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The fate of quinaldylglycyltaurine in the cat, rat, and rabbit
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
110,
The Fate of Quinaldylglycyltaurine MASAKO Division
of Clinical
Oncology,
in the Cat, Rat, and
KAIHARA
University Received
(1965)
316-319
J. M. PRICE2
AND
of Wisconsin December
Rabbit
Medical
School, Madison,
Wisconsin
28, 1964
The metabolic fate of the quinaldic acid moiety of quinaldylglycyltaurine has been studied in the rat, rabbit, and cat. This compound usually was excreted as quinaldic acid and quinaldylglycine by the rat, while the majority was eliminated unchanged or as quinaldylglycylglycine whether given orally or by subcutaneous injection to the cat. Quinaldic acid and quinaldylglycine, which have never been detected in cat urine, were found in the feces of two of the three cats. When quinaldylglycyltaurine was given orally to the rabbit, the major urinary metabolite was quinaldic acid, but if given by subcutaneous injection, it was excreted unchanged. Thus, the three species converted an oral dose of quinaldylglycyltaurine to quinaldic acid.
Previous studies have shown that when quinaldic acid was administered to the cat orally or by subcutaneous injection, the chief urinary metabolite was quinaldylglycyltaurine (1)) and most of the remainder of the dose was excreted as quinaldylglycylglycine (2). Neither the rat nor the rabbit excreted quinaldic acid conjugated with glycyltaurine or with glycylglycine (3, 4). The rat excreted quinaldic acid unchanged and as the glycine conjugat,e (3), while the rabbit excreted almost all of the compound in the nonconjugated form (4). The present study was undertaken to determine the ability of t’he cat, rat, and rabbit to degrade quinaldylglycyltaurine. For this purpose, radioactive quinaldylglycyltaurine was isolated from the urine of t,he cat after the administration of quinaldic acid-carboxyl-Cl*, and was given to the cat, rat, and rabbit orally and by subcutaneous injection. The distribution of urinary radioactivity among urinary quinaldic acid, quinaldylglycylglycine, quinaldylglycine, and quinaldylglycyltaurine was determined. 1 Supported in part by a grant from the National Institute of Arthritis and Metabolic Diseases (No. AM-01127), U.S. Public Health Service. 2 American Cancer Society-Charles S. Hayden Foundation Professor of Surgery in Cancer Research.
The data obtained are consistent with the interpretation that all three species converted an oral dose of quinaldylglycyltaurine to quinaldic acid. Quinaldic acid and quinaldylglycine, which have never been detected in cat urine, were found in the feces of 2 cats which failed to absorb a large fraction of the oral dose. PROCEDURE Reagents. Quinaldic acid carboxylG4 was synthesized by a previously described method (3). Quinaldylglycine and quinaldylglycylglycine were synthesized by the procedure described by Davis (5). Quinaldylglycyltaurine was available from materials used in previous studies (1). Quinaldylglycyltaurine-C14 was isolated from the urine of a cat which was given 10 mg (5,860,OOO cpm) of quinaldic acid-carboxyl-Cl4 daily in its food for 5 days. Urine was collected during those 5 days and also for the following 4 days. Quinaldylglycyltaurine-Cl4 was isolated as previously described (1). Experimental animals. Three adult male cats were purchased from local dealers. All cats were fed commercial canned cat food. Eight Holzman male albino rats weighing 170-200 gm were used; they were fed a stock grain diet. The rats were housed in groups of two, and the urine was collected from each pair of animals. Two male rabbits which had been used for previous studies (4) were used in this experiment. During urine collections 316
QUINALDYLGLYCYLTAURINE all animals were kept in stainless steel metabolism cages. The urine was collected under toluene and with glacial acetic acid, and both urine and feces were frozen until used. Administration of radioactive quinaldylglycyltour&e. The compound was administered in the quantities indicated in Table I. Oral supplements were administered via a stomach tube to the rat and rabbit. For the cat the compound was mixed with a small quantity of food which was ingested within a few minutes. Fractionation of urine or feces with Dowex 2acetate. An aliquot of urine or feces containing 13,000-189,000 cpm was used for fractionation of the radioactive constituents. Quinaldic acid and quinaldylglycine (6.4 #moles each) were added to the aliquot of sample before chromatography to insure accurate identification of the position of elution of these compounds as determined by a recording ultraviolet absorption meter (Gilson Medical Electronics). The urine was passed through a Dowex 2-acetate column measuring 1.2 X 16 cm, and gradient elution was carried out with acetic acid as previously described (l-3). When it was necessary to analyze feces, they TABLE OF Cl4 IN URINE AND FECES QUINALDYLGLYCYLTAURINE DISTRIBUTION
EXCRETION
1 3 5 7 1 3
& & & &
2 4 6 8
Oral Subcut. Oral Subcut. Oral Oral
Inject Inject.
‘3 excreted during first 48 hours (%I
-
BlOkS 110.7 110.7
CPm x 10-s
Urine
8.8 8.8 6.8 5.2
10.56 10.56 8.67 8.67 6.70 5.14
80.1 79.0 66.5 77.2 97.0 19.1
5.2
5.14
25.8
Feces
78.6 Oral
Cat 4
60.2 Cat 1 Cat 3 Rabbit Rabbit Rabbit Rabbit
1 2 1 2
Subcut. Subcut. Oral Oral Subcut. Subcut.
Inject. Inject.
Inject. Inject.
4.3 5.2 8.8 5.6 5.2 5.2
ADMINISTRXIYON AND
4.22 5.14 8.67 5.59 5.14 5.14
64.0 100.5 94.9 99.1 97.3 99.5
OF
T
i
Cl, recovered in fraction containing (%):
-
7-
P
Rats Rats Rats Rats Cat Cat
I
-
Dose given
nSethod of administration
were homogenized with water, the supernatant fluid was decanted after centrifugation, and the sediment was washed two times with water. The supernatant fluid and washings were pooled and made up to a known volume. An aliquot of this solution was passed through a 3 X 5 cm Dowex a-acetate column and eluted with 850 ml of 10 N acetic acid (to elute quinaldic acid and its glycine and glycylglycine conjugates) followed by 1000 ml of 2 N HCl (to elute quinaldylglycyltaurine) (1). The acetic acid and HCl were distilled from the fractions under reduced pressure. The residues were dissolved in water, and the result,ing solutions were rechromatographed on 1.2 X 16-cm columns of Dowex a-acetate with the same procedure as that used for the urine samples. If there were more than 3000 cpm in the quinaldic acid, quinaldylglycine, or quinaldylglycylglycine fractions from urine or feces, these compounds were recrystallized to constant specific activity with 30-60 mg of authentic carrier. Most of the quinaldylglycyltaurine fractions were not recrystallized because no suitable solvent is known which will permit repeated crystallization of small quinaldylglycyltaurine is samples. However, readily separated from quinaldic acid and its
DURING FIRST 48 HOURS AFTER TO RATS, CATS, AND RABBITS, OF Cl4 IN THE FRACTIONS
! Animal
317
METABOLISM
QAn
QAG
QAGT
43.7 12.0 67.5 41.6 4.2 0.3 11.1 0.4 18.9 1.5 0.2 88.2 89.3 6.2 9.9
44.7 87.9 22.2 11.4 0.9 5.2 13.0 0.7 2.4 0.3 1.0 9.4 9.2 2.2 2.9
0.1 1.5 35.4 82.5 80.7 77.6 76.6 75.2 96.3 98.1 1.2 2.3 87.1 85.4
2AGG
-b -
12.8 6.0 23.6 5.3
/ I
Total recovery of Cl’ in fractions (%)
88.5 99.9 91.2 88.4 100.4 92.2 101.7 101.3 96.5 103.4 99.3 98.8 100.8 95.5 98.2
a Abbreviations: &A, quinaldic acid; QAG, quinaldylglycine; QAGT, quinaldylglycyltaurine; QAGG, quinaldylglycylglycine; Subcut. Inject., subcutaneous injection. b Dashes indicate no detectable radioactivity in this fraction or a few counts which showed decreased specific activity upon repeated recrystallization with authentic carrier.
318
KAIHARA
AND
PRICE
NHCH2CONHCH2COOH
FIG. 1. Schematic
representation
of quinaldylglycyltaurine
other known conjugates (l), so there is no likelihood of contamination with these compounds. RESULTS
When 50 mg (29,000,OOOcpm) of quinaldic acid-carboxyl-Cl4 was given to a cat in 5 days, the cat excreted 79 % of the Cl4 in the urine. Fifty per cent of this radioactivity was isolated as quinaldylglycyltaurine, while 18 % was isolated as quinaldylglycylglycine, both of which were chemically and radiochemically pure. Most of the radioactivity in the urine or feces was accounted for in the first 48 hours after the administration of radioactive quinaldylglycyltaurine to the rat, cat, and rabbit (Table I). When the urine and feces were chromatographed on Dowex 2-acetate, the radioactivity was distributed as shown. Most of the quinaldylglycyltaurine was accounted for as quinaldic acid and quinaldylglycine whether bhis compound was given orally or by subcutaneous injection to the rats, while in the cat the compound was excreted as quinaldylglycyltaurine and quinaldylglycylglycine. The chief urinary metabolite in the rabbit was quinaldic acid when quinaldylglycyltaurine was administered orally, but most of t,he injected dose was excreted unchanged. Figure 1 shows the metabolic reactions of quinaldylglycyltaurine in these three species. DISCUSSION
In these and previous studies with the rabbit, no significant amount of conjugatlion
degradation
in the rat, rabbit,
and cat.
occurred when small doses of quinaldic acid or kynurenic acid were given (4). However, Ellinger and Matsuoka (6) found that large doses of quinaldic acid were conjugated with glycine by the rabbit. In the rat it was found that a dose of quinaldic acid could be almost completely accounted for in the urine as unchanged quinaldic acid or its glycine conjugate (3), and quinaldylglycyltaurine could not be detected in the urine after the administration of kynurenic acid-U4 (M. Kaihara, unpublished data). Quinaldylglycyltaurine was the major metabolite excreted by the cat after the ingestion of quinaldic acid or kynurenic acid (1). It was of interest to determine whether quinaldylglycyltaurine was further metabolized by the cat or whether degradation of this strongly acidic quinoline compound, which was not excreted by the rat or rabbit after the ingestion of quinaldic acid or kynurenic acid, occurred in these species. In the cat over 75 % of the radioactivity could be accounted for as unchanged quinaldylglycyltaurine, and 5-24 % was excreted in the urine as quinaldylglycylglycine. The feces of the cat contained no quinaldylglycylglycine, but some quinaldic acid and quinaldylglytine was found. Previous work (2) has shown that when quinaldylglycylglycine was given to the cat, the major metabolite usually was quinaldylglycyltaurine in the urine and quinaldic acid in the feces. Thus, in the cat an oral dose of quinaldic acid, quinaldyl-
QIJINALDYLGLYCYLTAURINE
glycylglycine, or quinaldylglycyltaurine is excreted ahnost completely in the form of t’he glycyltaurine and glycylglycine conjugates. The rat and rabbit degrade quinaldylglycyltaurine but apparently do not synthesize this conjugate. No change in the quinoline nucleus was observed in any of t’he three species of animals studied even t’hough they do not metabolize quinaldylglycyltaurine in the same manner. It would appear t,hat glycylglycine and glycyltaurine may be added to the relatively small list of compounds with which car-
METABOLISM
319
boxylic acids are known to be conjugated by animals prior to urinary excretion (7). REFERENCES 1. KAIHARA, RI., AND PRICE, J. hf., J. Biol. 236, 508 (1961). 2. KAIHARA, M., AND PRICE, J. RI., J. Biol.
Chem. Chem.
240, 454 (1965). 3. KAIHARA, M., J. Biol. Chem. 236, 136 (1960). 4. KAIHARA, M., AND PRICE, J. hf., J. Biol. Chem. 237, 1727 (1962). 5. DAVIS, J. W., JR., J. Org. Chem. 24.1691 (1959). 6. ELLINGER, A., AND MATSUOKA, Z., 2. Physiol. Chem. 109, 259 (1920). 7. WILLIAMS, R. T., “Detoxication Mechanisms,” 2nd edition, p. 348. Wiley, New York (1959).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
110,
The Fate of Quinaldylglycyltaurine MASAKO Division
of Clinical
Oncology,
in the Cat, Rat, and
KAIHARA
University Received
(1965)
316-319
J. M. PRICE2
AND
of Wisconsin December
Rabbit
Medical
School, Madison,
Wisconsin
28, 1964
The metabolic fate of the quinaldic acid moiety of quinaldylglycyltaurine has been studied in the rat, rabbit, and cat. This compound usually was excreted as quinaldic acid and quinaldylglycine by the rat, while the majority was eliminated unchanged or as quinaldylglycylglycine whether given orally or by subcutaneous injection to the cat. Quinaldic acid and quinaldylglycine, which have never been detected in cat urine, were found in the feces of two of the three cats. When quinaldylglycyltaurine was given orally to the rabbit, the major urinary metabolite was quinaldic acid, but if given by subcutaneous injection, it was excreted unchanged. Thus, the three species converted an oral dose of quinaldylglycyltaurine to quinaldic acid.
Previous studies have shown that when quinaldic acid was administered to the cat orally or by subcutaneous injection, the chief urinary metabolite was quinaldylglycyltaurine (1)) and most of the remainder of the dose was excreted as quinaldylglycylglycine (2). Neither the rat nor the rabbit excreted quinaldic acid conjugated with glycyltaurine or with glycylglycine (3, 4). The rat excreted quinaldic acid unchanged and as the glycine conjugat,e (3), while the rabbit excreted almost all of the compound in the nonconjugated form (4). The present study was undertaken to determine the ability of t’he cat, rat, and rabbit to degrade quinaldylglycyltaurine. For this purpose, radioactive quinaldylglycyltaurine was isolated from the urine of t,he cat after the administration of quinaldic acid-carboxyl-Cl*, and was given to the cat, rat, and rabbit orally and by subcutaneous injection. The distribution of urinary radioactivity among urinary quinaldic acid, quinaldylglycylglycine, quinaldylglycine, and quinaldylglycyltaurine was determined. 1 Supported in part by a grant from the National Institute of Arthritis and Metabolic Diseases (No. AM-01127), U.S. Public Health Service. 2 American Cancer Society-Charles S. Hayden Foundation Professor of Surgery in Cancer Research.
The data obtained are consistent with the interpretation that all three species converted an oral dose of quinaldylglycyltaurine to quinaldic acid. Quinaldic acid and quinaldylglycine, which have never been detected in cat urine, were found in the feces of 2 cats which failed to absorb a large fraction of the oral dose. PROCEDURE Reagents. Quinaldic acid carboxylG4 was synthesized by a previously described method (3). Quinaldylglycine and quinaldylglycylglycine were synthesized by the procedure described by Davis (5). Quinaldylglycyltaurine was available from materials used in previous studies (1). Quinaldylglycyltaurine-C14 was isolated from the urine of a cat which was given 10 mg (5,860,OOO cpm) of quinaldic acid-carboxyl-Cl4 daily in its food for 5 days. Urine was collected during those 5 days and also for the following 4 days. Quinaldylglycyltaurine-Cl4 was isolated as previously described (1). Experimental animals. Three adult male cats were purchased from local dealers. All cats were fed commercial canned cat food. Eight Holzman male albino rats weighing 170-200 gm were used; they were fed a stock grain diet. The rats were housed in groups of two, and the urine was collected from each pair of animals. Two male rabbits which had been used for previous studies (4) were used in this experiment. During urine collections 316
QUINALDYLGLYCYLTAURINE all animals were kept in stainless steel metabolism cages. The urine was collected under toluene and with glacial acetic acid, and both urine and feces were frozen until used. Administration of radioactive quinaldylglycyltour&e. The compound was administered in the quantities indicated in Table I. Oral supplements were administered via a stomach tube to the rat and rabbit. For the cat the compound was mixed with a small quantity of food which was ingested within a few minutes. Fractionation of urine or feces with Dowex 2acetate. An aliquot of urine or feces containing 13,000-189,000 cpm was used for fractionation of the radioactive constituents. Quinaldic acid and quinaldylglycine (6.4 #moles each) were added to the aliquot of sample before chromatography to insure accurate identification of the position of elution of these compounds as determined by a recording ultraviolet absorption meter (Gilson Medical Electronics). The urine was passed through a Dowex 2-acetate column measuring 1.2 X 16 cm, and gradient elution was carried out with acetic acid as previously described (l-3). When it was necessary to analyze feces, they TABLE OF Cl4 IN URINE AND FECES QUINALDYLGLYCYLTAURINE DISTRIBUTION
EXCRETION
1 3 5 7 1 3
& & & &
2 4 6 8
Oral Subcut. Oral Subcut. Oral Oral
Inject Inject.
‘3 excreted during first 48 hours (%I
-
BlOkS 110.7 110.7
CPm x 10-s
Urine
8.8 8.8 6.8 5.2
10.56 10.56 8.67 8.67 6.70 5.14
80.1 79.0 66.5 77.2 97.0 19.1
5.2
5.14
25.8
Feces
78.6 Oral
Cat 4
60.2 Cat 1 Cat 3 Rabbit Rabbit Rabbit Rabbit
1 2 1 2
Subcut. Subcut. Oral Oral Subcut. Subcut.
Inject. Inject.
Inject. Inject.
4.3 5.2 8.8 5.6 5.2 5.2
ADMINISTRXIYON AND
4.22 5.14 8.67 5.59 5.14 5.14
64.0 100.5 94.9 99.1 97.3 99.5
OF
T
i
Cl, recovered in fraction containing (%):
-
7-
P
Rats Rats Rats Rats Cat Cat
I
-
Dose given
nSethod of administration
were homogenized with water, the supernatant fluid was decanted after centrifugation, and the sediment was washed two times with water. The supernatant fluid and washings were pooled and made up to a known volume. An aliquot of this solution was passed through a 3 X 5 cm Dowex a-acetate column and eluted with 850 ml of 10 N acetic acid (to elute quinaldic acid and its glycine and glycylglycine conjugates) followed by 1000 ml of 2 N HCl (to elute quinaldylglycyltaurine) (1). The acetic acid and HCl were distilled from the fractions under reduced pressure. The residues were dissolved in water, and the result,ing solutions were rechromatographed on 1.2 X 16-cm columns of Dowex a-acetate with the same procedure as that used for the urine samples. If there were more than 3000 cpm in the quinaldic acid, quinaldylglycine, or quinaldylglycylglycine fractions from urine or feces, these compounds were recrystallized to constant specific activity with 30-60 mg of authentic carrier. Most of the quinaldylglycyltaurine fractions were not recrystallized because no suitable solvent is known which will permit repeated crystallization of small quinaldylglycyltaurine is samples. However, readily separated from quinaldic acid and its
DURING FIRST 48 HOURS AFTER TO RATS, CATS, AND RABBITS, OF Cl4 IN THE FRACTIONS
! Animal
317
METABOLISM
QAn
QAG
QAGT
43.7 12.0 67.5 41.6 4.2 0.3 11.1 0.4 18.9 1.5 0.2 88.2 89.3 6.2 9.9
44.7 87.9 22.2 11.4 0.9 5.2 13.0 0.7 2.4 0.3 1.0 9.4 9.2 2.2 2.9
0.1 1.5 35.4 82.5 80.7 77.6 76.6 75.2 96.3 98.1 1.2 2.3 87.1 85.4
2AGG
-b -
12.8 6.0 23.6 5.3
/ I
Total recovery of Cl’ in fractions (%)
88.5 99.9 91.2 88.4 100.4 92.2 101.7 101.3 96.5 103.4 99.3 98.8 100.8 95.5 98.2
a Abbreviations: &A, quinaldic acid; QAG, quinaldylglycine; QAGT, quinaldylglycyltaurine; QAGG, quinaldylglycylglycine; Subcut. Inject., subcutaneous injection. b Dashes indicate no detectable radioactivity in this fraction or a few counts which showed decreased specific activity upon repeated recrystallization with authentic carrier.
318
KAIHARA
AND
PRICE
NHCH2CONHCH2COOH
FIG. 1. Schematic
representation
of quinaldylglycyltaurine
other known conjugates (l), so there is no likelihood of contamination with these compounds. RESULTS
When 50 mg (29,000,OOOcpm) of quinaldic acid-carboxyl-Cl4 was given to a cat in 5 days, the cat excreted 79 % of the Cl4 in the urine. Fifty per cent of this radioactivity was isolated as quinaldylglycyltaurine, while 18 % was isolated as quinaldylglycylglycine, both of which were chemically and radiochemically pure. Most of the radioactivity in the urine or feces was accounted for in the first 48 hours after the administration of radioactive quinaldylglycyltaurine to the rat, cat, and rabbit (Table I). When the urine and feces were chromatographed on Dowex 2-acetate, the radioactivity was distributed as shown. Most of the quinaldylglycyltaurine was accounted for as quinaldic acid and quinaldylglycine whether bhis compound was given orally or by subcutaneous injection to the rats, while in the cat the compound was excreted as quinaldylglycyltaurine and quinaldylglycylglycine. The chief urinary metabolite in the rabbit was quinaldic acid when quinaldylglycyltaurine was administered orally, but most of t,he injected dose was excreted unchanged. Figure 1 shows the metabolic reactions of quinaldylglycyltaurine in these three species. DISCUSSION
In these and previous studies with the rabbit, no significant amount of conjugatlion
degradation
in the rat, rabbit,
and cat.
occurred when small doses of quinaldic acid or kynurenic acid were given (4). However, Ellinger and Matsuoka (6) found that large doses of quinaldic acid were conjugated with glycine by the rabbit. In the rat it was found that a dose of quinaldic acid could be almost completely accounted for in the urine as unchanged quinaldic acid or its glycine conjugate (3), and quinaldylglycyltaurine could not be detected in the urine after the administration of kynurenic acid-U4 (M. Kaihara, unpublished data). Quinaldylglycyltaurine was the major metabolite excreted by the cat after the ingestion of quinaldic acid or kynurenic acid (1). It was of interest to determine whether quinaldylglycyltaurine was further metabolized by the cat or whether degradation of this strongly acidic quinoline compound, which was not excreted by the rat or rabbit after the ingestion of quinaldic acid or kynurenic acid, occurred in these species. In the cat over 75 % of the radioactivity could be accounted for as unchanged quinaldylglycyltaurine, and 5-24 % was excreted in the urine as quinaldylglycylglycine. The feces of the cat contained no quinaldylglycylglycine, but some quinaldic acid and quinaldylglytine was found. Previous work (2) has shown that when quinaldylglycylglycine was given to the cat, the major metabolite usually was quinaldylglycyltaurine in the urine and quinaldic acid in the feces. Thus, in the cat an oral dose of quinaldic acid, quinaldyl-
QIJINALDYLGLYCYLTAURINE
glycylglycine, or quinaldylglycyltaurine is excreted ahnost completely in the form of t’he glycyltaurine and glycylglycine conjugates. The rat and rabbit degrade quinaldylglycyltaurine but apparently do not synthesize this conjugate. No change in the quinoline nucleus was observed in any of t’he three species of animals studied even t’hough they do not metabolize quinaldylglycyltaurine in the same manner. It would appear t,hat glycylglycine and glycyltaurine may be added to the relatively small list of compounds with which car-
METABOLISM
319
boxylic acids are known to be conjugated by animals prior to urinary excretion (7). REFERENCES 1. KAIHARA, RI., AND PRICE, J. hf., J. Biol. 236, 508 (1961). 2. KAIHARA, M., AND PRICE, J. RI., J. Biol.
Chem. Chem.
240, 454 (1965). 3. KAIHARA, M., J. Biol. Chem. 236, 136 (1960). 4. KAIHARA, M., AND PRICE, J. hf., J. Biol. Chem. 237, 1727 (1962). 5. DAVIS, J. W., JR., J. Org. Chem. 24.1691 (1959). 6. ELLINGER, A., AND MATSUOKA, Z., 2. Physiol. Chem. 109, 259 (1920). 7. WILLIAMS, R. T., “Detoxication Mechanisms,” 2nd edition, p. 348. Wiley, New York (1959).