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The effect of ethyl alcohol on rat liver tryptophan oxygenase
Lite 8ciencee vol. 11, Part II, pp " 1029-1028, 1972 . Printed in Cyreat Britain
Pergamon Prege
THE EFFECT OF ETHYL ALCOHOL ON RAT LIVER TRYPTDPHAN OXYGENASE Vishwanath M. Sardesai and Haydee S . Provido Robert S . Marx Laboratories, Department of Surgery . 9Payne State University, School of Medicine, 540 Eaat Cornfield, Detroit, !lichigan
(Received 10 August iß72 ; in final form 14 September 1972) SUMMARY The activity of liver tryptophan oxygenase was determined in rats administered intoxicating dose of ethanol . Following intraperitoneal injection of ethanol theta was a rise in the enzyme activity reaching 1®vela considerably above those of the control animals . Maximum enzyme activity was reached 5 hours after ethanol administration and thea returned to within contrc+l levels in about 9 hours . It is suggested that the observed increase in tryptophan .oxygenase in the liver may be the result of increased plasma adrenocortical hormone level following ethanol intoxication, or mny be secondary to the induction of ALA (delta-aminolevulinic acid) synthetase and consequent enchanced hems production . INTROD UC TION Tryptophan, in addition to its use as a substrate for protein synthesis, has two other major functions . muter,
One results in the production of neurotrana-
5-hydroxytryptamine (serotonin), much of which ie subsequently met
abolized to 5-hydroxyindole acetic acid
(HIAA) prior to excretion in the uriné.
Its second use is in the production of the vitamin, nicotinic acid .
The
latter substance is used, of course, in the production of nicotinamide adenine dinucleotide
(NAD) and nicotinamide adenine dinucleotlde phosphate (NADP),
compounds of major significance in the energy yielding reactions . There have been several reports during last few years indicating abnormalities of tryptophan metabolism in alcoholics .
Olaen et al
(1) found decreased
urinary excretion of HLAA in alcoholic patients than in normal controls both on control diets and after tryptophan loading .
1029
Rosenfeld (2) also observed
l0â!
Alcohol ~rd Liver Tryptop~an O~gg~ase
Vol. 11, No, a1
that normal subjects given 100 grams of alcohol* a:cretad less HIAA than in controls .
The alcoholic patients described by Olsan et al
(1) had normal
urinary excretion of N-methylnicotinamide, a major urinary metabolic product of nicotinic acid .
Thasa investigators concluded, therefore, that alcohol
does not cause a defect in the kynuranine pathway of tryptophan metabolism . However, Gabuzda and Davidson (3) observed that patients with cirrhosis of the liver associated with chronic alcoholism had increased urinary excretion of nicotinic acid and Nrmethylnicotinnmide .
Lérner et al (4) also reported that
chronic alcoholics, after the ingestion of 10 qms of DL tryptophan orally, had excessive urinary excretion of xanthurenic acid, an intermediate metabolite of tryptophan pathway leading to nicotinic acid .
it seems possible that after
the injection of alcohol more of the tryptophan may be metabolized via the kynureniae pathway (5) .
The present study was 8esigned to determine the eflect
o! an acute dose of alcohol to rats on the activity of liver tryptophan oxyjanase, the first and apparently the major rate limiting enzyme of the tryptophan-nicotinic acid pathway .
s b~ragus Dawley lemale study.
rats weighing between 175-200 grams were used in this
The animals were fasted for 18 hours before the intraperitonaal admin-
istration of 5 ml of ethanol (v/v) solution in saline or 5 ml of saline solution in control animals .
At the end of each time period after the injection
as indicated in the Table rats were sacrificed by dacnpitation and livers were quickly removed and washed with cold saline .
One gram of liver was homogenized
in 7 ml of cold 0 .14 M loCl containing 0 .0025 N NaOH . The tryptophan oxygeaase was determined by the method of lcaox and Auerbach (6) as codified by Berry and Smythe
(7) .
One ml of the liver homogenate was
aaaayed for the enzyus with and without the addition of 0.1 ~ of hamatin solu-
*
Alcohol cad ethanol are used syiwnymously in this paper.
Vol . 11, No. 21
tion .
Alcoàol and Liver Tryptophan O~genaee
10~
Tha hamatin solution was prepared immediately before use by dissolving
1 mg of twice-crystallised bovine heroin
(Sigma Chemical Company)
in 10 ml of
O .iN sodium hydroxide . RESOLTS The activity of tryptophan ozygenase without the addition of excess hematin is considered to be indicative of in vivo bolo enzyme activity and is therefore referred to as the "active" apoenzyme .
The addition of excess hema
tin reveals the additional amount of apoenzyme which is potentially active and is therefor® referred to as "total" apoenzyme.
The difference in activity with
and without the addition of excess activator is considered to be a measure of the amount of "inactive" apoenzyme in the liver homogenate .
TABLE I Effect of Alcohol on Liver Tryptophan Oxygenase Control *
Hours after Injection
Active
Total
Alcohol-Treated Active
Total
0
0.78 + 0.11
1 .22 + 0.12
0.78 + 0.05
1 .29 + 0.08
1
0.80 + 0 .06
1 .18 + 0 .11
0.75 + 0.06
1 .23 + 0.09
2
0.68 + 0.04
1 .16 + 0 .15
0.88 + 0.09
1 .50 + 0.15
3
0.79 + 0 .08
1 .23 + 0.07
0.95 + 0.15
2 .08 + 0.19
4
0 .62 + 0 .06
1 .18 + 0.10
1 .22 + 0 .19
2 .84 + 0 .31
5
0 .78 + 0 .09
1 .18 + 0.08
1.46 + 0.15
3.24 + 0 .39
6
0 .80 + 0 .10
1 .28 + 0 .15
1 .40 + 0 .22
2 .80 + 0 .28
9
0 .74 + 0 .08
1 .23 f 0 .10
0 .66 + 0 .06
1 .05 + 0 .08
* The activity is expressed as micromoles of Rynurenine formed par gram of liver per hour . Values are averages of 18 experiments with standard deviations . Duplicate runs were made on each tissue and agreed within + 10s. The data on the effect of alcohol on liver tryptophan oxygenase are presented in Table I .
Aa can be seen the admünistratioa of saline alone does
not result in appreciable change in the enzyme activity .
Following intraperi
toaeal injection of ethanol there 1s a üse is the enzyme activity, reaching
1028
Alcohol aad Liver Tryptophan O~genase
levels coaslderably above those of the control animals . active, total as sell ae inactive enzyme .
Vol. 11, No. 21 This is true for the
Maximum tryptophan oxygenase ac-
tivity was attained at 5 hours after the injection and then it began to decrease and reach value slightly below control levels 9 hours after alcohol administration . DISCUSSION Tryptophan oxygenase, which requires hems (or hematin) se a cofactor, catalyzes the first step in a series of reactions which leads to the conversion of tryptophan to nicotinic acid and then to pyridine nucleotide co enzymes NAD and NADP
(8) .
The oxidation of alcohol to acetaldehyde by the
enzyme alcohol dehydrogenase and the subsequent irreversible oxidation of acetaldehyde to acetate by aldehyde dehydrogenase requires the wnversion of NAD to NADH
(9) .
Also the nucleotide NADP is essential for the activity of
microsomal alcohol dehydrogenase (10) .
The observed increase in liver tryp-
tophan oxygenase could divert more of the tryptophan via the nicotinic acid pathway and help meet the increased demands for the nucleotides by the liver during alcohol oxidation.
Indeed, the total content of NAD plus NADH in both
liver and yeast cells is increased during the oxidation of ethanol (11) . It has been shown that administration of alcohol in doses leading to moderate degree of intoxication results in increased plasma corticosterone level (12) apparently due to stimulation of adrenal corticoid function . adrenocortical hormones or substances stimulating the adrenals
Since
(13,14) have
been shown to elevate the level of tryptophan oxygenase in rat liver, it is possible that the increased enzyme activity observed in this study may be due to the increased level of steroids during alcohol oxidation.
However, other
explanations are possible . Tryptophan oxygenase is a hems-requiring enzyme and both apoenzyme wad holoenzyme are normally present.
It has been postulated that factors that
increase hems availability to the enzyme increase its netivity and then lead to further induction of apoenzyme by a derepression mechanism or to accumulation
Vol. 11, No. 21
Alcohol and Liver Tryptophan O~genaee
of enzyme by protecting it against degradation (15) .
1027
The increase in the
activity of liver tryptophan oxygenase after the administration of allylieopropyl
acetamide (AIA) has been suggested to be secondary to the induction
of ALA (delta-aminolevulinic acid) synthetase activity, the first enzyme of the porphyrin-hems biosynthetic pathway, and consequent enhanced home production
(16) .
et al (17)
Interesting in this connection is the observation of Wetterberq that ALA administration to normal intact animals increases both
total liver tryptophan oxygenase activity and degree of saturation .
However,
in the data preaeslted herein the increase in the degree of saturation of the enzyme was not observed . Alcohol, like AIA, has been known to result in significant increase in hepatic ALA synthetase
(18) .
Whether or not alcohol induces tryptophan
pyrrolase because of the increased formation of hems secondary to the forma tion of ALA remains to be determined .
It has been suggested that NAD may be a
feedback inhibitor of tryptophan oxygenase (19)
and the induction of this
enzyme may be because of the lower level of liver NAD caused by alcohol oxidation
(19) .
However, Morland et al have recently reported that ethanol does
not increase the activity of this enzyme in isolated perfused rat liver (20) . Further work is in progress to determine the mechanism of tryptophan oxygenase induction during alcohol oxidation .
ACIQiOWLEDGEMENTS This work was supported by grants from the National Institutes of Health (MH13177), Michigan Heart Association, Detroit General Hospital Research Corportation and Grant-in-Aid from the office of Research Administration, Wayne State University . REFERENCES 1.
R. E . OLSEN, D . GURSEY and J. W. VFSTER, New England J. Med.,263,1169 (1960) .
Alcohol and Liver Tryptophan Oaßrgenaee
1028
Vol, il, No . 21
2.
G . ROSSNFELD, Proc . Soc . Ex~ Biol . Med ., 103, 144 (1960) .
3.
G . J . GABUZDA and C .S . DAVIDSON, Amer . J . Clin . Nutr ., 11, 502 (1962) .
4.
A . M, LERNER, L . M . DECARLI and C . S . DAVIDSON, Proc . Soc . Exp . Siol . Med ., 98, 841 (1958) .
5.
H . H, BItODIE, W . M . BDTLER, JR ., M, G . HORNING, R . P, MAICXEL and H . M . MALING, Am . J . Clin . Nutr ., 9, 432 (1961) .
6.
W . E . RNOX and V .H . AUEItHACH, J . Biol . Chem . ., 214, 307 (1955) .
7.
L . J, BERRY and D . S . SMYTFIE, J . Exp_ Med ., 118, 587 (1963) .
8.
0 . HAYAISHI, H . WICHI and A . ICHIYAMA, Advances in Enc . Req .,5,9 (1966) .
9.
C . S . LIEBER, Ann . Rev . Med ., 18, 35 (1967) .
10 .
C . S . LIEBER and L . M . DSCARLI, J . Biol . Chem . , 245, 2505 (1970) .
11 .
N . C . R . RAHLA and E . OURA, Proc . Soc . Exp . Hiol . Med ., 109, 908 (1962) .
12 .
F . W . ELLIS, J . Pharmac -8~ . Ther ., 153, 121 (1966) .
13 .
W . E . ~70Y, V . H . ADERBACH and C . C . LIN, Physiol . Revs ., 36, 164,(1956) .
14 .
O . GREENGARD and P . FEIGELSON, Natura, 190, 446 (1961) .
15 . . P . FEIGELSON, M .FEIGSLSON and O . GREEtiGARD, Recant Progr . in Hormone Res ., 18, 491 (1962) . 16 .
P . FEIGELSON, and O . GR88NGARD, Hiochim . Biophys . Acta , 52, 509 (1961) .
17 .
L . WSIIBRHEFäG, A . YUWILER and E . GSIS.ER, Life Sciencea , 8,part I,1047 (1969) .
18 .
H . C . SHANLSY, S . S . ZAIL aad S . M . JOIJHEitT, Lancet , 1, 70 (1968) .
19 .
1C . YAMAGQCHI, M . SHII~YADlA aad R . 1C . GHOLSON, Biochim . Hiopüys . Acta , 146, 102 (1967) .
20 .
J . MORLAt~, T . CHitISTOFFERSEN, J . B . OSNgS, P : O . SEGLBti and 1C . F . JERVELL Biocham . Pharmacol ., 21, 1849 (1972) .
Pergamon Prege
THE EFFECT OF ETHYL ALCOHOL ON RAT LIVER TRYPTDPHAN OXYGENASE Vishwanath M. Sardesai and Haydee S . Provido Robert S . Marx Laboratories, Department of Surgery . 9Payne State University, School of Medicine, 540 Eaat Cornfield, Detroit, !lichigan
(Received 10 August iß72 ; in final form 14 September 1972) SUMMARY The activity of liver tryptophan oxygenase was determined in rats administered intoxicating dose of ethanol . Following intraperitoneal injection of ethanol theta was a rise in the enzyme activity reaching 1®vela considerably above those of the control animals . Maximum enzyme activity was reached 5 hours after ethanol administration and thea returned to within contrc+l levels in about 9 hours . It is suggested that the observed increase in tryptophan .oxygenase in the liver may be the result of increased plasma adrenocortical hormone level following ethanol intoxication, or mny be secondary to the induction of ALA (delta-aminolevulinic acid) synthetase and consequent enchanced hems production . INTROD UC TION Tryptophan, in addition to its use as a substrate for protein synthesis, has two other major functions . muter,
One results in the production of neurotrana-
5-hydroxytryptamine (serotonin), much of which ie subsequently met
abolized to 5-hydroxyindole acetic acid
(HIAA) prior to excretion in the uriné.
Its second use is in the production of the vitamin, nicotinic acid .
The
latter substance is used, of course, in the production of nicotinamide adenine dinucleotide
(NAD) and nicotinamide adenine dinucleotlde phosphate (NADP),
compounds of major significance in the energy yielding reactions . There have been several reports during last few years indicating abnormalities of tryptophan metabolism in alcoholics .
Olaen et al
(1) found decreased
urinary excretion of HLAA in alcoholic patients than in normal controls both on control diets and after tryptophan loading .
1029
Rosenfeld (2) also observed
l0â!
Alcohol ~rd Liver Tryptop~an O~gg~ase
Vol. 11, No, a1
that normal subjects given 100 grams of alcohol* a:cretad less HIAA than in controls .
The alcoholic patients described by Olsan et al
(1) had normal
urinary excretion of N-methylnicotinamide, a major urinary metabolic product of nicotinic acid .
Thasa investigators concluded, therefore, that alcohol
does not cause a defect in the kynuranine pathway of tryptophan metabolism . However, Gabuzda and Davidson (3) observed that patients with cirrhosis of the liver associated with chronic alcoholism had increased urinary excretion of nicotinic acid and Nrmethylnicotinnmide .
Lérner et al (4) also reported that
chronic alcoholics, after the ingestion of 10 qms of DL tryptophan orally, had excessive urinary excretion of xanthurenic acid, an intermediate metabolite of tryptophan pathway leading to nicotinic acid .
it seems possible that after
the injection of alcohol more of the tryptophan may be metabolized via the kynureniae pathway (5) .
The present study was 8esigned to determine the eflect
o! an acute dose of alcohol to rats on the activity of liver tryptophan oxyjanase, the first and apparently the major rate limiting enzyme of the tryptophan-nicotinic acid pathway .
s b~ragus Dawley lemale study.
rats weighing between 175-200 grams were used in this
The animals were fasted for 18 hours before the intraperitonaal admin-
istration of 5 ml of ethanol (v/v) solution in saline or 5 ml of saline solution in control animals .
At the end of each time period after the injection
as indicated in the Table rats were sacrificed by dacnpitation and livers were quickly removed and washed with cold saline .
One gram of liver was homogenized
in 7 ml of cold 0 .14 M loCl containing 0 .0025 N NaOH . The tryptophan oxygeaase was determined by the method of lcaox and Auerbach (6) as codified by Berry and Smythe
(7) .
One ml of the liver homogenate was
aaaayed for the enzyus with and without the addition of 0.1 ~ of hamatin solu-
*
Alcohol cad ethanol are used syiwnymously in this paper.
Vol . 11, No. 21
tion .
Alcoàol and Liver Tryptophan O~genaee
10~
Tha hamatin solution was prepared immediately before use by dissolving
1 mg of twice-crystallised bovine heroin
(Sigma Chemical Company)
in 10 ml of
O .iN sodium hydroxide . RESOLTS The activity of tryptophan ozygenase without the addition of excess hematin is considered to be indicative of in vivo bolo enzyme activity and is therefore referred to as the "active" apoenzyme .
The addition of excess hema
tin reveals the additional amount of apoenzyme which is potentially active and is therefor® referred to as "total" apoenzyme.
The difference in activity with
and without the addition of excess activator is considered to be a measure of the amount of "inactive" apoenzyme in the liver homogenate .
TABLE I Effect of Alcohol on Liver Tryptophan Oxygenase Control *
Hours after Injection
Active
Total
Alcohol-Treated Active
Total
0
0.78 + 0.11
1 .22 + 0.12
0.78 + 0.05
1 .29 + 0.08
1
0.80 + 0 .06
1 .18 + 0 .11
0.75 + 0.06
1 .23 + 0.09
2
0.68 + 0.04
1 .16 + 0 .15
0.88 + 0.09
1 .50 + 0.15
3
0.79 + 0 .08
1 .23 + 0.07
0.95 + 0.15
2 .08 + 0.19
4
0 .62 + 0 .06
1 .18 + 0.10
1 .22 + 0 .19
2 .84 + 0 .31
5
0 .78 + 0 .09
1 .18 + 0.08
1.46 + 0.15
3.24 + 0 .39
6
0 .80 + 0 .10
1 .28 + 0 .15
1 .40 + 0 .22
2 .80 + 0 .28
9
0 .74 + 0 .08
1 .23 f 0 .10
0 .66 + 0 .06
1 .05 + 0 .08
* The activity is expressed as micromoles of Rynurenine formed par gram of liver per hour . Values are averages of 18 experiments with standard deviations . Duplicate runs were made on each tissue and agreed within + 10s. The data on the effect of alcohol on liver tryptophan oxygenase are presented in Table I .
Aa can be seen the admünistratioa of saline alone does
not result in appreciable change in the enzyme activity .
Following intraperi
toaeal injection of ethanol there 1s a üse is the enzyme activity, reaching
1028
Alcohol aad Liver Tryptophan O~genase
levels coaslderably above those of the control animals . active, total as sell ae inactive enzyme .
Vol. 11, No. 21 This is true for the
Maximum tryptophan oxygenase ac-
tivity was attained at 5 hours after the injection and then it began to decrease and reach value slightly below control levels 9 hours after alcohol administration . DISCUSSION Tryptophan oxygenase, which requires hems (or hematin) se a cofactor, catalyzes the first step in a series of reactions which leads to the conversion of tryptophan to nicotinic acid and then to pyridine nucleotide co enzymes NAD and NADP
(8) .
The oxidation of alcohol to acetaldehyde by the
enzyme alcohol dehydrogenase and the subsequent irreversible oxidation of acetaldehyde to acetate by aldehyde dehydrogenase requires the wnversion of NAD to NADH
(9) .
Also the nucleotide NADP is essential for the activity of
microsomal alcohol dehydrogenase (10) .
The observed increase in liver tryp-
tophan oxygenase could divert more of the tryptophan via the nicotinic acid pathway and help meet the increased demands for the nucleotides by the liver during alcohol oxidation.
Indeed, the total content of NAD plus NADH in both
liver and yeast cells is increased during the oxidation of ethanol (11) . It has been shown that administration of alcohol in doses leading to moderate degree of intoxication results in increased plasma corticosterone level (12) apparently due to stimulation of adrenal corticoid function . adrenocortical hormones or substances stimulating the adrenals
Since
(13,14) have
been shown to elevate the level of tryptophan oxygenase in rat liver, it is possible that the increased enzyme activity observed in this study may be due to the increased level of steroids during alcohol oxidation.
However, other
explanations are possible . Tryptophan oxygenase is a hems-requiring enzyme and both apoenzyme wad holoenzyme are normally present.
It has been postulated that factors that
increase hems availability to the enzyme increase its netivity and then lead to further induction of apoenzyme by a derepression mechanism or to accumulation
Vol. 11, No. 21
Alcohol and Liver Tryptophan O~genaee
of enzyme by protecting it against degradation (15) .
1027
The increase in the
activity of liver tryptophan oxygenase after the administration of allylieopropyl
acetamide (AIA) has been suggested to be secondary to the induction
of ALA (delta-aminolevulinic acid) synthetase activity, the first enzyme of the porphyrin-hems biosynthetic pathway, and consequent enhanced home production
(16) .
et al (17)
Interesting in this connection is the observation of Wetterberq that ALA administration to normal intact animals increases both
total liver tryptophan oxygenase activity and degree of saturation .
However,
in the data preaeslted herein the increase in the degree of saturation of the enzyme was not observed . Alcohol, like AIA, has been known to result in significant increase in hepatic ALA synthetase
(18) .
Whether or not alcohol induces tryptophan
pyrrolase because of the increased formation of hems secondary to the forma tion of ALA remains to be determined .
It has been suggested that NAD may be a
feedback inhibitor of tryptophan oxygenase (19)
and the induction of this
enzyme may be because of the lower level of liver NAD caused by alcohol oxidation
(19) .
However, Morland et al have recently reported that ethanol does
not increase the activity of this enzyme in isolated perfused rat liver (20) . Further work is in progress to determine the mechanism of tryptophan oxygenase induction during alcohol oxidation .
ACIQiOWLEDGEMENTS This work was supported by grants from the National Institutes of Health (MH13177), Michigan Heart Association, Detroit General Hospital Research Corportation and Grant-in-Aid from the office of Research Administration, Wayne State University . REFERENCES 1.
R. E . OLSEN, D . GURSEY and J. W. VFSTER, New England J. Med.,263,1169 (1960) .
Alcohol and Liver Tryptophan Oaßrgenaee
1028
Vol, il, No . 21
2.
G . ROSSNFELD, Proc . Soc . Ex~ Biol . Med ., 103, 144 (1960) .
3.
G . J . GABUZDA and C .S . DAVIDSON, Amer . J . Clin . Nutr ., 11, 502 (1962) .
4.
A . M, LERNER, L . M . DECARLI and C . S . DAVIDSON, Proc . Soc . Exp . Siol . Med ., 98, 841 (1958) .
5.
H . H, BItODIE, W . M . BDTLER, JR ., M, G . HORNING, R . P, MAICXEL and H . M . MALING, Am . J . Clin . Nutr ., 9, 432 (1961) .
6.
W . E . RNOX and V .H . AUEItHACH, J . Biol . Chem . ., 214, 307 (1955) .
7.
L . J, BERRY and D . S . SMYTFIE, J . Exp_ Med ., 118, 587 (1963) .
8.
0 . HAYAISHI, H . WICHI and A . ICHIYAMA, Advances in Enc . Req .,5,9 (1966) .
9.
C . S . LIEBER, Ann . Rev . Med ., 18, 35 (1967) .
10 .
C . S . LIEBER and L . M . DSCARLI, J . Biol . Chem . , 245, 2505 (1970) .
11 .
N . C . R . RAHLA and E . OURA, Proc . Soc . Exp . Hiol . Med ., 109, 908 (1962) .
12 .
F . W . ELLIS, J . Pharmac -8~ . Ther ., 153, 121 (1966) .
13 .
W . E . ~70Y, V . H . ADERBACH and C . C . LIN, Physiol . Revs ., 36, 164,(1956) .
14 .
O . GREENGARD and P . FEIGELSON, Natura, 190, 446 (1961) .
15 . . P . FEIGELSON, M .FEIGSLSON and O . GREEtiGARD, Recant Progr . in Hormone Res ., 18, 491 (1962) . 16 .
P . FEIGELSON, and O . GR88NGARD, Hiochim . Biophys . Acta , 52, 509 (1961) .
17 .
L . WSIIBRHEFäG, A . YUWILER and E . GSIS.ER, Life Sciencea , 8,part I,1047 (1969) .
18 .
H . C . SHANLSY, S . S . ZAIL aad S . M . JOIJHEitT, Lancet , 1, 70 (1968) .
19 .
1C . YAMAGQCHI, M . SHII~YADlA aad R . 1C . GHOLSON, Biochim . Hiopüys . Acta , 146, 102 (1967) .
20 .
J . MORLAt~, T . CHitISTOFFERSEN, J . B . OSNgS, P : O . SEGLBti and 1C . F . JERVELL Biocham . Pharmacol ., 21, 1849 (1972) .