627
Biochimica et Biophysica Acta, 428 (1976) 627--632 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27893
THE E F F E C T OF E T H A N O L INGESTION ON THE A L D E H Y D E D E H Y D R O G E N A S E S OF RAT L I V E R
N.J. GREENFIELD, R. PIETRUSZKO, G. LIN and D. LESTER
Center o f Alcohol Studies, Rutgers University, New Brunswick, N.J. 08903 (U.S.A.) (Received November 7th, 1975)
Summary The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity o f aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.
Introduction It has been reported that some alcoholics show significantly higher levels of acetaldehyde in the blood following ethanol ingestion than non-alcoholics [1--3]. One possible cause of the increase would be alterations in the activity of the aldehyde dehydrogenases responsible for acetaldehyde oxidation and removal consequent u p o n a continued insult from alcohol, and n o t predating the use of alcohol. There have been conflicting reports o f the effect of ethanol ingestion on the levels of acetaldehyde oxidation in animals. Some groups have reported no changes [ 4 - - 6 ] , while other groups have reported actual increases in activity [7,8]. H o r t o n [7], for example, reported a 50% increase in the level of acetaldehyde dehyrogenase in rat mitochondria following prolonged ingestion of ethanol using 0.026 mM acetaldehyde in his assay system. T o t t m a r et
628 al. [4] found no difference in aldehyde dehydrogenase levels of alcohol-fed and control rats, but their assay system used 5.0 mM acetaldehyde. Recently. Hasumura et al. [9], using an assay system including 0.060 mM acetaldehyde, have reported that acetaldehyde oxidation by intact hepatic mitochondria of rat liver is decreased by prolonged ethanol consumption, although the level of aldehyde dehydrogenase in disrupted mitochondria actually increased slightly. T o t t m a r et al. [10] and Horton and Barrett [11] have shown that there are at least two different aldehyde dehydrogenases with different subcellular distributions in rat liver tissue, one with a high g M for acetaldehyde and one with a low K M . The low K M enzyme is primarily located in the mitochondrial matrix while the enzyme with high K M for acetaldehyde is located primarily in the outer mitochondrial membranes and the microsomal fraction with some activity in the mitochondrial matrix [11]. Shum and Blair [12] have also separated two different NAD÷-dependent aldehyde dehydrogenases from rat liver cytosol with different affinities for acetaldehyde. In the previous work testing aldehyde dehydrogenase changes after ethanol feeding, assays of enzyme activity were n o t uniform and the experiments were carried out at widely different aldehyde concentrations. Using two substrate concentrations of propionaldehyde, we have measured the effect of alcohol consumption on the distribution and activity of aldehyde dehydrogenases in rat liver from pair-fed animals. Materials and Methods
Materials. Sprague-Dawley rats were obtained from Charles River Laboratories. NAD ÷, nitroblue tetrazolium and phenazine methosulphate were obtained from Sigma Chemical Co. Propionaldehyde was obtained from Eastman Organic Chemicals and redistilled before use. All other chemicals were reagent or enzyme grade. Rat liver fractionation. Sprague-Dawley rats were killed and the livers were fractionated into mitochondria, microsomes and postmicrosomal supernatant by the m e t h o d of Hogeboom [13]. The isolated mitochondria from 2 g of rat liver tissue were suspended in 5 ml of deionized water and frozen at --15°C and thawed twice to release the enzymes from the mitochondrial matrix. The membranes were separated from the soluble fraction by spinning at 20 000 X g in a Sorvall refrigerated centrifuge. The membranes then were dissolved in 5 ml of sodium deoxycholate, 2.6 mg/ml. The microsomal fraction from 2 g of rat liver was dissolved in 6 ml of 2.6 mg/ml sodium deoxycholate. Deoxycholate has been shown not to affect the activity of aldehyde dehydrogenases [11]. Assays o f enzymic activity: The enzymes were assayed for aldehyde dehydrogenase activity following the production of reduced NAD* by the change in absorbance at 340 nm, using a molar extinction coefficient of 6220 [14]. The assays were monitored in a Varian Techtron model 635 spectrophotometer at 25°C and were performed at two different propi0naldehyde concentrations (0.068 and 13.6 mM) in 0.09 M pyrophosphate/HC1 buffer, 0.1 M pyrazole, pH 9.0, containing 0.3 mg/ml NAD ÷. Assays w i t h o u t propionaldehyde were run as blank controls. The first concentration is sufficient to saturate the enzyme with a high affinity for propionaldehyde, and the second is sufficient to saturate the
629 enzyme with a low affinity for the substrate (unpublished results}. It has been shown by T o t t m a r et al. [10] and Horton and Barrett [11] that the activity of aldehyde dehydrogenase shows a biphasic dependence on acetaldehyde concentration attributed to one enzyme with an apparent Km value for acetaldehyde of less than 5 pM and another with a Km value for acetaldehyde of 1.5 mM. Assays with similar concentrations of acetaldehyde in place of the propionaldehyde gave similar results. Propionaldehyde was employed because it is more stable and less volatile than acetaldehyde and thus standard solutions are easier to prepare. The activity of the enzyme at low propionaldehyde concentration was subtracted from the activity at high concentration to give an estimate of the activity due to enzymes with low affinity for propionaldehyde. The effects o f ethanol feeding. To test the effects of ethanol feeding on aldehyde dehydrogenase activity, 14 pair-fed male Sprague-Dawley rats were utilized. The test rats were fed a diet containing ethanol at an average consumption of 12 g ethanol/kg/day. During the following day the control animals were given the same diet substituting isocaloric amounts of carbohydrate for the ethanol. These rats were maintained on the diets for approx. 60 days. The liquid diets used in this study were prepared from commercial liquid food, Nutrament (vanilla imitation flavor, Drackett Products Company, Cincinnati, Ohio), supplemented with casein hydrolysate (enzymatic, Sigma) and choline. With the addition of Dextri-Maltose (Mead Johnson Laboratories, Evansville, Ind.), ethanol and water, the liquid diets provided 1 kcal/ml. The composition of the control diet, expressed in kcal/100 ml was as follows: protein, 20%; fat, 13%; carbohydrate, 67% with adequate vitamins and minerals. In the experimental diet, 35% of the carbohydrate was replaced isocalorically with ethanol. Data treatment: Because the animals were pair fed and because the livers of each pair were fractionated and the fractions then assayed in parallel, Student's t test for correlated means was calculated to assess the statistical significance of the differences between activity values for the treated and untreated rats. Starch gel electrophoresis: The enzymes from the various liver fractions were separated via starch gel electrophoresis. The gel was made with Otto-Hiller starch and contained 5 mM sodium phosphate buffer, pH 7.0, while the buffer reservoirs contained 50 mM sodium phosphate buffer, pH 7.0. The gels were electrophoresed for 18 h at 100--150 V and were stained for enzyme activity in 0.1 M pyrophosphate/HC1, pH 9.0 buffer containing 0.2 mg/ml NAD, 13.6 mM propionaldehyde, nitrobluo tetrazolium and phenazine methosulphate. Results
The specific activities of aldehyde dehydrogenase in the alcohol-fed and control rats are shown in Table I. The total activity in each fraction/g of liver and the average protein concentrations of each fraction are shown in Table II. The effects of ethanol feeding on aldehyde dehydrogenase activity in rat liver differ with the cell fraction and substrate concentration. Although there is an average 60% increase in the specific activity of aldehyde dehydrogenase from the mitochondrial membranes of alcohol-fed rats when assayed at low propionaldehyde concentration (0.068 mM), this difference is significant only
630
TABLE I A C T I V I T Y O F A L D E H Y D E D E H Y D R O G E N A S E S P E R Mg P R O T E I N Assay m i x t u r e s c o n t a i n e d 0.09 M p y r o p b o s p h a t e / H C 1 buffer~ p H 9.0, 0.3 m g ] m l N A b +, 0.1 M p y r a z o l e a n d t h e d e s i g n a t e d p r o p i o n a l d e h y d e c o n c e n t r a t i o n . Assays w e r e p e r f o r m e d a t 25°C. P is t h e significance of the d i f f e r e n c e b e t w e e n r a t pairs c a l c u l a t e d b y S t u d e n t ' s t test for c o r r e l a t e d m e a n s . T h e e r r o r is the s t a n d a r d d e v i a t i o n , S e v e n a l c o h o l - f e d a n d seven pair-fed c o n t r o l m a l e S p r a g u e - D a w l e y rats were u s e d in t h e e x p e r i m e n t s . T h e r a t e s at t h e high p r o p i o n a l d e h y d e c o n c e n t r a t i o n are c o r r e c t e d for t h e c o n t r i b u t i o n due to t h e r a t e at the low p r o p i o n a i d e h y d e c o n c e n t r a t i o n . Propionaldehyde concentration
Rate of propionaldehyde oxidation (nmol/min/mg protein) mitochondriai extract
Control A l c o h o l fed
A l c o h o l fed
microsomes
0.068 mM 1
33.6 ± 9.2
6.5 ± 2.0
2.7 ± 2.0
~
38.7 ± 4.6
10.4 ± 4.5
1.4 ± 1.7
0.089
0.055
0.0004
' 13.6 m M ~
28.8 ± 11.4
34.7 ± 1 0 . 8
17.3 ± 8.0
l
10.2 ± 15.9
38.6 ± 9.8
13.9 -+ 6.7
0.013
0.63
0.35
p Control
mitochondrial membranes
p
supernatant
2.3±0.9 2.6±1.4 0.424
at the 0.055 level: 4 of the 7 pairs showed increases and 3 showed no change; none of the alcohol-fed rats exhibited decreases in activity. In the microsomes, on the other hand, there was a 58% decrease in activity when assayed at the low propionaldehyde concentration, significant at the 0.0004 level. Since the
TABLE II T O T A L A C T I V I T Y OF A L D E H Y D E D E H Y D R O G E N A S E PER g L I V E R Propionaldehyde concentration
R a t e of p r o p i o n a i d e h y d e o x i d a t i o n
(pmoles/min/g liver) * mitochondriai extract
Control A l c o h o l fed
A l c o h o l fed
microsomes
supernatant
0.068 mM 1
0.48 ± 0.18
0.15 ± 0.09
0 . 0 7 3 -+ 0 . 0 2 8
y
0 . 5 6 ± 0.11
0 . 2 2 ± 0.11
0.032 t 0.024
0 . 3 0 +- 0 . 1 7
0.096
0.056
0.011
0.529
0.375-+ 0 . 2 2
0.62-+ 0 . 1 9
0.71 -+ 0.31
0.25
0.61 ± 0 . 2 9
0.62 + 0.29
0.726
0.438
p Control
mitochondrial membranes
13.6 mM 7 ~
p
± 0.19
0.017
0.25 + 0.16
Protein concentration (mg/ml) Control
1 1 . 2 ± 3.8
8.6 ± 2.2
12.6 + 0.9
6.2 ± 1.4
A l c o h o l fed
1 3 . 8 -+ 5.2
7.9 ± 2.2
12.8 ± 1.6
6.0 -+ 1.8
p
0.285
0.364
0.418
00619
* T h e rates at l o w p r o p i o n a l d e h y d e c o n c e n t r a t i o n ( 0 . 0 6 8 m M ) w e r e s u b t r a c t e d f r o m the t o t a l r a t e s a t high p r o p i o n a l d e h y d e c o n c e n t r a t i o n (13.6 r a M ) to give an e s t i m a t e o f the r a t e s d u e to e n z y m e s w i t h l o w a f f i n i t y for p r o p i o n a l d e h y d e . F o r c o n d i t i o n s o f assay see T a b l e I.
631 activity levels in the microsomes at this concentration are low normally, the meaning of the decrease is unclear. The mitochondrial extracts and cytosolic supernatants showed no statistically significant changes at the low propionaldehyde concentration. At the higher concentrations of propionaldehyde (13.6 mM), the results were also complicated. The mitochondrial extracts showed a 58% decrease in enzyme specific activity in the alcohol-fed rats compared to the controls and this decrease was significant at the 0 . 0 1 3 level. The microsomes showed a smaller and nonsignificant decrease in the alcohol-fed rats and the mitochondrial membranes showed no change. No additional activity was detected in the supernatants when the assays were performed with the high rather than the low propionaldehyde concentration; however in this case, all the assays were performed on extracts which had been frozen for several days after the rats were killed and there was some enzyme decay during storage of this fraction. Because there are no statistically significant differences between the protein concentrations of the subcellular fractions of the paired animals (Table II), the aldehyde dehydrogenase activities of the various fractions, expressed per g liver, parallel those calculated per mg protein (Table I). Starch gel electrophoresis was utilized to determine whether alcohol feeding caused alterations in the number of electrophoretic distribution of aldehyde dehydrogenases. The various subcellular fractions were, therefore, separated and stained for enzyme activity. When such experiments were performed at pH 7.0 the mitochondrial matrix of the control rats showed at least two separate bands with enzymic activity that migrated to the anode. The post microsomal supernatant showed at least one anodal band. The enzymes from the mitochondrial membranes and microsomes which were solubilized with sodium deoxycholate showed one band that did not migrate and one very diffuse band which migrated to the anode. It is possible that the enzymes from the membranes and microsomes aggregated under conditions of electrophoresis. The enzymes from one of the control rats showed differences in electrophoretic mobility compared to the other control rats. The heterogeneity of the control gels obscured the effect, if any, of alcohol feeding on aldehyde dehydrogenase distribution. Discussion
Ethanol feeding appears to induce multiple effects on the distribution and activity of aldehyde dehydrogenases of rat liver. Horton [7] suggested that aldehyde dehydrogenase is induced in the mitochondria upon ethanol feeding to protect the membranes from the toxic effects of acetaldehyde. We found a substantial increase of mitochondrial membrane activity in about half of the rats tested. Deitrich and coworkers [15,16] have shown that phenobarbital induces aldehyde dehydrogenase activity in rat liver supernatants and that the response is genetic. The response to ethanol may also be genetic, different strains of rats showing different responses. Mice exhibit such differences in aldehyde dehydrogenase activity: Sheppard et al. [17] have shown that there is a 2--3-fold greater aldehyde-oxidizing capacity in the liver extracts of C57B1/6J mice compared to DBA/2J mice; the former strain prefers
632
10% ethanol to water while the latter avoids alcohol. Moreover, acetaldehyde levels are higher in the blood of the DBA/2J strain after ethanol injection than in the C57B1/6J strain (17). While an enzyme with high affinity for propionaldehyde is induced in the mitochondrial membranes, an enzyme with high affinity for propionaldehyde is repressed or destroyed in the microsomes and an enzyme with low affinity for propionaldehyde is also repressed or destroyed in the mitochondria. The total a m o u n t of acetaldehyde metabolised by these enzymes would be expected to be small, however, under physiological conditions, so the metabolic significance of these decreases remains to be established.
Acknowledgements This work was supported in part by grants from the National Institute on Alcohol Abuse and Alcoholism AA-00186, AA-00216 and AA-01849 and in part by a Bibmedical Science Support grant from Rutgers University. We would like to thank Joseph Eichenbaum for excellent technical assistance and Clinton Edson for access to some of his unpublished data.
References 1 F r e u n d , G. a n d O ' H o l l a x e n , P. ( 1 9 6 5 ) J. Lipid. Res. 6 , 4 7 1 - - 4 7 7 2 T r u i t t , J r . , E.B. ( 1 9 7 1 ) in B i o l o g i c a l A s p e c t s o f A l c o h o l , ( R o a c h , M . K . , M c I s s a c , W.M. a n d C r e a v e n , P.J., eds.), p p . 2 1 2 - - 2 3 2 , U n i v e r s i t y o f T e x a s Press, A u s t i n 3 K o r s t e n , M . A . , M a t s u z a k i , S., F e i n m a n , L. a n d L i e b e r , C.S. ( 1 9 7 5 ) N e w Engl. J. Med. 2 9 2 , 3 8 6 - - 3 8 9 4 T o t t m a r , S . O . C . , Kiessling, K . H . a n d F o r s l i n g , M. ( 1 9 7 4 ) A c t a P h a r m a c o l . T o x i c o l . 3 5 , 2 7 0 - - 2 7 6 5 R e d m o n d , G. a n d C o h e n , G. ( 1 9 7 1 ) S c i e n c e 1 1 7 , 3 8 7 - - 3 8 9 6 R a s k i n , N . H . a n d S o k o l o f f , L. ( 1 9 7 2 ) N a t u r e 2 3 6 , 1 3 8 - - 1 4 0 7 Horton, A.A. (1972) Bioehim. Biophys. Acta 253, 514--517 8 D a j a n i , R . M . , D a n i e l s k i , J . a n d O r t o n , J . M . ( 1 9 6 3 ) J. N u t r . S0, 1 9 6 - - 2 0 4 9 H a s u m u r a , Y., T e s c h k e , R, a n d L i e b e r , C.S. ( 1 9 7 5 ) S c i e n c e , 1 8 9 , 7 2 7 - - 7 2 9 10 T o t t m a r , S . O . C . , P e t t e r s o n , H. a n d Kiessling, K . H . ( 1 9 7 3 ) B i o c h e m . J. 1 3 5 , 5 7 7 - - 5 8 6 11 H o r t o n , A . A . a n d B a r r e t t , M.C. ( 1 9 7 5 ) A r c h . B i o c h e m . B i o p h y s . 1 6 7 , 4 2 6 - - 4 3 6 12 S h u m , J . G . a n d Blair, A . H . ( 1 9 7 2 ) C a n . J. B i o c h e m . 5 0 , 7 4 1 - - 7 4 8 13 H o g e b o o m , G . H . ( 1 9 5 7 ) M e t h o d s E n z y m o l . 1, 1 6 - - 1 9 14 K o r n b e r g , A. a n d H o r e c k e r , R . L . ( 1 9 5 3 ) B i o c h e m . P r e p . 3, 2 4 - - 2 8 15 D e i t r i c h , R . A . ( 1 9 7 1 ) S c i e n c e , 1 7 3 , 3 3 4 - - 3 3 6 16 D e i t r i c h , R . A . , Collins, A.C. a n d E r w i n , V . G . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 2 3 2 - - 7 2 3 6 17 S h e p p a r d , J . R . , A l b e r s h e i m , P. a n d M c C l e a r n , G.E. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 2 8 7 6 - - 2 8 8 2
Biochimica et Biophysica Acta, 428 (1976) 627--632 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27893
THE E F F E C T OF E T H A N O L INGESTION ON THE A L D E H Y D E D E H Y D R O G E N A S E S OF RAT L I V E R
N.J. GREENFIELD, R. PIETRUSZKO, G. LIN and D. LESTER
Center o f Alcohol Studies, Rutgers University, New Brunswick, N.J. 08903 (U.S.A.) (Received November 7th, 1975)
Summary The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity o f aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.
Introduction It has been reported that some alcoholics show significantly higher levels of acetaldehyde in the blood following ethanol ingestion than non-alcoholics [1--3]. One possible cause of the increase would be alterations in the activity of the aldehyde dehydrogenases responsible for acetaldehyde oxidation and removal consequent u p o n a continued insult from alcohol, and n o t predating the use of alcohol. There have been conflicting reports o f the effect of ethanol ingestion on the levels of acetaldehyde oxidation in animals. Some groups have reported no changes [ 4 - - 6 ] , while other groups have reported actual increases in activity [7,8]. H o r t o n [7], for example, reported a 50% increase in the level of acetaldehyde dehyrogenase in rat mitochondria following prolonged ingestion of ethanol using 0.026 mM acetaldehyde in his assay system. T o t t m a r et
628 al. [4] found no difference in aldehyde dehydrogenase levels of alcohol-fed and control rats, but their assay system used 5.0 mM acetaldehyde. Recently. Hasumura et al. [9], using an assay system including 0.060 mM acetaldehyde, have reported that acetaldehyde oxidation by intact hepatic mitochondria of rat liver is decreased by prolonged ethanol consumption, although the level of aldehyde dehydrogenase in disrupted mitochondria actually increased slightly. T o t t m a r et al. [10] and Horton and Barrett [11] have shown that there are at least two different aldehyde dehydrogenases with different subcellular distributions in rat liver tissue, one with a high g M for acetaldehyde and one with a low K M . The low K M enzyme is primarily located in the mitochondrial matrix while the enzyme with high K M for acetaldehyde is located primarily in the outer mitochondrial membranes and the microsomal fraction with some activity in the mitochondrial matrix [11]. Shum and Blair [12] have also separated two different NAD÷-dependent aldehyde dehydrogenases from rat liver cytosol with different affinities for acetaldehyde. In the previous work testing aldehyde dehydrogenase changes after ethanol feeding, assays of enzyme activity were n o t uniform and the experiments were carried out at widely different aldehyde concentrations. Using two substrate concentrations of propionaldehyde, we have measured the effect of alcohol consumption on the distribution and activity of aldehyde dehydrogenases in rat liver from pair-fed animals. Materials and Methods
Materials. Sprague-Dawley rats were obtained from Charles River Laboratories. NAD ÷, nitroblue tetrazolium and phenazine methosulphate were obtained from Sigma Chemical Co. Propionaldehyde was obtained from Eastman Organic Chemicals and redistilled before use. All other chemicals were reagent or enzyme grade. Rat liver fractionation. Sprague-Dawley rats were killed and the livers were fractionated into mitochondria, microsomes and postmicrosomal supernatant by the m e t h o d of Hogeboom [13]. The isolated mitochondria from 2 g of rat liver tissue were suspended in 5 ml of deionized water and frozen at --15°C and thawed twice to release the enzymes from the mitochondrial matrix. The membranes were separated from the soluble fraction by spinning at 20 000 X g in a Sorvall refrigerated centrifuge. The membranes then were dissolved in 5 ml of sodium deoxycholate, 2.6 mg/ml. The microsomal fraction from 2 g of rat liver was dissolved in 6 ml of 2.6 mg/ml sodium deoxycholate. Deoxycholate has been shown not to affect the activity of aldehyde dehydrogenases [11]. Assays o f enzymic activity: The enzymes were assayed for aldehyde dehydrogenase activity following the production of reduced NAD* by the change in absorbance at 340 nm, using a molar extinction coefficient of 6220 [14]. The assays were monitored in a Varian Techtron model 635 spectrophotometer at 25°C and were performed at two different propi0naldehyde concentrations (0.068 and 13.6 mM) in 0.09 M pyrophosphate/HC1 buffer, 0.1 M pyrazole, pH 9.0, containing 0.3 mg/ml NAD ÷. Assays w i t h o u t propionaldehyde were run as blank controls. The first concentration is sufficient to saturate the enzyme with a high affinity for propionaldehyde, and the second is sufficient to saturate the
629 enzyme with a low affinity for the substrate (unpublished results}. It has been shown by T o t t m a r et al. [10] and Horton and Barrett [11] that the activity of aldehyde dehydrogenase shows a biphasic dependence on acetaldehyde concentration attributed to one enzyme with an apparent Km value for acetaldehyde of less than 5 pM and another with a Km value for acetaldehyde of 1.5 mM. Assays with similar concentrations of acetaldehyde in place of the propionaldehyde gave similar results. Propionaldehyde was employed because it is more stable and less volatile than acetaldehyde and thus standard solutions are easier to prepare. The activity of the enzyme at low propionaldehyde concentration was subtracted from the activity at high concentration to give an estimate of the activity due to enzymes with low affinity for propionaldehyde. The effects o f ethanol feeding. To test the effects of ethanol feeding on aldehyde dehydrogenase activity, 14 pair-fed male Sprague-Dawley rats were utilized. The test rats were fed a diet containing ethanol at an average consumption of 12 g ethanol/kg/day. During the following day the control animals were given the same diet substituting isocaloric amounts of carbohydrate for the ethanol. These rats were maintained on the diets for approx. 60 days. The liquid diets used in this study were prepared from commercial liquid food, Nutrament (vanilla imitation flavor, Drackett Products Company, Cincinnati, Ohio), supplemented with casein hydrolysate (enzymatic, Sigma) and choline. With the addition of Dextri-Maltose (Mead Johnson Laboratories, Evansville, Ind.), ethanol and water, the liquid diets provided 1 kcal/ml. The composition of the control diet, expressed in kcal/100 ml was as follows: protein, 20%; fat, 13%; carbohydrate, 67% with adequate vitamins and minerals. In the experimental diet, 35% of the carbohydrate was replaced isocalorically with ethanol. Data treatment: Because the animals were pair fed and because the livers of each pair were fractionated and the fractions then assayed in parallel, Student's t test for correlated means was calculated to assess the statistical significance of the differences between activity values for the treated and untreated rats. Starch gel electrophoresis: The enzymes from the various liver fractions were separated via starch gel electrophoresis. The gel was made with Otto-Hiller starch and contained 5 mM sodium phosphate buffer, pH 7.0, while the buffer reservoirs contained 50 mM sodium phosphate buffer, pH 7.0. The gels were electrophoresed for 18 h at 100--150 V and were stained for enzyme activity in 0.1 M pyrophosphate/HC1, pH 9.0 buffer containing 0.2 mg/ml NAD, 13.6 mM propionaldehyde, nitrobluo tetrazolium and phenazine methosulphate. Results
The specific activities of aldehyde dehydrogenase in the alcohol-fed and control rats are shown in Table I. The total activity in each fraction/g of liver and the average protein concentrations of each fraction are shown in Table II. The effects of ethanol feeding on aldehyde dehydrogenase activity in rat liver differ with the cell fraction and substrate concentration. Although there is an average 60% increase in the specific activity of aldehyde dehydrogenase from the mitochondrial membranes of alcohol-fed rats when assayed at low propionaldehyde concentration (0.068 mM), this difference is significant only
630
TABLE I A C T I V I T Y O F A L D E H Y D E D E H Y D R O G E N A S E S P E R Mg P R O T E I N Assay m i x t u r e s c o n t a i n e d 0.09 M p y r o p b o s p h a t e / H C 1 buffer~ p H 9.0, 0.3 m g ] m l N A b +, 0.1 M p y r a z o l e a n d t h e d e s i g n a t e d p r o p i o n a l d e h y d e c o n c e n t r a t i o n . Assays w e r e p e r f o r m e d a t 25°C. P is t h e significance of the d i f f e r e n c e b e t w e e n r a t pairs c a l c u l a t e d b y S t u d e n t ' s t test for c o r r e l a t e d m e a n s . T h e e r r o r is the s t a n d a r d d e v i a t i o n , S e v e n a l c o h o l - f e d a n d seven pair-fed c o n t r o l m a l e S p r a g u e - D a w l e y rats were u s e d in t h e e x p e r i m e n t s . T h e r a t e s at t h e high p r o p i o n a l d e h y d e c o n c e n t r a t i o n are c o r r e c t e d for t h e c o n t r i b u t i o n due to t h e r a t e at the low p r o p i o n a i d e h y d e c o n c e n t r a t i o n . Propionaldehyde concentration
Rate of propionaldehyde oxidation (nmol/min/mg protein) mitochondriai extract
Control A l c o h o l fed
A l c o h o l fed
microsomes
0.068 mM 1
33.6 ± 9.2
6.5 ± 2.0
2.7 ± 2.0
~
38.7 ± 4.6
10.4 ± 4.5
1.4 ± 1.7
0.089
0.055
0.0004
' 13.6 m M ~
28.8 ± 11.4
34.7 ± 1 0 . 8
17.3 ± 8.0
l
10.2 ± 15.9
38.6 ± 9.8
13.9 -+ 6.7
0.013
0.63
0.35
p Control
mitochondrial membranes
p
supernatant
2.3±0.9 2.6±1.4 0.424
at the 0.055 level: 4 of the 7 pairs showed increases and 3 showed no change; none of the alcohol-fed rats exhibited decreases in activity. In the microsomes, on the other hand, there was a 58% decrease in activity when assayed at the low propionaldehyde concentration, significant at the 0.0004 level. Since the
TABLE II T O T A L A C T I V I T Y OF A L D E H Y D E D E H Y D R O G E N A S E PER g L I V E R Propionaldehyde concentration
R a t e of p r o p i o n a i d e h y d e o x i d a t i o n
(pmoles/min/g liver) * mitochondriai extract
Control A l c o h o l fed
A l c o h o l fed
microsomes
supernatant
0.068 mM 1
0.48 ± 0.18
0.15 ± 0.09
0 . 0 7 3 -+ 0 . 0 2 8
y
0 . 5 6 ± 0.11
0 . 2 2 ± 0.11
0.032 t 0.024
0 . 3 0 +- 0 . 1 7
0.096
0.056
0.011
0.529
0.375-+ 0 . 2 2
0.62-+ 0 . 1 9
0.71 -+ 0.31
0.25
0.61 ± 0 . 2 9
0.62 + 0.29
0.726
0.438
p Control
mitochondrial membranes
13.6 mM 7 ~
p
± 0.19
0.017
0.25 + 0.16
Protein concentration (mg/ml) Control
1 1 . 2 ± 3.8
8.6 ± 2.2
12.6 + 0.9
6.2 ± 1.4
A l c o h o l fed
1 3 . 8 -+ 5.2
7.9 ± 2.2
12.8 ± 1.6
6.0 -+ 1.8
p
0.285
0.364
0.418
00619
* T h e rates at l o w p r o p i o n a l d e h y d e c o n c e n t r a t i o n ( 0 . 0 6 8 m M ) w e r e s u b t r a c t e d f r o m the t o t a l r a t e s a t high p r o p i o n a l d e h y d e c o n c e n t r a t i o n (13.6 r a M ) to give an e s t i m a t e o f the r a t e s d u e to e n z y m e s w i t h l o w a f f i n i t y for p r o p i o n a l d e h y d e . F o r c o n d i t i o n s o f assay see T a b l e I.
631 activity levels in the microsomes at this concentration are low normally, the meaning of the decrease is unclear. The mitochondrial extracts and cytosolic supernatants showed no statistically significant changes at the low propionaldehyde concentration. At the higher concentrations of propionaldehyde (13.6 mM), the results were also complicated. The mitochondrial extracts showed a 58% decrease in enzyme specific activity in the alcohol-fed rats compared to the controls and this decrease was significant at the 0 . 0 1 3 level. The microsomes showed a smaller and nonsignificant decrease in the alcohol-fed rats and the mitochondrial membranes showed no change. No additional activity was detected in the supernatants when the assays were performed with the high rather than the low propionaldehyde concentration; however in this case, all the assays were performed on extracts which had been frozen for several days after the rats were killed and there was some enzyme decay during storage of this fraction. Because there are no statistically significant differences between the protein concentrations of the subcellular fractions of the paired animals (Table II), the aldehyde dehydrogenase activities of the various fractions, expressed per g liver, parallel those calculated per mg protein (Table I). Starch gel electrophoresis was utilized to determine whether alcohol feeding caused alterations in the number of electrophoretic distribution of aldehyde dehydrogenases. The various subcellular fractions were, therefore, separated and stained for enzyme activity. When such experiments were performed at pH 7.0 the mitochondrial matrix of the control rats showed at least two separate bands with enzymic activity that migrated to the anode. The post microsomal supernatant showed at least one anodal band. The enzymes from the mitochondrial membranes and microsomes which were solubilized with sodium deoxycholate showed one band that did not migrate and one very diffuse band which migrated to the anode. It is possible that the enzymes from the membranes and microsomes aggregated under conditions of electrophoresis. The enzymes from one of the control rats showed differences in electrophoretic mobility compared to the other control rats. The heterogeneity of the control gels obscured the effect, if any, of alcohol feeding on aldehyde dehydrogenase distribution. Discussion
Ethanol feeding appears to induce multiple effects on the distribution and activity of aldehyde dehydrogenases of rat liver. Horton [7] suggested that aldehyde dehydrogenase is induced in the mitochondria upon ethanol feeding to protect the membranes from the toxic effects of acetaldehyde. We found a substantial increase of mitochondrial membrane activity in about half of the rats tested. Deitrich and coworkers [15,16] have shown that phenobarbital induces aldehyde dehydrogenase activity in rat liver supernatants and that the response is genetic. The response to ethanol may also be genetic, different strains of rats showing different responses. Mice exhibit such differences in aldehyde dehydrogenase activity: Sheppard et al. [17] have shown that there is a 2--3-fold greater aldehyde-oxidizing capacity in the liver extracts of C57B1/6J mice compared to DBA/2J mice; the former strain prefers
632
10% ethanol to water while the latter avoids alcohol. Moreover, acetaldehyde levels are higher in the blood of the DBA/2J strain after ethanol injection than in the C57B1/6J strain (17). While an enzyme with high affinity for propionaldehyde is induced in the mitochondrial membranes, an enzyme with high affinity for propionaldehyde is repressed or destroyed in the microsomes and an enzyme with low affinity for propionaldehyde is also repressed or destroyed in the mitochondria. The total a m o u n t of acetaldehyde metabolised by these enzymes would be expected to be small, however, under physiological conditions, so the metabolic significance of these decreases remains to be established.
Acknowledgements This work was supported in part by grants from the National Institute on Alcohol Abuse and Alcoholism AA-00186, AA-00216 and AA-01849 and in part by a Bibmedical Science Support grant from Rutgers University. We would like to thank Joseph Eichenbaum for excellent technical assistance and Clinton Edson for access to some of his unpublished data.
References 1 F r e u n d , G. a n d O ' H o l l a x e n , P. ( 1 9 6 5 ) J. Lipid. Res. 6 , 4 7 1 - - 4 7 7 2 T r u i t t , J r . , E.B. ( 1 9 7 1 ) in B i o l o g i c a l A s p e c t s o f A l c o h o l , ( R o a c h , M . K . , M c I s s a c , W.M. a n d C r e a v e n , P.J., eds.), p p . 2 1 2 - - 2 3 2 , U n i v e r s i t y o f T e x a s Press, A u s t i n 3 K o r s t e n , M . A . , M a t s u z a k i , S., F e i n m a n , L. a n d L i e b e r , C.S. ( 1 9 7 5 ) N e w Engl. J. Med. 2 9 2 , 3 8 6 - - 3 8 9 4 T o t t m a r , S . O . C . , Kiessling, K . H . a n d F o r s l i n g , M. ( 1 9 7 4 ) A c t a P h a r m a c o l . T o x i c o l . 3 5 , 2 7 0 - - 2 7 6 5 R e d m o n d , G. a n d C o h e n , G. ( 1 9 7 1 ) S c i e n c e 1 1 7 , 3 8 7 - - 3 8 9 6 R a s k i n , N . H . a n d S o k o l o f f , L. ( 1 9 7 2 ) N a t u r e 2 3 6 , 1 3 8 - - 1 4 0 7 Horton, A.A. (1972) Bioehim. Biophys. Acta 253, 514--517 8 D a j a n i , R . M . , D a n i e l s k i , J . a n d O r t o n , J . M . ( 1 9 6 3 ) J. N u t r . S0, 1 9 6 - - 2 0 4 9 H a s u m u r a , Y., T e s c h k e , R, a n d L i e b e r , C.S. ( 1 9 7 5 ) S c i e n c e , 1 8 9 , 7 2 7 - - 7 2 9 10 T o t t m a r , S . O . C . , P e t t e r s o n , H. a n d Kiessling, K . H . ( 1 9 7 3 ) B i o c h e m . J. 1 3 5 , 5 7 7 - - 5 8 6 11 H o r t o n , A . A . a n d B a r r e t t , M.C. ( 1 9 7 5 ) A r c h . B i o c h e m . B i o p h y s . 1 6 7 , 4 2 6 - - 4 3 6 12 S h u m , J . G . a n d Blair, A . H . ( 1 9 7 2 ) C a n . J. B i o c h e m . 5 0 , 7 4 1 - - 7 4 8 13 H o g e b o o m , G . H . ( 1 9 5 7 ) M e t h o d s E n z y m o l . 1, 1 6 - - 1 9 14 K o r n b e r g , A. a n d H o r e c k e r , R . L . ( 1 9 5 3 ) B i o c h e m . P r e p . 3, 2 4 - - 2 8 15 D e i t r i c h , R . A . ( 1 9 7 1 ) S c i e n c e , 1 7 3 , 3 3 4 - - 3 3 6 16 D e i t r i c h , R . A . , Collins, A.C. a n d E r w i n , V . G . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 2 3 2 - - 7 2 3 6 17 S h e p p a r d , J . R . , A l b e r s h e i m , P. a n d M c C l e a r n , G.E. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 2 8 7 6 - - 2 8 8 2
