Upsala J M e d Sci 87: 99-109, 1982 The Amount of Adenine Nucleotides and Glycolytic Intermediates in Erythrocytes, Liver and Muscle Tissue Correlated with the Body Weight (Age ) in Wistar Rats Magnus H j e l m and Costa Arturson From the Department of Chernicul P a t h o l o g y , lnstitiite of C h i l l Htwlth tind H o s p i t a l for Sick Children, London. United Kingdom and the Biirn Center. University H o s p i t a l , Uppsalti, S w d e n ABSTRACT The c o n c e n t r a t i o n s o f a d e n i n e n u c l e o t i d e s i n t h e l i v e r and s k e l e t a l muscle i n c r e a s e d d u r i n g t h e m a t u r a t i o n p e r i o d ( d u r i n g t h e f i r s t 100 days o f l i f e ) and remained f a i r l y c o n s t a n t i n a d u l t r a t s . The c o n c e n t r a t i o n o f a d e n i n e n u c l e o - t i d e s i n e r y t h r o c y t e s d e c r e a s e d w i t h age. The c o n c e n t r a t i o n s o f hexose monophosphates i n c r e a s e d w i t h age i n l i v e r and The o b s e r v e d changes of m e t a b o l i t e s i n t h e d i f f e r e n t t i s s u e s w i t h i n c r e a s - m u s c l e t i s s u e and decreased w i t h age i n e r y t h r o c y t e s . i n g age o f r a t s c o u l d n o t be e x p l a i n e d by a changed w a t e r c o n t e n t . INTRODUCTION I n f o r m a t i o n a b o u t t h e n a t u r e and m a g n i t u d e o f age dependent changes o f t i s s u e m e t a b o l i t e s i s s c a n t y , and as concerns a d e n i n e n u c l e o t i d e s and g l y c o - l y t i c i n t e n n e d i a t e s a l m o s t l a c k i n g . Thus a number o f i n t r a c e l l u l a r m e t a b o l i t e s : a d e n i n e n u c l e o t i d e s (ATP, ADP, AMP), g l u c o s e 6-phosphate (G-6-P), f r u c t o s e 6-phosphate (F-6-P), were d e t e r m i n e d i n l i v e r and s k e l e t a l muscle and i n e r y t h - r o c y t e s , r e s p e c t i v e l y . The amount o f t h e m e t a b o l i t e s was p r i m a r i l y c o r r e l a t e d w i t h t h e body w e i g h t . S t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n s were t r a n s f o r m e d i n t o c u r v e s showing t h e r e l a t i o n s h i p between t h e amount o f t i s s u e m e t a b o l i t e and age, where age was de- r i v e d f r o m t h e mean c o r r e l a t i o n c u r v e between body w e i g h t and age f o r t h e t y p e o f r a t used. MATERIAL W h i t e male r a t s o f W i s t a r s t r a i n were used. D u r i n g t h e e x p e r i m e n t s a l l r a t s were k e p t s i n g l e i n m e t a b o l i c cages and d a i l y measurements o f body w e i g h t and t h e amount o f f o o d and w a t e r consumed were made. They were f e d on a s t a n d a r d d i e t supplemented w i t h v i t a m i n s , w i t h a c a l o r i c c o n t e n t o f 51 p e r c e n t c a r b o - h y d r a t e , 32 p e r c e n t f a t and 17 p e r c e n t p r o t e i n . The a n i m a l s r e c e i v e d food ad l i b i t u m and t h e y had f r e e access t o w a t e r . A l l r a t s were s t a r v e d 24 h o u r s 99 p r i o r t o s a c r i f i c e . The r a t s were divided in two groups: 4 9 animals were used f o r t h e determina- t i o n o f i n t r a c e l l u l a r m e t a b o l i t e s i n l i v e r and s k e l e t a l muscle and i n e r y t h r o - c y t e s . 17 animals were used f o r the measurement of the a p p a r e n t c e l l water i n the same type o f c e l l s . METHODS E t h e r a n a e s t h e s i a and removal of t i s s u e Rapid, l i g h t e t h e r a n a e s t h e s i a a s s o c i a t e d w i t h minimal d i s t u r b a n c e of t h e r a t s was achieved. The l i v e r was exposed through a m i d - l i n e , abdominal i n c i s i o n . Small p i e c e s of l i v e r were removed u s i n g precooled s t a i n l e s s s t e e l tongs which were immersed i n l i q u i d n i t r o g e n . Tissue from M . quadriceps femoris was taken by t h e same sampling procedure. 25-50 mg of l i v e r and muscle t i s s u e was o b t a i n - e d . The time r e q u i r e d f o r t h e i n c i s i o n t o t h e sampling v a r i e d between 15-30 s e - conds, on t h e average f a s t e r f o r t h e l i v e r . Blood (about 1 m l ) was c o l l e c t e d from t h e h e a r t by a s y r i n g e and immediately t r a n s f e r r e d i n t o 5 ml of 6 % ( v / v ) i c e c o l d p e r c h l o r i c a c i d . In o r d e r t o study t h e accuracy of t h e sampling procedure b i o p s i e s a l s o were s t o r e d i n v i t r o during c e r t a i n time i n t e r v a l s b e f o r e f r e e z i n g t h e specimen i n l i q u i d n i t r o g e n . E x t r a c t i o n of t i s s u e The frozen t i s s u e was prepared f o r a n a l y s i s of t h e d i f f e r e n t s u b s t a n c e s ac- cording t o Dale ( 5 ) e x c e p t t h a t l i q u i d n i t r o g e n was used i n s t e a d of l i q u i d ox- ygen and t h a t t h e p e r c h l o r i c a c i d e x t r a c t of the d i f f e r e n t t i s s u e s was brought t o pH 6 . 5 i n s t e a d of pH 3.5. Chemi c a l methods A T P , A D P and AMP were determined according t o Adam ( 1 ) by enzymatic methods a n d with s p e c t r o p h o t o m e t r i c r e a d i n g s . The methods a r e not a b s o l u t e s p e c i f i c f o r A T P , A D P and AMP as a l s o o t h e r t r i - , d i - and mononucleotides, i f p r e s e n t , may r e a c t . The e r r o r i n t r o d u c e d in t h e a c t u a l values a r e probably of minor import- a n c e , however, as t h e amount of ATP i n p e r c h l o r i c a c i d e x t r a c t s of l i v e r t i s s u e determined by t h e method given above and c o n t r o l l e d by t h e more s p e c i f i c hexo- k i n a s e methods ( 1 7 ) d i d n o t d i f f e r s i g n i f i c a n t l y ( M . 1 0 , n = 1 0 ) . G-6-P and F-6-P were determined e n z y m a t i c a l l y ( 1 4 ) and w i t h f l u o r i m e t r i c r e a d i n g s . Glycogen was converted t o glucose ( 2 1 ) a n d t h e glucose c o n c e n t r a t i o n was de- termined with t h e glucose o x i d a s e method of Hjelm and de Verdier ( 1 3 ) . Haemo- g l o b i n was determined by t h e cyanomethaemoglobin method and h a e m a t o c r i t values were o b t a i n e d with an I n t e r n a t i o n a l Micro-Capillary C e n t r i f u g e Model M B , I n t e r - n a t i o n a l Equipment Company, Nedham, HTS, Mass., USA. The h a e m a t o c r i t values were n o t c o r r e c t e d f o r t r a p p e d plasma ( 7 ) . The amount o f " f r e e - w a t e r " was e s t i m a t e d i n l i v e r and m u s c l e t i s s u e s by means o f d e h y d r a t i n g t i s s u e p i e c e s o f a p p r o x i m a t e l y e q u a l s i z e t o c o n s t a n t w e i g h t a t a t e m p e r a t u r e o f +105OC i n a t h e r m o s t a t c o n t a i n i n g a w a t e r a b s o r b i n g medi urn. C a l c u l a t i o n s The s t a t i s t i c a l method used f o r c o r r e l a t i o n s between m e t a b o l i t e s and body w e i g h t was p i e c e - w i s e l i n e a r r e g r e s s i o n ( 8 ) . The r e l a t i o n s h i p between age and body w e i g h t e s t a b l i s h e d by t h e l o c a l b r e e d e r of t h e M i s t a r r a t s , was used t o t r a n s f o r m t h e e x p e r i m e n t a l l y o b t a i n e d r e l a t i o n s h i p between t h e c o n t e n t o f meta- b o l i t e s and body w e i g h t i n t o one between t h e c o n t e n t of t h e m e t a b o l i t e s and age. RESULTS AND D I S C U S S I O N 1. M e t h o d o l o g i c a l a s p e c t s on t h e p r o c e d u r e f o r s a m p l i n g o f t i s s u e s The q u i t e c o n s i d e r a b l e change o f t h e c o n c e n t r a t i o n o f a number o f metabo- l i t e s i n l i v e r b i o p s i e s w i t h i n seconds i s shown i n F i g . 1. 2oor % loo+ 200 - 10 20 2ooL 100 Time, sec 200 r 100 P 200 10 20 Time, sec 10 20 Time, sec 100 10 20 Time ,sec F i g . 1. C o n c e n t r a t i o n s o f ATP, ADP, AMP, G-6-P and F-6-P i n l i v e r b i o p s i e s r e - moved u s i n g p r e c o o l e d s t a i n l e s s s t e e l t o n g s ( = l o 0 %) and t a k e n w i t h k n i f e and immersed i n l i q u i d n i t r o g e n a f t e r 3,5, 7, 10, 15 and 20 seconds r e s p e c t i v e l y . 101 The r e s u l t s t r e s s e s t h e i m p o r t a n c e o f an a c c u r a t e s a m p l i n g t e c h n i q u e o f t i s s u e s w i t h a r a p i d t u r n o v e r o f m e t a b o l i t e s i n o r d e r t o o b t a i n r e l e v a n t i n f o r m a t i o n a b o u t t h e c o n d i t i o n s i n v i v o . The i n c o n s i s t e n c y between v a l u e s f o r some t i s s u e m e t a b o l i t e s i n r a t s o f c o m p a r a t i v e w e i g h t s , c o m p i l e s f r o m t h e l i t e r a t u r e i n T a b l e 1, i n d i c a t e s t h a t t h e s a m p l i n g o f t i s s u e s m i g h t n o t always have been ac- c u r a t e and t h a t e a r l i e r r e s u l t s c a n n o t d i r e c t l y be used as r e f e r e n c e v a l u e s . T a b l e 1. The average c o n c e n t r a t i o n s o f a d e n i n e n u c l e o t i d e s i n m u s c l e t i s s u e , -issue and e r y t h r o c y t e s i n r a t s and some o t h e r s p e c i e s . (Range o f body w e i g h t 100-200). ~~ T i s s u e ATP ADP AMP ZPTP, ADP, AMP 1 ) Muscle ti s s ue I m a i , R i l e y & Berne ( 1 9 6 4 ) Pedersen & Sachs (1965) Scopes & Newbold (1968) Chaudry, Sayeed & Baue (1974) Berne & Rubio (1974) P r e s e n t i n v e s t i g a t i o n L i ve r t i s s ue Maass & Timm (1964) Kolousek, J i r a c e k , Z i c h a e t a1 (1965) Hems, Ross, B e r r y e t a1 (1966) Puddu, C a l d a r e r a & M a r c h e t t i (1967) Chaudry, Sayeed & Baue ( 1 9 7 4 ) Hirasawa, Chaudry & Baue (1978) Ozawa e t a1 (1981) P r e s e n t i n v e s t i g a t i o n E r y t h r o c y t e s ’ ) * ) Kolousek, J i r a c e k , Z i c h a e t a1 (1965) P r e s e n t i n v e s t i g a t i o n l ) 6 . 3 0.76 0.29 7.4 4.8 - - - 4 . 5 1.3 0.11 5.9 3.6 0.6 0.07 4.3 5.43 1.29 0.21 6.9 4.8 0.9 0.1 5.8 2.3 0.78 - 3.2 1.71 0.57 2.5 0.94 0.21 0.33 0.90 2.1 1.85 0.82 0.25 2.47 0.73 0.14 2.38 0.55 0.14 3.0 1.2 0.4 1.57 0.17 0.05 1.9 0.4 - - 5.4 3.7 3.3 2.9 3.3 3.1 4.6 1 . 8 - Moles x m u s c l e t i s s ue p e r m l o f e r y t h r o c y t e s o r p e r gram o f w e t w e i g h t o f l i v e r o r ’) The average h a e m a t o c r i t v a l u e assumed t o be 40 % 102 AT P moles 1061g we! weight L i v e , 3.5 3 . 0 - 2.0- 1.0- - - - - - _ / * I 100 200 300 g Body weigh! I A T P , A D P , A M P moles * I06/g of wet weight L l W , 2 . 5 - 2 . 0 - 1.5- 1.0- L . 0 - 3.0- 100 200 300 g Body weigh! 20 30 LO S O 6 0 7 0 A g e , days A T P m101es. 10s/q we! weight Muscle I * \ . \ 8 , 3.0- 2.0- 1.0- F i g . 2 . Concentrations of ATP and t h e sum of ATP, ADP and AMP i n l i v e r , s k e l e t a l muscle and e r y t h r o c y t e s c o r - r e l a t e d with body weight ( a g e ) . Re- g r e s s i o n l i n e w i t h 95 % confidence i n t e r v a l and 95 % t o l e r a n c e l i m i t f o r t h e measurements i s i n d i c a t e d . :: * ,' ,' 3.0 I \ ;\. Erythrocytes \ \ ._ - . \ 100 2 0 0 300 9 Body weight I ATP, A D P , A M P moles .106/rnl of erythrocytes Erythrocytes I '--- - - - - ~ - 100 2 0 0 300 g Body weight 2 0 30 LO 5 0 6 0 i o A g e . days 100 200 300 g Body weigh! 2 0 30 LO 50 6 0 7 0 A g e , d a y s 103 2 . T h e c o n c e n t r a t i o n of t i s s u e m e t a b o l i t e s c o r r e l a t e d w i t h body weight ( a g e ) 2.1. Adenine n u c l e o t i d e s i n muscle and l i v e r t i s s u e and i n e r y t h r o c y t e s The c o n c e n t r a t i o n of ATP i n t h e l i v e r i n c r e a s e d s i g n i f i c a n t l y during t h e ma- t u r a t i o n p e r i o d , i . e . during t h e f j r s t 100 days o f l i f e , and remained f a i r l y c o n s t a n t i n a d u l t r a t s ( F i g . 2 ) . P r e l i m i n a r y s t u d i e s i n r a t s showed an i n c r e a s - ed number of mitochondria i n l i v e r t i s s u e from animals w i t h a body weight of 400 g compared t o 60 g. The observed i n c r e a s e of t h e c o n t e n t o f ATP i n t h e l i v - e r of maturing r a t s may a c c o r d i n g l y be d u e t o an i n c r e a s e d number of mitochon- d r i a . The observed changes o f A D P and AMP and o f t h e sum of adenine n u c l e o t i d e s w i t h age ( F i g . 2 ) could a c c o r d i n g l y be secondary t o keeping t h e over a l l e q u i - l i b r i u m o f t h e a d e n y l a t e kinase reaction,(ATP x A M P ) / ( A D P ) 2 a t a c o n s t a n t l e v e l . of adenine n u c l e o t i d e s i n muscle t i s s u e changed with age i n a s i m i l a r way a s i n the l i v e r . I t i s , however, n o t known i f t h e number of mitochondria i n muscle t i s s u e v a r i e s with age. The s i t u a t i o n i s a l s o complicat- ed by t h e f a c t t h a t muscle t i s s u e i s composed of muscle f i b r e s of d i f f e r e n t t y p e s , which might c o n t a i n the same o r a d i f f e r e n t number of mitochondria per u n i t volume of tissue. T h u s a change of t h e r e l a t i v e frequency o f t h e types of muscle f i b r e s w i t h age m i g h t e x p l a i n t h e observed change of t h e over a l l con- tent of ATP. Work i s i n p r o g r e s s t o c l a r i f y t h i s problem. The c o n c e n t r a t i o n The c o n c e n t r a t i o n o f adenine n u c l e o t i d e s i n e r y t h r o c y t e s decreased slowly d u r i n g the maturation p e r i o d . In human e r y t h r o c y t e s HbF has a h i g h e r a f f i n i t y t o 2 , 3 diphosphoglycerate than HbA ( 6 ) . I n t a c t e r y t h r o c y t e s i n neonates c o n t a i n about twice as much of t h e phosphocompound a s i n a d u l t s t o a s s u r e t h e same oxy- gen r e l e a s e c a p a c i t y of e r y t h r o c y t e s i n neonates and a d u l t s ( 1 1 , 1 2 ) . The s i t u - a t i o n might be analogous f o r t h e binding o f ATP t o a f e t a l and a d u l t type of r a t haemoglobin. Then t h e d e c r e a s i n g c o n t e n t of ATP i n e r y t h r o c y t e s from young r a t s could be e x p l a i n e d by a concomitant d e c r e a s e of f e t a l r a t haemoglobin. However, l i t t l e i s known about r a t haemoglobins a t p r e s e n t and e s p e c i a l l y about t h e binding of o r g a n i c phosphocompounds t o t h e molecule. More experimental work i s necessary t o c l a r i f y t h e s i t u a t i o n . I t does n o t seem l i k e l y , however, t h a t the decreased c o n t e n t of ATP i s r e l a t e d t o a changed mean age of t h e c i r c u l a t - i n g e r y t h r o c y t e p o p u l a t i o n ( c f . 1 2 ) . n y l a t e kinase reaction,(ATP x A M P ) / ( A D P ) t o d e c r e a s e w i t h age in l i v e r and mus- c l e tissue, whereas the energy charge o f t h e a d e n y l a t e pool c a l c u l a t e d accord- i n g t o Atkinson ( 2 ) : ( A T P + 0.5 A D P ) / ( A T P + A D P + AMP) s t a y e d c o n s t a n t . No o b - vious changes of t h e s e two parameters w i t h age could be observed i n e r y t h r o c y t - e s ( F i g . 3 ) . There was a t r e n d f o r t h e apparent e u i l i b r i u m c o n s t a n t , K , f o r t h e ade- 1 104 O ’ l F i g . 3. The a p p a r e n t e q u i l i b r i u m c o n s t a n t , K , f o r t h e a d e n y l a t e kinase r e a c t i o n ( A T P x A M P ) / ( A D P ) 2 and t h e energy charge of t h e a d e n y l a t e pool i n l i v e r , s k e l e - t a l muscle and e r y t h r o c y t e s c o r r e l a t e d with body weight ( a g e ) . The t o t a l v a r i a n c e of A T P , A D P and AMP and of t h e a p p a r e n t e q u i l i b r i u m c o n s t a n t , K , around t h e r e g r e s s i o n l i n e s a g a i n s t age showed a c o n s i d e r a b l e v a r i a n c e , i n t h e o r d e r o f 2 10 t o t 15 % i n a l l t i s s u e s s t u d i e d ( F i g . 2 and 3 ) . The t o t a l v a r i a n c e around the r e g r e s s i o n l i n e f o r the energy charge a g a i n s t age i s con- s i d e r a b l y less and i n t h e o r d e r of ? 5 % ( F i g . 3 ) . A major p a r t of t h i s v a r i - ance could be e x p l a i n e d by f a c t o r s r e l a t e d t o t h e handling o f the specimens and t h e a n a l y t i c a l procedure. I f t h i s assumption i s c o r r e c t t h e i n t e r - i n d i v i d u a l ( b i o l o g i c a l ) v a r i a n c e o f t h e energy charge must be extremely low. I t a l s o f o l - lows t h a t the c o n s i d e r a b l e i n t e r - i n d i v i d u a l v a r i a n c e observed f o r t h e i n d i v i d u - a l adenine n u c l e o t i d e s could r e f l e c t a homeostatic mechanism by which the de- mand of keeping t h e energy charge w i t h i n a narrow a b s o l u t e l i m i t i s l i n k e d t o a varying i n t e r m e d i a r y energy metabolism, a d j u s t e d t o meet i n d i v i d u a l demands. I t s h o u l d , however, be p o i n t e d o u t t h a t t h e energy charge i s a lumped para- meter i n l i v e r and kidney t i s s u e as both l i v e r and muscle c e l l s r e p r e s e n t m u l t i - compartment systems with adenine n u c l e o t i d e s p r e s e n t both i n mitochondria and cytoplasma and r e l a t e d t o t h e c e l l membrane. 2 . 2 . Hexose monophosphates i n muscle and l i v e r t i s s u e and i n e r y t h r o c y t e s . In Fig. 4 the c o n c e n t r a t i o n o f the hexosemonophosphates glucose-&phosphate and fructose-6-phosphate a r e c o r r e l a t e d w i t h body weight. Evidently t h e r e i s an i n c r e a s e o f t h e m e t a b o l i t e s i n both muscle and l i v e r t i s s u e , most pronounced i n young r a t s . How t h i s f i n d i n g s should be explained i s u n c e r t a i n . Though t h e n u m - b e r of measurements i n e r y t h r o c y t e s i s l i m i t e d t h e r e s u l t i n d i c a t e s t h a t t h e r e i s a d e c r e a s e o f t h e amount of t h e hexosemonophosphates, a l s o i n young animals. This might be r e l a t e d t o t h e d i f f e r e n t binding o f the compound t o a f e t a l and I05 a d u l t type of r a t haemoglobin. 0 0 0 0 0 % 0 O O O 0 00 8 0 oo 00 0 0 0 8 - i j G-6-P + F-6-P Muscle Liver Erythrocytes moles = 1 0 7 9 tissue x Id’ 10 a 6 L 2 100 200 300 Loo 500 100 200 300 LOO 500 Body weight, g Body weight, g Body weight, g Fig. 4. Concentration of G-6-P and F-6-P in l i v e r , s k e l e t a l muscle a n d erythro- cytes correlated with body weight (age). 2 . 3 . Glycogen in l i v e r t i s s u e The amount of l i v e r glycogen has been correlated with body weight a n d the age of the animal i n Fig. 5. There i s a pronounced increase of glycogen during the f i r s t seventy days of l i f e a n d then a decrease of the compound. There i s n o obvious explanation f o r the finding. However, i t seems of importance t o con- s i d e r t h i s r e s u l t in f u t u r e experimental s i t u a t i o n s where l i v e r glycogen i s de- termined e i t h e r in r a t s of d i f f e r e n t age or during longitudinal s t u d i e s . 2 . 4 . Water in muscle and l i v e r t i s s u e and in erythrocytes The content o f water i n the three types of c e l l s investigated has been de- termined and correlated t o the body weight or aae. I t appears from Fig. 6 a n d 7 t h a t the small changes observed are n o t a t a l l of such a magnitude t o explain the changes of metabolites as a r e s u l t of a changed water content of the invest- igated c e l l types. 2 . 5 . Other f a c t o r s influencing the amount of metabolites in the t i s s u e in- ves t i gated Other f a c t o r s t h a n those discussed above might be of importance t o explain the present findings, e.g. the e f f e c t of l i g h t e t h e r anaesthesia. This form of anaesthesia might in the absence of a blocking compound have released adrena- 106 l i n e , a c t i v a t i n g t h e a d e n y l a t e c y l c l a s e system and with secondary e f f e c t s on t h e i n t e r m e d i a r y metabolism e s p e c i a l l y of t h e l i v e r . I t would be necessary t o c a r r y o u t a d d i t i o n a l work t o e l u c i d a t e t h i s problem. Glycogen mg/g w e t w e q h t 1 100 i 60 ! 20 1 Liver I I 1 1 . ~~ 100 200 300 400 500 Body wecght, g Fig. 5 . The amount o f glycogen i n the l i v e r c o r r e l a t e d with body weight ( a g e ) . Apparent water content, % Liver tissue Muscle tissue __il 1 ' I I 100 200 300 400 500 Body weight, g MCHC 36 i 40 1 MCH 22 1 16 1 ~ L - 1 1 I i 100 200 300 LOO 500 Body weight. g F i g . 6 . M C H C , M C V and MCH i n e r y t h r o c y t e s c o r r e l a t e d w i t h body weight ( a g e ) . Fig. 7 . The a p p a r e n t water c o n t e n t o f l i v e r and muscle t i s s u e c o r r e l a t e d w i t h body weight ( a g e ) . I07 ACKNOWLEDGEMENT S u p p o r t e d b y t h e Swedish M e d i c a l Research C o u n c i l ( P r o j e c t Nos. 40X-676 and 40Y-2370). 1. 2. 3. 4. 5 . 6. 7. 8 . 9. 10. 11. 12. 13. 14. 15. REFERENCES Adam, H . : Adenosin-5 - t r i p h o s p h a t e and A d e n o s i n - 5 - d i p h o s p h a t e i n H.-U. Bergmeyer ( e d ) . Methoden d e r e n z y m a t i s c h e n Analyse, V e r l a g Chemie GmbH, Weinheim 1962, p . 539 a n d 573. A t k i n s o n , D. E . : The e n e r g y c h a r g e o f t h e a d e n y l a t e p o o l as a r e g u l a t o r y p a r a m e t e r . I n t e r a c t i o n w i t h feedback m o d i f i e r s . Biochem 7:4030, 1968. Berne, R . M. & Rubio, R . : Adenine n u c l e o t i d e m e t a b o l i s m i n t h e h e a r t . C i r c . Res. 34:109, 1974. Chaudry, I. H . , Sayeed, M. M. and Baue, A . E . : E f f e c t o f h e m o r r h a g i c shock on t i s s u e a d e n i n e n u c l e o t i d e s i n c o n s c i o u s r a t s . Can J Phys Pharm 52:131, 1974. Dale, R . A . : E f f e c t s o f s a m p l i n g p r o c e d u r e s on t h e c o n t e n t o f some i n t e r - m e d i a t e m e t a b o l i t e s o f g l y c o l y s i s i n r a t t i s s u e s . J P h y s i o l 181:701, 1965. Garby, L . and de V e r d i e r , C . H . : A f f i n i t y o f human h e m o g l o b i n A t o 2 , 3 - d i - p h o s p h o g l y c e r a t e . E f f e c t o f hemoglobin c o n c e n t r a t i o n and o f pH. Scand J C l i n Lab I n v e s t 27:345, 1971. Garby, L. and V u i l l e , J.-C.: The amount o f t r a p p e d plasma i n a h i g h speed m i c r o c a p i l l a r y h e m o t o c r i t c e n t r i f u g e . Scand J C l i n Lab I n v e s t 13:642, 1961. G r o t h , T., F a l k , H. and H j e l m , M.: A programme f o r e s t i m a t i o n o f t r e n d s and r e f e r e n c e i n t e r v a l s o f b i o m e d i c a l v a r i a b l e s . To be p u b l i s h e d 1982. Hems, K., R O S S , B. D., B e r r y , M. N. and Krebs, H . A . : Gluconeogenesis i n t h e p e r f u s e d r a t l i v e r . Biochem J 101:284, 1966. Hirasawa, H., Chaudry, I. H. and Baue, A. E . : Improved h e p a t i c f u n c t i o n and s u r v i v a l w i t h a d e n o s i n e t r i p h o s p h a t e - m a g n e s i u m c h l o r i d e a f t e r h e p a t i c i s - chemia. S u r g e r y 83:655, 1978. H j e l m , M.: The c o n c e n t r a t i o n o f some n u c l e o t i d e s and g l y c o l y t i c m e t a b o l i t e s i n human e r y t h r o c y t e s o f d i f f e r e n t ages. F o l i a Haemat ( L e i p z i g ) 89:392, 1968. H j e l m , M.: The mode o f e x p r e s s i n g t h e c o n t e n t o f i n t r a c e l l u l a r components o f human e r y t h r o c y t e s w i t h s p e c i a l r e f e r e n c e t o a d e n i n e n u c l e o t i d e s . Scand J Haemat 6:56, 1969. H j e l m , M. and de V e r d i e r , C.-H.: A m e t h o d o l o g i c a l s t u d y o f t h e e n z y m a t i c d e t e r m i n a t i o n o f g l u c o s e i n b l o o d . Scand J Lab C l i n I n v e s t 5:415, 1963. H o h o r s t , H. J.: D-glucose-6-phosphate and D - f r u c t o s e - 6 - p h o s p h a t e i n H . U. Bergmeyer ( e d ) . Methoden d e r e n z y m a t i s c h e n A n a l y s e . V e r l a g Chemie, GmbH, Weinheim 1962, p. 134. I m a i , S . , R i l e y , A. L. and Berne, R. M.: E f f e c t s o f i s c h e m i a on a d e n i n e nu- c l e o t i d e s i n c a r d i a c and s k e l e t a l muscle. C i r c Kes 9:443, 1964. I08 16. Kolousek, J. , J i r a c e k , V. , Z i c h a , B. e t . a l . : The i n f l u e n c e o f x - r a d i a t i o n , m e t h i o n i n e s u l p h o x i m i n e , b u r n s , and c y t o s t a t i c a g e n t s TS 160 ( S p o f a ) on t h e u r i n a r y e x c r e t i o n o f i n o r g a n i c phosphates and ammonia and on t h e l e v e l o f a d e n i n e n u c l e o t i d e s i n b l o o d and l i v e r o f r a t s . Neoplasma ( B r a t i s l ) 12:565, 1965. 17. Lamprecht, W. and T r a u t s c h o l d , L . : Adenosine-5-triphosphate: D e t e r m i n a t i o n w i t h h e x o k i n a s e and g l u c o s e - 6 - p h o s p h a t e dehydrogenase i n H.-U. Bergmeyer (ed!. Methods o f e n z y m a t i c a n a l y s i s . New York. Academic P r e s s I n c . 1965, Chap. 5, pp 543-552. 18. Maass, H. and Timm, M. : N u k l e o s i d p h o s p h a t g e h a l t i n v e r s c h i e d e n e n Organen d e r R a t t e nach G a n z k o r p e r b e s t r a h l u n g . S t r a h l e n t h e r a p i e 123:64, 1964. 19. Ozawa, K . , Yamaoka, Y . , Kimura, K . , Kamiyama, Y . , Sato, M. Ukikusa, M. and Tobe, T. : C i r c u l a t i n g h e p a t o d e p r e s s a n t f a c t o r s d e c r e a s i n g t h e e n e r g y c h a r g e l e v e l s o f t h e remnant l i v e r a f t e r hepatectomy. E u r . s u r g . Res. 13:444, 1981. 20. Pedersen, P . L. and Sachs, J . : Hexosephosphate f o r m a t i o n and t h e r e g u l a t i o n o f g l y c o l y s i s i n muscle. A r c h Biochem 112:548, 1965. 21. P f l e i d e r e r , G.: Glykogen Bestimmung a l s D-glucose m i t Hexokinase, P y r u v a t - k i n a s e und Lactat-Dehydrogenase i n H. U. Bergmeyer ( e d ) . Methoden d e r en- z y m a t i s c h e n Analyse, V e r l a g Chemie GmbH, Weinheim 1962, p. 59. f a t t y l i v e r . Biochem J 102:163, 1967. c l e . Biochem J 109:197, 1968. 22. Puddu, P., C a l d a r e r a , C. M. and M a r c h e t t i , M.: S t u d i e s on e t h i o n i n e i n d u c e d 23. Scopes, R. K . and Newbold, R. P . : Postmortem g l y c o l y s i s i n ox s k e l e t a l mus- Address f o r r e p r i n t s : Gosta A r t u r s o n , M. D. B u r n C e n t e r U n i v e r s i t y H o s p i t a l S-750 14 Uppsala SWEDEN I09