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Renal ammonia metabolic response in the rat to repeated ethanol loading
Toxicology Letters, 22 (1984) 339-342
339
Elsevier
TOXLett.
1276
RENAL
AMMONIA
REPEATED
METABOLIC
ETHANOL
RESPONSE
(Ammonia; ammoniagenesis; urea synthesis; ethanol-caused hyperammonemia)
V. MOHANACHARI,
M.M.
IN THE
RAT TO
LOADING
REDDY
urea cycle enzymes;
glutamine;
and K. INDIRA
Department of Zoology, Sri Venkatesware University, Tirupati-517 502, Andhra Pradesh (India) (Received
February
(Revision
received
(Accepted
April
29th, April
30th,
1984) 23rd,
1984)
1984)
SUMMARY Lactate
dehydrogenase
(LDH),
glutamate
dehydrogenase
(GDH),
succinate AMP
dehydrogenase deaminase,
(SDH),
ornithine
aspartate
aminotransferase
transcarbamylase
glutamine synthetase (GS) activities were increased in the kidney loading. The significance of these findings is discussed.
(OTC),
of the rat during
(AAT),
arginase
repeated
and
ethanol
INTRODUCTION
Kidney has low alcohol dehydrogenase [l] and hence is very vulnerable for alcohol toxicity. Excessive ingestion of ethanol causes acute renal failure [2]. Although the effects of ethanol on kidney structure and function have been investigated [3, 41, the precise biochemical changes that occur in impaired renal function are still obscure. Earlier studies indicated altered ammonia metabolism on ethanol treatment in the rat [5]. In the present study the activities of some of the renal enzymes associated with ammonia metabolism and energy production, in control and ethanol-dosed rats, are reported.
Abbreviations: thetase;
LDH,
AAT,
aspartate
lactate
aminotransferase;
dehydrogenase;
OTC,
GDH, glutamate dehydrogenase; ornithine transcarbamylase;
dehydrogenase.
0378-4274/84/$
03.00
0 Elsevier
Science
Publishers
B.V.
GS, glutamine synSDH, succinate
340
MATERIALS
AND
METHODS
Wistar strain male albino rats aged 3 months (200 + 20 g), were treated i.p. with saline-diluted ethanol (5 g/kg body wt.) per day for 5 consecutive days. Control animals received the same volume of physiological saline i.p. Details of animal maintenance and food intake were described in our earlier paper [6]. 5 days after treatment, the animals were killed, the kidney was excised quickly and homogenized in required media, centrifuged at 5000 x g for 20 min and the supernatant used for the following enzymic assays. Sucrose homogenates were used for assaying LDH (EC 1.1.1.27), SDH (EC 1.3.99.1) by the method of Nachlas et al. [7], GDH (EC 1.4.1.2) by the method of Lee and Lardy [8], AAT (EC 2.6.1.1) by the method of Reitman and Frankel as suggested in the Sigma Technical Bulletin [9] and GS activity by the method of Rowe et al. [lo]. AMP deaminase (EC 3.5.4.6) was assayed in distilled water homogenates by the method of Setlow et al. [ll]. OTC (EC 2.1.3.3) activity by the method of Huggins et al. [12] and arginase (EC 3.5.3.1) by the method of Beruter et al. [13] were assayed in cetyltrimethylammoniumbromide extracts. The protein concentration in the homogenates was analysed by the method of Lowry et al. [14].
I
TABLE
METABOLIC ENZYME
CHANGES
ACTIVITIES
IN KIDNEY
DURING
ARE REPRESENTED
Enzyme
REPEATED IN UNITWmg
Control
ETHANOL
LOADING
IN RATS
PROTEIN/MIN Experimental
% Change over control
Lactate
dehydrogenase
0.1372kO.004
0.2371 f 0.002
72.7
0.03 11 + 0.001
0.07
I 1 f 0.002
128.6
0.028 + 0.001
0.0402 f 0.001
43.5
(pm01 of formazan) Succinate
dehydrogenase
(pm01 of formazan) Aspartate aminotransferase (pm01 of pyruvate) Glutamate
0.0029 + 0.0001
0.0056 + 0.0003
93.1
(am01 of formazan) AMP deaminase
dehydrogenase
0.0038 + 0.0002
0.0046 + 0.0001
19.1
(pm01 of ammonia) Ornithine transcarbamylase
0.0227 + 0.009
0.0554 f 0.007
144.0
(pm01 of citrulline) Arginase
0.0277 f 0.003
0.0462 + 0.007
66.8
0.005 1+ 0.0003
0.0143 + 0.002
184.5
(pm01 of urea) Glutamine synthetase (cm01 of y-glutamyl Values
are mean and
hydroxymate) + S.D. of 8 samples
and all experimental
values are significant
(P
341
RESULTS
AND DISCUSSION
The changes
in different
enzymic
activities
are summarized
in Table
I.
Changes in LDH and SDH activities representing glycoIytic path way and TCA cycle operation LDH and SDH showed significantly increased activity. The higher % increase in SDH activity (128.6) as compared with the increase in LDH activity (72.7) suggests high succinate oxidation, perhaps by an increased inflow of TCA cycle intermediates from other fuels.
Changes in the transamination
and deamination patterns
AAT activity was increased indicating increased formation of oxaloacetate and glutamate. The oxaloacetate thus formed is channelled into TCA cycle while glutamate is oxidatively deaminated to ammonia and 2-ketoglutarate by GDH. As expected, the GDH activity was found to be increased leading to ammonia production. The increased levels of cyclic GMP during alcoholism [ 151 may be responsible for increased GDH activity [16]. In addition to glutamate-based ammonia production, the purine-based ammonia production as mediated by AMP deaminase also increased. The higher % increase in GDH activity compared with that of AMP deaminase suggests increased ammonia production from glutamate. Nevertheless the combined raised activities of GDH and AMP deaminase may be responsible for the elevated ammonia levels of the kidney [5] during alcohol toxicity.
Changes in ammonia detoxifying
enzyme activities
Excess ammonia stimulates urea and glutamine synthesis. The levels of OTC and arginase were elevated indicating active conversion of ammonia to urea. But the urea level was low in the kidney [5] indicating its possible entry into the vascular flow, since kidney has a significant role in elevating blood urea level [3]. Another reason for the low level of urea in kidney may be its increased rate of urinary excretion [17]. Similarly, while GS activity was increased, the glutamine concentration was low [5] due to its rapid utilization in several metabolic functions [ 181. Low glutamine levels may also be due to high renal glutaminase activity. ACKNOWLEDGEMENTS
Dr. V. Mohanachari
is grateful
for the grant
support
from UGC,
New Delhi.
342
REFERENCES
1 J.H.
Exton,
Progress
in endocrinology
and metabolism,
gluconeogenesis,
Metabolism,
21 (1972)
945-990. 2 R.R. Chose, S.K. Gupta Med. J., 280 (1980). 3 D.H.
Vanthiel,
J.J.M
and M.J.
von Bertele,
Little and R. Lester,
Acute
Alcohol:
renal failure
after a beer drinking
its effect on the kidney,
binge,
Metabolism,
Br.
26 (1977)
857-865. 4 G.R.
Cherrik
and C.M.
nicotinamide
adenine
Leevy, The effect
dinucleotide
of ethanol
in liver, kidney
metabolism
and heart,
on level of oxidized
Biochim.
Biophys.
and reduced
Acta,
109 (1965)
29-37. 5 V. Mohanachari, loading,
K. Satyavelu
Toxicol.
Lett.,
6 V. Mohanachari, monia
K. Satyavelu
metabolism,
7 M.M. Nachlas, succinate
Biochem.
9 Sigma
Technical
glutamic
Seligman,
J. Biol. Chem.,
Influence
in various
Bulletin,
pyruvic
fate of ammonia
in rat after ethanol
of thyroid
organs (1979),
induced
alterations
in rat hepatic
am-
A calorimetric
method
for the determination
of
235 (1960) 499-504.
hormones
on L-glycerophosphate
of rat, J. Biol. Chem.,
The quantitative
transaminases
Ethanol
32 (1983) 2825-2827.
and A.M.
activity,
dehydrogenases
Metabolic
Reddy and K. Indira, Pharmacol.,
S.P. Margulins
dehydrogenase
8 Y.L. Lee and H.A. Lardy, other
Reddy and K. Indira,
20 (1984) 225-228.
calorimetric
dehydrogenases
and
240 (1957) 1427-1432.
estimation
of glutamic-oxaloacetate
and
No. 505.
10 W.B. Rowe, R.N. Ronzio, V.P. Wellner and A. Meister, Glutamine synthetase (sheep brain), Methods in Enzymology, Vol. XVIIA, Academic Press, New York, 1970, pp. 900-910. 11 B. Setlow,
R. Burger
guanosine
and J.M.
triphosphates
Lowenstein,
on activity
Chem., 241 (1966) 1244-1245. 12 A.K. Huggins, G. Skutsch and E. Baldwin. Biochem.
Physiol.,
13 J. Beruter,
J.P.
15 A.W.K.
reagent,
Colombo
Gore,
Petit and I.B. Alix, Effect
L-glutamic
Biochem. of ethanol
Pharmacol.,
metabolism
on Glutamine,
Purification
Relationship
acid dehydrogenase,
rat liver in vivo, Biochem. Glutamine
of adenosine enzyme,
in teleostean
and
J. Biol.
fish, Comp.
and properties Protein
of arginase
measurement
from
human
with the Folin
193 (1951) 265-275.
Heubusch,
16 M.G.
Symposium
I, The effects of the regulated
urea cycle enzymes
J., 175 (1978) 449-454. A.L. Farr and R.J. Randall,
with chlorodiazepoxide,
17 M.A.
18 H. Krebs,
Ornithine
and C. Bachmann,
J. Biol. Chem.,
Chan and P.H.
of ethanol
deaminase
distribution
28 (1969) 587-602.
liver and erythrocytes, Biochem. 14 O.H. Lowry, N.J. Rosebrough, phenol
Adenylate
and the organ
in
levels and the interaction
31 (1982) 85-89.
Int. J. Biochem., dose on amino
13 (1981) 879-886.
acid and urea concentrations
in the fed
28 (1979) 2591-2596.
in animal
Academic
of brain cyclic nucleotides Pharmacol.,
Press,
body,
in J. Mora
New York,
and R. Palactos
1980, pp. 319.
(Eds.),
International
339
Elsevier
TOXLett.
1276
RENAL
AMMONIA
REPEATED
METABOLIC
ETHANOL
RESPONSE
(Ammonia; ammoniagenesis; urea synthesis; ethanol-caused hyperammonemia)
V. MOHANACHARI,
M.M.
IN THE
RAT TO
LOADING
REDDY
urea cycle enzymes;
glutamine;
and K. INDIRA
Department of Zoology, Sri Venkatesware University, Tirupati-517 502, Andhra Pradesh (India) (Received
February
(Revision
received
(Accepted
April
29th, April
30th,
1984) 23rd,
1984)
1984)
SUMMARY Lactate
dehydrogenase
(LDH),
glutamate
dehydrogenase
(GDH),
succinate AMP
dehydrogenase deaminase,
(SDH),
ornithine
aspartate
aminotransferase
transcarbamylase
glutamine synthetase (GS) activities were increased in the kidney loading. The significance of these findings is discussed.
(OTC),
of the rat during
(AAT),
arginase
repeated
and
ethanol
INTRODUCTION
Kidney has low alcohol dehydrogenase [l] and hence is very vulnerable for alcohol toxicity. Excessive ingestion of ethanol causes acute renal failure [2]. Although the effects of ethanol on kidney structure and function have been investigated [3, 41, the precise biochemical changes that occur in impaired renal function are still obscure. Earlier studies indicated altered ammonia metabolism on ethanol treatment in the rat [5]. In the present study the activities of some of the renal enzymes associated with ammonia metabolism and energy production, in control and ethanol-dosed rats, are reported.
Abbreviations: thetase;
LDH,
AAT,
aspartate
lactate
aminotransferase;
dehydrogenase;
OTC,
GDH, glutamate dehydrogenase; ornithine transcarbamylase;
dehydrogenase.
0378-4274/84/$
03.00
0 Elsevier
Science
Publishers
B.V.
GS, glutamine synSDH, succinate
340
MATERIALS
AND
METHODS
Wistar strain male albino rats aged 3 months (200 + 20 g), were treated i.p. with saline-diluted ethanol (5 g/kg body wt.) per day for 5 consecutive days. Control animals received the same volume of physiological saline i.p. Details of animal maintenance and food intake were described in our earlier paper [6]. 5 days after treatment, the animals were killed, the kidney was excised quickly and homogenized in required media, centrifuged at 5000 x g for 20 min and the supernatant used for the following enzymic assays. Sucrose homogenates were used for assaying LDH (EC 1.1.1.27), SDH (EC 1.3.99.1) by the method of Nachlas et al. [7], GDH (EC 1.4.1.2) by the method of Lee and Lardy [8], AAT (EC 2.6.1.1) by the method of Reitman and Frankel as suggested in the Sigma Technical Bulletin [9] and GS activity by the method of Rowe et al. [lo]. AMP deaminase (EC 3.5.4.6) was assayed in distilled water homogenates by the method of Setlow et al. [ll]. OTC (EC 2.1.3.3) activity by the method of Huggins et al. [12] and arginase (EC 3.5.3.1) by the method of Beruter et al. [13] were assayed in cetyltrimethylammoniumbromide extracts. The protein concentration in the homogenates was analysed by the method of Lowry et al. [14].
I
TABLE
METABOLIC ENZYME
CHANGES
ACTIVITIES
IN KIDNEY
DURING
ARE REPRESENTED
Enzyme
REPEATED IN UNITWmg
Control
ETHANOL
LOADING
IN RATS
PROTEIN/MIN Experimental
% Change over control
Lactate
dehydrogenase
0.1372kO.004
0.2371 f 0.002
72.7
0.03 11 + 0.001
0.07
I 1 f 0.002
128.6
0.028 + 0.001
0.0402 f 0.001
43.5
(pm01 of formazan) Succinate
dehydrogenase
(pm01 of formazan) Aspartate aminotransferase (pm01 of pyruvate) Glutamate
0.0029 + 0.0001
0.0056 + 0.0003
93.1
(am01 of formazan) AMP deaminase
dehydrogenase
0.0038 + 0.0002
0.0046 + 0.0001
19.1
(pm01 of ammonia) Ornithine transcarbamylase
0.0227 + 0.009
0.0554 f 0.007
144.0
(pm01 of citrulline) Arginase
0.0277 f 0.003
0.0462 + 0.007
66.8
0.005 1+ 0.0003
0.0143 + 0.002
184.5
(pm01 of urea) Glutamine synthetase (cm01 of y-glutamyl Values
are mean and
hydroxymate) + S.D. of 8 samples
and all experimental
values are significant
(P
341
RESULTS
AND DISCUSSION
The changes
in different
enzymic
activities
are summarized
in Table
I.
Changes in LDH and SDH activities representing glycoIytic path way and TCA cycle operation LDH and SDH showed significantly increased activity. The higher % increase in SDH activity (128.6) as compared with the increase in LDH activity (72.7) suggests high succinate oxidation, perhaps by an increased inflow of TCA cycle intermediates from other fuels.
Changes in the transamination
and deamination patterns
AAT activity was increased indicating increased formation of oxaloacetate and glutamate. The oxaloacetate thus formed is channelled into TCA cycle while glutamate is oxidatively deaminated to ammonia and 2-ketoglutarate by GDH. As expected, the GDH activity was found to be increased leading to ammonia production. The increased levels of cyclic GMP during alcoholism [ 151 may be responsible for increased GDH activity [16]. In addition to glutamate-based ammonia production, the purine-based ammonia production as mediated by AMP deaminase also increased. The higher % increase in GDH activity compared with that of AMP deaminase suggests increased ammonia production from glutamate. Nevertheless the combined raised activities of GDH and AMP deaminase may be responsible for the elevated ammonia levels of the kidney [5] during alcohol toxicity.
Changes in ammonia detoxifying
enzyme activities
Excess ammonia stimulates urea and glutamine synthesis. The levels of OTC and arginase were elevated indicating active conversion of ammonia to urea. But the urea level was low in the kidney [5] indicating its possible entry into the vascular flow, since kidney has a significant role in elevating blood urea level [3]. Another reason for the low level of urea in kidney may be its increased rate of urinary excretion [17]. Similarly, while GS activity was increased, the glutamine concentration was low [5] due to its rapid utilization in several metabolic functions [ 181. Low glutamine levels may also be due to high renal glutaminase activity. ACKNOWLEDGEMENTS
Dr. V. Mohanachari
is grateful
for the grant
support
from UGC,
New Delhi.
342
REFERENCES
1 J.H.
Exton,
Progress
in endocrinology
and metabolism,
gluconeogenesis,
Metabolism,
21 (1972)
945-990. 2 R.R. Chose, S.K. Gupta Med. J., 280 (1980). 3 D.H.
Vanthiel,
J.J.M
and M.J.
von Bertele,
Little and R. Lester,
Acute
Alcohol:
renal failure
after a beer drinking
its effect on the kidney,
binge,
Metabolism,
Br.
26 (1977)
857-865. 4 G.R.
Cherrik
and C.M.
nicotinamide
adenine
Leevy, The effect
dinucleotide
of ethanol
in liver, kidney
metabolism
and heart,
on level of oxidized
Biochim.
Biophys.
and reduced
Acta,
109 (1965)
29-37. 5 V. Mohanachari, loading,
K. Satyavelu
Toxicol.
Lett.,
6 V. Mohanachari, monia
K. Satyavelu
metabolism,
7 M.M. Nachlas, succinate
Biochem.
9 Sigma
Technical
glutamic
Seligman,
J. Biol. Chem.,
Influence
in various
Bulletin,
pyruvic
fate of ammonia
in rat after ethanol
of thyroid
organs (1979),
induced
alterations
in rat hepatic
am-
A calorimetric
method
for the determination
of
235 (1960) 499-504.
hormones
on L-glycerophosphate
of rat, J. Biol. Chem.,
The quantitative
transaminases
Ethanol
32 (1983) 2825-2827.
and A.M.
activity,
dehydrogenases
Metabolic
Reddy and K. Indira, Pharmacol.,
S.P. Margulins
dehydrogenase
8 Y.L. Lee and H.A. Lardy, other
Reddy and K. Indira,
20 (1984) 225-228.
calorimetric
dehydrogenases
and
240 (1957) 1427-1432.
estimation
of glutamic-oxaloacetate
and
No. 505.
10 W.B. Rowe, R.N. Ronzio, V.P. Wellner and A. Meister, Glutamine synthetase (sheep brain), Methods in Enzymology, Vol. XVIIA, Academic Press, New York, 1970, pp. 900-910. 11 B. Setlow,
R. Burger
guanosine
and J.M.
triphosphates
Lowenstein,
on activity
Chem., 241 (1966) 1244-1245. 12 A.K. Huggins, G. Skutsch and E. Baldwin. Biochem.
Physiol.,
13 J. Beruter,
J.P.
15 A.W.K.
reagent,
Colombo
Gore,
Petit and I.B. Alix, Effect
L-glutamic
Biochem. of ethanol
Pharmacol.,
metabolism
on Glutamine,
Purification
Relationship
acid dehydrogenase,
rat liver in vivo, Biochem. Glutamine
of adenosine enzyme,
in teleostean
and
J. Biol.
fish, Comp.
and properties Protein
of arginase
measurement
from
human
with the Folin
193 (1951) 265-275.
Heubusch,
16 M.G.
Symposium
I, The effects of the regulated
urea cycle enzymes
J., 175 (1978) 449-454. A.L. Farr and R.J. Randall,
with chlorodiazepoxide,
17 M.A.
18 H. Krebs,
Ornithine
and C. Bachmann,
J. Biol. Chem.,
Chan and P.H.
of ethanol
deaminase
distribution
28 (1969) 587-602.
liver and erythrocytes, Biochem. 14 O.H. Lowry, N.J. Rosebrough, phenol
Adenylate
and the organ
in
levels and the interaction
31 (1982) 85-89.
Int. J. Biochem., dose on amino
13 (1981) 879-886.
acid and urea concentrations
in the fed
28 (1979) 2591-2596.
in animal
Academic
of brain cyclic nucleotides Pharmacol.,
Press,
body,
in J. Mora
New York,
and R. Palactos
1980, pp. 319.
(Eds.),
International