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The effect of ethanol metabolism on levels of oxidized and reduced nicotinamide-adenine dinucleotide in liver, kidney, and heart
BIOCI-IIMICA ET BIOPHYSICA ACTA
29
BBA 25349 T H E E F F E C T OF E T H A N O L METABOLISM ON LEVELS OF O X I D I Z E D AND R E D U C E D N I C O T I N A M I D E - A D E N I N E D I N U C L E O T I D E IN L I V E R , K I D N E Y , AND H E A R T G. R. C H E R R I C K * AND C. M. L E E V Y
Division of Hepatic Metabolism and Nutrition, Department of Medicine, New Jersey College of Medicine and Dentistry, Jersey City, N.J. (U.S.A.) (Received N o v e m b e r i8th, 1964)
S U MMARY
I. Hepatic generation of reduced nicotinamide-adenine dinucleotide during ethanol metabolism was found to be a function both of the ethanol dose used and time following ethanol administration. Negligible decrease in the hepatic ratio of oxidized to reduced nicotinamide-adeuine dinucleotide resulted from 2 g per kg ethanol; 3 and 9 g per kg ethanol caused the ratio to fall 41% and 68 %, respectively. This effect was observed at m a x i m u m 4 h after ethanol administration. 2. Ethanol metabolism caused a significant decrease ill the ratio of oxidized to reduced nicotinamide-adenine dinucleotide of kidney and heart but had no effect on this ratio in blood or brain. Since neither kidney nor heart oxidizes in vivo significant amounts of ethanol, these changes are attributed to utilization b y these organs of metabolites with nicotinamide-adenine dinucleotide reducing potential which are derived from ethanol. 3. Administration of DL-lactate in quantities which produced blood lactate levels equivalent to those obtained with ethanol, caused significant decrease in the ratio of oxidized to reduced nicotinamide-adenine dinucleotide in liver and kidney but not in heart. The change in the ratio of oxidized to reduced nicotinamide-adenine dinucleotide in kidney, resulting from ethanol metabolism, is due at least in part, to a lactate effect. A lactate effect during ethanol metabolism, m a y potentiate the effect of other metabolites in determining the ratio in heart; it m a y secondarily influence the ratio in liver.
INTRODUCTION
I t has been shown previously that ethanol oxidation causes a decrease in the hepatic N A D + : N A D H ratio 1-4. The nature of that effect has not been entirely clear. In the original demonstration, the ratio changed in fed rats solely because of a decrease in NAD+ concentration1; two subsequent papers demonstrated a more-orless reciprocal rise in N A D H levels and fall in NAD + levels in such animals 2, 4. Moreover, while SMITE AND NEWMAN2 found that the giving of 3 g ethanol per kg body weight reduced the ratio to unity in fed rats and reversed it in fasting rats, RAII~A * P r e s e n t address: St. L u k e ' s Hospital Center, N e w Y o r k City, N.J., U.S.A.
Biochim. Biophys. Aeta, lO 7 (1965) 29-37
3°
Go R. CHERRICK, C. M. LEEVY
AND OLmA~ reported a greater fall in the ratio in fed animals than in fasting animals The variability of this literature, also seen in grea±ly differing values reported for d~c hepatic N A D + : N A D H ratio in control animals'2-% probably derives in part from ~he various methods of tissue handling and pyridine nucleotide extraction which hav<~ been employed. The present investigations were motivated by the demonstration that NADH generation has important metabolic consequences<<< They were designed to determine whether the ethanot effect on the hepatic N A D + : N A D H ratio is dose and time dependent; whether ethanol aiso affects the N A D + : N A D H ratio in kidney, heart, brain, and blood; and to assess the role of lactate in the production of MAD+: NADH-ratio decrements in liver and extrahepatic tissues during ethanol metabolism MATERIALSAND METHODS
Animals a~d experime~zts Male Sprague-Dawley rats, weighing I75-225 g were maintained on Purina Laboratory Chow and water ad libitum. They were not fasted prior to study. Animals were given, by stomach tube, either 2, 3, 6, or 9 g ethanol (50 %, v/v) per kg; equivalent volumes of saline were given to control animals. Those given the 6-g dose or equivalent saline were sacrificed at hourly intervals for a period of 20 h; the rest were sacrificed 4 h after treatment. Hepatic MAD+ and N A D H levels were determined in one-half of each group; serum ethanol and blood lactate levels were measured in the other half. Ischemia has been shown to decrease hepatic levels of NAD + and increase hepatic levels of NADHL It was therefore decided not to bleed the same animals in which hepatic levels of these coenzymes were to be determined. MAD + and N A D H levels were also measured in kidney, heart, brain and blood of animals sacrificed 4 h after treatment with 6 g ethanoI per kg or equivalent saline. The effect of lactate metabolism on MAD + and NADH levels of liver, kidney, and heart was determined in tissue removed 4 h after rats had been intubated with either 3.5 g sodium DL-lactate per kg (in a solution containing I7. 4 % sodium L-lactate and I2.6 To sodium D-Iactate, w/v) or equivalent volumes of saline. Tissue preparatio~ Rats were killed by cervical dislocation. Liver, kidney, and heart were removed within 2o sec and brain within 4o see. Blood was drawn from the inferior vena cava into a heparinized syringe within 40 sec. Tissues were quickly blotted, dropped into liquid N 2 and weighed at --2o °. Samples for NAD+ assay were homogenized at • : too in ice cold o.o2 N H 2 S 0 4 - o . I M Na2SO~ and heated at 60 ° for 45 rain; samples for N A D H assay were similarly prepared in o.o2 N N a O H - 5 - Io 4 M cysteine and heated at 6o ° for zo mill. Blood was diluted I : Ioo in the same acid solution] but the alkaline solution was o.I M Na2COa-o.o2 M NaHCO a (refs. 8, 9). Enzymes used in NAD+ and N A D H analysis ~-NADH (Sigma Chem. Co., St. Louis, Me.) was assayed spectrophotometrically. L-Glutamic acid dehydrogenase (EC 1.4.I.2 ) (Mann Research Laboratories, Inc., New York, N.Y.) was assayed by a modification of the method of LowRY et ail< Rabbit skeIetal muscle and beef-heart lactate dehydrogenase (EC IoI. z:27) (WorthingBiochim. Biophys° Acts, Io7 (I965) 29-37
EFFECT OF ETKANOL ON
NAD+ AND N A D H
LEVELS
31
ton Biochemical Corp., Freehold, N.J.) were assayed b y the method of WACKE~ et al. n. The latter enzyme was purified of NAD according to NEILANDS1~.
Analysis of NAD+ and N A D H NAD+ and N A D H were measured b y the enzymatic cycling method of LowRY et al. 8 at tissue dilutions of I : I o o o or more. Fluorometric measurements were made with the Farrand photoelectric fluorometer, using the following Corning filters: primary 7-37, secondary 3-72 and 5-61. Alcohol dehydrogenase assay Liver, kidney, and heart homogellates were tested for alcohol dehydrogenase (EC I . I . i . I ) activity according to the method of BONNICI~SENAND BRINK13. Ethanol assay Serum ethanol was determined in duplicate by a colorimetric assay (Worthingtoll Biochemical Corp., Freehold, N.J.) in which oxidation of ethanol to acetaldehyde is coupled with reduction of sodium 2,6-dichlorophenolindophenol. Lactate assay Blood lactate was determined in duplicate according to the method of BARKER AND SUMMEESON 14.
RESULTS
Animals intubated with saline in volumes equivalent to the 4 ethanol doses used in these experiments did not differ significantly, either among themselves or from an untreated group, in hepatic content of NAD + and NADH. The latter animals are shown as the control group in both Fig. 2 and Fig. 3-The N A D + : N A D H ratio in these animals was 6. 9. As seen in Fig. 2, 2 g ethanol per kg had a negligible effect on hepatic: NAD + and N A D H levels but a marked influence on blood lactate levels in a paired group of rats. The N A D + : N A D H ratio decreased (P < o.ooi) following the 3 g per k g dose and fell again (P < o.oi) after each of the 2 higher doses. Maximum ethanol effect occurred after the 9 g per kg dose, which caused the N A D + : N A D H ratio, to fal[ to 2.2 and the blood lactate to rise almost 3.5-fold (Fig. 2).The ratio decreased b o t h as the result of a rise in N A D H and a fall in NAD + levels. As shown in Fig. 2, m a x i m u m effect of 6 g ethanol per kg on hepatic NAD + and N A D H levels was seen 4 h after its administration and resulted in a N A D + : N A D H ratio of 3. This coincided with the maximum-observed blood ethanol concentratiom Blood lactate continued to rise, during a time in which the N A D + : N A D H ratio was; returning toward control value, and was seen at m a x i m u m 8 h after ethanol administration. I6 h after ethanol treatment, the N A D + : N A D H ratio w as no longer significantly different from control value; blood lactate, however, was almost twice that of control animals. 6 g ethanol per kg caused a fall in the NAD+: N A D H ratio of kidney (P < o.ooi)? and heart (P < o.oi), as well as liver, 4 h after dose administration. T h a t ratio in. brain and blood was not affected significantly (Table I). Administration of 3-5 g sodium DL-lactate also evoked a fall in the N A D + : N A D H ratio of liver (P < o.ooi) Biochim. Biophys. dcta, lo 7 (1965) 2 9 - 3 7
32
C, M. LEEV~
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EXT
PATHWAYS OF
RA M I T
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ELECTRON FLOW FROM EXTRAMITOCHONDRIAL NADH
Fig. I. ~ o r m a t i o n a n d o x i d a t i o n of e x t r a m _ t o c h o n d r i a l N A D H g e n e r a t e d d u r i n g e t h a n o l m e t a b o l i s m , TABLE I EF~'ECT OF ~THANOL OXIDATION ON T I S S U ~ ~}~2~]~)÷ AND ~ A ~ - ~
LI~VELS
R a t s were g i v e n e t h a n o l (6 g per kg) or e q u i v a l e n t v o l u m e s of saline b y s t o m a c h t u b e 4 h before sacrificel V a l u e s are e x p r e s s e d in # m o l e s p e r k g w e t w e i g h t as m e a n : S.E. of t h e m e a n . i n bl ood s a m p l e s N A D + a n d N A D H are e x p r e s s e d in ffmoles pe r 1 w h o l e blood, c o r r e c t e d t o h e m a t o c r k of 47 %"
Tissue
TT,eatme~et
Number of a~¢imals
ArAD +
Liver Liver Kidney I~idney Heart Heart Brain Brain Blood Blood
Saline Ethanol Saline Ethanol Saline Ethanol Saline Ethanol Saline Ethanol
6 6 9 9 9 9 6 6 9 9
729 685 562 596 6o0 578 396 387 ix2 H6
Biochim. Biophys. Aota, lO 7 (I965) 29-37
~ ± ± ~ ~ ~ ~ ~± z
48 46 38 52 29 41 22 27 3 6
~'\\4 D H
N A D + 4~r,~ IDH
NAD+ : NA D H ~atio
i o 4 _. i I 226 ± z8 94 ± 9
833 9 II 656 752 719 736 478 464 I95 2o5
7.oi i - o.I9 3,o3 ~ o.1~ 5.98 _~_ 0.40 3.82 _: o.25 5.o4 _ : o . z 9 3.6C-> ~ o.s 4 4.83 ~ 0,27 5.03 4_: 0.32 1.35 -3:o . i z z.3o -- o.2~
1 5 6 ~- I 5
119 i58 82 77 83 89
~ 2_ ~. c __ ~
i3 8 zI I4 3 4
~ 34 7_ 51 :h 45 ~ 58 ~ 32 ~ 46 ~j:28 ~ 39 7=- 3 -- 8
EFFECT OF ETHANOL ON N A D +
AND N A D H
LEVELS
33
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iiliii iiiii 9~/kg
CONTROL
3g/kg ETHANOL
Sg/N 9 DOSE
9g/kg
GIVEN
Fig. 2. Effect of ethanol dosage on hepatic N A D + a n d N A D H Ievels and s e r u m - e t h a n o I and bloodlactate concentrations in male S p r a g u e - D a w I e y rats. E a c h value is the m e a n ± S.E. of the m e a n obtained f r o m analysis of 6 animals. T A B L E II EFFECT
OF LACTATE
METABOLISM
ON TISSUE
NAD+
AND
NADH
LEVELS
R a t s were given DL-lactate (3.5 g per kg) or equivalent volumes of saline b y s t o m a c h t u b e 4 h before sacrifice. Blood ]actate was i i . i :k 1.8 mg % in 5 saline-treated r a t s and 37.8 ± 2.6 trig % in 5 l a c t a t e - t r e a t e d r a t s at time of sacrifice. Values are in #moles per kg wet weight (mean ± S.E. of t h e mean). Tissue
Treatment
Numbee of animals
NAD +
NADH
NAD + + NADH
NAD+: NADH ratio
Liver Liver Kidney Kidney Heart Heart
Saline Lactate Saline Lactate Saline Lactate
5 5 6 6 9 9
66o 625 512 485 563 515
9o I59 1Io 164 96 92
75o :~ 29
7-33 3.93 4-65 2.96 5.86 5-59
~ 25 i 22 ~ 27 ~ 32 _~ 23 :~ 19
~_ 6 -c 9 4- I2 ~- 8 ~_ 6 = 8
784 ~ 15 622 649 659 6o7
~~ ~ :"
28 I8 2I 15
~ o.i6 ~- o.I4 ----_o.19 ± o.12 -~ 0.23 ~ o.15
and kidney (P < o.oI) ; the ratio, however, did not change significantly (P = o.4o) in heart (Table II). Alcohol dehydrogenase activity, determined in liver homogenates of 6 male Biochim. Biophys. Acla, Io 7 (1965) 29-37
34
G. m
C~ISSRZCK~ C. Si. L~:SVv
S p r a g u e - D a w i e y rats, was o.6 4 ~ o.o8 p~mole per g wet weight per minute. Aetivi1:} of t h a t e n z y m e could not be d e t e c t e d in h o m o g e n a t e s of k i d n e y a n d heart, i d e n t i c a l l y processed a n d analyzed. DISCUSSION T h e N A D + and N A D H levels r e p o r t e d in this p a p e r were d e t e r m i n e d from analysis of u n f r a c t i o n a t e d tissue. A g r e a t e r effect of ethanol on the N A D 4 - : X A D H ratio w o u l d have been a p p a r e n t had the soluble e x t r a m i t o c h o n d r i a l portion of the c y t o p l a s m been a n a l y z e d exclusively. Mitochondrial N A D H is b o u n d or has r e a d y access to the electron t r a n s p o r t s y s t e m a n d undergoes r a p i d o x i d a t i o n which is coupled w i t h p h o s p h a t e esterification ls,16. R e m o v a l of m i t o c h o n d r i a l N A D H is further facilitated b y a t r a n s h y d r o g e n a s e , present in liver, which p e r m i t s t h e transfer of an electron from N A D H to N A D P + (refs. 17, I8/. Inclusion of m i t o c h o n d r i a l N A D ~- and N A D H . which are m a i n t a i n e d at a r e l a t i v e l y high o x i d a t i o n : reduetior_ ratio, gives an overall view of cellular m e t a b o l i s m b u t de-emphasizes t h e ethanol m e t a b o l i c effect. The values for fota! h e p a t i c N A D in control animals are similar to those found b y others << ~9,20. T h e N A D - : N A D H ratios are higher t h a n those generally reported b u t are in keeping with the ratios found b y i n v e s t i g a t o r s who use t h e same m e t h o d s of tissue p r e p a r a t i o n a n d e x t r a c t i o n 7,19. These higher ratios are p a r t i a l l y a t t r i b u t a b l e to a r e d u c t i o n of post-morte~ change effected b y r a p i d h a n d l i n g a n d freezing of tissue 7 Values for ~otal N A D in k i d n e y , heart, brain, a n d blood (Table I agree well w i t h those of GLOCK AND McLEAN 20. I t is e v i d e n t t h a t t h e e t h a n o l effect on the h e p a t i c N A D + : N A D H ratio is doser e l a t e d ; the p h e n o m e n o n is no~ all-or-none (Fig. 21. The reasons for this are implicit in t h e locus of N A D H f o r m a t i o n and the m e c h a n i s m s b y which it is oxidized. D u r i n g e t h a n o l oxidation, N A D H generation occurs in the soluble e x t r a m i t o c h o n d r i a l portlor, of the c y t o p l a s m , m which alcohol d e h y d r o g e n a s e has been isolated2t Liver m~tochondria are i m p e r m e a b l e to NADH~2,2a. which can therefore be oxidized b v d-~e m i t o c h o n d r i a l f l a v o p r o t e i n - c y t o c h r o m e electron t r a n s p o r t system only i n d i r e c t l y (Fig. z). This indirect flow of electrons occurs when N A D H p a r t i c i p a t e s in the reduction of c y t o p l a s m i c m e t a b o l i t e s which t h e n become s u b s t r a t e s for m i t o c h o n d f i a l oxidation. Two such reactions are the reduction of d i h y d r o x y a c e t o n e p h o s p h a t e Le z_c~_glycerophosphate.~< 25 a n d the reduction of a c e t o a c e t a t e to /%hydroxybutyrate~-< 3Aitochondrial o x i d a t i o n is l i m i t e d b v two r e l a t i v e l y slow, r a t e - d e t e r m i n i n g processes : d i s e n g a g e m e n t of the r e d u c e d molecule from one complex a n d d i s e n g a g e m e n t a n d t r a n s f e r of esterified p h o s p h a t e to ADP~< As the dose of e t h a n o l is increased, it a p p e a r s t h a t a b n i l d - u o of subscrate with reducing p o t e n t i a l , such a s / ~ - h y d r o x y b u t y r a t e a1:d z - ~ - g l y c e r o p h o s p h a t e m a y exceed the c a p a c i t y of the r a t e - l i m i t e d m i t o c h o n d r i a l oxidizing system. This then causes a slowing of electron flow from N A D H . resulting in a decrease of the N A D + : N A D H ratio. A further insight into the dose relationship of e t h a n o l to the h e p a t i c NAD~ N A D H ratio is afforded b v consideration of l a c t a t e metabolism. E x t r a m i t o c h o n d r i a ] N A D H can also be reoxidized b y p a r t i c i p a t i n g in the r e d u c t i o n of p y r u v a r e ~ lactate~7,zs As the dose of e t h a n o l is increased, blood l a c t a t e rises ~Fig. 2 : lactate. when given to r a t s in a m o u n t s which p r o d u c e b l o o d - l a c t a t e levels c o m p a r a b l e to those resulting from ethanol m e t a b o l i s m causes t h e r e d u c t i o n of h e p a t i c N A D + (Table Ii'~. Biocl~im. Bio1~hys. Hct~, zo7 (z965) °-9-37
EFFECT OF ETHANOL ON N A D + AND N A D H
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ETHANOL
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Fig. 3- Effect of 6 g ethanol per kg on hepatic N A D + and N A D H levels and s e r u m - e t h a n o l and blood-lactate concentrations during all I8-h period. E a c h value is the m e a n -~ S.E. of the m e a n o b t a i n e d f r o m analysis of 6 animals.
This example of the reduction of NAD+ by a metabolite arising from the oxidation of N A D H emphasizes the cyclical nature of the process. It seems probable that wheI1 lactate accumulation reaches a certain level, feedback to NAD + shifts the equilibrium in favor of a lower N A D + : N A D H ratio. It is also possible that ethanol, within a certain concentration range, may reduce hepatic blood flow and cellular oxygenation sufficiently to inhibit the oxidation of N A D H via the electron transport system• The change of hepatic N A D + : N A D H ratio with respect to time after a single dose of ethanol (Fig. 3) can be explained as a representation in time of the same metabolic events which determined the hepatic N A D + : N A D H ratios produced by different doses of ethanol after a fixed interval. The present observations of the effect of ethanol metabolism on hepatic NAD+ and N A D H levels differ from those previously reported in two ways : since the control NAD+: N A D H ratio is considerably higher than that found by all 1, 2, 4 but REBOUCAS AND ISSELBACHER 3, the ratio found after ethanol treatment is also considerably higher. RAIKA AND OURA 4, who made observations from 30 min to 4 h after ethanol administration, found that the hepatic NAD +: NADH ratio was decreased and maintained at a "fairly steady level" during that time. The reasons for that difference from our data are not apparent. Other investigators ~9,3o have detected the oxidation of small amounts of ethanol by kidney in vitro. Heart has not been reported to be a site for significant ethanol oxidation. We were unable to find alcohol dehydrogenase activity in either organ with methods which accurately assay crystalline alcohol dehydrogenase and which Biochim. Biophys. Acta, lO 7 (I965) 29-37
.36
G. R, GHERRICK, C. )L LEEV\f
demonstrate the enzyme i~ liver. The depression of the N A D + : N A D H ratio induc<::d in kidney by ethanol (Table I) cannot result from low rates of ethanol oxidatio;-~ in that organ; even the change in hepatic N A D + : N A D H ratio in rats metabolizing a>: much as z g ethanol per kg was trivial (Fig. z). The effect of ethanol on levets of NAD + and N A D H in kidney is aitributab]e, at least in part, to lactate utilization by that organ. When rats were given sufficient Ds> lactate to produce blood lactate levels comparable to those resulting from 6 g ethanol per ks, a significant fal! in the N A D ÷ : N A D H ratio was found (Table tI). The proportion of >- and L-lactate isomers in blood of animals treated with >5-!acta.te may nol have been equivalent to the proportion of lactate isomers in biood of animals treated with ethanol. Since mammals preferentially metabolize the >-isomeral, a< an even greater effect on the NAD+ and NADH levels probably would have resulted had the lactate-treated animals been given that isomer exclusively. The effect of ethanol on levels of NAD + and N A D H in heart (Table i) probably results additive]y from the utilization by that organ of several metabolites with reducing potential, elaborated during ethanol metabolism, of which lactate is one; the effect is not evoked by lactate alone (Table iI). It appears that heart has more effective mechanisms than kidney for reoxidizing cytoplasmic NADH. Isolated "intact" heart mitochondria show a relatively high rate of NADH oxidation, in contrast with mito..chondria of liver or kidney~ This is not taken_ to mean that heart mitochondria are actually permeable in rive to N A D H but rather that the mitochondriaI m.embrane is "labi!e"~< Reducing metabolites may pass through it, i~a rive, with ~anusual ease. ACKNOWLEDGEMENTS
The authors are grateful to Dr. H. B. BuRc~ and Dr. O. H. LowRY for valuable help and advice. Expert technical assistance was rendered by Mr. W. Ge~STZ,La_~>% Mr. W. GLA~Z.~A>-,and Mr. D. MARCIANO.This work was supported in part by grants MH-o554z-o3 and T1 AM 5e36-o 4 from the U.S. Public Health Service, and a grant from the New jersey State Department of Health, Division of Chrordc Illness Control REFERENCES I O. ]F'ORSANDER, N . R A I H A AND H . SL'OhlaLAINEX, Z. Ph¢esioh Cl~e~o<, 312 (1958) e 4 3 . 2 M. E . S>IIT~ i n n H . W . N~Wh~A~, J. Biol. Chem., 2 3 4 ( I 9 5 9 ) I 5 4 4 . 3 G. REBOUCAS AND K . J . ISSELBaCHER. ]. Cli~z Iv¢vesl 4 o ( 1 9 6 ! ) ~355. 4 N . C. R . R A I ~ A .XXD E . O
Biochi,n. Bi@hys. ~ida, io 7 (:~965) ~9-37
EFFECT OF ETHANOL ON N A D + AND N A D H
LEVELS
37
17 N. O. KAPLAN, S. P, COLOWICK, L. J. ZATMAN AND M. M. CIOTTI, dr. Biol. Chem., 205 (I953) 3 I. 18 A. iVI. STEIN~ N. O. KAPLAN AND IV[. M. CIOTTI, J. Biol. Chem., 234 (I959) 979. 19 L. GARCIA-BuNUEL, D. B. MCDOUGAL, JR., H. B. BURCH, E. M. JONES AND 1~. TOUHILL, dr. Neurochem., 9 (1962) 589. 20 ~. E. GLOCK A N D P. MCLEAN, Biochem. J., 61 (1955) 388. 21 A. NYBERG, J. SCHLTBERTH AND L. ANGGARD, Aeta Chem. Seand., 7 (1953) 117o22 G. E. BOXER AND T. M. DEVLIN, Science, 134 (I96I) I495. 23 D. E. GREEN AND F. L. CRANE, Prec. Intern. Syrup. Enzyme Chem., Tokyo, Kyoto, I957, P a n Pacific Press, Tokyo, 1958, p. 275. 24 R. W. ESTABROOI< AND ]3. SACI
Biochim. Biophys. Aeta, IO7 (I965) 2 9 - 3 7
29
BBA 25349 T H E E F F E C T OF E T H A N O L METABOLISM ON LEVELS OF O X I D I Z E D AND R E D U C E D N I C O T I N A M I D E - A D E N I N E D I N U C L E O T I D E IN L I V E R , K I D N E Y , AND H E A R T G. R. C H E R R I C K * AND C. M. L E E V Y
Division of Hepatic Metabolism and Nutrition, Department of Medicine, New Jersey College of Medicine and Dentistry, Jersey City, N.J. (U.S.A.) (Received N o v e m b e r i8th, 1964)
S U MMARY
I. Hepatic generation of reduced nicotinamide-adenine dinucleotide during ethanol metabolism was found to be a function both of the ethanol dose used and time following ethanol administration. Negligible decrease in the hepatic ratio of oxidized to reduced nicotinamide-adeuine dinucleotide resulted from 2 g per kg ethanol; 3 and 9 g per kg ethanol caused the ratio to fall 41% and 68 %, respectively. This effect was observed at m a x i m u m 4 h after ethanol administration. 2. Ethanol metabolism caused a significant decrease ill the ratio of oxidized to reduced nicotinamide-adenine dinucleotide of kidney and heart but had no effect on this ratio in blood or brain. Since neither kidney nor heart oxidizes in vivo significant amounts of ethanol, these changes are attributed to utilization b y these organs of metabolites with nicotinamide-adenine dinucleotide reducing potential which are derived from ethanol. 3. Administration of DL-lactate in quantities which produced blood lactate levels equivalent to those obtained with ethanol, caused significant decrease in the ratio of oxidized to reduced nicotinamide-adenine dinucleotide in liver and kidney but not in heart. The change in the ratio of oxidized to reduced nicotinamide-adenine dinucleotide in kidney, resulting from ethanol metabolism, is due at least in part, to a lactate effect. A lactate effect during ethanol metabolism, m a y potentiate the effect of other metabolites in determining the ratio in heart; it m a y secondarily influence the ratio in liver.
INTRODUCTION
I t has been shown previously that ethanol oxidation causes a decrease in the hepatic N A D + : N A D H ratio 1-4. The nature of that effect has not been entirely clear. In the original demonstration, the ratio changed in fed rats solely because of a decrease in NAD+ concentration1; two subsequent papers demonstrated a more-orless reciprocal rise in N A D H levels and fall in NAD + levels in such animals 2, 4. Moreover, while SMITE AND NEWMAN2 found that the giving of 3 g ethanol per kg body weight reduced the ratio to unity in fed rats and reversed it in fasting rats, RAII~A * P r e s e n t address: St. L u k e ' s Hospital Center, N e w Y o r k City, N.J., U.S.A.
Biochim. Biophys. Aeta, lO 7 (1965) 29-37
3°
Go R. CHERRICK, C. M. LEEVY
AND OLmA~ reported a greater fall in the ratio in fed animals than in fasting animals The variability of this literature, also seen in grea±ly differing values reported for d~c hepatic N A D + : N A D H ratio in control animals'2-% probably derives in part from ~he various methods of tissue handling and pyridine nucleotide extraction which hav<~ been employed. The present investigations were motivated by the demonstration that NADH generation has important metabolic consequences<<< They were designed to determine whether the ethanot effect on the hepatic N A D + : N A D H ratio is dose and time dependent; whether ethanol aiso affects the N A D + : N A D H ratio in kidney, heart, brain, and blood; and to assess the role of lactate in the production of MAD+: NADH-ratio decrements in liver and extrahepatic tissues during ethanol metabolism MATERIALSAND METHODS
Animals a~d experime~zts Male Sprague-Dawley rats, weighing I75-225 g were maintained on Purina Laboratory Chow and water ad libitum. They were not fasted prior to study. Animals were given, by stomach tube, either 2, 3, 6, or 9 g ethanol (50 %, v/v) per kg; equivalent volumes of saline were given to control animals. Those given the 6-g dose or equivalent saline were sacrificed at hourly intervals for a period of 20 h; the rest were sacrificed 4 h after treatment. Hepatic MAD+ and N A D H levels were determined in one-half of each group; serum ethanol and blood lactate levels were measured in the other half. Ischemia has been shown to decrease hepatic levels of NAD + and increase hepatic levels of NADHL It was therefore decided not to bleed the same animals in which hepatic levels of these coenzymes were to be determined. MAD + and N A D H levels were also measured in kidney, heart, brain and blood of animals sacrificed 4 h after treatment with 6 g ethanoI per kg or equivalent saline. The effect of lactate metabolism on MAD + and NADH levels of liver, kidney, and heart was determined in tissue removed 4 h after rats had been intubated with either 3.5 g sodium DL-lactate per kg (in a solution containing I7. 4 % sodium L-lactate and I2.6 To sodium D-Iactate, w/v) or equivalent volumes of saline. Tissue preparatio~ Rats were killed by cervical dislocation. Liver, kidney, and heart were removed within 2o sec and brain within 4o see. Blood was drawn from the inferior vena cava into a heparinized syringe within 40 sec. Tissues were quickly blotted, dropped into liquid N 2 and weighed at --2o °. Samples for NAD+ assay were homogenized at • : too in ice cold o.o2 N H 2 S 0 4 - o . I M Na2SO~ and heated at 60 ° for 45 rain; samples for N A D H assay were similarly prepared in o.o2 N N a O H - 5 - Io 4 M cysteine and heated at 6o ° for zo mill. Blood was diluted I : Ioo in the same acid solution] but the alkaline solution was o.I M Na2COa-o.o2 M NaHCO a (refs. 8, 9). Enzymes used in NAD+ and N A D H analysis ~-NADH (Sigma Chem. Co., St. Louis, Me.) was assayed spectrophotometrically. L-Glutamic acid dehydrogenase (EC 1.4.I.2 ) (Mann Research Laboratories, Inc., New York, N.Y.) was assayed by a modification of the method of LowRY et ail< Rabbit skeIetal muscle and beef-heart lactate dehydrogenase (EC IoI. z:27) (WorthingBiochim. Biophys° Acts, Io7 (I965) 29-37
EFFECT OF ETKANOL ON
NAD+ AND N A D H
LEVELS
31
ton Biochemical Corp., Freehold, N.J.) were assayed b y the method of WACKE~ et al. n. The latter enzyme was purified of NAD according to NEILANDS1~.
Analysis of NAD+ and N A D H NAD+ and N A D H were measured b y the enzymatic cycling method of LowRY et al. 8 at tissue dilutions of I : I o o o or more. Fluorometric measurements were made with the Farrand photoelectric fluorometer, using the following Corning filters: primary 7-37, secondary 3-72 and 5-61. Alcohol dehydrogenase assay Liver, kidney, and heart homogellates were tested for alcohol dehydrogenase (EC I . I . i . I ) activity according to the method of BONNICI~SENAND BRINK13. Ethanol assay Serum ethanol was determined in duplicate by a colorimetric assay (Worthingtoll Biochemical Corp., Freehold, N.J.) in which oxidation of ethanol to acetaldehyde is coupled with reduction of sodium 2,6-dichlorophenolindophenol. Lactate assay Blood lactate was determined in duplicate according to the method of BARKER AND SUMMEESON 14.
RESULTS
Animals intubated with saline in volumes equivalent to the 4 ethanol doses used in these experiments did not differ significantly, either among themselves or from an untreated group, in hepatic content of NAD + and NADH. The latter animals are shown as the control group in both Fig. 2 and Fig. 3-The N A D + : N A D H ratio in these animals was 6. 9. As seen in Fig. 2, 2 g ethanol per kg had a negligible effect on hepatic: NAD + and N A D H levels but a marked influence on blood lactate levels in a paired group of rats. The N A D + : N A D H ratio decreased (P < o.ooi) following the 3 g per k g dose and fell again (P < o.oi) after each of the 2 higher doses. Maximum ethanol effect occurred after the 9 g per kg dose, which caused the N A D + : N A D H ratio, to fal[ to 2.2 and the blood lactate to rise almost 3.5-fold (Fig. 2).The ratio decreased b o t h as the result of a rise in N A D H and a fall in NAD + levels. As shown in Fig. 2, m a x i m u m effect of 6 g ethanol per kg on hepatic NAD + and N A D H levels was seen 4 h after its administration and resulted in a N A D + : N A D H ratio of 3. This coincided with the maximum-observed blood ethanol concentratiom Blood lactate continued to rise, during a time in which the N A D + : N A D H ratio was; returning toward control value, and was seen at m a x i m u m 8 h after ethanol administration. I6 h after ethanol treatment, the N A D + : N A D H ratio w as no longer significantly different from control value; blood lactate, however, was almost twice that of control animals. 6 g ethanol per kg caused a fall in the NAD+: N A D H ratio of kidney (P < o.ooi)? and heart (P < o.oi), as well as liver, 4 h after dose administration. T h a t ratio in. brain and blood was not affected significantly (Table I). Administration of 3-5 g sodium DL-lactate also evoked a fall in the N A D + : N A D H ratio of liver (P < o.ooi) Biochim. Biophys. dcta, lo 7 (1965) 2 9 - 3 7
32
C, M. LEEV~
@, R, C H E R R i C K ,
T
il
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# HYDRO×YBUTYRATE
ACETOACETATE
D I H Y D R O X Y A G E T O N E - P ~-6LYCERO~P
/
.......................
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NAOH
ALCO.OLZZ
!
. ......
ACETYL Co A
~.AO+~'["~CE~
EXT
PATHWAYS OF
RA M I T
OC H O N
DRIAL
SPACE
ELECTRON FLOW FROM EXTRAMITOCHONDRIAL NADH
Fig. I. ~ o r m a t i o n a n d o x i d a t i o n of e x t r a m _ t o c h o n d r i a l N A D H g e n e r a t e d d u r i n g e t h a n o l m e t a b o l i s m , TABLE I EF~'ECT OF ~THANOL OXIDATION ON T I S S U ~ ~}~2~]~)÷ AND ~ A ~ - ~
LI~VELS
R a t s were g i v e n e t h a n o l (6 g per kg) or e q u i v a l e n t v o l u m e s of saline b y s t o m a c h t u b e 4 h before sacrificel V a l u e s are e x p r e s s e d in # m o l e s p e r k g w e t w e i g h t as m e a n : S.E. of t h e m e a n . i n bl ood s a m p l e s N A D + a n d N A D H are e x p r e s s e d in ffmoles pe r 1 w h o l e blood, c o r r e c t e d t o h e m a t o c r k of 47 %"
Tissue
TT,eatme~et
Number of a~¢imals
ArAD +
Liver Liver Kidney I~idney Heart Heart Brain Brain Blood Blood
Saline Ethanol Saline Ethanol Saline Ethanol Saline Ethanol Saline Ethanol
6 6 9 9 9 9 6 6 9 9
729 685 562 596 6o0 578 396 387 ix2 H6
Biochim. Biophys. Aota, lO 7 (I965) 29-37
~ ± ± ~ ~ ~ ~ ~± z
48 46 38 52 29 41 22 27 3 6
~'\\4 D H
N A D + 4~r,~ IDH
NAD+ : NA D H ~atio
i o 4 _. i I 226 ± z8 94 ± 9
833 9 II 656 752 719 736 478 464 I95 2o5
7.oi i - o.I9 3,o3 ~ o.1~ 5.98 _~_ 0.40 3.82 _: o.25 5.o4 _ : o . z 9 3.6C-> ~ o.s 4 4.83 ~ 0,27 5.03 4_: 0.32 1.35 -3:o . i z z.3o -- o.2~
1 5 6 ~- I 5
119 i58 82 77 83 89
~ 2_ ~. c __ ~
i3 8 zI I4 3 4
~ 34 7_ 51 :h 45 ~ 58 ~ 32 ~ 46 ~j:28 ~ 39 7=- 3 -- 8
EFFECT OF ETHANOL ON N A D +
AND N A D H
LEVELS
33
-50
..+ ...... .I
4O0-
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300-
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-
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iiliii iiiii 9~/kg
CONTROL
3g/kg ETHANOL
Sg/N 9 DOSE
9g/kg
GIVEN
Fig. 2. Effect of ethanol dosage on hepatic N A D + a n d N A D H Ievels and s e r u m - e t h a n o I and bloodlactate concentrations in male S p r a g u e - D a w I e y rats. E a c h value is the m e a n ± S.E. of the m e a n obtained f r o m analysis of 6 animals. T A B L E II EFFECT
OF LACTATE
METABOLISM
ON TISSUE
NAD+
AND
NADH
LEVELS
R a t s were given DL-lactate (3.5 g per kg) or equivalent volumes of saline b y s t o m a c h t u b e 4 h before sacrifice. Blood ]actate was i i . i :k 1.8 mg % in 5 saline-treated r a t s and 37.8 ± 2.6 trig % in 5 l a c t a t e - t r e a t e d r a t s at time of sacrifice. Values are in #moles per kg wet weight (mean ± S.E. of t h e mean). Tissue
Treatment
Numbee of animals
NAD +
NADH
NAD + + NADH
NAD+: NADH ratio
Liver Liver Kidney Kidney Heart Heart
Saline Lactate Saline Lactate Saline Lactate
5 5 6 6 9 9
66o 625 512 485 563 515
9o I59 1Io 164 96 92
75o :~ 29
7-33 3.93 4-65 2.96 5.86 5-59
~ 25 i 22 ~ 27 ~ 32 _~ 23 :~ 19
~_ 6 -c 9 4- I2 ~- 8 ~_ 6 = 8
784 ~ 15 622 649 659 6o7
~~ ~ :"
28 I8 2I 15
~ o.i6 ~- o.I4 ----_o.19 ± o.12 -~ 0.23 ~ o.15
and kidney (P < o.oI) ; the ratio, however, did not change significantly (P = o.4o) in heart (Table II). Alcohol dehydrogenase activity, determined in liver homogenates of 6 male Biochim. Biophys. Acla, Io 7 (1965) 29-37
34
G. m
C~ISSRZCK~ C. Si. L~:SVv
S p r a g u e - D a w i e y rats, was o.6 4 ~ o.o8 p~mole per g wet weight per minute. Aetivi1:} of t h a t e n z y m e could not be d e t e c t e d in h o m o g e n a t e s of k i d n e y a n d heart, i d e n t i c a l l y processed a n d analyzed. DISCUSSION T h e N A D + and N A D H levels r e p o r t e d in this p a p e r were d e t e r m i n e d from analysis of u n f r a c t i o n a t e d tissue. A g r e a t e r effect of ethanol on the N A D 4 - : X A D H ratio w o u l d have been a p p a r e n t had the soluble e x t r a m i t o c h o n d r i a l portion of the c y t o p l a s m been a n a l y z e d exclusively. Mitochondrial N A D H is b o u n d or has r e a d y access to the electron t r a n s p o r t s y s t e m a n d undergoes r a p i d o x i d a t i o n which is coupled w i t h p h o s p h a t e esterification ls,16. R e m o v a l of m i t o c h o n d r i a l N A D H is further facilitated b y a t r a n s h y d r o g e n a s e , present in liver, which p e r m i t s t h e transfer of an electron from N A D H to N A D P + (refs. 17, I8/. Inclusion of m i t o c h o n d r i a l N A D ~- and N A D H . which are m a i n t a i n e d at a r e l a t i v e l y high o x i d a t i o n : reduetior_ ratio, gives an overall view of cellular m e t a b o l i s m b u t de-emphasizes t h e ethanol m e t a b o l i c effect. The values for fota! h e p a t i c N A D in control animals are similar to those found b y others << ~9,20. T h e N A D - : N A D H ratios are higher t h a n those generally reported b u t are in keeping with the ratios found b y i n v e s t i g a t o r s who use t h e same m e t h o d s of tissue p r e p a r a t i o n a n d e x t r a c t i o n 7,19. These higher ratios are p a r t i a l l y a t t r i b u t a b l e to a r e d u c t i o n of post-morte~ change effected b y r a p i d h a n d l i n g a n d freezing of tissue 7 Values for ~otal N A D in k i d n e y , heart, brain, a n d blood (Table I agree well w i t h those of GLOCK AND McLEAN 20. I t is e v i d e n t t h a t t h e e t h a n o l effect on the h e p a t i c N A D + : N A D H ratio is doser e l a t e d ; the p h e n o m e n o n is no~ all-or-none (Fig. 21. The reasons for this are implicit in t h e locus of N A D H f o r m a t i o n and the m e c h a n i s m s b y which it is oxidized. D u r i n g e t h a n o l oxidation, N A D H generation occurs in the soluble e x t r a m i t o c h o n d r i a l portlor, of the c y t o p l a s m , m which alcohol d e h y d r o g e n a s e has been isolated2t Liver m~tochondria are i m p e r m e a b l e to NADH~2,2a. which can therefore be oxidized b v d-~e m i t o c h o n d r i a l f l a v o p r o t e i n - c y t o c h r o m e electron t r a n s p o r t system only i n d i r e c t l y (Fig. z). This indirect flow of electrons occurs when N A D H p a r t i c i p a t e s in the reduction of c y t o p l a s m i c m e t a b o l i t e s which t h e n become s u b s t r a t e s for m i t o c h o n d f i a l oxidation. Two such reactions are the reduction of d i h y d r o x y a c e t o n e p h o s p h a t e Le z_c~_glycerophosphate.~< 25 a n d the reduction of a c e t o a c e t a t e to /%hydroxybutyrate~-< 3Aitochondrial o x i d a t i o n is l i m i t e d b v two r e l a t i v e l y slow, r a t e - d e t e r m i n i n g processes : d i s e n g a g e m e n t of the r e d u c e d molecule from one complex a n d d i s e n g a g e m e n t a n d t r a n s f e r of esterified p h o s p h a t e to ADP~< As the dose of e t h a n o l is increased, it a p p e a r s t h a t a b n i l d - u o of subscrate with reducing p o t e n t i a l , such a s / ~ - h y d r o x y b u t y r a t e a1:d z - ~ - g l y c e r o p h o s p h a t e m a y exceed the c a p a c i t y of the r a t e - l i m i t e d m i t o c h o n d r i a l oxidizing system. This then causes a slowing of electron flow from N A D H . resulting in a decrease of the N A D + : N A D H ratio. A further insight into the dose relationship of e t h a n o l to the h e p a t i c NAD~ N A D H ratio is afforded b v consideration of l a c t a t e metabolism. E x t r a m i t o c h o n d r i a ] N A D H can also be reoxidized b y p a r t i c i p a t i n g in the r e d u c t i o n of p y r u v a r e ~ lactate~7,zs As the dose of e t h a n o l is increased, blood l a c t a t e rises ~Fig. 2 : lactate. when given to r a t s in a m o u n t s which p r o d u c e b l o o d - l a c t a t e levels c o m p a r a b l e to those resulting from ethanol m e t a b o l i s m causes t h e r e d u c t i o n of h e p a t i c N A D + (Table Ii'~. Biocl~im. Bio1~hys. Hct~, zo7 (z965) °-9-37
EFFECT OF ETHANOL ON N A D + AND N A D H
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ETHANOL
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/ kg)
Fig. 3- Effect of 6 g ethanol per kg on hepatic N A D + and N A D H levels and s e r u m - e t h a n o l and blood-lactate concentrations during all I8-h period. E a c h value is the m e a n -~ S.E. of the m e a n o b t a i n e d f r o m analysis of 6 animals.
This example of the reduction of NAD+ by a metabolite arising from the oxidation of N A D H emphasizes the cyclical nature of the process. It seems probable that wheI1 lactate accumulation reaches a certain level, feedback to NAD + shifts the equilibrium in favor of a lower N A D + : N A D H ratio. It is also possible that ethanol, within a certain concentration range, may reduce hepatic blood flow and cellular oxygenation sufficiently to inhibit the oxidation of N A D H via the electron transport system• The change of hepatic N A D + : N A D H ratio with respect to time after a single dose of ethanol (Fig. 3) can be explained as a representation in time of the same metabolic events which determined the hepatic N A D + : N A D H ratios produced by different doses of ethanol after a fixed interval. The present observations of the effect of ethanol metabolism on hepatic NAD+ and N A D H levels differ from those previously reported in two ways : since the control NAD+: N A D H ratio is considerably higher than that found by all 1, 2, 4 but REBOUCAS AND ISSELBACHER 3, the ratio found after ethanol treatment is also considerably higher. RAIKA AND OURA 4, who made observations from 30 min to 4 h after ethanol administration, found that the hepatic NAD +: NADH ratio was decreased and maintained at a "fairly steady level" during that time. The reasons for that difference from our data are not apparent. Other investigators ~9,3o have detected the oxidation of small amounts of ethanol by kidney in vitro. Heart has not been reported to be a site for significant ethanol oxidation. We were unable to find alcohol dehydrogenase activity in either organ with methods which accurately assay crystalline alcohol dehydrogenase and which Biochim. Biophys. Acta, lO 7 (I965) 29-37
.36
G. R, GHERRICK, C. )L LEEV\f
demonstrate the enzyme i~ liver. The depression of the N A D + : N A D H ratio induc<::d in kidney by ethanol (Table I) cannot result from low rates of ethanol oxidatio;-~ in that organ; even the change in hepatic N A D + : N A D H ratio in rats metabolizing a>: much as z g ethanol per kg was trivial (Fig. z). The effect of ethanol on levets of NAD + and N A D H in kidney is aitributab]e, at least in part, to lactate utilization by that organ. When rats were given sufficient Ds> lactate to produce blood lactate levels comparable to those resulting from 6 g ethanol per ks, a significant fal! in the N A D ÷ : N A D H ratio was found (Table tI). The proportion of >- and L-lactate isomers in blood of animals treated with >5-!acta.te may nol have been equivalent to the proportion of lactate isomers in biood of animals treated with ethanol. Since mammals preferentially metabolize the >-isomeral, a< an even greater effect on the NAD+ and NADH levels probably would have resulted had the lactate-treated animals been given that isomer exclusively. The effect of ethanol on levels of NAD + and N A D H in heart (Table i) probably results additive]y from the utilization by that organ of several metabolites with reducing potential, elaborated during ethanol metabolism, of which lactate is one; the effect is not evoked by lactate alone (Table iI). It appears that heart has more effective mechanisms than kidney for reoxidizing cytoplasmic NADH. Isolated "intact" heart mitochondria show a relatively high rate of NADH oxidation, in contrast with mito..chondria of liver or kidney~ This is not taken_ to mean that heart mitochondria are actually permeable in rive to N A D H but rather that the mitochondriaI m.embrane is "labi!e"~< Reducing metabolites may pass through it, i~a rive, with ~anusual ease. ACKNOWLEDGEMENTS
The authors are grateful to Dr. H. B. BuRc~ and Dr. O. H. LowRY for valuable help and advice. Expert technical assistance was rendered by Mr. W. Ge~STZ,La_~>% Mr. W. GLA~Z.~A>-,and Mr. D. MARCIANO.This work was supported in part by grants MH-o554z-o3 and T1 AM 5e36-o 4 from the U.S. Public Health Service, and a grant from the New jersey State Department of Health, Division of Chrordc Illness Control REFERENCES I O. ]F'ORSANDER, N . R A I H A AND H . SL'OhlaLAINEX, Z. Ph¢esioh Cl~e~o<, 312 (1958) e 4 3 . 2 M. E . S>IIT~ i n n H . W . N~Wh~A~, J. Biol. Chem., 2 3 4 ( I 9 5 9 ) I 5 4 4 . 3 G. REBOUCAS AND K . J . ISSELBaCHER. ]. Cli~z Iv¢vesl 4 o ( 1 9 6 ! ) ~355. 4 N . C. R . R A I ~ A .XXD E . O
Biochi,n. Bi@hys. ~ida, io 7 (:~965) ~9-37
EFFECT OF ETHANOL ON N A D + AND N A D H
LEVELS
37
17 N. O. KAPLAN, S. P, COLOWICK, L. J. ZATMAN AND M. M. CIOTTI, dr. Biol. Chem., 205 (I953) 3 I. 18 A. iVI. STEIN~ N. O. KAPLAN AND IV[. M. CIOTTI, J. Biol. Chem., 234 (I959) 979. 19 L. GARCIA-BuNUEL, D. B. MCDOUGAL, JR., H. B. BURCH, E. M. JONES AND 1~. TOUHILL, dr. Neurochem., 9 (1962) 589. 20 ~. E. GLOCK A N D P. MCLEAN, Biochem. J., 61 (1955) 388. 21 A. NYBERG, J. SCHLTBERTH AND L. ANGGARD, Aeta Chem. Seand., 7 (1953) 117o22 G. E. BOXER AND T. M. DEVLIN, Science, 134 (I96I) I495. 23 D. E. GREEN AND F. L. CRANE, Prec. Intern. Syrup. Enzyme Chem., Tokyo, Kyoto, I957, P a n Pacific Press, Tokyo, 1958, p. 275. 24 R. W. ESTABROOI< AND ]3. SACI
Biochim. Biophys. Aeta, IO7 (I965) 2 9 - 3 7