by a d h e s i v e tapes on Kodak No-Screen M e d i c a l X-ray f i l m f o l l o w i n g a n a e s t h e s i a by e t h e r . P r o p e r p o s i t i o n i n g was e s s e n t i a l t o a l l o w s t a n d a r d i z e d p r o j e c t i o n of the bones from b o t h d o r s i - v e n t r a l and l a t e r a l v i e w s . A n i m a l s were x-rayed w i t h a P i c k e r P o r t a b l e X-ray machine at a d i s t a n c e of 69.5 cm u s i n g 8 seconds exposure at 40 kvp and 15 mA. The x-ray f i l m s were manually p r o c e s s e d a c c o r d i n g t o m a n u f a c t u r e r ' s recommended ti m e s f o r d e v e l o p i n g and f i x i n g ( P i c k e r C h e m i c a l s , I n c . , C h e r r y H i l l , N.Y., U.S.A.). S k e l e t a l m a t u r i t y was a s s e s s e d from r a d i o g r a p h s based on s e q u e n t i a l changes of m a t u r i t y i n d i c a t o r s . Each bone undergoes an o r d e r l y sequence of d e v e l o p m e n t a l changes from i t s i n c e p t i o n as an o s s i f i c a t i o n c e n t r e , p r o g r e s s i n g through s t a g e s as e p i p h y s i s and i t s subsequent f u s i o n t o d i a p h y s i s . O s s i f i c a t i o n c e n t r e s of bones were a s s i g n e d n u m e r i c a l s c o r e s by comparison t o d e s c r i p t i v e c r i t e r i a and accompanying s t a n d a r d i l l u s t r a t i o n s ( F i g u r e 2 ). Three r e g i o n a l bone m a t u r i t y s c o r e s were d e r i v e d from the r a t i n g s of 11 bones i n the f o r e l i m b , 10 i n the h i n d l i m b and 6 i n the c a u d a l v e r t e b r a e . The sum of t h e s e s c o r e s y i e l d e d the t o t a l bone m a t u r i t y s c o r e . R a t i n g of bone development n e c e s s i t a t e d the a r b i t r a r y d i v i s i o n of an e s s e n t i a l l y c o n t i n u o u s p r o c e s s i n t o d i s c r e t e s t a g e s . To improve the r e l i a b i l i t y of bone s c o r e s , a l l r a t i n g s were performed by the i n v e s t i g a t o r and r e p e a t e d t h r i c e . 28 FIGURE 2 Bone Scoring in the Rat. Bones a r e r a t e d i n the f o l l o w i n g o r d e r : I. Forelimb Bone Maturity Score 1. R i g h t humerus 2. L e f t humerus 3. L e f t r a d i u s 4. L e f t u l n a 5. L e f t m e t a c a r p a l I I 6. L e f t m e t a c a r p a l I I I 7. L e f t m e t a c a r p a l IV 8. L e f t p r o x i m a l phalange I I 9. L e f t p r o x i m a l phalange I I I 10. L e f t p r o x i m a l phalange IV II. Hind-limb Bone Maturity Score 1 1 . R i g h t femur 12. L e f t femur 13. R i g h t t i b i a 14. L e f t t i b i a 15. L e f t calcaneum 16. R i g h t m e t a t a r s a l I I 17. R i g h t m e t a t a r s a l I I I 18. R i g h t m e t a t a r s a l IV 19. R i g h t p r o x i m a l phalange I I 20. R i g h t p r o x i m a l phalange I I I 21. R i g h t p r o x i m a l phalange IV III. T a i l Bone Maturity Score 22. Caudal v e r t e b r a e 8 + 9 23. Caudal v e r t e b r a e 1 0 + 1 1 24. Caudal v e r t e b r a e 1 2 + 1 3 25. Caudal v e r t e b r a e 1 4 + 1 5 26. Caudal v e r t e b r a e 16 + 17 27. Caudal v e r t e b r a e 18 29 E. Assay of Plasma Thyroxine and Triiodothyronine T o t a l plasma t h y r o x i n e (T4) and t r i i o d o t h y r o n i n e (T3) were determined by radioimmunoassay (Amerlex T-3 and T-4 RIA K i t s , Amersham C o r p o r a t i o n , O n t a r i o , Canada). The t e c h n i q u e i s based on the p r i n c i p l e of c o m p e t i t i v e p r o t e i n - b i n d i n g assay ( N e l s o n , 1979). Serum t h y r o x i n e and t r i i o d o t h y r o n i n e compete w i t h t h e i r r e s p e c t i v e r a d i o a c t i v e c o u n t e r p a r t s f o r a l i m i t e d number of b i n d i n g s i t e s on s p e c i f i c a n t i b o d i e s . They d i s p l a c e the r a d i o -a c t i v e a n t i g e n s t o form n o n r a d i o a c t i v e a n t i g e n - a n t i b o d y complexes. By measuring the p r o p o r t i o n of r a d i o a c t i v e bound a n t i g e n s i n s t a n d a r d s e r a c o n t a i n i n g known amounts of > the hormones, the c o n c e n t r a t i o n s of t h y r o x i n e and t r i i o d o t h y r o n i n e i n the plasma samples were d e t e r m i n e d . Radioimmunoassays of the plasma samples from s e l e c t e d pups i n a l l t r e a t m e n t groups were run i n the same assay t o a v o i d i n t e r a s s a y v a r i a t i o n . I n t r a - a s s a y c o e f f i c i e n t of v a r i a t i o n was 3.6% f o r t h y r o x i n e and 6.6% f o r t r i i o d o t h y r o n i n e . The c r o s s -r e a c t i v i t y of the T3 a n t i s e r a was l e s s than 0.3% f o r L - t h y r o x i n e and t h a t of the T4 a n t i s e r a was l e s s than 7.5% f o r L - t r i i o d o t h y r o n i n e . The s e n s i t i v i t y of the RIA K i t s was 0.3 ug T4/100 ml and 0.1 ng T3/ml. The assay r e q u i r e d 25 u l a l i q u o t s of u n d i l u t e d plasma samples and these were de t e r m i n e d i n d u p l i c a t e . F. S t a t i s t i c a l Analysis of Results Measurements of m a t e r n a l food i n t a k e and body weight 30 d u r i n g pregnancy among the c o n t r o l and e x p e r i m e n t a l groups were examined s t a t i s t i c a l l y by r e p e a t e d measures model a n a l y s i s of v a r i a n c e f o r the f a c t o r s of time u s i n g the BMDP S t a t i s t i c a l S o f t w a r e . D i f f e r e n c e s i n m a t e r n a l body weight d u r i n g l a c t a t i o n were a n a l y z e d by one way a n a l y s i s of v a r i a n c e (SPSS-X). In the case of s i g n i f i c a n t d i f f e r e n c e s , S c h e f f e ' s m u l t i p l e comparison t e s t was done t o i d e n t i f y the s o u r c e s of d i f f e r e n c e s among t r e a t m e n t group means. Data on the pups' body weight d u r i n g growth and d e v e l o p -ment were a n a l y z e d f o r the e f f e c t of t r e a t m e n t by one way a n a l y s i s of v a r i a n c e (SPSS-X). Test of s i g n i f i c a n c e f o r d i f f e -r e n c e s i n mean body w e i g h t s of the l o n g e s t s u r v i v i n g a n i m a l s from each t r e a t m e n t group was performed by means of a n a l y s i s of v a r i a n c e w i t h r e p e a t e d measures f o r the f a c t o r of time (SPSS-X). V a l u e s f o r t o t a l bone s c o r e s were examined by r e p e a t e d measures a n a l y s i s of v a r i a n c e f o r the f a c t o r of time (SPSS-X). Trend a n a l y s i s of plasma s i l i c o n d a ta was accom-p l i s h e d by a n a l y s i s of v a r i a n c e u s i n g o r t h o g o n a l p o l y n o m i a l breakdown of the sum of squares f o r time and group i n t e r a c t i o n . D i f f e r e n c e s i n plasma s i l i c o n l e v e l s among t r e a t m e n t groups were a n a l y z e d by one way a n a l y s i s of v a r i a n c e (SPSS-X). In the case of s i g n i f i c a n t d i f f e r e n c e s , p o s t hoc t e s t u s i n g S c h e f f e ' s p r o c e d u r e t o compare a l l p o s s i b l e p a i r s of means was performed. 31 CHAPTER IV RESULTS A. Food Intake of Dams Table I I p r e s e n t s the food i n t a k e of c o n t r o l and hypo-t h y r o i d dams f e d ad l i b i t u m from day 1 t o day 20 of g e s t a t i o n . Rats i n the h y p o t h y r o i d group r e c e i v e d 0.05% (w/v) 6 - p r o p y l - 2 -t h i o u r a c i l i n the d r i n k i n g f l u i d s t a r t i n g on day 12 of g e s t a -t i o n . Food consumption of r a t s i n the h y p e r t h y r o i d group was not measured. Presumably, the food i n t a k e of c o n t r o l dams would r e f l e c t t h o s e of h y p e r t h y r o i d r a t s s i n c e b o t h groups were s i m i l a r l y t r e a t e d throughout pregnancy. A l t h o u g h food i n t a k e i n c r e a s e d s i g n i f i c a n t l y over the c o u r s e of pregnancy (p<0.05), 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 between c o n t r o l and hypo-t h y r o i d dams i n the t o t a l q u a n t i t y of food consumed. These d i f f e r e n c e s remained i n s i g n i f i c a n t even when the i n i t i a l food i n t a k e was h e l d as a c o v a r i a t e t o d i s c o u n t f o r p o s s i b l e e f f e c t s of d i f f e r e n c e s i n body s i z e of the a n i m a l s on the o v e r a l l food i n t a k e . S i m i l a r l y , no s i g n i f i c a n t d i f f e r e n c e s between the two groups c o u l d be d e t e c t e d when o n l y the food i n t a k e c o r r e s p o n -d i n g t o the p e r i o d of p r o p y l t h i o u r a c i l a d m i n i s t r a t i o n (days 12-20) was examined or when the i n i t i a l food i n t a k e was a l s o c o n s i d e r e d as c o v a r i a t e . B. Body Weight of Dams 1. Pregnancy 32 T A B L E II Food Consumption of C o n t r o l and H y p o t h y r o i d Rats D u r i n g Pregnancy. Mean + s t a n d a r d d e v i a t i o n C o n t r o l H y p o t h y r o i d * Number of R a t s : (6) (5) Days of G e s t a t i o n : grams/2 days grams/2 days 1 - 2 44.0 + 8.2 42.8 + 3.8 3 - 4 50.7 + 6.0 49.9 + 3.6 5 - 6 50.6 + 3.8 48.8 + 5.8 7 - 8 54.7 + 4.5 52.7 + 4.4 9 - 1 0 56.8 + 4.4 51.2 + 5.1 1 1 - 1 2 57.8 + 4.4 55.2 + 5.7 13 - 14 55.9 + 7.2 52.5 + 7. 1 15 - 16 56.9 + 8.6 54.5 + 5.7 17 - 18 62.9 + 5.7 56.5 + 5.5 19 - 20 61.7 + 10.4 54.0 + 5.5 * Not s i g n i f i c a n t l y (p<0.05) d i f f e r e n t from c o n t r o l 33 F i g u r e 3 i l l u s t r a t e s the weekly changes i n body weight of dams from the c o n t r o l , hypo- and h y p e r t h y r o i d groups d u r i n g pregnancy. Dams i n the h y p e r t h y r o i d group were s i m i l a r l y t r e a t e d as c o n t r o l r a t s throughout pregnancy and l a c t a t i o n . However, the i n i t i a l body weight of the two groups of a n i m a l s was s i g n i f i c a n t l y (p<0.05) d i f f e r e n t on days 1 and 7 of g e s t a -t i o n . The d i f f e r e n c e i n body weight was no l o n g e r s t a t i s t i c a l -l y s i g n i f i c a n t by day 14 of g e s t a t i o n . T o t a l and i n c r e m e n t a l weight g a i n s as w e l l as the body weight a t t a i n e d a t the end of g e s t a t i o n were s i m i l a r f o r both c o n t r o l and h y p e r t h y r o i d groups. No s i g n i f i c a n t e f f e c t s of p r o p y l t h i o u r a c i l a d m i n i s t r a -t i o n on the body weight of h y p o t h y r o i d dams were observed a t any time d u r i n g pregnancy. T o t a l and i n c r e m e n t a l weight g a i n s as w e l l as the f i n a l body weight a c h i e v e d by h y p o t h y r o i d dams a t the end of g e s t a t i o n were not s t a t i s t i c a l l y d i f f e r e n t from those of c o n t r o l a n i m a l s . 2. Lactation Data on weekly body weight of dams from the c o n t r o l , hypo-and h y p e r t h y r o i d groups d u r i n g l a c t a t i o n a r e d i s p l a y e d i n F i g u r e 4. P r o g r e s s i v e l y d e c r e a s i n g sample s i z e s among the t h r e e groups r e f l e c t t he number of l i t t e r s s a c r i f i c e d and a c o r r e s p o n d i n g number of dams e x c l u d e d from body weight measure-ments a t each o b s e r v a t i o n p o i n t . As e x p e c t e d , the body weight of dams from c o n t r o l and h y p e r t h y r o i d groups were not s i g n i f i -c a n t l y d i f f e r e n t s i n c e n e i t h e r group r e c e i v e d any tr e a t m e n t 34 F IGURE 3 Weekly Body Weight of Dams During Pregnancy. Each p o i n t r e p r e s e n t s mean + s t a n d a r d d e v i a t i o n of 6 dams i n c o n t r o l and 12 dams each i n h y p e r t h y r o i d and h y p o t h y r o i d groups. * denotes s i g n i f i c a n t l y (p<0.05) h i g h e r body weight of h y p e r t h y r o i d dams than c o n t r o l dams on days 1 (F r a t i o = 9 . 1 4 ) and 7 (F r a t i o = 6 . 0 l ) o n l y u s i n g S c h e f f e ' s m u l t i p l e - r a n g e t e s t . 35 FIGURE 4 Weekly Body Weight of Dams During L a c t a t i o n (or u n t i l the l i t t e r s of young were s a c r i f i c e d ) . Each bar r e p r e s e n t s mean + s t a n d a r d d e v i a t i o n of a n i m a l s as l i s t e d below: d a y l day7 day!4 day21 day28 No. of c o n t r o l dams 6 6 3 3 3 No. of hypothyroid dams 12 12 9 6 3 No. of h y p e r t h y r o i d dams 12 12 9 6 3 * denotes s i g n i f i c a n t l y (p<0.05) lower body weight of h y p o t h y r o i d dams as compared w i t h c o n t r o l dams on day 14 o n l y (F ra t i o = 5 . 6 7 ) u s i n g S c h e f f e ' s m u l t i p l e - r a n g e t e s t . 36 400 -co E co Ui O LU > o o m 300 77, 200 -100 --C O N T R O L H Y P O T H Y R O I D H Y P E R T H Y R O I D 7 14 21 T I M E O F L A C T A T I O N ( d a y s ) 28 d u r i n g the study p e r i o d . H y p o t h y r o i d dams demonstrated s i g n i -f i c a n t l y reduced body weight (p<0.05) s p e c i f i c a l l y on day 14 of l a c t a t i o n o n l y , when compared w i t h c o n t r o l a n i m a l s . C. Body Weight of Pups F i g u r e 5 d e p i c t s the d a i l y body weight of r a t pups from p o s t n a t a l day 2 t o day 28. S i x pups each from the c o n t r o l , hypo- and h y p e r t h y r o i d groups were s e l e c t e d from the l o n g e s t s u r v i v i n g l i t t e r s . A c t u a l sample s i z e s were much l a r g e r than t a b u l a t e d , but the t h r e e groups v a r y i n numbers. E q u a l sample s i z e s of s e l e c t e d pups a l l o w e d s t a t i s t i c a l a n a l y s i s of the d a t a f o r an e f f e c t of t r e a t m e n t and time on body weight. The a n a l y s i s of v a r i a n c e assumed t h a t the s e l e c t e d pups i n the samples were r e p r e s e n t a t i v e of each of the t h r e e groups and t h a t the samples were of e q u a l s i z e s . Data a n a l y s i s showed t h a t t h e r e were s i g n i f i c a n t r e d u c t i o n s i n the body weight of hypo- and h y p e r t h y r o i d pups as compared w i t h c o n t r o l pups due to the independent and i n t e r a c t i v e e f f e c t s of tr e a t m e n t and time on body weight (p<0.05). In o r d e r t o a s s e s s the e f f e c -t i v e n e s s of t r e a t m e n t s employed i n the p r e s e n t s t u d y , the e n t i r e sample from each of the t h r e e groups was s u b j e c t t o a n a l y s i s of v a r i a n c e a t s p e c i f i c time p o i n t s . The r e s u l t s showed t h a t hypo- and h y p e r t h y r o i d pups e x h i b i t e d s i g n i f i c a n t l y (p<0.00l) r e t a r d e d body weight by days 2 and 10 r e s p e c t i v e l y as compared w i t h c o n t r o l pups. The body weight of c o n t r o l and h y p e r t h y r o i d pups on day 2 were 6.68 + 0.14 g and 6.60 + 0.06 g 37 FIGURE 5 D a i l y Body Weight of Rat Pups From Day 2 t o Day 28 P o s t n a t a l l y . Each p o i n t r e p r e s e n t s mean + s t a n d a r d d e v i a t i o n of 6 r a t s . * denotes s i g n i f i c a n t l y reduced (p<0.05) body weight of both hypo- a'nd h y p e r t h y r o i d pups as compared w i t h c o n t r o l pups due t o an e f f e c t of t r e a t m e n t (F r a t i o = 2 0 l 1 . 6 1 ) and time (F rat i o = 5 5 . 0 4 ) . 38 • -) 1 1 —I 1 I 1 1 1 1 I —H ) I 2 4 6 8 10 12 m 16 18 20 22 24 26 28 A G E (days) r e s p e c t i v e l y . These v a l u e s were s i g n i f i c a n t l y h i g h e r than 5.99 + 0.06 g of body weight o b s e r v e d i n h y p o t h y r o i d pups. D i f f e r e n c e s i n body weight between the c o n t r o l and h y p e r t h y r o i d groups, which were 21.02 + 0.42 and 18.77 + 0.23 grams r e s p e c -t i v e l y , reached s t a t i s t i c a l s i g n i f i c a n c e (p<0.00l) by day 10. The h i g h e r weight advantage of h y p e r t h y r o i d pups over the hypo-t h y r o i d pups, which was observed a t b i r t h , p e r s i s t e d throughout the p e r i o d of o b s e r v a t i o n i n the p r e s e n t s t u d y . D. Skeletal Maturity Scores T o t a l bone s c o r e s from l o n g i t u d i n a l assessment of s k e l e t a l m a t u r i t y i n s e l e c t e d r a t pups i n each of c o n t r o l , hypo- and h y p e r t h y r o i d groups a r e shown i n F i g u r e 6. No s i g n i f i c a n t e f f e c t of sex on bone s c o r e s c o u l d be e s t a b l i s h e d (p>0.05), t h e r e f o r e d a t a on male and female r a t s a re p r e s e n t e d t o g e t h e r . There were s i g n i f i c a n t main and i n t e r a c t i v e e f f e c t s of t r e a t -ments and time on bone s c o r e s among the t h r e e groups. C o n t r o l pups e x h i b i t e d s i g n i f i c a n t l y h i g h e r (p<0.05) bone s c o r e s than h y p o t h y r o i d r a t s by p o s t n a t a l day 14, whereas h y p e r t h y r o i d pups d i s p l a y e d s i g n i f i c a n t l y h i g h e r s c o r e s as compared w i t h c o n t r o l r a t s by day 7 (p<0.05). At 14 days of age, h y p e r t h y r o i d r a t s showed advanced bone age comparable t o t h a t of 21 days o l d c o n t r o l a n i m a l s . E. Plasma S i l i c o n Concentrations Plasma c o n c e n t r a t i o n s of s i l i c o n i n the c o n t r o l , hypo- and 39 FIGURE 6 Bone M a t u r i t y Scores At Weekly I n t e r v a l s During the P o s t n a t a l Development of Rat Pups. Each bar r e p r e s e n t s mean + s t a n d a r d d e v i a t i o n of 6 r a t s . * denotes s i g n i f i c a n t l y (p<0.05) h i g h e r bone s c o r e s of h y p e r t h y r o i d pups by p o s t n a t a l day 7 and s i g n i f i c a n t l y lower bone s c o r e s of h y p o t h y r o i d pups by p o s t n a t a l day 14 when compared w i t h c o n t r o l pups as a r e s u l t of treatment (F ratio=1019.948) and time (F r a t i o = 5 7 0 5 . 6 0 9 ) . 40 300 7 14 21 28 A G E ( d a y s ) h y p e r t h y r o i d r a t pups a t d i f f e r e n t ages a r e p r e s e n t e d i n F i g u r e 7. A n a l y s i s of t h e s e v a l u e s showed s i g n i f i c a n t changes (p<0.05) i n plasma s i l i c o n l e v e l s as a f u n c t i o n of age i n each of the t h r e e groups. In the c o n t r o l group, the plasma v a l u e s of s i l i c o n averaged 0.668 + 0.050 ppm on p o s t n a t a l day 6 and rose t o a maximum c o n c e n t r a t i o n of 0.970 + 0.221 ppm by day 12. Plasma s i l i c o n s u b s e q u e n t l y d e c l i n e d t o 0.247 + 0.125 ppm by the time of weaning on day 20, where i t remained a t 0.260 + 0.029 ppm on day 25. C o n c e n t r a t i o n s of plasma s i l i c o n i n both hypo- and h y p e r t h y r o i d groups f o l l o w e d much the same p a t t e r n . S i l i c o n was p r e s e n t a t low l e v e l s of 0.404 + 0.031 and 0.328 + 0.025 ppm i n the two groups r e s p e c t i v e l y on day 7. However, a l t h o u g h plasma s i l i c o n c o n c e n t r a t i o n i n c r e a s e d i n both groups by day 14, the responses were r e l a t i v e l y b l u n t e d . These l e v e l s c o r r e s p o n d e d t o 0.365 + 0.076 and 0.466 + 0.118 ppm i n hypo-and h y p e r t h y r o i d pups r e s p e c t i v e l y and were lower than the peak c o n c e n t r a t i o n of 0.970 + 0.221 ppm observed i n c o n t r o l pups. The method of s a m p l i n g a p p l i e d i n the p r e s e n t study d i d not per m i t s t a t i s t i c a l a n a l y s i s of plasma s i l i c o n v a l u e s because of d i f f e r e n c e s i n the time of s a c r i f i c e among the t h r e e groups. I t may be p o s s i b l e t o s t a t i s t i c a l l y compare the mean of two d i f f e r e n t plasma v a l u e s on days 6 and 9 i n the c o n t r o l group t o the plasma s i l i c o n l e v e l found on day 7 i n both the hypo- and h y p e r t h y r o i d groups. The r e s u l t s i n d i c a t e d t h a t s i l i c o n l e v e l s of c o n t r o l pups over the age i n t e r v a l of 6 and 9 days were s i g n i f i c a n t l y h i g h e r than l e v e l s found i n 7 day o l d hypo- and 41 h y p e r t h y r o i d pups. L a s t l y , the method of o r t h o g o n a l p o l y n o m i a l s was a p p l i e d t o d a t a from the c o n t r o l group. The f u n c t i o n a l r e l a t i o n s h i p between plasma s i l i c o n c o n c e n t r a t i o n and age of c o n t r o l a n i m a l s c o u l d be d e s c r i b e d by a power s e r i e s of the f o l l o w i n g : Y= 35.444 - 15.139X + 2.4495x2 - 0.18478x3 + 0.0065355x4 where x i s the age a t s a c r i f i c e . F. Plasma Thyroxine and Triiodothyronine Plasma c o n c e n t r a t i o n s of t h y r o x i n e and t r i i o d o t h y r o n i n e a t v a r i o u s ages i n s e l e c t e d r a t pups from the c o n t r o l , hypo- and h y p e r t h y r o i d groups are p r e s e n t e d i n T a b l e 3. S m a l l sample s i z e s p r e c l u d e d the s t a t i s t i c a l a n a l y s i s of d i f f e r e n c e s i n mean plasma v a l u e s a t t r i b u t e d t o t r e a t m e n t , age and sex. The i n t e n d e d use of these r e s u l t s was t o demonstrate e l e v a t e d l e v e l s of the t h y r o i d hormones i n t h y r o x i n e - i n j e c t e d pups w i t h advanced s k e l e t a l m a t u r i t y , and c o n v e r s e l y , lower l e v e l s of the hormones i n p r o p y l t h i o u r a c i 1 - e x p o s e d a n i m a l s w i t h d e l a y e d bone age. The v a l u e s o b t a i n e d were w i t h i n the e x p e c t e d ranges i n a l l c a s e s . 42 FIGURE 7 Plasma C o n c e n t r a t i o n of S i l i c o n i n Rat Pups At D i f f e r e n t Ages i n C o n t r o l , H y p e r t h y r o i d and H y p o t h y r o i d Groups. Each p o i n t r e p r e s e n t s mean + s t a n d a r d e r r o r of the mean of a number of r a t pups as i n d i c a t e d i n the b r a c k e t . Plasma l e v e l of s i l i c o n v a r i e s s i g n i f i c a n t l y (p<0.05) w i t h age i n each of c o n t r o l , hypo- and h y p e r t h y r o i d group. 43 4 3o-TABLE III Plasma Levels of T4 and T3 of Selected Rats At Different Ages Group No. Pups Age T4 ug/ml T3 ng/ml C o n t r o l 2 1 5 5. ,48 0. 58 2 20 5. ,22 0. 54 2 30 4 . 35 0. 85 H y p o t h y r o i d 2 7 3. .01 0. 32 2 1 4 2, . 1 5 0. 22 2 21 2, .07 0. 23 2 28 1 . .75 0. 20 H y p e r t h y r o i d 2 7 31 . 64 5. 75 2 1 4 44. .62 5. 70 2 21 34. .53 5. 07 2 28 36, .66 5. 1 9 44 CHAPTER V DISCUSSION The r e s u l t s of t h i s s tudy demonstrate t h a t plasma c o n c e n t r a t i o n s of s i l i c o n i n normal e u t h y r o i d r a t s v a r y w i t h age d u r i n g the e a r l y p o s t n a t a l p e r i o d . A l t e r a t i o n s i n t h y r o i d f u n c t i o n i n f l u e n c e the r a t e of s k e l e t a l development and the p a t t e r n of change of plasma s i l i c o n w i t h age. H y p e r t h y r o i d s t a t e s a c c e l e r a t e s k e l e t a l development, w h i l e h y p o t h y r o i d s t a t e s r e t a r d s k e l e t a l m a t u r a t i o n r e l a t i v e t o u n t r e a t e d c o n t r o l s . Both c o n d i t i o n s a r e accompanied by g e n e r a l l y d e p r e s s e d plasma s i l i c o n v a l u e s and by a s h i f t towards e a r l i e r and l a t e r ages a t which the maximum s i l i c o n l e v e l s o c c u r r e d . D i f f e r e n c e s i n the time of s a c r i f i c e used i n the p r e s e n t study do not p e r m i t a q u a n t i t a t i v e e v a l u a t i o n of the d a t a f o r a r e l a t i o n s h i p between plasma s i l i c o n c o n c e n t r a t i o n s , s k e l e t a l development and c a l c i f i c a t i o n , and age. The d i s c u s s i o n which f o l l o w s , w i l l a d d r ess t h r e e i s s u e s c e n t r a l t o the s t u d y . The f i r s t i s s u e r e l a t e s t o the c h o i c e of a n a l y t i c a l method f o r s i l i c o n d e t e r m i n a t i o n i n plasma samples i n terms of i t s s u i t a b i l i t y , and s e n s i t i v i t y and r e p r o d u c i b i l i t y of the measurements. The second i s s u e concerns the d e v e l o p m e n t a l p a t t e r n of s i l i c o n changes w i t h age. The l a s t i s s u e d e a l s w i t h the r o l e of t h y r o i d s t a t u s i n plasma l e v e l s of s i l i c o n d u r i n g the e a r l y p o s t n a t a l weeks which c o i n c i d e w i t h p e r i o d s of r a p i d bone development i n r a t s . In 45 t h i s r e s p e c t , e v i d e n c e s are p r e s e n t e d from assessments of somatic growth, s k e l e t a l development and plasma l e v e l s of t h y r o x i n e and t r i i o d o t h y r o n i n e t o v a l i d a t e hypo- and h y p e r t h y -r o i d i s m i n the t r e a t e d o f f s p r i n g s . The d i s c u s s i o n w i l l c l o s e w i t h some s u g g e s t i o n s f o r f u t u r e c o n s i d e r a t i o n . A. Method of Plasma S i l i c o n Determination S i l i c o n d e t e r m i n a t i o n i n t h i s s t u d y was performed by f u r n a c e atomic a b s o r p t i o n s p e c t r o m e t r y . The most w i d e l y used a n a l y t i c a l method f o r d e t e r m i n i n g s i l i c o n i n b i o l o g i c a l samples i s c o l o r i m e t r y ( K i n g , 1939). The method r e q u i r e s p r e l i m i n a r y dr y a s h i n g or wet d i g e s t i o n of the b i o l o g i c a l sample and subsequent c o n v e r s i o n t o reduced s i l i c o m o l y b d a t e . I t i s s u b j e c t t o i n t e r f e r e n c e by phosphate, i r o n and p r o t e i n ( K i n g , 1939; A u s t i n e t a l . , 1972; I h n a t , 1982). There i s concern t h a t p r e l i m i n a r y a s h i n g p r o c e d u r e may c o n t r i b u t e t o s i l i c o n l o s s i n the presence of f l u o r i d e or l o s s of s i l i c o n as s i l i c o n c a r b i d e (Schwarz, 1977). Reagent grade p o t a s s i u m permanganate and ammonium molybdate have been shown t o be p r i m a r y sources of s i l i c o n c o n t a m i n a t i o n i n reagent b l a n k s ( I h n a t , 1982). In the p r e s e n t s t u d y , f u r n a c e atomic a b s o r p t i o n s p e c t r o m e t r y was con-s i d e r e d the method of c h o i c e . The proce d u r e i s s u i t a b l e f o r use i n plasma a n a l y s i s of s i l i c o n i n n e o n a t a l r a t s as o n l y m i c r o -l i t e r volumes of the plasma f i l t r a t e a r e r e q u i r e d . M i n i m a l sample p r e p a r a t i o n i s needed, t h e r e b y e l i m i n a t i n g e x t r a n e o u s s o u r c e s of c o n t a m i n a t i o n from r e a g e n t s and r e p e a t e d h a n d l i n g . 46 The method a f f o r d s h i g h s e n s i t i v i t y w i t h d e t e c t i o n l i m i t s i n the 0.9 ng (Lo and C h r i s t i a n , 1977), and 0.2 ng r e g i o n s ( T a d d i a , 1986). Under the c o n d i t i o n of t h i s s t u d y , the method of f u r n a c e atomic a b s o r p t i o n was not s u f f i c i e n t l y s e n s i t i v e t o d i s c e r n d i f f e r e n c e s i n plasma c o n c e n t r a t i o n s of s i l i c o n below 0.2 ppm. As plasma a n a l y s e s a r e s u b j e c t t o m a t r i x i n t e r f e r e n c e s , the method of s t a n d a r d a d d i t i o n s was employed. Deuterium background c o r r e c t i o n was a p p l i e d d e s p i t e low background i n t e r f e r e n c e from smoke emanating from h e a t e d samples. L i n e a r i t y of response was obser v e d over the range of 0 t o 5 ppm of aqueous s i l i c o n s t a n d a r d s . R e l a t i v e s t a n d a r d d e v i a t i o n w i t h 10 u l of 2 ppm s i l i c o n s t a n d a r d s o l u t i o n was 2.6%. Plasma samples i n t h i s study were d i l u t e d t e n f o l d t o reduce the v i s c o -s i t y and a l l o w more complete d e l i v e r y of a l i q u o t s by manual m i c r o d i S p e n s e r . C o e f f i c i e n t of v a r i a t i o n f o r 20 u l (1:10) plasma samples was 3.2%. Recovery of s i l i c o n was performed i n the absence of c e r t i f i e d s t a n d a r d plasma sample f o r s i l i c o n . The r e c o v e r i e s a t 2 ppm s i l i c o n s t a n d a r d i n 20 u l a l i q u o t s of plasma were 86 + 1%. At lower c o n c e n t r a t i o n s , poor r e c o v e r y of added s t a n d a r d r e q u i r e d the use of l a r g e r a l i q u o t s . These underwent s e q u e n t i a l d r y i n g between i n j e c t i o n s u n t i l the d e s i r e d volume was d i s p e n s e d i n t o the sample p o r t and the program of h e a t i n g was a l l o w e d t o p r o g r e s s t o c o m p l e t i o n . The temperature program s e l e c t e d f o r s i l i c o n d e t e r m i n a t i o n i n the p r e s e n t study i s c o n s i s t e n t w i t h programs used by o t h e r 47 i n v e s t i g a t o r s (Lo and C h r i s t i a n , 1978; T a d d i a , 1986). T a d d i a (1986) r e p o r t e d g r e a t e r absorbances w i t h 10 u l a l i q u o t s of aqueous s o l u t i o n s from p y r o l y t i c g r a p h i t e tubes a t a s h i n g temperature of 1300 degrees C e l s i u s . H i gher a s h i n g temperature used i n the p r e s e n t study was n e c e s s a r y p o s s i b l y due t o the l a r g e r a l i q u o t s a n a l y z e d . In t h i s s t u d y , the use of maximum power h e a t i n g t o e l e v a t e t e m p e r a t u r e s from the c h a r r i n g t o the a t o m i z a t i o n s t e p s i s s u p p o r t e d by the f i n d i n g s of Taddia (1986) t h a t the maximum s e n s i t i v i t y i s a c h i e v e d when the maximum ground s t a t e atom d e n s i t y i s o b t a i n e d . The study a l s o demon-s t r a t e s h i g h e r s e n s i t i v i t y by a f a c t o r of 90 when the i n t e r n a l gas f l o w i s stopped c o m p l e t e l y d u r i n g a t o m i z a t i o n which reduced the d i f f u s i o n a l l o s s e s of s i l i c o n atoms. The a l t e r n a t e mode of reduced i n t e r n a l gas f l o w d u r i n g a t o m i z a t i o n was used i n the p r e s e n t s t u d y i n orde r t o i n c r e a s e the u s e f u l l i f e t i m e of the g r a p h i t e t u b e s . S e n s i t i v i t y and p r e c i s i o n of s i l i c o n d e t e r m i n a t i o n i n plasma samples by f u r n a c e atomic a b s o r p t i o n s p e c t r o m e t r y may be f u r t h e r improved by the use of an a u t o m a t i c s a m p l i n g system. P r e c i s i o n c r i t i c a l l y depends on the r e p r o d u c i b l e p o s i t i o n i n g of the sample i n the f u r n a c e and on complete d e l i v e r y of the v i s c o u s sample w i t h o u t sample suspending from around the sample i n t r o d u c t i o n p o r t (Lo and C h r i s t i a n , 1978). Automatic s a m p l i n g d e v i c e s a r e equipped w i t h p r e c i s i o n s a m p l i n g and f l u s h i n g pumps ( P e r k i n - E l m e r O p e r a t o r ' s Manual). V i s c o u s samples may be a n a l y z e d w i t h o u t p r i o r d i l u t i o n , which may improve the o v e r a l l 48 p r e c i s i o n and a c c u r a c y of the d e t e r m i n a t i o n s . F u r t h e r m o r e , the computer module of the sa m p l i n g system can be programmed t o d i s p e n s e and a n a l y z e p r e - s e l e c t e d volumes of sample a l i q u o t s and added s t a n d a r d s . T h i s w i l l f r e e the o p e r a t o r from r e l a t i v e -l y l o n g w a i t between sample i n t r o d u c t i o n s which was a p p r o x i -m a t e l y 3 minutes and 6 seconds i n t h i s s t u d y . When the method of s t a n d a r d a d d i t i o n s i s a p p l i e d t o plasma a n a l y s i s , the time of a n a l y s i s i s i n c r e a s e d s i g n i f i c a n t l y . In the p r e s e n t s t u d y , i t was not un u s u a l t o complete a n a l y s i s on 2 - 4 d i f f e r e n t plasma samples a t the end of the day, depending on the i n i t i a l s i l i c o n c o n c e n t r a t i o n of u n s p i k e d samples. B. Plasma S i l i c o n Concentrations in Control Pups I t i s r e c o g n i z e d t h a t s i l i c o n d e t e r m i n a t i o n i s s u b j e c t t o e n v i r o n m e n t a l c o n t a m i n a t i o n and t e c h n i c a l d i f f i c u l t i e s . E l a b o r a t e p r e c a u t i o n s were ta k e n at each s t e p from sample c o l l e c t i o n t o plasma s i l i c o n d e t e r m i n a t i o n i n o r d e r t o m i n i m i z e c o n t a m i n a t i o n s . Yet the p r e s e n t study found h i g h l y e l e v a t e d plasma c o n c e n t r a t i o n s of s i l i c o n i n n e o n a t a l r a t s . For reason of p o s s i b l e c o n t a m i n a t i o n , plasma s i l i c o n v a l u e s f o r newborn and 3 day o l d pups have been e x c l u d e d from c o n s i d e r a t i o n . I t i s p o s s i b l e t h a t the b l o o d samples were i n a d v e r t e n t l y c o n t a m i n a t e d d u r i n g c o l l e c t i o n i n the i n i t i a l s t a g e of the e x p e r i m e n t . I h n a t (1982) demonstrated t h a t p o l y p r o p y l e n e v e s s e l s and commonly used a n t i c o a g u l a n t s such as sodium h e p a r i n c o n t r i b u t e n e g l i g i b -l e e x t r a n e o u s s i l i c o n . As the c o n t a c t time of plasma sample 49 w i t h the s u r f a c e of the g l a s s v e s s e l i n c r e a s e d t o one day, e x t e n s i v e l e a c h i n g of s i l i c o n from b o r o s i l i c a t e of g l a s s con-t r i b u t e d up t o 39.8% of the t o t a l s i l i c o n . In the p r e s e n t s t u d y , the maximum p e r i o d of c o n t a c t w i t h the g l a s s h e m a t o c r i t tubes was i n s u f f i c i e n t • t o account f o r the e l e v a t e d plasma s i l i c o n l e v e l s i n neonates. P o s s i b l e s o u r c e s of c o n t a m i n a t i o n may o r i g i n a t e from c o n t a c t of b l o o d w i t h the s k i n on the c a r c a s s of the an i m a l f o l l o w i n g d e c a p i t a t i o n , c o n t a c t of the sample w i t h p l a s t i c e n e used t o s e a l the c a p i l l a r y tube or c o n t a c t of the c a p i l l a r y tubes w i t h the m e t a l of the c e n t r i -fuge. S i l i c o n c o n t a m i n a t i o n i n subsequent samples was m i n i -mized when the b l o o d was c o l l e c t e d by c a r d i a c p u n c t u r e from opened c h e s t c a v i t y and drawn d i r e c t l y i n t o h e p a r i n i z e d p l a s t i c s y r i n g e s . The p r e s e n t study demonstrates t h a t the plasma s i l i c o n c o n c e n t r a t i o n s of r a t s d u r i n g normal p o s t n a t a l development were a l t e r e d w i t h t i m e . S i l i c o n c o n c e n t r a t i o n rose from 0.668 + 0.050 ppm on day 6 t o a maximum of 0.970 + 0.221 ppm on day 12. S i l i c o n l e v e l s then d e c l i n e d t o 0.247 + 0.125 ppm a t about the time of weaning where i t remained a t low b a s e - l i n e , a d u l t l e v e l s . The p a t t e r n of s i l i c o n change w i t h age may be d e s c r i b e d by the f o l l o w i n g power s e r i e s , Y = 35.444 - I5.139x + 2.4495x2 - 0.18478x3 + 0.0065355x4 where x i s the age of s a c r i f i c e . However, the f u n c t i o n a l p a r a -meters r e l a t e d t o the change i n plasma s i l i c o n w i t h time c o u l d 50 not be r e l a t e d t o s p e c i f i c b i o l o g i c e v e n t s . N e v e r t h e l e s s , C a r l i s l e (1980b) has demonstrated t h a t the plasma s i l i c o n l e v e l c l o s e l y p a r a l l e l s s i l i c o n c o n c e n t r a t i o n i n the t i b i a l p e r i o s -teum of the c h i c k . I t i s p o s s i b l e t h a t plasma s i l i c o n concen-t r a t i o n may be c o r r e l a t e d w i t h bone m a t u r i t y s c o r e , which i s based on r a d i o g r a p h i c changes i n bone m i n e r a l i z a t i o n w i t h m a t u r i t y . Hughes and Tanner (1970) have d e s c r i b e d a d i s t i n c t peak i n the r a t e of increment i n bone s c o r e s of the b l a c k -hooded r a t s on p o s t n a t a l day 9 f o r females and on day 11 f o r male r a t s . In the p r e s e n t s t u d y , the h i g h e s t increment i n bone s c o r e s of the Sprague-Dawley r a t s was observed between days 7 and 14, w h i l e the peak c o n c e n t r a t i o n of s i l i c o n was observed on day 12. D i f f e r e n c e s i n the peak o c c u r r e n c e s of the maximal v e l o c i t y i n bone s c o r e s between t h e s e two s t u d i e s may be e x p l a i n e d by the method of s a m p l i n g , s t r a i n d i f f e r e n c e s and sex dimorphism of s k e l e t a l development. Perphaps sex d i f f e r e n c e s i n peak c o n c e n t r a t i o n s of s i l i c o n may a l s o account f o r the l a r g e s t a n d a r d e r r o r of the mean v a l u e observed on day 12. I f bone m a t u r a t i o n i s r e l a t e d t o plasma l e v e l s of s i l i c o n , then bone s c o r e s may be c o r r e l a t e d w i t h plasma s i l i c o n c o n c e n t r a -t i o n s d u r i n g t h i s p e r i o d of development. However, the data i n t h i s study do not p e r m i t s t a t i s t i c a l a n a l y s i s of the c o r r e l a -t i o n between bone s c o r e s and plasma s i l i c o n l e v e l s i n bone m a t u r a t i o n due t o d i f f e r e n c e s i n s a m p l i n g between the. c o n t r o l , and hypo- and h y p e r t h y r o i d groups. Up t o the p r e s e n t time t h e r e have been no comparable 51 s t u d i e s p u b l i s h e d , i n which plasma s i l i c o n has been d e t e r m i n e d i n r a t s a t v a r i o u s p e r i o d s d u r i n g the e a r l y p o s t n a t a l l i f e . S e v e r a l i n v e s t i g a t o r s have r e p o r t e d serum or plasma s i l i c o n v a l u e s i n a d u l t r a t s , but the r e p o r t e d f i g u r e s show a wide range. L e V i e r (1975) r e p o r t e d serum l e v e l s of 0.2 + 0.3 ppm, w h i l e Charnot and Pe r e s (1971a) found plasma s i l i c o n c o n c e n t r a -t i o n s t o be 6.19 + 1.41 and 6.59 + 1.09 mg/1 i n males and females r e s p e c t i v e l y . Aumonier and Q u i l i c h i n i (1962) r e p o r t e d serum s i l i c o n t o be i n the range of 0.61 - 1.4 ppm. C a r l i s l e (1974b) r e p o r t e d a p p r o x i m a t e l y 1 ppm f o r b l o o d s i l i c o n , and Schwarz (1974) found whole b l o o d s i l i c o n t o be 2 ppm. Plasma c o n c e n t r a t i o n s of s i l i c o n d e t e r m i n e d as 0.260 + 0.050 ppm a t weaning i n the p r e s e n t s t u d y , i s s i m i l a r t o l e v e l s r e p o r t e d by L e V i e r (1975). The wide range of r e p o r t e d v a l u e s f o r s i l i c o n i l l u s t r a t e s the d i f f i c u l t y of s i l i c o n m i c r o d e t e r m i n a t i o n i n b i o l o g i c a l t i s s u e s . Lower v a l u e s a r e r e p o r t e d as the s e n s i t i -v i t y of a n a l y t i c a l p r o c e d u r e s improves and as c o n t a m i n a t i o n of samples i s r i g o r o u s l y c o n t r o l l e d . S e v e r a l i n v e s t i g a t o r s a l s o r e p o r t e d h i g h e r s i l i c o n c o n c e n t r a t i o n i n plasma than i n serum of human b l o o d (Lo and C h r i s t i a n , 1978) and a wide range of s i l i c o n v a l u e s i n r e d b l o o d c e l l s ( A u s t i n et a l . , 1973). In the p r e s e n t s t u d y , i t i s p o s s i b l e t h a t hemolyzed r e d b l o o d c e l l s i n c e r t a i n plasma samples may have c o n t r i b u t e d t o h i g h e r v a l u e s of s i l i c o n and g r e a t e r v a r i a b i l i t y i n the r e p o r t e d mean s i l i c o n v a l u e s of the pups. A p p l i c a t i o n of d i f f e r e n t a n a l y t i -c a l methods i n c l u d i n g c o l o r i m e t r y ( C a r l i s l e , 1974b; Charnot and 52 P e r e s , 1971a; L e V i e r , 1975) and e m i s s i o n s p e c t r o m e t r y may a l s o c o n t r i b u t e t o the v a r i a b i l i t y i n the r e p o r t e d plasma s i l i c o n v a l u e s . C. Effectiveness of Treatment With Propylthiouracil and L-Thyroxine 1. Maternal Influences During Pregnancy and Lactation In t h i s s t u d y , t h y r o i d e x c e s s and t h y r o i d p r i v i a i n the o f f s p r i n g s of dams were compared f o r a l t e r a t i o n s i n p o s t n a t a l p a t t e r n of plasma s i l i c o n l e v e l s w i t h age. Treatments of the o f f s p r i n g s w i t h p r o p y l t h i o u r a c i l and t h y r o x i n e were e f f e c t i v e i n i n d u c i n g hypo- and h y p e r t h y r o i d i s m r e s p e c t i v e l y i n the p o s t -n a t a l p e r i o d . D u r i n g pregnancy, the dams from h y p e r t h y r o i d group r e c e i v e d no t r e a t m e n t and were e s s e n t i a l l y the same as the c o n t r o l dams. The s t u d y shows t h a t t r e a t m e n t w i t h p r o p y l -t h i o u r a c i l d i d not a f f e c t the o v e r a l l food consumption of h y p o t h y r o i d dams d u r i n g pregnancy. The N a t i o n a l Academy of S c i e n c e s (1978) has e s t a b l i s h e d n u t r i e n t r e q u i r e m e n t s f o r growth and g e s t a t i o n i n Sprague-Dawley l a b o r a t o r y r a t s . Food i n t a k e of pregnant r a t s a v e r a g e s 20 grams per day based on a mean body weight of 300 grams. In the p r e s e n t s t u d y , c o n t r o l and h y p o t h y r o i d a n i m a l s consumed an average of 27.6 grams and 25.9 grams r e s p e c t i v e l y of Purina. Rat Chow per day throughout g e s t a t i o n . The mean body weight of c o n t r o l and h y p o t h y r o i d dams a t the s t a r t of g e s t a t i o n were 255 grams and 258 grams r e s p e c t i v e l y . The r e s u l t s i n d i c a t e d t h a t b oth groups of dams 53 a t comparable mean body weight consumed food a t or above the amounts n e c e s s a r y t o meet the n u t r i e n t r e q u i r e m e n t s of pregnancy, based on the n u t r i e n t a n a l y s i s of P u r i n a Rat Chow. The absence of d i f f e r e n c e s among the t h r e e groups i n t o t a l m a t e r n a l weight g a i n s d u r i n g pregnancy f u r t h e r s u p p o r t s the c o n c l u s i o n t h a t the c o u r s e of pregnancy was s i m i l a r f o r a l l groups. T h i s i s d e s p i t e the s t a t i s t i c a l l y h i g h e r mean body weight of r a t s i n h y p e r t h y r o i d group d u r i n g the f i r s t week of g e s t a t i o n . W h i l e h y p e r t h y r o i d r a t s were h e a v i e r a t the s t a r t of pregnancy, the weekly i n c r e m e n t a l and t o t a l weight g a i n e d as w e l l as the f i n a l weight a t t a i n e d p r i o r t o p a r t u r i t i o n were s i m i l a r t o c o n t r o l r a t s . In the p r e s e n t study p r o p y l t h i o u r a c i l e x e r t e d no o v e r a l l e f f e c t on the body weight of h y p o t h y r o i d dams d u r i n g pregnancy or l a c t a t i o n . The f i n d i n g i s i n agree-ment w i t h the o b s e r v a t i o n i n Sprague-Dawley r a t s g i v e n the same dose of p r o p y l t h i o u r a c i l i n d r i n k i n g water from about the t h i r d day of g e s t a t i o n ( D u s s a u l t and Walker, 1978). The N a t i o n a l Academy of S c i e n c e s (1978) has r e p o r t e d t h a t the t o t a l weight g a i n e d d u r i n g g e s t a t i o n i s d i r e c t l y r e l a t e d t o the s i z e of the l i t t e r and v a r i e s from 125 grams to 155 grams. In t h i s s t u d y , the mean weight g a i n s of dams i n c o n t r o l , h y p o t h y r o i d and h y p e r t h y r o i d groups were 162.9 +30.6 g, 147.6 + 28.5 g and 140.3 + 19.9 g r e s p e c t i v e l y . D u r i n g l a c t a t i o n , the dams from a l l t h r e e groups showed n e i t h e r a l a r g e g a i n or a l a r g e l o s s i n body weight (+10 grams) w i t h l a r g e v a r i a t i o n s a s s o c i a t e d w i t h the mean body weight i n 54 each group. T h i s i s c o n s i s t e n t w i t h the i n f o r m a t i o n p r o v i d e d by the N a t i o n a l Academy of S c i e n c e s (1978). 2. Body Weight of Pups The o f f s p r i n g s of dams g i v e n p r o p y l t h i o u r a c i l or t h y r o x i n e d u r i n g the p o s t n a t a l p e r i o d showed abnormal l e v e l s of t h y r o i d hormones r e l a t i v e t o c o n t r o l pups. Body weight i s a s e n s i t i v e i n d i c a t o r of reduced t h y r o i d f u n c t i o n i n the r a t (Greenberg, N a j j a r and B l i z z a r d , 1974). In the p r e s e n t s t u d y h y p o t h y r o i d pups exposed t o p r o p y l t h i o u r a c i l i n u t e r o m a n i f e s t e d s i g n i f i -c a n t l y d e c r e a s e d body weight a t b i r t h . The r e s u l t s of t h i s study a re a t v a r i a n c e w i t h those of Hamburgh, Lynn and Weiss (1964) which showed normal i n t r a u t e r i n e growth and m a t u r a t i o n i n p r o p y l t h i o u r a c i l - t r e a t e d r a t s . D i f f e r e n c e s i n dosages, r o u t e and time of a d m i n i s t r a t i o n of p r o p y l t h i o u r a c i l may e x p l a i n the d i s c r e p a n c y between these r e s u l t s . S i m i l a r l y , D u s s a u l t and Walker (1978) obse r v e d no r e d u c t i o n i n the body weight of h y p o t h y r o i d a n i m a l s t r e a t e d b e f o r e b i r t h . The reason f o r t h i s i s not r e a d i l y a p p a r e n t . T h e i r t r e a t m e n t p r o t o c o l , which was begun from around the t h i r d day of g e s t a t i o n , was s i m i l a r t o the one employed i n the p r e s e n t s t u d y . I t i s p o s s i b l e t h a t the o m i s s i o n of an adjustment p e r i o d p r i o r t o the s t a r t of the tre a t m e n t may account f o r the l a c k of an e f f e c t of p r o p y l t h i o u r a c i l on body w e i g h t . C o n t r a r y t o past work, F r o e l i c h and Meserve (1982) have r e c e n t l y demonstrated t h a t i n t r a u t e r i n e somatic growth i s c l e a r l y dependent on an 55 e u t h y r o i d s t a t e . Moreover, p o s t n a t a l growth r e t a r d a t i o n a s s o c i a t e d w i t h h y p o t h y r o i d i s m i s most severe when t h i o u r a c i l i s o f f e r e d b oth p r e - and p o s t n a t a l l y as compared t o i t s a d m i n i s t r a t i o n e i t h e r p r e n a t a l l y or p o s t n a t a l l y a l o n e . T h i s i s sup p o r t e d by r e c e n t s t u d i e s ( M o r r e a l e de Escobar e t a l . , 1985) which show t h a t term f e t u s e s from t h y r o i d e c t o m i z e d r a t s d i s p l a y e d low c o n c e n t r a t i o n of T4 and T3 both p r i o r t o and f o l l o w i n g onset of f e t a l t h y r o i d f u n c t i o n . A l t e r e d t h y r o i d f u n c t i o n was accompanied by d e v e l o p m e n t a l d e l a y which p e r s i s t e d and was not compensated a t term. In the p r e s e n t s t u d y , the s i g n i f i c a n t l y d e p r e s s e d growth r a t e e v i d e n t a t b i r t h i n hypo-t h y r o i d pups was m a i n t a i n e d throughout the p o s t n a t a l p e r i o d u n t i l about day 23 when the a n i m a l s c o m p l e t e l y c e a s e d growing. The f i n d i n g i s c o n s i s t e n t w i t h the r e p o r t e d o b s e r v a t i o n of an almost complete c e s s a t i o n of growth from day 21 when p r o p y l t h i -o u r a c i l t r e a t m e n t was t e r m i n a t e d u n t i l day 57 when the experiment was t e r m i n a t e d ( D u s s a u l t and Walker, 1978). Other i n v e s t i g a t o r s have found c o n s i s t e n t l y reduced mean body weight i n h y p o t h y r o i d r a t s which were s i g n i f i c a n t l y lower than those of c o n t r o l s ( B u r s t e i n e t a l . , 1979). Mean body weight of 25 day o l d h y p o t h y r o i d r a t s i n the p r e s e n t s t u d y was 29.7 grams which r e p r e s e n t e d 41.5% of the body weight of c o n t r o l a n i m a l s . Higher body weight has been r e p o r t e d at 38.2 grams f o r t h i o u r a -c i l - t r e a t e d r a t s ( F r o e l i c h and Meserve, 1982) and a t 38.0 grams f o r r a t s t h y r o i d e c t o m i z e d on p o s t n a t a l day 1 (Ge e l and T i m i r a s , 1970). Varma, Murray and Stanbury (1978) have demonstrated 56 t h a t the growth and development of the f e t u s and newborn a r e not a f f e c t e d by the m a t e r n a l t h y r o i d s t a t u s of r a d i o t h y r o i d e c -t o m i z e d r a t s . N e v e r t h e l e s s , Shaumbaugh and W i l b e r (1974) have shown t h a t n e o n a t a l u n d e r n u t r i t i o n r e s u l t s i n h y p o t h y r o i d i s m d u r i n g the p e r i o d of the n u t r i t i o n a l d e p r i v a t i o n . T h e r e f o r e , the p o s s i b i l i t y cannot be e x c l u d e d t h a t i n a n i t i o n secondary to inadequate n u r s i n g a s s o c i a t e d w i t h m a t e r n a l h y p o t h y r o i d i s m may be a c o n t r i b u t i n g f a c t o r i n the s e v e r i t y of the d e p r e s s e d growth r a t e of the o f f s p r i n g s . H y p e r t h y r o i d a n i m a l s t r e a t e d w i t h d a i l y subcutaneous i n j e c t i o n s of L - t h y r o x i n e i n the p r e s e n t study d i s p l a y e d s i g n i f i c a n t l y r e t a r d e d mean body weight as compared w i t h c o n t r o l r a t s by p o s t n a t a l day 10. The degree of r e t a r d a t i o n i n body weight was not as severe as t h a t o b s e r v e d i n h y p o t h y r o i d pups. Mean body weight of 25 day o l d h y p e r t h y r o i d pups was 58.5 grams which r e p r e s e n t s 81.7% of the body weight of c o n t r o l r a t s . In c o n t r a s t , o t h e r i n v e s t i g a t o r s have d e s c r i b e d s i m i l a r body weight between c o n t r o l and h y p e r t h y r o i d r a t s t r e a t e d w i t h 0.4 T4 ug/g of body weight d u r i n g the p e r i o d of t r e a t m e n t from b i r t h u n t i l 12 days of age ( D u s s a u l t , Coulombe and Walker, 1982). The r e s u l t of the p r e s e n t study i s i n agreement w i t h those of Varma and Crawford (1979), Hamburgh, Lynn and Weiss (1964) and D u s s a u l t , Coulombe and Walker (1982). I t i s f u r t h e r c o r r o b o r a t e d by the f i n d i n g s of E a y r s and Holmes (1964) t h a t n e o n a t a l h y p e r t h y r o i d i s m r e s u l t s i n d e c r e a s e d body w e i g h t , which p e r s i s t s a f t e r t r e a t m e n t has been d i s c o n t i n u e d . 57 Subsequent works have demonstrated a p e r s i s t e n c e of reduced body weight i n n e o n a t a l h y p e r t h y r o i d i s m (Bakke e t a l . , 1975). I t i s h y p o t h e s i z e d t h a t h y p e r t h y r o i d i s m i n the n e o n a t a l p e r i o d r e s u l t s i n permanent r e s e t t i n g of the r e g u l a t o r y s e t p o i n t f o r p i t u i t a r y t h y r o i d s t i m u l a t i n g hormone s e c r e t i o n and i n c r e a s e d s e n s i t i v i t y t o the feedback i n h i b i t o r y e f f e c t s of the t h y r o i d hormones. 3. Skeletal Maturation of Pups P e r t u r b a t i o n s i n t h y r o i d f u n c t i o n have been shown t o a f f e c t the m a t u r a t i o n of s k e l e t a l system. In t h i s s t u d y , hypo-t h y r o i d pups demonstrated d e l a y e d bone development on p o s t n a t a l day 7 which became s t a t i s t i c a l l y s i g n i f i c a n t compared t o c o n t r o l s by day 14. H y p e r t h y r o i d pups d i s p l a y e d s i g n i f i c a n t l y a c c e l e r a t e d s k e l e t a l m a t u r a t i o n r e l a t i v e t o c o n t r o l s by day 7. The f i n d i n g s of t h i s study a r e s u p p o r t e d by o t h e r works on h y p o t h y r o i d i s m ( G e l o s o e t a l . , 1968; Hamburgh, 1968) and hyper-t h y r o i d i s m ( R i e k s t n i e c e and A s l i n g , 1966; Becks e t a l . , 1950) i n t r e a t e d a n i m a l s . 4. Plasma Levels of Thyroid Hormones in Pups As e x p e c t e d , t h y r o i d d y s f u n c t i o n i n e x p e r i m e n t a l a n i m a l s i s r e f l e c t e d i n abnormal c o n c e n t r a t i o n of the t h y r o i d hormones. The p r e s e n t study demonstrates lower plasma l e v e l s of T4 and T3 i n h y p o t h y r o i d pups as compared w i t h c o n t r o l a n i m a l s . B u r s t e i n et a l . (1979) r e p o r t e d serum l e v e l s of T4 t o be l e s s 58 than 1 ug/100 ml i n 14 day o l d r a t s t r e a t e d w i t h p r o p y l t h i o u r a c i l b e f o r e and a f t e r b i r t h . D u s s a u l t and Walker (1978) a l s o r e p o r t e d t h a t they c o u l d not d e t e c t serum l e v e l s of T4 and T3 by p o s t n a t a l day 5 i n pups s i m i l a r l y t r e a t e d p r e n a t a l l y . A l t h o u g h plasma l e v e l s of t h y r o i d hormones i n t h i s s t u d y were h i g h e r than the p u b l i s h e d v a l u e s , the s i g n i f i c a n t l y r e t a r d e d growth r a t e and s k e l e t a l development i n d i c a t e d t h a t p r o p y l t h i o u r a c i l a d m i n i s t e r e d i n the m a t e r n a l d r i n k i n g f l u i d was e f f e c t i v e i n i n d u c i n g h y p o t h y r o i d i s m of the o f f s p r i n g s . C o n v e r s e l y , plasma T4 and T3 c o n c e n t r a t i o n s of h y p e r t h y r o i d r a t s were markedly e l e v a t e d d u r i n g the p e r i o d of t h y r o x i n e a d m i n i s t r a t i o n . T h i s i s i n a c c o r d a n c e w i t h the f i n d i n g s of D u s s a u l t , Coulombe and Walker (1982). E v i d e n c e of s i g n i f i c a n t l y d e pressed body w e i g h t , advanced s k e l e t a l m a t u r a t i o n and e l e v a t e d plasma c o n c e n t r a t i o n s of t h y r o i d hormones i n h y p e r t h y r o i d a n i m a l s i n d i c a t e t h a t t h y r o x i n e t r e a t m e n t i n t h i s study was e f f e c t i v e i n i n d u c i n g h y p e r t h y r o i d i s m i n the o f f s p r i n g s . D. Plasma S i l i c o n Concentrations in Hypo- and Hyperthyroid Pups Plasma s i l i c o n l e v e l s of h y p o t h y r o i d and h y p e r t h y r o i d pups were g e n e r a l l y lower than those of c o n t r o l pups, p a r t i c u l a r l y d u r i n g the e a r l y p o s t n a t a l p e r i o d s . The h i g h e s t v a l u e s f o r plasma s i l i c o n were observed between days 7 and 14 i n h y p o t h y r o i d group and on day 14 i n h y p e r t h y r o i d group. T h i s 59 c o n t r a s t e d w i t h the peak o b s e r v e d i n the c o n t r o l group which was m a n i f e s t e d on day 12 at t w i c e the c o n c e n t r a t i o n s p r e s e n t i n t r e a t e d pups. The two t r e a t e d groups were sampled a t weekly i n t e r v a l , w h i l e the c o n t r o l group was sampled a t 3-day i n t e r v a l s over t h i s p e r i o d . The method of s a m p l i n g i n t r e a t e d pups does not p r e c l u d e the p o s s i b i l i t y t h a t s i l i c o n c o n c e n t r a -t i o n may have r i s e n s h a r p l y and s u b s e q u e n t l y d e c l i n e d between s a m p l i n g p o i n t s . In the l a t e p o s t n a t a l p e r i o d , s i l i c o n concen-t r a t i o n s of b o t h the hypo- and h y p e r t h y r o i d groups d e c l i n e d t o 0.105 + 0.017 ppm and 0.111 + 0.022 ppm r e s p e c t i v e l y by day 28. These l e v e l s were lower than the s i l i c o n c o n c e n t r a t i o n observed i n c o n t r o l pups on day 25 a t 0.260 + 0.029 ppm. S t a t i s t i c a l a n a l y s i s i n d i c a t e d t h a t f o r each of the e x p e r i m e n t a l t r e a t m e n t s t h e r e were s i g n i f i c a n t a l t e r a t i o n s i n plasma s i l i c o n c o n c e n t r a -t i o n s w i t h age. I t would appear t h a t the p a t t e r n s of changes i n s i l i c o n w i t h age i n both the hypo- and h y p e r t h y r o i d pups a r e d i f f e r e n t from c o n t r o l r a t s . However, the d a t a i n t h i s s t u d y do not p e r m i t s t a t i s t i c a l c omparison of the r e l a t i o n s h i p because of d i f f e r e n c e s i n the time of s a c r i f i c e used. In t h i s s t u d y , the h i g h e s t v a l u e s f o r plasma s i l i c o n i n c o n t r o l , hypo- and h y p e r t h y r o i d pups were o b s e r v e d on day 12, between days 7 and 14, and on day 14 r e s p e c t i v e l y . The p e r i o d between days 7 and 14 a l s o c o r r e s p o n d e d t o the p e r i o d of maximum v e l o c i t y i n bone m a t u r i t y s c o r e s f o r a l l t h r e e groups of r a t s . I f plasma s i l i c o n i s r e l a t e d t o s k e l e t a l development, then the o b s e r v e d peak i n plasma s i l i c o n of 12 day o l d c o n t r o l 60 pups c o r r e s p o n d e d t o a bone s c o r e of about 150. H y p o t h y r o i d pups e x h i b i t e d s i g n i f i c a n t l y r e t a r d e d bone development, and a t t a i n e d a maximum s c o r e of about 114 by p o s t n a t a l day 14 and then ceased f u r t h e r s k e l e t a l development. The a c c l e r a t e d ske-l e t a l development of h y p e r t h y r o i d pups was r a t e d a t 207 by day 14. I t i s p o s s i b l e t h a t the peak v a l u e i n plasma s i l i c o n may not occur i n h y p o t h y r o i d r a t s s i n c e bone development f a i l e d t o re a c h the s t a g e of m a t u r a t i o n comparable to t h a t observed i n c o n t r o l r a t s on day 12. The peak v a l u e f o r plasma s i l i c o n i n h y p e r t h y r o i d pups may have o c c u r r e d e a r l i e r than day 12, when the bone s c o r e of 150 was reached and s u b s e q u e n t l y was not d e t e c t e d i n t h i s s t u d y . C o n v e r s e l y , i f plasma s i l i c o n r e l a t e s t o bone development, then the plasma s i l i c o n c o n c e n t r a t i o n s of pups a t e q u i v a l e n t bone s c o r e s would be e x p e c t e d t o be s i m i l a r . In t h i s s t u d y , the 14 day o l d h y p e r t h y r o i d pups were s i m i l a r t o 21 day o l d c o n t r o l r a t s i n bone age. However, plasma s i l i c o n c o n c e n t r a t i o n s of these r a t s a t 0.466 + 0.118 and 0.247 + 0.125 ppm r e s p e c t i v e l y were d i f f e r e n t a t s i m i l a r bone ages. The r e s u l t s of t h i s s t u d y i n d i c a t e t h a t t h y r o i d f u n c t i o n appears t o i n f l u e n c e p o s t n a t a l p a t t e r n of plasma s i l i c o n . S i l i c o n v a l u e s were g e n e r a l l y lower i n t h y r o i d e x c e s s and t h y r o i d p r i v i a r e l a t i v e t o e u t h y r o i d c o n d i t i o n . P a t t e r n s of p o s t n a t a l s i l i c o n l e v e l s i n hypo- and h y p e r t h y r o i d i s m were a l t e r e d towards e a r l i e r and l a t e r ages a t which the maximum l e v e l s of plasma s i l i c o n were a t t a i n e d as compared w i t h normal e u t h y r o i d c o n d i t i o n . The mechanism of t h y r o i d c o n t r o l over 61 plasma s i l i c o n i s not known. I t i s s p e c u l a t e d t h a t plasma s i l i c o n i s r e l a t e d t o s k e l e t a l development. There i s e v i d e n c e t h a t s i l i c o n p l a y s a r o l e i n bone m i n e r a l i z a t i o n ( C a r l i s l e , 1970b; 1974b) and t h a t bone s i l i c o n i n c r e a s e s t o a maximum as c a l c i f i c a t i o n p r o g r e s s e s and s u b s e q u e n t l y d e c l i n e s t o v e r y low l e v e l s when c a l c i f i c a t i o n i s completed ( C a r l i s l e , 1969). In t h i s s t u d y , the peak v a l u e s of plasma s i l i c o n i n a l l t h r e e groups of pups c o i n c i d e d w i t h the p e r i o d of maximum v e l o c i t y i n bone m a t u r i t y s c o r e s of each group and, presumably, i n the r a t e of bone m i n e r a l i z a t i o n . The method of s a m p l i n g used i n t h i s s t udy does n o t - a l l o w s t a t i s t i c a l comparison of d a t a on plasma s i l i c o n and bone age. T h e r e f o r e , under the e x p e r i m e n t a l c o n d i -t i o n s employed i n t h i s s t u d y , i t cannot be c o n c l u d e d t h a t p o s t -n a t a l s i l i c o n l e v e l s i s r e l a t e d t o s k e l e t a l development. E. Suggestions For Future Consideration The p r e s e n t study may be improved by m a i n t a i n i n g e q u a l number of pups and s a c r i f i c i n g pups of the same ages i n each group to a l l o w f o r s t a t i s t i c a l comparison and c o r r e l a t i o n of parameters among c o n t r o l and e x p e r i m e n t a l groups. In view of the r a p i d and l a r g e i n c r e m e n t s i n plasma s i l i c o n c o n c e n t r a t i o n s o b s e r v e d i n the f i r s t two weeks of p o s t n a t a l l i f e , more f r e q u e n t s a m p l i n g over t h i s p e r i o d may be n e c e s s a r y t o a c c u r a -t e l y c h a r a c t e r i z e the d e v e l o p m e n t a l p a t t e r n of plasma s i l i c o n changes w i t h i n c r e a s i n g age. M i l d e r s t a t e s of hypo- and h y p e r -t h y r o i d i s m which induce s m a l l changes i n the r a t e of bone 62 development, may p r o v i d e d a t a s u i t a b l e f o r c o r r e l a t i o n a l s t u d i e s between plasma s i l i c o n l e v e l s and bone m a t u r i t y s c o r e s . I f s i l i c o n p l a y s a r o l e i n bone m i n e r a l i z a t i o n which b e g i n s i n u t e r o and c o n t i n u e s p o s t n a t a l l y , then t r e a t m e n t w i t h p r o p y l -t h i o u r a c i l and t h y r o x i n e may be d e l i n e a t e d p r e - and p o s t n a t a l l y t o d i s c e r n the r o l e of s i l i c o n i n bone f o r m a t i o n a c c o r d i n g l y . The mechanism by which s i l i c o n a c c e l e r a t e s bone m i n e r a l i z a t i o n i s not known. S i n c e s i l i c o n i s u b i q u i t o u s , m e t a b o l i c s t u d i e s which c o r r e l a t e s m a t e r n a l d i e t a r y i n t a k e w i t h i n t e s t i n a l a b s o r p t i o n as w e l l as s i l i c o n c o n t e n t of m i l k w i t h plasma l e v e l s of s i l i c o n i n young pups over t h i s p e r i o d would be of i n t e r e s t . 63 REFERENCES A l e x a n d e r , G.B., Heston, W.M. and I l e r , R.K.J. 1954. 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