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Effects of ethanol on mitochondrial oxidations
i[4
BIOCHIM1CA ET B~[OPHYSICAACTA s s £ 25o05
E F F E C T S OF E T H A N O L ON M ~ [ T O C H O N D R I A L O X I D A T I O N S B. o. CHRISTOPHERSEN Norsh Hydro's Ins~il~,tefor Ca~cef Research, Mo~febdlo, Oslo (Norzvc~y)
(Received August 3oth, i963)
SUMMARY X. Effects of ethanol on mitochondriat oxidations i s vitro have been studied: 2. Ethanol in relatively low concentrations was found to stimulate the O2 uptake of rat-liver mitochondria with fi-hydroxybutyrate as the substrate. With pyruvate~ ~-ketoglutarate and succinate as substrates ethanol depressed the O~ uptake. 3, With /?-hydroxybutyrate the O, uptake increased with increasing ethanol concentration to reach a maximum of 13o-x35 % of the controI value at o.5-0.6 M ethanol. The increased O~ uptake coMd be accounted for by a stoichiometric increase in the acetoacetate formation. 4. Sonic disruption of the mitochondria prior to incubation nearly abolished the stimulating effect of ethanol on the O.~ consumption. 5. The activity of/?-hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate :NAD oxidorednctase, EC I.I.I.3o) was not affected by ethanol in concentrations which stimulated the mitochondrial O a uptake with/~-hydroxybutyrate. 6. The P/O ratio was not lowered by ethanol concentrations which increased the/~-hydroxybutyrate oxidation. 7. It is suggested that ethanol increases the oxidation of the/~-hydroxybutyrate, by facilitating its transport into the mitochondria.
INTRODUCTION Recently KIESSLiNG A N D TILANDER1 reported that liver mitochondria isolated from rats given a prolonged alcohol feeding, have a reduced capacity to oxidize pyruvate and succinate. In the present work the effect of ethanol on mitochondrial oxidations in vitro has been studied. The experiments were prompted by the observation that ethanol, used as a vehicle for water-insoluble steroids, proved to have distinct effects on the rate of oxidation of various substrates. The oxidation of/3-hydroxybutyrate was found to be stimulated by relatively small concentrations of ethanol, while the oxidation of other substrates was depressed. In the present communication these data are reported as well as experiments carried out to elucidate the mechanism of the observed effects. ~fATERIALS AND METHODS Preparatio~ of mitocho~dria
The general method of HOGEBOOSIAND SCHNEIDERas modified by ~3REM);Rz \vas used. Rat-liver mitochondria were prepared by homogenization in ice cold o.25 3'~ Biochim. Biophys. Actc~, 86 (I964) ~4-~o
EFFECTS OF ETHANOL ON MITOCHONDRIALOXIDATIONS
15
sucrose containing 5 mM neutralized E D T A and 5 mM Tris buffer (pH 7-4). Nuclei and cell debris were removed b y centrifugation at 700 and 800 × g twice. The mitochondria were sedimented at 20 ooo x g for 15 rain and washed once b y resuspension in homogenizing medium and recentrifugation. The mitochondria were finally resuspended in the homogenizing medium in a concentration corresponding to approx. o.75 g fresh liver per ml. The average content of mitochondrial protein was 9-11.5 mg per ml suspension. The mitochondria were used in the experiments within I h after the preparation. Sonication of mitochondria In certain experiments the 02 uptake of mitochondria which had been exposed to sonic;ation was measured. Aliquots (8 ml each) of the freshly prepared mitochondrial suspension were treated b y an MSE ultrasonic disintegrator at 18-2o keycles. The sonication was carried out under ice cooling in 3o-sec periods, interrupted b y 2-rain pauses to avoid temperature rise. Assays The 02 uptake was measured with Warburg technique at 30 o with air as the atmosphere and 30% K O H in the center well. The standard incubation mixture was in principle as described b y WEINBACHa. Tris buffer was used in the place of glycylglycine buffer. The standard incubation mixture contained 8o/*moles Tris buffer (pH 7.4), 30/,moles phosphate buffer (pH 7.4), IO/,moles MgC12, 4 °/,moles NaF, 5/,moles ADP, 0.5 mg hexokinase (Sigma Grade III), 2/,moles DPN, 0.03/,moles cytoehrome c. The mitochondriai suspension (0.5 ml) was added last. The total volume was 2 ml. When ethanol was used, it was added as a 96 % solution before the addition of the mitochondria. The reaction mixture containing all components except the substrate, was preincubated for lO rain to allow time for gas equilibrium and adjustment of the manometers. The reaction was started by tipping in the substrate. The substrates were used as the sodium salts. Acetoacetate was measured according to WALKER4. The phosphate uptake was calculated from the disappearance of Pi, measured b y the method of Iq'ISKE AND SUBBARow 5. Protein was measured according to LowRY et al.*. The /3-hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD oxidoreductase, EC 1.1.1.3o) activity was assayed according to LEI-ININGER et al. 7 b y measuring spectrophotometrically at 34 ° m / , the D P N H formed. Mitochondria isolated by the standard method were suspended in o.125 M KC1 containing 0.02 M Tris buffer (pH 7.4)- The suspension contained (per ml) mitochondria from approx. 7 ° mg fresh liver. Aliquots of IO ml were exposed to sonieation at 18-2o kcycles for 6 rain under ice-cooling. The sonication was carried out in 3o-sec periods, interrupted by 2-rain pauses to avoid temperature rise. The enzyme activity of the freshly prepared preparations was measured within lO rain. All the reagents were obtained commercially. RESULTS Effect of ethanol on ~-hydroxybutyrate oxidation The stimulating effect of 0.3 M ethanol on the 02 uptake when fi-hydroxybutyrate was used as substrate, is shown in Fig. Ia. The effect was nearly constant throughout Biochim. Biophys. dcla, 86 (1964) 14-2o
I6
t3. O~ C H R ] S T O P H E R S E N
the incubation period. The effect was found to be highly reprodncible. Thus in a series of five experiments with mitochondria prepared from different rats and on differen',: days, the average O 2uptake in the presence of o.3 N! ethanol was found to be ~23 ~: 3 % of that of the control. Fig. ~b shows the effect of increasing ethanol concentration on the O~ uptake with /~-hydroxybutyrate as substrate. The initially stimulating effect subsides at higher concentrations and finalty a depression is observed. U follows from the data in Table I that in the concentration range from zero to o.45 M ethanol the O~ uptake increases approximately linearly with increasing ethanol concentration. The small O2 uptake in the absence of substrate was not increased by ethanol (Fig. Is) demonstrating that the ethanol effect is not due to increased endogenous c~
12C ~.
I
bo ~ O ~
/°/° i /°o/O
°t
5 4c
]
4
2O 40 Ethanol concn. (M) Time (rain) F i g . i a . E f f e c t of e t h a n o l o n t h e m i t o c h o n d r i a l O.~ u p t a k e a s a f u n c t i o n of i n c u b a t i o n t i m e w i t h ~-hydroxybutyrate a s s u b s t r a t e . T h e s t a n d a r d i n c u b a t i o n m i x t u r e w a s u s e d . 5 ° l~moles of /%hydroxybutyrate w a s t i p p e d i n a f t e r p r e i n c u b a t i o n f o r IO r a i n . @ - - O , c o n t r o l , n o e t h a n o l ; O - - O , o.3 M e t h a n o l p r e s e n t ; A . - - A , i n c u b a t i o n w i t h n o s u b s t r a t e a n d n o e t h a n o l ; ~ - - Q , no s u b s t r a t e w i t h o. 3 M e t h a n o l . I b . E f f e c t of i n c r e a s i n g e t h a n o l c o n c e n t r a t i o n o n t h e O 2 u p t a k e w i t h ~-hydroxybutyrate as substrate. The standard incubation mixture was used and the substrate a d d e d a s d e s c r i b e d i n t h e l e g e n d t o F i g . i s . I n c u b a t i o n p e r i o d 6o m i n . TABLE
I
~FF~CT OF INCRgASINO ~T~ANOL CO>rCENTR_~TIO~'S O>r TH~ ©2 UPTAKE X~D ACETOACETAT~ FORS~ATIO~ WIT~ ~-HVnROX'ZBUT'ZR.~TE .~S SUBSTRAT~ The standard incubation mixture was used and ~-hydroxybutyrate added as described in the l e g e n d t o F i g . I s . T h e i n c u b a t i o n p e r i o d w a s 6o m i n . A f t e r ~che l a s t m a n o m e t e r r e a d i n g t h e v e s s e l s w e r e q u i c k l y c o o l e d a n d t h e r e a c t i o n s s t o p p e d b y t h e a d d i t i o n of o . 3 5 m l of 25 % t r i c h l o r o a c e t i c acid and acetoacetate was determined. Preparation No. I Ethanol concn. (M)
Oo uptake ( l~atoms)
Acetoace¢ate formed (#mo l,es)
o O.li o.15 o.I9 0.3o 0.45
8.0 9.3
8. 5 9.3 -9.7 lO. 3 11.2
9.4 1o.o lO.6
Preparation No. I f 02 uptake (#atoms)
6.6 -7 .2 -8. 4 9.0
Acetoacetate formed (~umoles)
6.6 -7.3 -8.2 9.1
respiration or to oxidation of the ethanol added. Direct evidence that the increased 02 uptake in the presence of ethanol, can be accounted for by increased utilization of the substrate, was obtained by, correlating the 02 consumption with the acetoacetate concurrently formed (Table I). Thus, it is apparent that the increased O 2 B i o c T d m . B g o p h ) , s . A c t a , 8 6 ( I 9 6 4 ) ~4--2o
EFFECTS OF ETHANOL ON MITOCHONDRIAL OXIDATIONS
17
consumption is associated with a stoichrometric increase in the amount of acetoacetate formed.
The effect of ethanol on the oxidation of other substrates With pyruvate, c~-ketoglutarate and succinate as substrates the O~ uptake decreased in the presence of increasing ethanol concentrations (Fig. 2). The experiments indicate that ethanol has a substrate-selective effect, ranging from a moderate, but significant stimulation of mitochondrial oxidation of ~-hydroxybutyrate to increasing degrees of depression with the substrates succinate, c~-ketoglutarate and pyruvate. I00 [
t
~--o
60
~
~
,
o
~o
~1
K o 0115 I I I 0.45 Ethanol conch. (M)
'
o16
'
,.~
'
E t hanoi 'concn. (M)
]Fig. 3. T h e P / O ratio in t h e p r e s e n c e of Fig. 2. Effect of i n c r e a s i n g ethanol conceni n c r e a s i n g e t h a n o l c o n c e n t r a t i o n s w i t h /~trations on the 02 uptake with pyruvate, h y d r o x y b u t y r a t e as s u b s t r a t a . T h e s t a n d a r d ce-ketoglutarate a n d s u c c i n a t e as s u b s t r a t e s . i n c u b a t i o n m i x t u r e w a s used. /~-HydroxyT h e s t a n d a r d i n c u b a t i o n m i x t u r e w a s used. 20 # m o l e s of t h e s u b s t r a t e (the o x i d a t i o n of b u t y r a t e was a d d e d as described in t h e legend to Fig. ia. I n c u b a t i o n period 50 rain. p y r u v a t e w a s s p a r k e d b y 2 / 2 m o l e s of malate) w a s t i p p e d in a f t e r p r e i n c u b a t i o n for IO rain. I n c u b a t i o n period 60 rain. E ] - - [ 3 , pyruva%e, O 2 u p t a k e of control 96 /~1; 0 - - 0 , ~-ketog l u t a r a t e , O 2 u p t a k e of control 93/zl; O - - O , succinate, O 3 u p t a k e of control 131/21.
Mechanism of the effect of ethanol on ~-hydroxybutyrate oxidation The observed effects of ethanol might conceivably be caused b y interference with any of a number of processes, such as the passage of substrate molecules into the mitochondria, the oxidations at substrate level or the various reactions in the terminal respiratory chain. However, since the terminal respiratory chain is a common p a t h in the oxidation of all the substrates tested (with the exception of succinate), the observed effects can hardly be explained b y an effect of ethanol on this chain alone. The stimulating effect of ethanol on ~-hydroxybutyrate oxidation can not be explained b y a possible uncoupling effect of ethanol. Fig. 3 demonstrates that the P/O ratio was not significantly lowered by ethanol in the concentration range where ethanol had a definitely stimulating effect on the 0 3 consumption. 0 n l y at higher ethanol concentrations a gradual decrease of the P/O ratio was found (Fig. 3)An a t t e m p t to investigate whether ethanol affects the enzymic reaction performing the substrate-level oxidation of ~-hydroxybutyrate was carried out. The D P N H formed b y oxidation of the substrate, was measured spectrophotometrically, the reoxidation of D P N H b y the respiratory chain being blocked b y NaCN. In these experiments ~-hydroxybutyrate dehydrogenase from sonically disrupted mitochondria was used because it was confirmed that in intact mitochondria added D P N was not Biochim. Biophys. Acta, 86 (i964) 1 4 - 2 o
18
B. 0 . C E R i S T O P I - I E R S E N
reduced by the fl-hydroxybutyrate dehydrogenase, possibly because DPN m a y not be able to penetrate the external mitoehondrial membraneL "Fable If shows that ethanol had no significant effects under these conditions. The experiments do not: TABLE ~-IIYDROXYBIJTYRATE~DEI~IYDROGENASE AT VARYING
Ii
ACTIVITY ~THANOL
MI~ASURED
SPECTROPHOTOMETRICALLY
CONCENTRATIONS
T h e e n z y m e s u s p e n s i o n w a s p r e p a r e d as d e s c r i b e d in t h e s e c t i o n MATERIALS AND ~ ' [ E T H O D S , a n d e n z y m e c o r r e s p o n d i n g t o a p p r o x : I 5 m g f r e s h l i v e r u s e d p e r celi. T h e t e s t s y s t e m c o n t a i n e d o . o 4 M T r i s b u f f e r ( p H 8.5), o . o 5 M n i c o t i n a m i d e , o . o i M¢ c y s t e i n e , o . o o 3 M D P N , o . o 2 M ~)L-~hydroxybutyrate, o . o o i M N a C N a n d e n z y m e ( a d d e d l a s t ) in a t o t a l v o l u m e of 3.o ml. T h e e t h a n o ! , w h e n u s e d , w a s a d d e d a s a 9 6 % s o ! u t i o n . T h e i n c r e a s e i n a b s o r b a n c y a t 34 ° m/~ w a s r e a d a t ~ - m i n i n t e r v a l s i n a i - c m cell a t 2 o °. T h e v a l u e s w e r e c o r r e c t e d f o r t h e s l i g h t c h a n g e s o c c u r r i n g i n t h e c o n t r o l cells n o t c o n t a i n i n g s u b s t r a t e . T h e r e s u l t s w e r e e x p r e s s e d in } t m o l e s D P N H f o r m e d p e r g l i v e r p e r h a n d b a s e d o n t h e i n c r e a s e i n a b s o r b a n e y t h e f i r s t 5 i n i n a f t e r t h e a d d i t i o n of the enzyme. Ethanol conch.
#moles D P N H formed per g liver per h
(M/
Preparation No. f
Preparation No. II
o.o o.I 0.3 o.6
I32 -I34 136
i49 i49 I43 t44
TABLE THE
iII
]gFFECT OF SONICATION ON THE STIMULATING ACTION OF ETHANOL ON THE MITOCI-tONDRIAL OXIDATION OF /~-HYDROXYBUTYRATE
T h e e f f e c t of e t h a n o l o n m i t o c h o n d r i a e x p o s e d t o s o n i c a ~ i o n w a s c o m p a r e d w i t h t h e e f f e c t o n untreated mitochondria. !n the control experiments the standard incubation mixture containing o.5 m l m i t o c h o n d r i a l s u s p e n s i o n w a s u s e d a n d f l - h y d r o x y b u t y r a t e a d d e d a s d e s c r i b e d in t h e l e g e n d t o F i g . i a . A l i q u o t s of t h e s a m e m i t o c h o n d r i a l s u s p e n s i o n a s u s e d i n t h e c o n t r o l s , w e r e s o n i c a t e d f o r 3 m i n a s d e s c r i b e d i n t i l e s e c t i o n MATERIALS AND METHODS. o . 5 - m l p o r t i o n s of t h e sonicated mitochondrial suspensions were incubated in exactly the same way as the controls_ E t h a n o l w a s u s e d i n a c o n c e n t r a t i o n of o . 3 o M. 4-, w i t h e t h a n o l ; - - , w i t h o u t e t h a n o l ; A , d i f f e r e n c e between experiments with and without ethanol. Preparation No. I Incubation period (rain)
Ethanol
No sonicalion
O~ uptake
(#l)
A
32 Io
Sonication
O,a upt~tke
O~l)
40
-
-
67
@
Ioo 99
60 +
I22
58 I3
o 58
9I 4
97
o 43
89
93 23
43
76
85
I
Io
3
68 17
~o8
A
26
6o
82
(/.d~ 25
5° 4
i8
Souicetgon
O~ uptake
5 34
62
82
4°
(#l)
A
29
58 12
+
O~ uptake
2 36
55 2o
No sonication
A
34 8
c_
Preparation No. l I
o 68
B{oohim. B~ophys. Acta, 8 6 ( I 9 6 4 ) ~ 4 - 2 o .
EFFECTS OF ETHANOL ON MITOCI{ONDRIAL OXIDATIONS
19
support the possibility that the stimulating effect of ethanol on the mitochondrial oxidation of /3-hydroxybutyrate is caused by a stimulation of the substrate-level oxidation. It was pointed out above that the effect of ethanol on the/3-hydroxybutyrate oxidation might be caused b y stimulating action on the transport of the substrate to the enzyme sites. Information on this question was sought by comparing the effect of ethanol on mitochondria prepared as intact as possible, with its effect on mitochondria disrupted by sonication. Table I I I shows that sonication nearly abolished the stimulating effect of ethanol on fi-hydroxybutyrate oxidation. This finding is compatible with the view that ethanol renders the substrate more accessible to the enzyme sites, possibly b y facilitating the transport of the substrate into the mitochondria. DISCUSSION
The results reported here indicate that incubation of mitochondria with ethanol has distinct effects on their ability to oxidize different substrates. While the oxidation of /~-hydroxybutyrate was stimulated, the oxidation of c~-ketoglutarate, pyruvare and succinate was depressed. These effects seem not to have been taken into consideration by previous investigators who have used ethanol in work with mitochondria, The observed effects of ethanol on the mitochondrial oxidation of pyruvate and succinate are of interest in relation to the finding ot previous authors 1 that mitochondria isolated from rats given a prolonged alcohol feeding, had a reduced capacity to oxidize the same substrates. It should be noted that the lowest ethanol concentrations found here to have substrate-selective effects are near the levels to be expected in acute alcohol intoxication in man. The interpretation offered here that ethanol stimulates the oxidation of ~-hydrox y b u t y r a t e by facilitating its transport into the mitochondria is based mainly on the finding that sonication of the mitochondria prior to incubation, abolished the stimulating effect of ethanol. The lack of effect of ethanol on the jO-hydroxybutyratedehydrogenase activity as such is in agreement with such a mechanism. The present data do not permit an adequate interpretation of the mechanism of the depressing effect of ethanol on the oxidation of pyruvate, ~-ketoglutarate and succinate. However, it is interesting that JOHNSON AND LARDY8 have observed substrate-selective effects on mitochondrial oxidations by varying the tonicity of the medium, a procedure which is thought to affect the permeability of mitochondria. In view of the fact that the mitochondrial membranes are rich in lipids, it is perhaps not surprising that ethanol m a y affect the properties of these membranes. The results reported above suggest that ethanol and perhaps other lipid-sohible substances used in this way m a y be of value as experimental tools in work on the permeability of the mitochondrial membranes. ACKNOWLEDGEMENTS
The author is grateful to Professor A. PIHL and T. SANNER for help and interest in the work. This study was supported b y the Norwegian Research Council for the Science and Humanities and N. A. Stangs Legat. Biochim. Biophys. Actc~, 86 (1964) 14-2o
20
B, O. CH~ISTOPHERSEN REFERENCES
1 ~ . - H . KIESSL~NG AND K. TILANDER, Expll, Cell Res, 3 ° (1963) 476 2 j. 13I~EM~, J. Biol. Chem., 237 (I962) 2228. a E. C. WEXNBACH, J. Biol. Chem., 234 (I959) I58oP. G. WALK~R, Biochem. J., 58 (I954) 699. 5 C. H . ~:?isIzE AND Y. SUBBA~:~OW,J . Biol. Chem., 66 (1925) 375. 6 0 . H. LOWRY, N. J. ROSEBROL'GH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., x93 (!95 x) 265o A. L. L~HXlNGER, H. C. StrnnUTH ANn J. B. W I s ~ ; Ji Biol. Chem., 235 (~96o) 2450. 8 D. JOHNSON ~
Biochim. Biophys. Acta, 86 (1964) T4--2o
BIOCHIM1CA ET B~[OPHYSICAACTA s s £ 25o05
E F F E C T S OF E T H A N O L ON M ~ [ T O C H O N D R I A L O X I D A T I O N S B. o. CHRISTOPHERSEN Norsh Hydro's Ins~il~,tefor Ca~cef Research, Mo~febdlo, Oslo (Norzvc~y)
(Received August 3oth, i963)
SUMMARY X. Effects of ethanol on mitochondriat oxidations i s vitro have been studied: 2. Ethanol in relatively low concentrations was found to stimulate the O2 uptake of rat-liver mitochondria with fi-hydroxybutyrate as the substrate. With pyruvate~ ~-ketoglutarate and succinate as substrates ethanol depressed the O~ uptake. 3, With /?-hydroxybutyrate the O, uptake increased with increasing ethanol concentration to reach a maximum of 13o-x35 % of the controI value at o.5-0.6 M ethanol. The increased O~ uptake coMd be accounted for by a stoichiometric increase in the acetoacetate formation. 4. Sonic disruption of the mitochondria prior to incubation nearly abolished the stimulating effect of ethanol on the O.~ consumption. 5. The activity of/?-hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate :NAD oxidorednctase, EC I.I.I.3o) was not affected by ethanol in concentrations which stimulated the mitochondrial O a uptake with/~-hydroxybutyrate. 6. The P/O ratio was not lowered by ethanol concentrations which increased the/~-hydroxybutyrate oxidation. 7. It is suggested that ethanol increases the oxidation of the/~-hydroxybutyrate, by facilitating its transport into the mitochondria.
INTRODUCTION Recently KIESSLiNG A N D TILANDER1 reported that liver mitochondria isolated from rats given a prolonged alcohol feeding, have a reduced capacity to oxidize pyruvate and succinate. In the present work the effect of ethanol on mitochondrial oxidations in vitro has been studied. The experiments were prompted by the observation that ethanol, used as a vehicle for water-insoluble steroids, proved to have distinct effects on the rate of oxidation of various substrates. The oxidation of/3-hydroxybutyrate was found to be stimulated by relatively small concentrations of ethanol, while the oxidation of other substrates was depressed. In the present communication these data are reported as well as experiments carried out to elucidate the mechanism of the observed effects. ~fATERIALS AND METHODS Preparatio~ of mitocho~dria
The general method of HOGEBOOSIAND SCHNEIDERas modified by ~3REM);Rz \vas used. Rat-liver mitochondria were prepared by homogenization in ice cold o.25 3'~ Biochim. Biophys. Actc~, 86 (I964) ~4-~o
EFFECTS OF ETHANOL ON MITOCHONDRIALOXIDATIONS
15
sucrose containing 5 mM neutralized E D T A and 5 mM Tris buffer (pH 7-4). Nuclei and cell debris were removed b y centrifugation at 700 and 800 × g twice. The mitochondria were sedimented at 20 ooo x g for 15 rain and washed once b y resuspension in homogenizing medium and recentrifugation. The mitochondria were finally resuspended in the homogenizing medium in a concentration corresponding to approx. o.75 g fresh liver per ml. The average content of mitochondrial protein was 9-11.5 mg per ml suspension. The mitochondria were used in the experiments within I h after the preparation. Sonication of mitochondria In certain experiments the 02 uptake of mitochondria which had been exposed to sonic;ation was measured. Aliquots (8 ml each) of the freshly prepared mitochondrial suspension were treated b y an MSE ultrasonic disintegrator at 18-2o keycles. The sonication was carried out under ice cooling in 3o-sec periods, interrupted b y 2-rain pauses to avoid temperature rise. Assays The 02 uptake was measured with Warburg technique at 30 o with air as the atmosphere and 30% K O H in the center well. The standard incubation mixture was in principle as described b y WEINBACHa. Tris buffer was used in the place of glycylglycine buffer. The standard incubation mixture contained 8o/*moles Tris buffer (pH 7.4), 30/,moles phosphate buffer (pH 7.4), IO/,moles MgC12, 4 °/,moles NaF, 5/,moles ADP, 0.5 mg hexokinase (Sigma Grade III), 2/,moles DPN, 0.03/,moles cytoehrome c. The mitochondriai suspension (0.5 ml) was added last. The total volume was 2 ml. When ethanol was used, it was added as a 96 % solution before the addition of the mitochondria. The reaction mixture containing all components except the substrate, was preincubated for lO rain to allow time for gas equilibrium and adjustment of the manometers. The reaction was started by tipping in the substrate. The substrates were used as the sodium salts. Acetoacetate was measured according to WALKER4. The phosphate uptake was calculated from the disappearance of Pi, measured b y the method of Iq'ISKE AND SUBBARow 5. Protein was measured according to LowRY et al.*. The /3-hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD oxidoreductase, EC 1.1.1.3o) activity was assayed according to LEI-ININGER et al. 7 b y measuring spectrophotometrically at 34 ° m / , the D P N H formed. Mitochondria isolated by the standard method were suspended in o.125 M KC1 containing 0.02 M Tris buffer (pH 7.4)- The suspension contained (per ml) mitochondria from approx. 7 ° mg fresh liver. Aliquots of IO ml were exposed to sonieation at 18-2o kcycles for 6 rain under ice-cooling. The sonication was carried out in 3o-sec periods, interrupted by 2-rain pauses to avoid temperature rise. The enzyme activity of the freshly prepared preparations was measured within lO rain. All the reagents were obtained commercially. RESULTS Effect of ethanol on ~-hydroxybutyrate oxidation The stimulating effect of 0.3 M ethanol on the 02 uptake when fi-hydroxybutyrate was used as substrate, is shown in Fig. Ia. The effect was nearly constant throughout Biochim. Biophys. dcla, 86 (1964) 14-2o
I6
t3. O~ C H R ] S T O P H E R S E N
the incubation period. The effect was found to be highly reprodncible. Thus in a series of five experiments with mitochondria prepared from different rats and on differen',: days, the average O 2uptake in the presence of o.3 N! ethanol was found to be ~23 ~: 3 % of that of the control. Fig. ~b shows the effect of increasing ethanol concentration on the O~ uptake with /~-hydroxybutyrate as substrate. The initially stimulating effect subsides at higher concentrations and finalty a depression is observed. U follows from the data in Table I that in the concentration range from zero to o.45 M ethanol the O~ uptake increases approximately linearly with increasing ethanol concentration. The small O2 uptake in the absence of substrate was not increased by ethanol (Fig. Is) demonstrating that the ethanol effect is not due to increased endogenous c~
12C ~.
I
bo ~ O ~
/°/° i /°o/O
°t
5 4c
]
4
2O 40 Ethanol concn. (M) Time (rain) F i g . i a . E f f e c t of e t h a n o l o n t h e m i t o c h o n d r i a l O.~ u p t a k e a s a f u n c t i o n of i n c u b a t i o n t i m e w i t h ~-hydroxybutyrate a s s u b s t r a t e . T h e s t a n d a r d i n c u b a t i o n m i x t u r e w a s u s e d . 5 ° l~moles of /%hydroxybutyrate w a s t i p p e d i n a f t e r p r e i n c u b a t i o n f o r IO r a i n . @ - - O , c o n t r o l , n o e t h a n o l ; O - - O , o.3 M e t h a n o l p r e s e n t ; A . - - A , i n c u b a t i o n w i t h n o s u b s t r a t e a n d n o e t h a n o l ; ~ - - Q , no s u b s t r a t e w i t h o. 3 M e t h a n o l . I b . E f f e c t of i n c r e a s i n g e t h a n o l c o n c e n t r a t i o n o n t h e O 2 u p t a k e w i t h ~-hydroxybutyrate as substrate. The standard incubation mixture was used and the substrate a d d e d a s d e s c r i b e d i n t h e l e g e n d t o F i g . i s . I n c u b a t i o n p e r i o d 6o m i n . TABLE
I
~FF~CT OF INCRgASINO ~T~ANOL CO>rCENTR_~TIO~'S O>r TH~ ©2 UPTAKE X~D ACETOACETAT~ FORS~ATIO~ WIT~ ~-HVnROX'ZBUT'ZR.~TE .~S SUBSTRAT~ The standard incubation mixture was used and ~-hydroxybutyrate added as described in the l e g e n d t o F i g . I s . T h e i n c u b a t i o n p e r i o d w a s 6o m i n . A f t e r ~che l a s t m a n o m e t e r r e a d i n g t h e v e s s e l s w e r e q u i c k l y c o o l e d a n d t h e r e a c t i o n s s t o p p e d b y t h e a d d i t i o n of o . 3 5 m l of 25 % t r i c h l o r o a c e t i c acid and acetoacetate was determined. Preparation No. I Ethanol concn. (M)
Oo uptake ( l~atoms)
Acetoace¢ate formed (#mo l,es)
o O.li o.15 o.I9 0.3o 0.45
8.0 9.3
8. 5 9.3 -9.7 lO. 3 11.2
9.4 1o.o lO.6
Preparation No. I f 02 uptake (#atoms)
6.6 -7 .2 -8. 4 9.0
Acetoacetate formed (~umoles)
6.6 -7.3 -8.2 9.1
respiration or to oxidation of the ethanol added. Direct evidence that the increased 02 uptake in the presence of ethanol, can be accounted for by increased utilization of the substrate, was obtained by, correlating the 02 consumption with the acetoacetate concurrently formed (Table I). Thus, it is apparent that the increased O 2 B i o c T d m . B g o p h ) , s . A c t a , 8 6 ( I 9 6 4 ) ~4--2o
EFFECTS OF ETHANOL ON MITOCHONDRIAL OXIDATIONS
17
consumption is associated with a stoichrometric increase in the amount of acetoacetate formed.
The effect of ethanol on the oxidation of other substrates With pyruvate, c~-ketoglutarate and succinate as substrates the O~ uptake decreased in the presence of increasing ethanol concentrations (Fig. 2). The experiments indicate that ethanol has a substrate-selective effect, ranging from a moderate, but significant stimulation of mitochondrial oxidation of ~-hydroxybutyrate to increasing degrees of depression with the substrates succinate, c~-ketoglutarate and pyruvate. I00 [
t
~--o
60
~
~
,
o
~o
~1
K o 0115 I I I 0.45 Ethanol conch. (M)
'
o16
'
,.~
'
E t hanoi 'concn. (M)
]Fig. 3. T h e P / O ratio in t h e p r e s e n c e of Fig. 2. Effect of i n c r e a s i n g ethanol conceni n c r e a s i n g e t h a n o l c o n c e n t r a t i o n s w i t h /~trations on the 02 uptake with pyruvate, h y d r o x y b u t y r a t e as s u b s t r a t a . T h e s t a n d a r d ce-ketoglutarate a n d s u c c i n a t e as s u b s t r a t e s . i n c u b a t i o n m i x t u r e w a s used. /~-HydroxyT h e s t a n d a r d i n c u b a t i o n m i x t u r e w a s used. 20 # m o l e s of t h e s u b s t r a t e (the o x i d a t i o n of b u t y r a t e was a d d e d as described in t h e legend to Fig. ia. I n c u b a t i o n period 50 rain. p y r u v a t e w a s s p a r k e d b y 2 / 2 m o l e s of malate) w a s t i p p e d in a f t e r p r e i n c u b a t i o n for IO rain. I n c u b a t i o n period 60 rain. E ] - - [ 3 , pyruva%e, O 2 u p t a k e of control 96 /~1; 0 - - 0 , ~-ketog l u t a r a t e , O 2 u p t a k e of control 93/zl; O - - O , succinate, O 3 u p t a k e of control 131/21.
Mechanism of the effect of ethanol on ~-hydroxybutyrate oxidation The observed effects of ethanol might conceivably be caused b y interference with any of a number of processes, such as the passage of substrate molecules into the mitochondria, the oxidations at substrate level or the various reactions in the terminal respiratory chain. However, since the terminal respiratory chain is a common p a t h in the oxidation of all the substrates tested (with the exception of succinate), the observed effects can hardly be explained b y an effect of ethanol on this chain alone. The stimulating effect of ethanol on ~-hydroxybutyrate oxidation can not be explained b y a possible uncoupling effect of ethanol. Fig. 3 demonstrates that the P/O ratio was not significantly lowered by ethanol in the concentration range where ethanol had a definitely stimulating effect on the 0 3 consumption. 0 n l y at higher ethanol concentrations a gradual decrease of the P/O ratio was found (Fig. 3)An a t t e m p t to investigate whether ethanol affects the enzymic reaction performing the substrate-level oxidation of ~-hydroxybutyrate was carried out. The D P N H formed b y oxidation of the substrate, was measured spectrophotometrically, the reoxidation of D P N H b y the respiratory chain being blocked b y NaCN. In these experiments ~-hydroxybutyrate dehydrogenase from sonically disrupted mitochondria was used because it was confirmed that in intact mitochondria added D P N was not Biochim. Biophys. Acta, 86 (i964) 1 4 - 2 o
18
B. 0 . C E R i S T O P I - I E R S E N
reduced by the fl-hydroxybutyrate dehydrogenase, possibly because DPN m a y not be able to penetrate the external mitoehondrial membraneL "Fable If shows that ethanol had no significant effects under these conditions. The experiments do not: TABLE ~-IIYDROXYBIJTYRATE~DEI~IYDROGENASE AT VARYING
Ii
ACTIVITY ~THANOL
MI~ASURED
SPECTROPHOTOMETRICALLY
CONCENTRATIONS
T h e e n z y m e s u s p e n s i o n w a s p r e p a r e d as d e s c r i b e d in t h e s e c t i o n MATERIALS AND ~ ' [ E T H O D S , a n d e n z y m e c o r r e s p o n d i n g t o a p p r o x : I 5 m g f r e s h l i v e r u s e d p e r celi. T h e t e s t s y s t e m c o n t a i n e d o . o 4 M T r i s b u f f e r ( p H 8.5), o . o 5 M n i c o t i n a m i d e , o . o i M¢ c y s t e i n e , o . o o 3 M D P N , o . o 2 M ~)L-~hydroxybutyrate, o . o o i M N a C N a n d e n z y m e ( a d d e d l a s t ) in a t o t a l v o l u m e of 3.o ml. T h e e t h a n o ! , w h e n u s e d , w a s a d d e d a s a 9 6 % s o ! u t i o n . T h e i n c r e a s e i n a b s o r b a n c y a t 34 ° m/~ w a s r e a d a t ~ - m i n i n t e r v a l s i n a i - c m cell a t 2 o °. T h e v a l u e s w e r e c o r r e c t e d f o r t h e s l i g h t c h a n g e s o c c u r r i n g i n t h e c o n t r o l cells n o t c o n t a i n i n g s u b s t r a t e . T h e r e s u l t s w e r e e x p r e s s e d in } t m o l e s D P N H f o r m e d p e r g l i v e r p e r h a n d b a s e d o n t h e i n c r e a s e i n a b s o r b a n e y t h e f i r s t 5 i n i n a f t e r t h e a d d i t i o n of the enzyme. Ethanol conch.
#moles D P N H formed per g liver per h
(M/
Preparation No. f
Preparation No. II
o.o o.I 0.3 o.6
I32 -I34 136
i49 i49 I43 t44
TABLE THE
iII
]gFFECT OF SONICATION ON THE STIMULATING ACTION OF ETHANOL ON THE MITOCI-tONDRIAL OXIDATION OF /~-HYDROXYBUTYRATE
T h e e f f e c t of e t h a n o l o n m i t o c h o n d r i a e x p o s e d t o s o n i c a ~ i o n w a s c o m p a r e d w i t h t h e e f f e c t o n untreated mitochondria. !n the control experiments the standard incubation mixture containing o.5 m l m i t o c h o n d r i a l s u s p e n s i o n w a s u s e d a n d f l - h y d r o x y b u t y r a t e a d d e d a s d e s c r i b e d in t h e l e g e n d t o F i g . i a . A l i q u o t s of t h e s a m e m i t o c h o n d r i a l s u s p e n s i o n a s u s e d i n t h e c o n t r o l s , w e r e s o n i c a t e d f o r 3 m i n a s d e s c r i b e d i n t i l e s e c t i o n MATERIALS AND METHODS. o . 5 - m l p o r t i o n s of t h e sonicated mitochondrial suspensions were incubated in exactly the same way as the controls_ E t h a n o l w a s u s e d i n a c o n c e n t r a t i o n of o . 3 o M. 4-, w i t h e t h a n o l ; - - , w i t h o u t e t h a n o l ; A , d i f f e r e n c e between experiments with and without ethanol. Preparation No. I Incubation period (rain)
Ethanol
No sonicalion
O~ uptake
(#l)
A
32 Io
Sonication
O,a upt~tke
O~l)
40
-
-
67
@
Ioo 99
60 +
I22
58 I3
o 58
9I 4
97
o 43
89
93 23
43
76
85
I
Io
3
68 17
~o8
A
26
6o
82
(/.d~ 25
5° 4
i8
Souicetgon
O~ uptake
5 34
62
82
4°
(#l)
A
29
58 12
+
O~ uptake
2 36
55 2o
No sonication
A
34 8
c_
Preparation No. l I
o 68
B{oohim. B~ophys. Acta, 8 6 ( I 9 6 4 ) ~ 4 - 2 o .
EFFECTS OF ETHANOL ON MITOCI{ONDRIAL OXIDATIONS
19
support the possibility that the stimulating effect of ethanol on the mitochondrial oxidation of /3-hydroxybutyrate is caused by a stimulation of the substrate-level oxidation. It was pointed out above that the effect of ethanol on the/3-hydroxybutyrate oxidation might be caused b y stimulating action on the transport of the substrate to the enzyme sites. Information on this question was sought by comparing the effect of ethanol on mitochondria prepared as intact as possible, with its effect on mitochondria disrupted by sonication. Table I I I shows that sonication nearly abolished the stimulating effect of ethanol on fi-hydroxybutyrate oxidation. This finding is compatible with the view that ethanol renders the substrate more accessible to the enzyme sites, possibly b y facilitating the transport of the substrate into the mitochondria. DISCUSSION
The results reported here indicate that incubation of mitochondria with ethanol has distinct effects on their ability to oxidize different substrates. While the oxidation of /~-hydroxybutyrate was stimulated, the oxidation of c~-ketoglutarate, pyruvare and succinate was depressed. These effects seem not to have been taken into consideration by previous investigators who have used ethanol in work with mitochondria, The observed effects of ethanol on the mitochondrial oxidation of pyruvate and succinate are of interest in relation to the finding ot previous authors 1 that mitochondria isolated from rats given a prolonged alcohol feeding, had a reduced capacity to oxidize the same substrates. It should be noted that the lowest ethanol concentrations found here to have substrate-selective effects are near the levels to be expected in acute alcohol intoxication in man. The interpretation offered here that ethanol stimulates the oxidation of ~-hydrox y b u t y r a t e by facilitating its transport into the mitochondria is based mainly on the finding that sonication of the mitochondria prior to incubation, abolished the stimulating effect of ethanol. The lack of effect of ethanol on the jO-hydroxybutyratedehydrogenase activity as such is in agreement with such a mechanism. The present data do not permit an adequate interpretation of the mechanism of the depressing effect of ethanol on the oxidation of pyruvate, ~-ketoglutarate and succinate. However, it is interesting that JOHNSON AND LARDY8 have observed substrate-selective effects on mitochondrial oxidations by varying the tonicity of the medium, a procedure which is thought to affect the permeability of mitochondria. In view of the fact that the mitochondrial membranes are rich in lipids, it is perhaps not surprising that ethanol m a y affect the properties of these membranes. The results reported above suggest that ethanol and perhaps other lipid-sohible substances used in this way m a y be of value as experimental tools in work on the permeability of the mitochondrial membranes. ACKNOWLEDGEMENTS
The author is grateful to Professor A. PIHL and T. SANNER for help and interest in the work. This study was supported b y the Norwegian Research Council for the Science and Humanities and N. A. Stangs Legat. Biochim. Biophys. Actc~, 86 (1964) 14-2o
20
B, O. CH~ISTOPHERSEN REFERENCES
1 ~ . - H . KIESSL~NG AND K. TILANDER, Expll, Cell Res, 3 ° (1963) 476 2 j. 13I~EM~, J. Biol. Chem., 237 (I962) 2228. a E. C. WEXNBACH, J. Biol. Chem., 234 (I959) I58oP. G. WALK~R, Biochem. J., 58 (I954) 699. 5 C. H . ~:?isIzE AND Y. SUBBA~:~OW,J . Biol. Chem., 66 (1925) 375. 6 0 . H. LOWRY, N. J. ROSEBROL'GH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., x93 (!95 x) 265o A. L. L~HXlNGER, H. C. StrnnUTH ANn J. B. W I s ~ ; Ji Biol. Chem., 235 (~96o) 2450. 8 D. JOHNSON ~
Biochim. Biophys. Acta, 86 (1964) T4--2o