Acetate metabolism and nembutal anesthesia

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Acetate metabolism and Nembutal anesthesia* A specific biochemical effect of the drug p e n t o b a r b i t a l (Nembutal) has been d e m o n s t r a t e d for liver h o m o g e n a t e s I where t h e d r u g at 5" lO-4 M levels decreased b o t h p h o s p h o r y l a t i o n and oxygen c o n s u m p t i o n , and grossly changed P : O ratios. Because of the d e m o n s t r a t e d effect of N e m b u t a l u p o n p h o s p h o r y l a t i o n and because of the close association of p h o s p h o r y l a t i o n reactions with the energy r e q u i r e m e n t s of lipide synthesis we h a v e studied the effect of this drug u p o n lipogenesis. Adult male Spragne-Dawley rats, 2oo-23o g in weight, were carefully trained to feeding for 2 weeks b y being given IO to 12 g P u r i n a chow at 8 :oo a.m. and again at 5 :oo p.m. This a m o u n t of food was regularly eaten in I hour. Animals were used exactly i h o u r after the m o r n i n g I - h o u r eating period and are t h u s described as i h o u r p o s t p r a n d i a l animals. This rigorous control of fasting a n d feeding is required because of the wide fluctuations in lipogenesis caused b y feeding 2, Five of the r a t s received i o / z c while 4 received 2o #c of acetate-I-14C 3 at the end of the I h o u r p o s t p r a n d i a l period. The 5 r a t s receiving t h e io #c were anesthetized. Animals anesthetized were given 3.5 m g per IOO g b o d y weight of N e m b u t a l . The anesthesia was given 45 m i n u t e s after the end of the eating period, t h u s 15 m i n u t e s before the tracer acetate was given at the I h o u r p o s t p r a n d i a l time. This tracer dose of acetate was given intraperitoneally and the r a t placed i m m e d i a t e l y in a m e t a b o l i s m c h a m b e r 4. CO2-free air at a rate of 1o 1 per h o u r was d r a w n p a s t the animal, a shielded Geiger-Miiller t u b e and t h r o u g h a N a O H absorber s y s t e m in series. A cont i n u o u s record of r e s p i r a t o r y 14CO2 activities was t h u s made. A m a n u a l record was also m a d e of the accumulated c o u n t s appearing on the decimal scaler at I- or 2-minute intervals. The counting rate of the gas p h a s e c o u n t i n g w a s used for s u b s e q u e n t 14CO2 time course curve calculations. After 4 h o u r s the r a t s were chloroformed and dissected. Tissues were digested in alcoholic K O H and saponifiable (fatty acid) and non-saponifiable (cholesterol) fractions were isolated as before 5. Aliquots of NaOH-Na2CO 3 solutions from t h e COe absorbers or f r o m the wet c o m b u s t i o n e of lipide fractions were plated as BaCO a and counted as infinitely thick samples or were corrected to this thickness. D a t a on per cent incorporation of label into f a t t y acid and cholesterol fractions of liver, gut, carcass and skin are n o t presented in detail. I0,00C Because of the range of values found and .c because of the m i n i m u m size of the animal groups, the d a t a were treated statistically ~5,00C using confidence limits of 9 5 % . The 7" E values (test of significance between m e a n differences) found, indicated t h a t no E tissue showed statistically different responses to acetate-i-14C incorporation with N e m b u t a l anesthesia w h e n c o m p a r e d '~ 1,00C to n o n - N e m b u t a l i z e d controls. T values of < less t h a n i. I were found for the f a t t y acids 5oc and of less t h a n 1.8 for cholesterol fracU tions. A T of less t h a n 2.3 is considered to render differences insignificant. If one a s s u m e s a u n i f o r m distribution of the N e m b u t a l dose t h r o u g h o u t the tissues, t h e n the 3-5 mg given per ioo g body weight m i g h t result in tissue concentrao 1~2b3b4bsb6'o 12b tions of 1.4. lO-5 M. This figure is close to IV~nutes the a m o u n t s h o w n to affect oxidation and Fig. I. Time course curves of r e s p i r a t o r y 14CO2 p h o s p h o r y l a t i o n of liver homogenates, i.e. activity following acetate-i-14C a d m i n i s t r a t i o n to 5" lO-5 M. The lack of effect on lipogenesis anesthetized (O O) and non-anesthetized s h o w n here m a y be due to the low tissue (× x ) rats. These c o u n t i n g rares m a y be concentration of the drug t h a t was actuconverted to the equivalent c o u n t i n g rate of inally attained, b u t to make a comparison finitely thick BaCO 3 samples b y multiplying b y 5-3. of in vivo drug concentrations with those available in h o m o g e n a t e e x p e r i m e n t s is p r o b a b l y not valid. The d a t a presented here do n o t rule

8b

~o

This investigation was s u p p o r t e d largely b y c o n t r a c t No. AT(45-I)-225 of the United States Atomic E n e r g y Commission. A p o r t i o n of this material was presented at the meeting of the American Society of Biological Chemistry, at San Francisco, April lO-14, 1955P a r t of the d a t a was t a k e n f r o m a thesis s u b m i t t e d b y WALLACE R. WILLIAMS in June, 1955, to the D e p a r t m e n t of Biochemistry of the University of Oregon Medical School, in partial fulfillm e n t of the r e q u i r e m e n t s for the degree of Master of Science.

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o u t some drug interference w i t h p h o s p h o r y l a t i o n and oxidation for these entities could not be specifically measured. Additional positive information on the effect of N e m b u t a l u p o n the metabolic fate of acetate1-14C is afforded b y the CO s and 14CO~ d a t a collected. I n Fig. I are presented time course curves for the specific activity of the 14CO~ exhaled during the first 2 h o u r s of the 4-hour experimental period. Curve A shows the average time course of 14CO~ of animals receiving acetate intraperitoneally w i t h o u t N e m b u t a l anesthesia and curve B the average response of 2 of the anesthetized animals. Non-anesthetized animals show a m a x i m u m specific activity of 14CO2 in 8 to io m i n u t e s and t h e n a decrease to one-half the m a x i m u m in 26 minutes. This early peak is similar to t h a t seen for 14CO2 when r a t s were injected intraperitoneally w i t h bicarbonate 7. The " t u r n o v e r " or decay of 14CO2 activities is, however, prolonged over t h a t seen with bicarbonate dosage. The anesthetized animals showed a real delay in the appearance of a 14CO2 m a x i m u m specific activity, for highest values were n o t reached until 30 to 35 minutes. The s u b s e q u e n t decrease to one-half required a b o u t 38 minutes. The effects of N e m b u t a l s h o w n here m a y be due to a n u m b e r of factors. A decreased rate of m e t a b o l i s m of acetate to CO s does not seem to be the prime cause of the 14CO~ changes 7 for we h a v e found t h a t i n t r a v e n o u s a d m i n i s t r a t i o n of the tracer to anesthetized a n i m a l s resulted in an early (3 to 4 minutes) 14CO~ peak and a s u b s e q u e n t decay to one-half in 39 minutes. If acetate is metabolized at a n o r m a l rate b y the anesthetized rat, the delay seen in z4CO~ clearance m i g h t be caused b y delayed a b s o r p t i o n of the tracer from the peritoneal space. I n a previous p a p e r s it was concluded t h a t N e m b u t a l does not affect the peritoneal a b s o r p t i o n rate of bicarbonate. I n the case of the p r e s e n t e x p e r i m e n t it appears possible t h a t the changes seen in the 14CO2 time course curve are due to a decreased s u p p l y of tracer to the tissues caused b y a delayed peritoneal a b s o r p t i o n rate. As seen in the case of the animals injected w i t h bicarbonate, w h e n anesthetized animals arouse, a brief period of h y p e r v e n t i l a t i o n occurs followed b y a more s t e a d y rate of breathing, causing the s h a r p break in the a p p r o p r i a t e curve of Fig. 1. D u r i n g the r e m a i n d e r of the e x p e r i m e n t the I4CO2 specific activities decline to half values in a b o u t 31 minutes. The a u t h o r s acknowledge with t h a n k s assistance rendered b y Dr. EDWARD S. WEST. Valuable technical assistance was rendered b y Mr. JACK VERNETXI. WALLACE R. WILLIAMS Department o/Biochemistry, University o/ Oregon Medical School, J . T . VAN BRUGGEN

Portland, Ore. (U.S.A.) z T. M. BRODY AND J. A. BAIN, Prec. Soc. Exptl. Biol. Med., 77 (1951) 5 °.

Federation Prec., 14 (1955) 295. 3 j. T. VAN BRUGGEN,C. K. CLAYCOMB AND T. T. HUTCItENS, Nucleonics, 7, No. 3 (195o) 45. 4 j . T. VAN BRUGGEN AND T. T. HUTCHENS, Atomic Energy Commission Report, available as A E C U 3128. J. T. VAN BRUGGEN, T. T. HOTCHENS, C. K. CLAYCOMB, "W. J. CATHEV AND E. S. WEST, J. Biol. Chem., I96 (1952) 389 . 6 C. K. CLAYCOMB, T. T. HDTCHENS AND J. T. VAN BRUGGEN, Nucleonics, 7, No. 3 (195 o) 38'~ \V. R. \VILLIAMS AND J. T. VAN BRUGGEN, to be published. s j . T. VAN BRUGGEN, Proc. Soc. Exptl. Biol. Med., 91 (1956) 14o. Received April i7th, 1956 2 j . T. VAN BRUGGEN, R. M. COCKBURN AND W. R. WILLIAMS,

Hypoxanthine, cofactor for cysteine oxidation by liver preparations* Previous studies from this l a b o r a t o r y have s h o w n t h a t h y p o x a n t h i n e is a cofactor for the oxidation of sulfite b y a partially purified e n z y m e p r e p a r a t i o n from dog liver 1, 2 which also catalyzes the aerobic oxidation of dimercaptolipoate and of cysteine 3 as well as the reduction of methylene blue a n d o t h e r dyes b y these c o m p o u n d s . Evidence has been presented which strongly suggests t h a t t h e initial step in sulfite oxidation is cleavage of lipoate to lipoate hemithiosulfonate followed b y hydrolysis to sulfate and dimercaptolipoate 3. The h y p o x a n t h i n e r e q u i r e m e n t has been t h o u g h t to relate to the s u b s e q u e n t oxidation of reduced lipoate. The following e x p e r i m e n t s offer f u r t h e r evidence of the participation of h y p o x a n t h i n e in such systems. The enzyme p r e p a r a t i o n was t h a t previously designated F - I I I A ~ which h a d been dialyzed for 72 h o u r s against o.o5 M phosphate, p H 7.8. As s h o w n in Fig. 1, the rate of m e t h y l e n e blue reduction b y cysteine was more t h a n doubled * These studies were s u p p o r t e d b y G r a n t RG-9I from the National I n s t i t u t e s of H e a l t h and by c o n t r a c t AT-(4o-I)-289 between Duke University and the U.S. Atomic E n e r g y Commission.