15.3. 1975
387
Specialia
T h e c o m b is b u i l t of steel needles, set i n t o a c o p p e r h e a d on a long h a n d l e (Figure 2). M e a s u r e m e n t s of t h e needles a r e : l e n g t h 25 m m , d i a m e t e r 0.5 ram, t i p d i a m e t e r 50-150 F m (according t o t h e n a t u r e of t r e a t e d tissue). T h e h e a d is 26 m m wide, 4 m m t h i c k . T h e h a n d l e is a b o u t 120 m m long. A space of 0.5 m m was left b e t w e e n e a c h t w o successive needles. T h e whole i n s t r u m e n t is c h r o m e nickel p l a t e d a n d c a n b e d r y - a i r sterilized. T h e c o m b s were used successfully to free t e r m i n a l villi a n d peeling s y n c y t i o t r o p h o b l a s t i c c o v e r i n g s f r o m h u m a n t e r m p l a c e n t a s , as t h e first step in a p r o c e d u r e d e v e l o p e d in o u r l a b o r a t o r y for i s o l a t i o n of s y n c y t i o t r o p h o b l a s t ~. R e c e n t l y t h e c o m b s were used successfully to segregate l y m p h a t i c tissue f r o m l y m p h a t i c o r g a n s i n o r d e r to free
i n d i v i d u a l l y m p h o i d cells. S i m i l a r l y conceived i n s t r u m e n t was a l r e a d y used for t e a s i n g l y m p h o c y t e s f r o m l y m p h n o d e s s; h o w e v e r , t h i s was b u i l t b y 2 a n d 3 needles o n l y a n d its efficiency was l i m i t e d . I t seems t h a t t h e d e s c r i b e d c o m b s m a y b e of h e l p in d i s s o c i a t i n g procedures, w h e r e a q u i c k s e p a r a t i o n of a l a r g e r a m o u n t of i n d i v i d u a l cells f r o m flexible tissues is necessary.
Rdsumd. Des peignes s p 6 c i a u x form6s d ' u n e r a n g 6 e d'aiguilles ins6r6es d a n s u n e poign6e o n t servi & dissocier des tissus. Ces peignes utilis6s p a r p a i r e o n lib6r6 r a p i d e m e n t de n o m b r e u s e s cellules p l a c e n t a i r e s et l y m p h a tiques intactes. T. KASPI
I T. KASPt and L. NEBEL, Obstet. Gynec. 43, 549 (1974). 2 B. R. BLOOM and B. BENNETT, in In Vitro Methods in CellMediated Immunity (Eds. B. R. BLOOMand P. R. GLADE; Academic Press, New York and London 1970), p. 248.
Department o/Embryology and Teratology, Tel Aviv University, Faculty o/ Medicine, Ch. Sheba Medical Center, Tel-Hashomer (Israel), 2 July 1974.
PRO EXPERIMENTIS
Factors Influencing the Serum Activity in Mice after Intravenous and Intraperitoneal Injection of ~4C Orotic Acid I
I n t r a v e n o u s i n j e c t i o n is easy to p e r f o r m in t h e r a t a n d gives reliable results. I n s m a l l e r a n i m a l s i.p. i n j e c t i o n is o f t e n preferred, b u t a t e c h n i q u e for i.v. i n j e c t i o n i n t o t h e tails of mice h a s b e e n d e s c r i b e d ~. D u r i n g a s t u d y of orotie acid i n c o r p o r a t i o n i n t o m o u s e l i v e r n u c l e o t i d e s a n d R N A , we f o u n d c o n s i d e r a b l e a m o u n t s of t h e isotope left in t h e tails a f t e r i n j e c t i o n i n t o t h e tail v e i n 2. W e also f o u n d it difficult to j u d g e t h e success of tail v e i n i n j e c t i o n s a n d w a n t e d a m o r e reliable c r i t e r i u m t h a n v i s u a l e x a m i n a t i o n . F u r t h e r m o r e , we f o u n d a 3-5-fold difference in s e r u m a c t i v i t y b e t w e e n i.p. a n d i.v. i n j e c t e d mice. D u e t o t h e i n s t a b i l i t y of t h e n u c l e o t i d e s d u r i n g anoxia, b l o o d h a d t o 10E
Series o--oa
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10~ i
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be s a m p l e d in t h e p e r i t o n e a l c a v i t y a f t e r l i v e r excision. B l o o d s a m p l e d in t h e p e r i t o n e a l c a v i t y s e e m e d t o be c o n t a m i n a t e d b y t h e i.p. a d m i n i s t e r e d isotope u p to 60 m i n a f t e r injection. I n o r d e r f u r t h e r t o e v a l u a t e t h e i n f l u e n c e of i n j e c t i o n t e c h n i q u e a n d b l o o d s a m p l i n g t e c h n i q u e on s e r u m a c t i v i t y in mice, we h a v e a n a l y z e d s e r u m f r o m b l o o d s a m p l e d in t h e p e r i t o n e a l c a v i t y or f r o m t h e b r a c h i a l vessels 2 t o 60 m i n a f t e r i.p. or i.v. a d m i n i s t r a t i o n of (6-1aC) orotic acid. Materials and methods. A n i m a l s . Male N M R I mice (from A n t i c i m e x , U p p s a l a , Sweden) w e i g h i n g 28-30 g were used. T h e y 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 r e g a r d i n g l i g h t a n d t e m p e r a t u r e a n d were g i v e n s t a n d a r d food a n d w a t e r a d l i b i t u m . A s s a y procedures. T h e isotope (6-14C) o r o t m acid, spec. a c t i v i t y 61 m C i / m M ( A m e r s h a m ) was a d m i n i s t e r e d i n 75 tzl 0.9% NaC1 (1.75 tzCi) as a single 15 sec i.v. or i.p. injection. T h e i n j e c t i o n of a n e x a c t v o l u m e of 75 [zl was a s s u r e d b y use of a r e p e a t i n g d i s p e n s e r ( H a m i l t o n Comp.). T h e a n i m a l s were sacrificed a t 2, 5, 10, 20 or 60 rain a f t e r t h e injection. A n o x y g e n - e t h e r a t m o s p h e r e was used to r e d u c e t i s s u e anoxia. Blood was collected e i t h e r in t h e p e r i t o n e u m a f t e r l i v e r excision or f r o m t h e b r a c h i a l vessels before l i v e r excision. T h e b l o o d was c o a g u l a t e d a n d t h e r a d i o a c t i v i t y in t h e acid soluble f r a c t i o n of t h e s e r u m was d e t e r m i n e d i n a P a c k a r d s c i n t i l l a t o r for 20 min. 3 a l t e r n a t i v e series were c o m p a r e d : a) i.p. i n j e c t i o n c o m b i n e d w i t h b l o o d s a m p l i n g f r o m t h e b r a c h i a l vessels; b) i.p. i n j e c t i o n c o m b i n e d w i t h b l o o d s a m p l i n g f r o m t h e p e r i t o n e a l c a v i t y a f t e r l i v e r excision; c) i.v. i n j e c t i o n in t h e t a i l c o m b i n e d w i t h blood s a m p l i n g f r o m t h e p e r i t o n e a l c a v i t y a f t e r l i v e r excision. T h e t a i l s were h y d r o l y z e d in 3 M K O H , a n d t h e r a d i o a c t i v i t y in e a c h acidified h y d r o l y s a t e was d e t e r m i n e d . Tails f r o m t a i l v e i n i n j e c t e d a n i m a l s were g e n t l y w a s h e d in w a t e r a f t e r b l o o d c o a g u l a t i o n a t t h e site of p u n c t u r e .
Time after injection
Cpm in total serum after injection of (6-t4C) orotic acid. Only the successfully injected animals ( + + + ) from series o were considered. Semilogarithmie scale.
1 S. BERGSTROM,Lab. Anim. Sci. 21, 600 (1971). L. LEWAN, I. PETERSEN and T. YNGNER,Hoppe Seyler's Z. physiol. Chem., in press (1975).
288
EXPERIENTI& 31[3
Specialia
Cpm in serum and tails of tail vein injected animals (series c) and in tails of i.p. injected animals (series * and b). Time after injection (rain}
Success rate b
cpm after i.v. injection ( • 10-~)
epm after i.p. injection ( • 10-s)
Serum c
Tail a
Tail Total
Washable
2 2 2 5 5 5 10 10 10
+ _u + + + + ++ +++ +++ 0 + + + + + 0
117 170 39 64 77 39 28 51 32
180 188 247 58 91 247 50 242 247
14 9 23 9 4 33 16 12 22
20 20 20 20
+ + + + + + 0
32 23 25 42
30 17 91 203
4 1 4 56
60 60 60 60
+ + + ++ + 0
14 11 14 12
41 57 49 78
13 1 3 29
3-5
6-8
7-9
4-5
2-3
The total dose injected was equivalent to 670 • 108 cpm. b + + +, The isotope was clearly flushed along the vein and at withdrawal of the eanula the vein was rapidly filled with blood. + +, After injection, vein filling;was slower and a certairt diseolouration of the tail was noticed. + , Vein filling after injeetiorL quite slow and clear diseolouration caused by a subcutaneous fluid buildup. 0, Very slow vein filling pronounced discolouration and outflow of isotope at the site of puncture, o 5.6 ml/100 g body weight% a Highest and lowest values found at analysis of 6 animals. T h e r a d i o a c t i v i t y of t h e w a t e r was m e a s u r e d t o e s t i m a t e t h e o u t f l o w of i s o t o p e f r o m t h e vein t o t h e outside of t h e tails a t w i t h d r a w a l of t h e canula. Results and discussion. A f t e r tail v e i n i n j e c t i o n large a m o u n t s of i s o t o p e r e m a i n e d in t h e tails a n d increased t h e r a d i o a c t i v i t y up t o 50 t i m e s t h a t f o u n d in tails f r o m i.p. i n j e c t e d mice (Table). A f t e r i.p. a d m i n i s t r a t i o n , t h e isotope r e m a i n e d in t h e p e r i t o n e a l c a v i t y a n d increased t h e r a d i o a c t i v i t y of Lp. s a m p l e d blood s e r u m as c o m p a r e d to s e r u m collected t r a m t h e b r a c h i a l vessels (Figure). "When a similar a m o u n t of orotic acid was a d m i n i s t e r e d i.p. in 200 ~1 i n s t e a d of in 75 al, t h e c o n t a m i n a t i o n b y u n a b s o r b e d isotope was still m o r e p r o n o u n c e d 2. Blood s a m p l e d f r o m t h e b r a c h i a l vessels of i.p. i n j e c t e d a n i m a l s a n d b l o o d s a m p l e d in t h e p e r i t o n e a l c a v i t y a f t e r tail v e i n i n j e c t i o n should n o t be c o n t a m i n a t e d b y local a c c u m u l a t i o n of isotope. T h e r a d i o a c t i v i t y f o u n d in t h e s e sera was in general q u i t e similar. H o w e v e r , a f t e r 2 rain t h e s e r u m a c t i v i t y was h i g h e r a f t e r i.v. i n j e c t i o n t h a n a f t e r i.p. injection. This m a y i n d i c a t e a m o r e r a p i d isotope d i s t r i b u t i o n a f t e r i.v. injection. A f t e r 20 a n d 60 rain t h e s e r u m a c t i v i t y was also h i g h e r a f t e r i.v. i n j e c t i o n t h a n a f t e r i.p, injection. A t t h e s e s t a g e s t h e a c c u m u l a t i o n of i s o t o p e in t h e tails, still o b v i o u s a t 60 min, m a y h a v e c o n t r i b u t e d t o a n increased level of s e r u m a c t i v i t y . T h e a t t e m p t t o j u d g e t h e success of t h e tail v e i n i n j e c t i o n s b y visual e x a m i n a t i o n is s h o w n in t h e Table. I n general, i n j e c t i o n s w h i c h were c o n s i d e r e d successful r e s u l t e d in h i g h s e r u m isotope levels a n d r e l a t i v e l y low levels in t h e tails 2 a n d 5 m i n a f t e r a d m i n i s t r a t i o n . A f t e r 10 t o 60 rain, s e r u m activities were also r e l a t i v e l y h i g h a f t e r unsuccessful injections. The h i g h r a d i o a c t i v i t y in t h e tails of t h e s e unsuccessfully i n j e c t e d a n i m a l s p o s s i b l y f u n c t i o n e d as a r e s e v o i r m a i n t a i n i n g a p r o p o r t i o n a l l y h i g h level of s e r u m a c t i v i t y . I t was possible t o w a s h only a m i n o r a m o u n t of t h e a d m i n i s t e r e d isotope f r o m t h e tails of i.v. i n j e c t e d animals, p r o v i n g t h a t t h e isotope m u s t h a v e b e e n t r a p p e d w i t h i n t h e tail tissue.
Our results show t h a t if t h e i n j e c t i o n v o l u m e s are small, a s u b s t a n c e m a y be a d m i n i s t e r e d e i t h e r i.v. or i.p. in mice w h e n a r a p i d d i s t r i b u t i o n is desirable. U n d e r c i r c u m s t a n c e s w h e r e a slow u p t a k e a n d d i s t r i b u t i o n are f a v o u r e d , t h e s u b s t a n c e should be g i v e n i.p. in a r e l a t i v e l y large volume. A slow u p t a k e a n d d i s t r i b u t i o n of t h e isotope can also be o b t a i n e d a f t e r i.m. i n j e c t i o n 3 or i n j e c t i o n of isotope a d s o r b e d o n t o a c t i v a t e d c h a r c o a l a. Care m u s t be t a k e n n o t to h a v e b l o o d s a m p l e s a n d p e r i t o n e a l organs cont a m i n a t e d b y local a c c u m u l a t i o n s of t h e i n j e c t e d subs t a n c e 3 W h e n anoxic effects are of no i m p o r t a n c e t o t h e e x p e r i m e n t a l results, b l o o d s a m p l i n g f r o m t h e brachial vessels, f r o m t h e r e t r o o r b i t a l sinus, t h r o u g h cardiac p u n c t u r e or b y r e s e c t i o n of t h e tail can be used. I t is, h o w e v e r , i m p o r t a n t t o h a v e in m i n d t h a t i s o t o p e a c t i v i t y m i g h t v a r y b e t w e e n s a m p l i n g sites due t o d i f f e r e n t tissue clearance c a p a c i t i e s a.
Zusammenfassung, l~C-Orots~ure w u r d e m i t zwei vers c h i e d e n e n I n j e k t i o n s m e t h o d e n in MXuse injiziert u n d die Serumaktivit~Lt n a c h 2, 5, 10, 20 u n d 60 rain gemessen. N a c h i.p. Inj e k t i o n w u r d e Blur, das aus der Bauchh61e n a c h E n t f e r n u n g der L e b e r e n t n o m m e n w o r d e n war, m i t n o c h n i c h t a b s o r b i e r t e m I s o t o p g e m i s c h t . Bei i.v. S c h w a n z i n j e k t i o n v e r b l i e b bei n i c h t p e r f e k t e r Inj e k t i o n eine e r h e b l i c h e Menge der Orots/iure i m S c h w a n z g e w e b e u n d ergab e i n e n k o n t i n u i e r l i c h e n I s o t o p e n z u s c h u s s z u m Serum. T. YNGNER, L. LEWAN a n d I. PETERSEN
Institute o/Zoophysiology, University of Lurid, Helgonaviigen 3b, S-223 62 Lund (Sweden), 22 October 1974. 3 IV[.G. ORD and L. A. STOCKEN, Biochem. J. 132, 47 (1973). 4 G. C. RvssEv and R. G. TASANEV,Analyt. Bioehem. 54, 115 {1973). 5 C. G. POTTER, Experientia 30, 25 (1974). 6 A. C. RICHES, J. G. S~ARP, D. B. THOMAS and S. V. SMITH, J. Physiol., Loud. 228, 279 (1973).
387
Specialia
T h e c o m b is b u i l t of steel needles, set i n t o a c o p p e r h e a d on a long h a n d l e (Figure 2). M e a s u r e m e n t s of t h e needles a r e : l e n g t h 25 m m , d i a m e t e r 0.5 ram, t i p d i a m e t e r 50-150 F m (according t o t h e n a t u r e of t r e a t e d tissue). T h e h e a d is 26 m m wide, 4 m m t h i c k . T h e h a n d l e is a b o u t 120 m m long. A space of 0.5 m m was left b e t w e e n e a c h t w o successive needles. T h e whole i n s t r u m e n t is c h r o m e nickel p l a t e d a n d c a n b e d r y - a i r sterilized. T h e c o m b s were used successfully to free t e r m i n a l villi a n d peeling s y n c y t i o t r o p h o b l a s t i c c o v e r i n g s f r o m h u m a n t e r m p l a c e n t a s , as t h e first step in a p r o c e d u r e d e v e l o p e d in o u r l a b o r a t o r y for i s o l a t i o n of s y n c y t i o t r o p h o b l a s t ~. R e c e n t l y t h e c o m b s were used successfully to segregate l y m p h a t i c tissue f r o m l y m p h a t i c o r g a n s i n o r d e r to free
i n d i v i d u a l l y m p h o i d cells. S i m i l a r l y conceived i n s t r u m e n t was a l r e a d y used for t e a s i n g l y m p h o c y t e s f r o m l y m p h n o d e s s; h o w e v e r , t h i s was b u i l t b y 2 a n d 3 needles o n l y a n d its efficiency was l i m i t e d . I t seems t h a t t h e d e s c r i b e d c o m b s m a y b e of h e l p in d i s s o c i a t i n g procedures, w h e r e a q u i c k s e p a r a t i o n of a l a r g e r a m o u n t of i n d i v i d u a l cells f r o m flexible tissues is necessary.
Rdsumd. Des peignes s p 6 c i a u x form6s d ' u n e r a n g 6 e d'aiguilles ins6r6es d a n s u n e poign6e o n t servi & dissocier des tissus. Ces peignes utilis6s p a r p a i r e o n lib6r6 r a p i d e m e n t de n o m b r e u s e s cellules p l a c e n t a i r e s et l y m p h a tiques intactes. T. KASPI
I T. KASPt and L. NEBEL, Obstet. Gynec. 43, 549 (1974). 2 B. R. BLOOM and B. BENNETT, in In Vitro Methods in CellMediated Immunity (Eds. B. R. BLOOMand P. R. GLADE; Academic Press, New York and London 1970), p. 248.
Department o/Embryology and Teratology, Tel Aviv University, Faculty o/ Medicine, Ch. Sheba Medical Center, Tel-Hashomer (Israel), 2 July 1974.
PRO EXPERIMENTIS
Factors Influencing the Serum Activity in Mice after Intravenous and Intraperitoneal Injection of ~4C Orotic Acid I
I n t r a v e n o u s i n j e c t i o n is easy to p e r f o r m in t h e r a t a n d gives reliable results. I n s m a l l e r a n i m a l s i.p. i n j e c t i o n is o f t e n preferred, b u t a t e c h n i q u e for i.v. i n j e c t i o n i n t o t h e tails of mice h a s b e e n d e s c r i b e d ~. D u r i n g a s t u d y of orotie acid i n c o r p o r a t i o n i n t o m o u s e l i v e r n u c l e o t i d e s a n d R N A , we f o u n d c o n s i d e r a b l e a m o u n t s of t h e isotope left in t h e tails a f t e r i n j e c t i o n i n t o t h e tail v e i n 2. W e also f o u n d it difficult to j u d g e t h e success of tail v e i n i n j e c t i o n s a n d w a n t e d a m o r e reliable c r i t e r i u m t h a n v i s u a l e x a m i n a t i o n . F u r t h e r m o r e , we f o u n d a 3-5-fold difference in s e r u m a c t i v i t y b e t w e e n i.p. a n d i.v. i n j e c t e d mice. D u e t o t h e i n s t a b i l i t y of t h e n u c l e o t i d e s d u r i n g anoxia, b l o o d h a d t o 10E
Series o--oa
,'--,b
10[
co
E o
10~ i
~
i
i
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i
60rain
be s a m p l e d in t h e p e r i t o n e a l c a v i t y a f t e r l i v e r excision. B l o o d s a m p l e d in t h e p e r i t o n e a l c a v i t y s e e m e d t o be c o n t a m i n a t e d b y t h e i.p. a d m i n i s t e r e d isotope u p to 60 m i n a f t e r injection. I n o r d e r f u r t h e r t o e v a l u a t e t h e i n f l u e n c e of i n j e c t i o n t e c h n i q u e a n d b l o o d s a m p l i n g t e c h n i q u e on s e r u m a c t i v i t y in mice, we h a v e a n a l y z e d s e r u m f r o m b l o o d s a m p l e d in t h e p e r i t o n e a l c a v i t y or f r o m t h e b r a c h i a l vessels 2 t o 60 m i n a f t e r i.p. or i.v. a d m i n i s t r a t i o n of (6-1aC) orotic acid. Materials and methods. A n i m a l s . Male N M R I mice (from A n t i c i m e x , U p p s a l a , Sweden) w e i g h i n g 28-30 g were used. T h e y 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 r e g a r d i n g l i g h t a n d t e m p e r a t u r e a n d were g i v e n s t a n d a r d food a n d w a t e r a d l i b i t u m . A s s a y procedures. T h e isotope (6-14C) o r o t m acid, spec. a c t i v i t y 61 m C i / m M ( A m e r s h a m ) was a d m i n i s t e r e d i n 75 tzl 0.9% NaC1 (1.75 tzCi) as a single 15 sec i.v. or i.p. injection. T h e i n j e c t i o n of a n e x a c t v o l u m e of 75 [zl was a s s u r e d b y use of a r e p e a t i n g d i s p e n s e r ( H a m i l t o n Comp.). T h e a n i m a l s were sacrificed a t 2, 5, 10, 20 or 60 rain a f t e r t h e injection. A n o x y g e n - e t h e r a t m o s p h e r e was used to r e d u c e t i s s u e anoxia. Blood was collected e i t h e r in t h e p e r i t o n e u m a f t e r l i v e r excision or f r o m t h e b r a c h i a l vessels before l i v e r excision. T h e b l o o d was c o a g u l a t e d a n d t h e r a d i o a c t i v i t y in t h e acid soluble f r a c t i o n of t h e s e r u m was d e t e r m i n e d i n a P a c k a r d s c i n t i l l a t o r for 20 min. 3 a l t e r n a t i v e series were c o m p a r e d : a) i.p. i n j e c t i o n c o m b i n e d w i t h b l o o d s a m p l i n g f r o m t h e b r a c h i a l vessels; b) i.p. i n j e c t i o n c o m b i n e d w i t h b l o o d s a m p l i n g f r o m t h e p e r i t o n e a l c a v i t y a f t e r l i v e r excision; c) i.v. i n j e c t i o n in t h e t a i l c o m b i n e d w i t h blood s a m p l i n g f r o m t h e p e r i t o n e a l c a v i t y a f t e r l i v e r excision. T h e t a i l s were h y d r o l y z e d in 3 M K O H , a n d t h e r a d i o a c t i v i t y in e a c h acidified h y d r o l y s a t e was d e t e r m i n e d . Tails f r o m t a i l v e i n i n j e c t e d a n i m a l s were g e n t l y w a s h e d in w a t e r a f t e r b l o o d c o a g u l a t i o n a t t h e site of p u n c t u r e .
Time after injection
Cpm in total serum after injection of (6-t4C) orotic acid. Only the successfully injected animals ( + + + ) from series o were considered. Semilogarithmie scale.
1 S. BERGSTROM,Lab. Anim. Sci. 21, 600 (1971). L. LEWAN, I. PETERSEN and T. YNGNER,Hoppe Seyler's Z. physiol. Chem., in press (1975).
288
EXPERIENTI& 31[3
Specialia
Cpm in serum and tails of tail vein injected animals (series c) and in tails of i.p. injected animals (series * and b). Time after injection (rain}
Success rate b
cpm after i.v. injection ( • 10-~)
epm after i.p. injection ( • 10-s)
Serum c
Tail a
Tail Total
Washable
2 2 2 5 5 5 10 10 10
+ _u + + + + ++ +++ +++ 0 + + + + + 0
117 170 39 64 77 39 28 51 32
180 188 247 58 91 247 50 242 247
14 9 23 9 4 33 16 12 22
20 20 20 20
+ + + + + + 0
32 23 25 42
30 17 91 203
4 1 4 56
60 60 60 60
+ + + ++ + 0
14 11 14 12
41 57 49 78
13 1 3 29
3-5
6-8
7-9
4-5
2-3
The total dose injected was equivalent to 670 • 108 cpm. b + + +, The isotope was clearly flushed along the vein and at withdrawal of the eanula the vein was rapidly filled with blood. + +, After injection, vein filling;was slower and a certairt diseolouration of the tail was noticed. + , Vein filling after injeetiorL quite slow and clear diseolouration caused by a subcutaneous fluid buildup. 0, Very slow vein filling pronounced discolouration and outflow of isotope at the site of puncture, o 5.6 ml/100 g body weight% a Highest and lowest values found at analysis of 6 animals. T h e r a d i o a c t i v i t y of t h e w a t e r was m e a s u r e d t o e s t i m a t e t h e o u t f l o w of i s o t o p e f r o m t h e vein t o t h e outside of t h e tails a t w i t h d r a w a l of t h e canula. Results and discussion. A f t e r tail v e i n i n j e c t i o n large a m o u n t s of i s o t o p e r e m a i n e d in t h e tails a n d increased t h e r a d i o a c t i v i t y up t o 50 t i m e s t h a t f o u n d in tails f r o m i.p. i n j e c t e d mice (Table). A f t e r i.p. a d m i n i s t r a t i o n , t h e isotope r e m a i n e d in t h e p e r i t o n e a l c a v i t y a n d increased t h e r a d i o a c t i v i t y of Lp. s a m p l e d blood s e r u m as c o m p a r e d to s e r u m collected t r a m t h e b r a c h i a l vessels (Figure). "When a similar a m o u n t of orotic acid was a d m i n i s t e r e d i.p. in 200 ~1 i n s t e a d of in 75 al, t h e c o n t a m i n a t i o n b y u n a b s o r b e d isotope was still m o r e p r o n o u n c e d 2. Blood s a m p l e d f r o m t h e b r a c h i a l vessels of i.p. i n j e c t e d a n i m a l s a n d b l o o d s a m p l e d in t h e p e r i t o n e a l c a v i t y a f t e r tail v e i n i n j e c t i o n should n o t be c o n t a m i n a t e d b y local a c c u m u l a t i o n of isotope. T h e r a d i o a c t i v i t y f o u n d in t h e s e sera was in general q u i t e similar. H o w e v e r , a f t e r 2 rain t h e s e r u m a c t i v i t y was h i g h e r a f t e r i.v. i n j e c t i o n t h a n a f t e r i.p. injection. This m a y i n d i c a t e a m o r e r a p i d isotope d i s t r i b u t i o n a f t e r i.v. injection. A f t e r 20 a n d 60 rain t h e s e r u m a c t i v i t y was also h i g h e r a f t e r i.v. i n j e c t i o n t h a n a f t e r i.p, injection. A t t h e s e s t a g e s t h e a c c u m u l a t i o n of i s o t o p e in t h e tails, still o b v i o u s a t 60 min, m a y h a v e c o n t r i b u t e d t o a n increased level of s e r u m a c t i v i t y . T h e a t t e m p t t o j u d g e t h e success of t h e tail v e i n i n j e c t i o n s b y visual e x a m i n a t i o n is s h o w n in t h e Table. I n general, i n j e c t i o n s w h i c h were c o n s i d e r e d successful r e s u l t e d in h i g h s e r u m isotope levels a n d r e l a t i v e l y low levels in t h e tails 2 a n d 5 m i n a f t e r a d m i n i s t r a t i o n . A f t e r 10 t o 60 rain, s e r u m activities were also r e l a t i v e l y h i g h a f t e r unsuccessful injections. The h i g h r a d i o a c t i v i t y in t h e tails of t h e s e unsuccessfully i n j e c t e d a n i m a l s p o s s i b l y f u n c t i o n e d as a r e s e v o i r m a i n t a i n i n g a p r o p o r t i o n a l l y h i g h level of s e r u m a c t i v i t y . I t was possible t o w a s h only a m i n o r a m o u n t of t h e a d m i n i s t e r e d isotope f r o m t h e tails of i.v. i n j e c t e d animals, p r o v i n g t h a t t h e isotope m u s t h a v e b e e n t r a p p e d w i t h i n t h e tail tissue.
Our results show t h a t if t h e i n j e c t i o n v o l u m e s are small, a s u b s t a n c e m a y be a d m i n i s t e r e d e i t h e r i.v. or i.p. in mice w h e n a r a p i d d i s t r i b u t i o n is desirable. U n d e r c i r c u m s t a n c e s w h e r e a slow u p t a k e a n d d i s t r i b u t i o n are f a v o u r e d , t h e s u b s t a n c e should be g i v e n i.p. in a r e l a t i v e l y large volume. A slow u p t a k e a n d d i s t r i b u t i o n of t h e isotope can also be o b t a i n e d a f t e r i.m. i n j e c t i o n 3 or i n j e c t i o n of isotope a d s o r b e d o n t o a c t i v a t e d c h a r c o a l a. Care m u s t be t a k e n n o t to h a v e b l o o d s a m p l e s a n d p e r i t o n e a l organs cont a m i n a t e d b y local a c c u m u l a t i o n s of t h e i n j e c t e d subs t a n c e 3 W h e n anoxic effects are of no i m p o r t a n c e t o t h e e x p e r i m e n t a l results, b l o o d s a m p l i n g f r o m t h e brachial vessels, f r o m t h e r e t r o o r b i t a l sinus, t h r o u g h cardiac p u n c t u r e or b y r e s e c t i o n of t h e tail can be used. I t is, h o w e v e r , i m p o r t a n t t o h a v e in m i n d t h a t i s o t o p e a c t i v i t y m i g h t v a r y b e t w e e n s a m p l i n g sites due t o d i f f e r e n t tissue clearance c a p a c i t i e s a.
Zusammenfassung, l~C-Orots~ure w u r d e m i t zwei vers c h i e d e n e n I n j e k t i o n s m e t h o d e n in MXuse injiziert u n d die Serumaktivit~Lt n a c h 2, 5, 10, 20 u n d 60 rain gemessen. N a c h i.p. Inj e k t i o n w u r d e Blur, das aus der Bauchh61e n a c h E n t f e r n u n g der L e b e r e n t n o m m e n w o r d e n war, m i t n o c h n i c h t a b s o r b i e r t e m I s o t o p g e m i s c h t . Bei i.v. S c h w a n z i n j e k t i o n v e r b l i e b bei n i c h t p e r f e k t e r Inj e k t i o n eine e r h e b l i c h e Menge der Orots/iure i m S c h w a n z g e w e b e u n d ergab e i n e n k o n t i n u i e r l i c h e n I s o t o p e n z u s c h u s s z u m Serum. T. YNGNER, L. LEWAN a n d I. PETERSEN
Institute o/Zoophysiology, University of Lurid, Helgonaviigen 3b, S-223 62 Lund (Sweden), 22 October 1974. 3 IV[.G. ORD and L. A. STOCKEN, Biochem. J. 132, 47 (1973). 4 G. C. RvssEv and R. G. TASANEV,Analyt. Bioehem. 54, 115 {1973). 5 C. G. POTTER, Experientia 30, 25 (1974). 6 A. C. RICHES, J. G. S~ARP, D. B. THOMAS and S. V. SMITH, J. Physiol., Loud. 228, 279 (1973).
