Upsala J Med Sci 88: 3 3 4 1 , 1983 Intraarterial and Intraportal in vivo Catheterization of the Regenerating Rat Liver Effects upon body and liver weights and DNA synthesis Lillernor Lewan', Herman Arnneus', Ola Forsberg3 and Kerstin Lundberg4 Department of Zoophysiology', University of Lund, Lund, and Department of Physical Biolo y 2 , Central Hospital, Vaster&, and Department of Experimental Medicine4, Pharmacia A B , Uppsala, Sweden The Gustaf Werner Institute, University of Uppsala, Uppsala, and Department of Surgery B , ABSTRACT A method f o r c o n c o m i t a n t p a r t i a l hepatectomy and c a t h e t e r i z a t i o n of t h e a r t e r i a l and p o r t a l systems of t h e l i v e r i n t h e r a t i s d e s c r i b e d . C a t h e t e r s were i n s e r t e d i n t o t h e g a s t r o d u o d e n a l a r t e r y and t h e i l e o c o l i c v e i n . Continuous s a l i n e p e r f u s i o n was performed d u r i n g 36 h o u r s . I n c a t h e t e r i z e d r a t s r e c o v e r y of l i v e r and body w e i g h t l a g g e d b e h i n d t h a t of n o n - c a t h e t e r i z e d r a t s . The more e x t e n s i v e s u r g e r y and t h e p r e s e n c e of c a t h e t e r s a l s o c a u s e d d e c r e a s e d i n c o r p o r a - t i o n of 3H-thymidine i n t o l i v e r DNA 24 h o u r s p o s t o p e r a t i v e l y . The v a r i a t i o n i n t h y m i d i n e i n c o r p o r a t i o n between a n i m a l s was l a r g e . It was shown t h a t by pre- l a b e l l i n g l i v e r DNA w i t h "C-thymidine t h e r a t s c a n s e r v e a s t h e i r own c o n t r o l s d u r i n g a c u t e e x p e r i m e n t s i n v o l v i n g 'H-thymidine, t h u s r e d u c i n g t h e i n c o n s i s - t a n c y of i n d i v i d u a l v a r i a t i o n . INTRODUCTION P a r t i a l r e s e c t i o n of 213 of t h e l i v e r i n r a t s i n d u c e s h y p e r t r o p h y and h y p e r p l a s i a of t h e r e m a i n i n g l i v e r t i s s u e . T h i s i s c h a r a c t e r i z e d by changes i n enzyme a c t i v i t i e s , i n RNA- and DNA-synthesis, and i n m i t o t i c a c t i v i t y . Almost a l l c e l l s a r e s t i m u l a t e d t o s y n t h e s i z e DNA and p a s s t h r o u g h t h e c e l l c y c l e 1-2 t i m e s . R e s t o r a t i o n of o r g a n w e i g h t is u s u a l l y s e e n w i t h i n a week. The c o u r s e of t i m e f o r t h e s e e v e n t s a r e w e l l known ( 2 , 5 , 8 , 1 3 , 1 4 ) . I n t h e p r e s e n t s t u d y w e d e s c r i b e a model d e s i g n e d t o a n a l y z e t h e e f f e c t s of blood f l o w m o d i f i c a t i o n s on l i v e r c e l l k i n e t i c s i n u n a e s t h e t i z e d r a t s . We e v a l u a t e d t h e e f f e c t s of c o n c o m i t a n t c a t h e t e r i z a t i o n of t h e h e p a t i c a r t e r y a n d p o r t a l v e i n i n c o n n e c t i o n w i t h p a r t i a l hepatectomy on body and l i v e r w e i g h t s , a s w e l l as t h e e f f e c t of c a t h e t e r i z a t i o n upon i n c o r p o r a t i o n of 3H-thymidine and 14C-thymidine i n t o l i v e r DNA, u s i n g e a c h animal a s i t s own c o n t r o l . MATERIAL AND METHODS Animals Young, a d u l t male Sprague Dawley r a t s weighing 170-210 g (SPF q u a l i t y , 3-832859 33 L i v e r of Fig. 1. Schematic view of c a t h e t e r s i n t h e i l e o - c o l i c v e i n , i n t h e gastroduodenal a r t e r y and of t h e f r e e c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . For d e t a i l s of t h e a r t e r i a l and p o r t a l systems r e f e r t o Green ( 7 ) . from pump t o a r t e r i a l \ and p o r t a l c a t h e t e r p e r i t : t e r :oneal F i g . 2 . C a t h e t e r i z e d r a t i n cage. 34 Anticimex, Sweden) were u s e d f o r t h e e x p e r i m e n t . The a n i m a l s were k e p t u n d e r c o n s t a n t c o n d i t i o n s and had f r e e access t o s t a n d a r d l a b o r a t o r y d i e t b e f o r e and a f t e r s u r g e r y . S u r g i c a 1 p r o c e d u r e s The a n i m a l s were a n a e s t h e t i z e d w i t h a n i p i n j e c t i o n of Mebumal, 30 mg/kg body w e i g h t . A f t e r m i d l i n e i n c i s i o n and r e t r a c t i o n of t h e l i v e r , t h e o p e r a t i o n w a s performed under a microscope ( 4 x). A p o l y e t h y l e n e c a n n u l a ( I n t r a m e d i c PE 50) w i t h a narrow t i p was i n s e r t e d i n t o t h e g a s t r o d u o d e n a l a r t e r y i n t h e r e t r o g r a d e d i r e c t i o n , a c c o r d i n g t o L e i v e s t a d and Malt ( 1 2 ) . The i l e o c o l i c v e i n was c a t h e t e r i z e d i n t h e a n t e r o g r a d e d i r e c t i o n . To a l l o w i n t r a p e r i t o n e a l i n f u s i o n , t h e t i p of a t h i r d c a t h e t e r w a s p o s i t i o n e d i n t h e p e r i t o n e a l c a v i t y , f i g . 1. A f t e r c a t h e t e r i z a t i o n t h e median and l e f t l a t e r a l l o b e s of t h e l i v e r were r e s e c t e d a c c o r d i n g t o H i g g i n s and Anderson ( 9 ) . I n s e p a r a t e e x p e r i m e n t s p a r t i a l hepatectomy w a s accomplished b e f o r e c a t h e t e r i z a t i o n . The c a t h e t e r s were p u l l e d s u b c u t a n e o u s l y t o t h e neck and t h e abdominal wound w a s s t i t c h e d . A small p l a s t i c v i a l a t t a c h e d t o t h e neck s e c u r e d t h e c a t h e t e r s from damage. From t h e v i a l t o t h e t o p of t h e c a g e , t h e c a t h e t e r s were p r o t e c t e d by a m e t a l h e l i x , f i g . 2 . The i n t r a v a s c u l a r c a t h e t e r s were c o n n e c t e d t o pumps (Brown-Melsungen, t y p e 71100) which s u p p l i e d 1 m1/24 h r of F y s k o s a l / H e p a r i n (500 I E Hep/100 c c F y s k ) . The p e r i t o n e a l c a t h e t e r was f i l l e d w i t h t h e same s o l u t i o n and c l o s e d a t i t s f r e e end. F i v e r a t s s u b j e c t e d t o p a r t i a l hepatectomy o n l y s e r v e d as c o n t r o l s . P a r t i a l hepatectomy a l o n e was u s u a l l y accomplished w i t h i n 5 m i n u t e s . Cathe- t e r i z a t i o n r e q u i r e d a n o t h e r 15-25 m i n u t e s . To minimize e f f e c t s of d i u r n a l v a r i a - t i o n s , o p e r a t i v e work was r e s t r i c t e d t o a 1-2 hour p e r i o d i n t h e morning. One group of r a t s w e r e d e t a c h e d from t h e pumps 36 h o u r s a f t e r t h e o p e r a t i o n . The metal h e l i x was removed, t h e c a t h e t e r s were f l u s h e d , m e l t e d s h u t , c o i l e d and p u t i n t o p l a s t i c v i a l s . T h i s group a n d t h e c o n t r o l g r o u p of non c a t h e t e r i z e d r a t s were weighed d a i l y . Seven days a f t e r o p e r a t i o n t h e r a t s were k i l l e d and t h e l i v e r s removed and weighed. Animals aimed f o r r a d i o n u c l i d e s t u d i e s were g i v e n r a d i o a c t i v e thymidine 24 h u r s a f t e r t h e o p e r a t i o n v i a t h e p e r i t o n e a l c a t h e t e r o r i n t h e c a t h e t e r i n t h e i l e o c o l i c v e i n . Slow f l u s h i n g w i t h 5 i.ll a t a t i m e was accomplished w i t h a r e p e a t i n g d i s p e n s e r (Hamilton Comp) u n t i l 100 i.ll c o n t a i n i n g 185 kBq ( 5 pCi) of (methyl-I4C) thymidine (185 GBq, Amersham) a n d / o r 100 i.l1 c o n t a i n i n g 3 . 7 MBq (100 pCi) ( m e t h ~ l - ~ H ) thymidine (555-1110 GBq, Amersham) w a s a d m i n i s t e r e d . For d o u b l e l a b e l l i n g , where e a c h a n i m a l s e r v e d a s i t s own c o n t r o l , t4C-thymidine was i n j e c t e d f i r s t and 3H-thymidine one o r two h o u r s l a t e r . One hour a f t e r t h e l a s t i n j e c t i o n t h e r a t s were k i l l e d and t h e l i v e r s were removed,and t h e d i f f e r e n t p a r t s of t h e r e g e n e r a t i n g l o b e were i d e n t i f i e d , f r o z e n 35 on s o l i d C O z , and s t o r e d a t - 7 O O C u n t i l a n a l y s i s . o t h e r , one group of a n i m a l s r e c e i v e d 3H-thymidine o n l y and a n o t h e r group 3H- thymidine and 14C-thymidine c o n c o m i t a n t l y . The same d o s e s as d e s c r i b e d above were used. To e n s u r e t h a t t h e two i n j e c t i o n s of t h y m i d i n e d i d n o t i n t e r f e r e w i t h e a c h To show m e t a b o l i c e f f e c t s on thymidine i n c o r p o r a t i o n i n l i v e r DNA one group of a n i m a l s was g i v e n 3H-thymidine 24 h o u r s a f t e r p a r t i a l hepatectomy and one hour l a t e r 14C-thymidine mixed w i t h 0 . 0 0 1 o r 0 . 2 mmol "cold" t h y m i d i n e . Number Body w e i g h t of r a t s M SD 0 1 2 3 4 5 6 7 day 5 w i t h 202g 100% 90% 92% 95% 97% 100% - 1 0 7 % l ) no c a t h . + + + + + + + 4 w i t h 206g 100% 87% 87% 89% 90% 91% - 98% c a t h . -5 -2 -2 -3 -4 -4 -4 24 -6 -3 -4 - 4 -3 -3 -2 23 + + + + + + + I A n a l y s i s of DNA Specimens t a k e n from t h r e e d i f f e r e n t p a r t s of t h e r e g e n e r a t i n g l o b e were a n a l y z e d by two methods. DNA was e x t r a c t e d a c c o r d i n g t o Schmidt-Tannhauser as m o d i f i e d by Munro & F l e c k ( 1 6 ) and q u a n t i f i e d a c c o r d i n g t o Burton ( 4 ) . DNA w a s a l s o i s o l a t e d by p r o t e i n a s e - K a c c o r d i n g t o Kasche-Amneus (10) and q u a n t i f i e d by W - s p e c t r o p h o t o m e t r y . R a d i o a c t i v i t y was a n a l y s e d i n a s c i n t i l l a t i o n c o u n t e r (Mark 11) and DNA s p e c i f i c r a d i o a c t i v i t y d e t e r m i n e d . The two methods f o r DNA a n a l y s e s were i n good agreement. Only r e s u l t s from a n a l y s e s performed a c c o r d i n g t o t h e Schmidt- Tannhauser method are r e p o r t e d h e r e . F o r c a l c u l a t i o n of t h e r a t i o between t h e s p e c i f i c 3H- and 1 4 C - a c t i v i t y i n DNA, t h e measured CPM/mg DNA w a s d i v i d e d by t h e CPM of i n j e c t e d 3H-thymidine a n d 14C-thymidine, r e s p e c t i v e l y . L i v e r w e i g h t M SD day 7 7 . 4gL) ?0.3 6.4g 2) 20.9 RESULTS The e f f e c t s of s u r g e r y and c a t h e t e r i z a t i o n upon t o t a l body and l i v e r w e i g h t s a r e g i v e n i n T a b l e 1. D i f f e r e n c e s between c a t h e t e r i z e d and non-cathe- t e r i z e d r a t s were small, b u t r e c o v e r y i n r a t s w i t h c a t h e t e r s l a g g e d b e h i n d of t h e c o n t r o l group. T a b l e 1. E f f e c t of c a t h e t e r i z a t i o n on body a n d l i v e r w e i g h t 1-7 d a y s a f t e r p a r t i a l hepatectomy. C a t h e t e r s were c o n n e c t e d t o t h e pumps f o r 36 h o u r s . Mean f s t a n d a r d d e v i a t i o n . t h a t 36 N e i t h e r p r e - n o r c o a d m i n i s t r a t i o n o f 14C-thymidine i n t e r f e r r e d s i g n i f i c a n t l y w i t h t h e i n c o r p o r a t i o n of 3H-thymidine i n t o l i v e r DNA ( T a b l e 2 ) . T a b l e ' 2 . E f f e c t of pre- o r c o a d m i n i s t r a t i o n of 14C-thymidine ( 1 0 - 4 mmol) on 3H- thymidine ( 5 x mmol) i n c o r p o r a t i o n i n t o l i v e r DNA 24 h o u r s a f t e r p a r t i a l hepatectomy. R a d i o n u c l i d e s were a d m i n i s t e r e d t h r o u g h a per- manent c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . Mean * s t a n d a r d d e v i a t i o n , r e l a t i v e v a l u e s . Number of a n i m a l s 4 3 5 ~~ Hours a f t e r p a r t i a l hepatectomy CPM p e r mg DNA p e r i n j e c t e d CPM i n j e c t i o n of 14c 3H k i l l e d 'I 4C-thymidine 3H-thymidine M SD M S D - 24 25 - 2.43?0.61 24 24 25 3.55?0.55 2 . 9020. 72 23 24 25 4.17?0.72 3.08:O. 70 I n T a b l e 3 , two g r o u p s of r a t s , h a v i n g e i t h e r a n i n t r a p e r i t o n e a l c a t h e t e r o n l y (A1) o r h a v i n g an i n t r a p e r i t o n e a l c a t h e t e r as w e l l as c a t h e t e r s i n t h e g a s t r o d u o d e n a l a r t e r y and i l e o c o l i c v e i n (B ) are compared. Both g r o u p s were T a b l e 3. E f f e c t of c a t h e t e r s i n t h e g a s t r o d u o d e n a l a r t e r y and t h e i l e o - c o l i c v e i n on t h y m i d i n e i n c o r p o r a t i o n i n t o l i v e r DNA 24 h o u r s a f t e r p a r t i a l hepatectomy. I4C-thymidine o r 3H-thymidine was a d m i n i s t e r e d t h r o u g h a permanent c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . Mean s t a n d a r d d e v i a t i o n . 1 I n j e c t i o n of I k i l l e d I M ? SD r a d i o n u c l i d e C a t h e t e r s i n SD % of 1 A 1 c a v i t y B 1 A2 B2 p e r i t o n e a l c a v i t y p e r i t o n e a 1 g a s t roduodena 1 a. i l e o - c o l i c v . per i t one: 1 c a v i t y p e r i t o n e a l c a v i t y g a s t r o d u o d e n a l a. i l e o - c o l i c v . 23 23 24 24 Radio- n u c l i d e (numb er of r a t s ) ( 5 r a t s ) 4C-thymidine 'C-thymidine ( 3 r a t s ) 3H-thymid i n e (12 r a t s ) 3H- t hymid i n e (7 r a t s ) 25 4.17 * 0.72* 17 25 2 . 3 8 0.49* 2 1 25 2.82 * 0.68** 24 25 1.75 0.86** 49 Hours a f t e r p a r t i a l CPM p e r mg DNA p e r hepatectomy i n j e c t e d CPM * The d i f f e r e n c e i s s i g n i f i c a n t 0 . 0 1 > p > 0.002 I 1 I 1 1 1 ** 1 1 0.02 > p > 0 . 0 1 g i v e n 14C-thymidine t h r o u g h t h e i n t r a p e r i t o n e a l c a t h e t e r . The e x p e r i m e n t was r e p e a t e d i n two similar g r o u p s , A2 a n d B2, g i v e n 3H-thymidine ( T a b l e 3 ) . Rats w i t h i n t r a v a s c u l a r c a t h e t e r s had a l a r g e r v a r i a t i o n i n t h y m i d i n e i n c o r p o r a t i o n i n t o DNA of t h e r e g e n e r a t i n g l i v e r , and l e s s i n c o r p o r a t i o n compared t o a n i m a l s 37 T a b l e 4. V a r i a t i o n i n DNA l a b e l l i n g i n g r o u p s of r a t s and i n i n d i v i d u a l r a t s . 14C-thymidine w a s a d m i n i s t e r e d 23 h o u r s and 3H-thymidine 24 h o u r s p o s t o p e r a t i v e l y a n d t h e r a t s were k i l l e d one hour l a t e r . CPM p e r mg DNA p e r i n j e c t e d CPM ' 4 c 3H Mean SD Mean - SD + 2.19 0.66 2.10 -+ 0.90 p e r i t o n e a l p e r i t o n e a l ly.;s) /cavity 3 H / ' 4 C i n DNA Mean SD 1 . 0 3 0.16 p e r i t o n e a l p e r i t o n e a l c a v i t y c a v i t y g a s t roduodenal a r t e r y i l e o - c o l i c ( 3 r a t s ) i l e o - c o l i c i l e o - c o l i c I e i n e i n ( 8 r a t s ) v e i n 2 . 1 3 1 . 7 9 1 . 4 6 0.56 CPM p e r mg DNA p e r i n j e c t e d CPM 2) 4 ) 0 . 4 3 0.13 + 1) 0.79 - 0.49 0.29 0.04 0.36 _t 0.01 3) M ~ S D 4.17?0.72 2.38+0.49 2.19?0.66 SD : of M 1 7 2 1 30 3H M%D 3.08:O. 7C l . 8 S 2 0 . 5 4 ? . l O - + O . 90 SD : of M 23 29 4 3 T a b l e 5. E f f e c t of a d d i t i o n of "coldll t h y m i d i n e on i n c o r p o r a t i o n 3 H / ' 4 C i n DNA i n e a c h r a t of I 1 1 6 1 6 - of r a d i o a c t i v e thymidine i n r e g e n e r a t i n g rat l i v e r . A l l i n j e c t i o n s were made t h r o u g h a c a t h e t e r i n t h e i l e o - c o l i c v e i n . 0.001 o r 0.02 mnol l ' c o l d l l thymidine w a s mixed w i t h t h e i n j e c t e d 3H-thymidine. I4C-thymidine was adminis- t e r e d 23 h o u r s and 3H-thymidine w i t h c o l d thymidine 24 h o u r s p o s t - o p e r a t i v e l y and t h e r a t s were k i l l e d one hour l a t e r . "Cold" thymidine mn01/100 1.11 A - (8 r a t s ) B 0.001 (2 r a t s ) c 0.2 ( 2 r a t s ) . ~~ .~ ~~~~ ~ 1) D i f f e r e n c e a s compared t o 3 H - a c t i v i t y i n group A i s n o t s i g n i f i c a n t . 0 . 1 > p > 0.05 ( S t u d e n t ' s t - t e s t ) 0.05 > p > 0.02 1 1 I 1 11 I ' 3H/14C i n group A i s s i g n i f i c a n t . 1 1 I 1 I 1 3 H - a c t i v i t y i n g r o u p A i s s i g n i f i c a n t . 1 1 1 1 1 1 3H/14C i n group A i s s i g n i f i c a n t . 2) 3) 4) 0.002 > p > 0.001 0.001 > p. 38 w i t h o u t i n t r a v a s c u l a r c a t h e t e r s . The s p e c i f i c a c t i v i t y of DNA w a s lower a f t e r i n t r a v a s c u l a r a d m i n i s t r a t i o n of l a b e l l e d thymidine t h a n a f t e r i n t r a p e r i t o n e a l a d m i n i s t r a t i o n i n a n i m a l s w i t h o u t i n t r a v a s c u l a r c a t h e t e r s ( T a b l e 4 ) . The s t a n d a r d d e v i a t i o n of t h e DNA s p e c i f i c a c t i v i t y i n t h e d i f f e r e n t groups of a n i m a l s w a s 17-43% of t h e mean, whereas t h e s t a n d a r d d e v i a t i o n of t h e H/ C of DNA i n e a c h a n i m a l was 1-16% of t h e mean. Thus, w i t h a p r e t r e a t m e n t i n j e c t i o n of l a b e l l e d thymidine t h e l e v e l of i n c o r p o r a t i o n i n t o DNA f o r e a c h a n i m a l c o u l d be d e t e r m i n e d . 3 1 4 The e f f e c t of a n i n t e r f e r i n g s u b s t a n c e c o u l d b e d e m o n s t r a t e d a t t h e t i m e of t h e second i n j e c t i o n of l a b e l l e d thymidine ( T a b l e 5 ) . The a d d i t i o n of " c o l d " thymidine t o t h e i n j e c t e d 3H-thymidine d e c r e a s e d t h e i n c o r p o r a t i o n of l a b e l i n t o DNA i n agreement w i t h known m e t a b o l i c e f f e c t s ( 6 , l l ) . DISCUSS I O N The e x p e r i m e n t a l u s e of t h e c o n t i n u o u s l y p e r f u s e d r a t l i v e r i n v i v o h a s been p r e v i o u s l y d e s c r i b e d ( 1 2 ) . I n t h i s s t u d y w e have shown t h a t t h e p o r t a l and t h e a r t e r i a l systems c a n be c o n c o m i t a n t l y p e r f u s e d i n r a t s i n c o m b i n a t i o n w i t h p a r t i a l hepatectomy. P a r t i a l hepatectomy s t i m u l a t e s t h y m i d i n e p h o s p h o r y l a t i o n and DNA s y n t h e s i s i n l i v e r c e l l s . Thus, t h e e x p e r i m e n t a l s y s t e m d e s c r i b e d i s of i n t e r e s t f o r i n v e s t i g a t i o n s of c e l l k i n e t i c s . Because of t h e d u a l blood f l o w t h e l i v e r i s more c o m p l i c a t e d t h a n o t h e r models. It o f f e r s , however, i n t e r e s t i n g p o s s i b i l i t i e s i n t h e s t u d y of e f f e c t s of blood f l o w r e s t r i c t i o n and compensa- t o r y mechanisms. I n t h e p r e s e n t s t u d y , i n f u s i o n was i n t e r r u p t e d 36 h o u r s p o s t o p e r a t i v e l y b u t i t c o u l d have b e e n c o n t i n u e d w i t h o u t d i f f i c u l t y . A t l a t e r times f o l l o w i n g p a r t i a l hepatectomy, however, o p e r a t e d r a t s seem t o b e no more s e n s i t i v e t o t h e e x p e r i m e n t a l s i t u a t i o n t h a n are o r d i n a r y r a t s , which t o l e r a t e i n f u s i o n q u i t e w e l l ( 1 2 ) . A p r o l o n g e d a n d c o m p l i c a t e d o p e r a t i v e p r o c e d u r e a n d / o r t h e p r e s e n c e of c a t h e t e r s a f f e c t e d body and l i v e r weight a s w e l l a s l i v e r c e l l meta- b o l i s m . L i v e r DNA s y n t h e s i s was d e c r e a s e d o r d e l a y e d and t h e v a r i a t i o n b e t - ween a n i m a l s i n c r e a s e d . These d i f f e r e n c e s show t h a t i t i s e s s e n t i a l t o u s e c a t h e t e r i z e d r a t s g i v e n sham i n j e c t i o n s a s c o n t r o l s . I n t h i s s t u d y t h e incon- s i s t a n c y of i n d i v i d u a l v a r i a t i o n w a s d e c r e a s e d when a n i m a l s were used as t h e i r own c o n t r o l s u s i n g t h e d o u b l e l a b e l l i n g t e c h n i q u e . The c h o i c e of l a b e l l e d t h y m i d i n e i n t h i s s e r i e s of e x p e r i m e n t s was r e l a t e d t o t h e v a s t documentation of t h e f a t e of t h i s p r e c u r s o r i n t h e r e g e n e r a t i n g l i v e r ( 1 3 ) . It i s w e l l known t h a t t h e d i u r n a l rhythm of t h e r a t i s r e f l e c t e d i n l i v e r m e t a b o l i s m d u r i n g t h e r e g e n e r a t i v e p r o c e s s (1) and t h a t a g e and stress f a c t o r s i n f l u e n c e DNA s y n t h e s i s ( 3 ) . We c o u l d p o s s i b l y have improved t h e synchrony o f l i v e r r e g e n e r a t i o n by u s i n g a r e g u l a r f e e d i n g s c h e d u l e (17) t h u s d e c r e a s i n g v a r i a t i o n i n t h y m i d i n e i n c o r p o r a t i o n between a n i m a l s . We do n o t 39 t h i n k , however, t h a t t h e v a r i a t i o n between t h e r a t s due t o c a t h e t e r i z a t i o n would b e d e c r e a s e d s i g n i f i c a n t l y . T h e r e f o r e , t h e d o u b l e l a b e l l i n g method s h o u l d b e a v a l u a b l e t o o l i n improving t h e s i g n i f i c a n c e of t e s t r e s u l t s . It i s p o s s i b l e t h a t t h e i n t e r v a l between i n j e c t i o n s c a n be p r o l o n g e d , b u t b e c a u s e of d i u r n a l v a r i a t i o n t h i s must b e i n v e s t i g a t e d f u r t h e r . High c o n c e n t r a t i o n s of "cold" t h y m i d i n e i n j e c t e d w i t h 3H-thymidine d i l u t e s t h e r a d i o a c t i v e m o l e c u l e s r e s u l t i n g i n d e c r e a s e d l a b e l l i n g o f DNA ( 1 1 ) . Thymidine a l s o d i s t u r b s d e o x y n u c l e o t i d e metabolism and n u c l e i c a c i d s y n t h e s i s r e s u l t i n g i n growth i n h i b i t i o n (6,111. The n e g a t i v e e f f e c t of added t h y m i d i n e on t h e i n c o r p o r a t i o n of 3H-thymidine i n t o DNA was obvious i n t h i s s t u d y . We c o n s i d e r DNA e s p e c i a l l y w e l l s i u t e d f o r t h e d e t e r - m i n a t i o n of f i n a l r e s u l t s of m e t a b o l i c a l t e r a t i o n s b e c a u s e of i t s v e r y low t u r n o v e r . Other compounds w i t h a low t u r n o v e r s u c h a s c e r t a i n RNAs and p r o t e i n s s h o u l d a l s o b e u s e f u l . 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Sehulte-Hermann, R.: Role o f F e e d i n g and L i g h t S c h e d u l e s f o r t h e Timing of t h e D i u r n a l Rhythm of C e l l P r o l i f e r a t i o n i n t h e L i v e r . In: L i v e r R e g e n e r a t i o n a f t e r E x p e r i m e n t a l I n j u r y (ed. L e s c h , R . & R e u t t e r , W . ) S t r a t t o n , NY, 1 9 7 5 . Address f o r r e p r i n t s : L Lewan, PhD Department of Zoophysiology Helgonavagen 3b S-223 62 LLJND Sweden 41