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Metabolism of p-anisic acid by the rat
Life Sciences Vol. 10, Part II, pp. 1255-1259, 1971 . Printed in Great Britain
Pergamon Press
Mrrreanr .r.~ 0~ p-ANI3IC ACID BY TAE RAT Michael H. Cramer* and William R. Michael The Procter 8e Gamble Compaq, Miami Valley Laboratories P. 0. Box 39175, Cincinnati, Ohio 45239
(Received 28 June 1971; in final form 11 October 1971) Summar~r The rat metabolized and excreted in the urine over 85~, of a 100 mg IP dose of pAA within 24 hours . The only oxidation product of pAA metabolimm vas pHHA . Both pAA end pHBA were excreted primarily as conjugates . IN
1932,
wick reported that men excreted p-aniaic acid (pAA ;p-methoxy-benzoic
acid) as the glucuronide conjugate and the glycine conjugate in approximately equal proportions (1) .
Bray et el ., in
1955,
rabbits primarily as the glucuronide (2) . as the glycine conjugate ; less thaw as the free acid .
5`~
found that pAA vas excreted by
Most of the remai_^~pr vas excreted
of a single dose appeared in the urine
Administration of glycine concurrently with pAA increased
the proportion of the glycine conjugate in the urine. Axalrod has shorn that pAA could be 0-demethylated by enzymes in rabbit liver microsames (3) .
Qaick reported that dogs excreted p-hydro~grbenzoic acid
(pHHA), the product of pAA 0-demethylation, as the glucuronide while man ex crated the compound as the free acid and the glycine conjugate (1) .
Hrey et
al ., found that rabbits excreted pHBA primarily as the free acid but also as the glycine and glucuronide conjuga-tes ; a small fraction of a single dose was excreted as the ethereal sulfate (4) . Metabolism studies with p-methoxybenzoylscopolemiae, an experimental anticholinergic drug, have revealed that rat liver, akin and serum ere capable of hydrolyzing the drug to scopolamine end pAA (5) .
*Present address :
This study vas undertaken
University of Houston, College of Pharmacy, Hauston, Texas 77004.
1255
125 8
Metabolism ad p-Anisic Acid
Vol. 10, No. 21
to determine the fate of one of these metabolic products, pAA,
in the rat .
Materials and Methods pAA was purchased from Chemicals Procureme~ Labs, College Point, New York, and recrystallized three times from methyl ethyl ketone before use. pHHA, for use as n standard for analysis, was purchased from Metheson, Coleman and Hell, Cincinnati .
All thin-layer chromatography (TLC) was done using
silica gel F-254 precoated analytical glass plates with fluorescent indicator (Hrinkmana Instruments, Westbury, N. Y.) . An aqueous solution of pAA was prepared using a slight excess of NaOH . After the pH was adjusted to 7.0 with HC1, the solution was diluted with deionized water to contain 100 m8 PAA per ml .
Five male Sprague-Dawley rats
(Sprague-Dawley, Inc ., Madison, Wisconsin), 210-270 g were administered intraperitoneal (IP)
injections of 1 ml of the pAA solution (100 mg pAA per rat) .
Urine was collected under dry ice for 24 hours following injection.
Free
access to water, but no food, was permitted during the collection period.
All
urine samples were diluted to 50 ml i®nediately prior to use . Aliquots (25 ul) of diluted urine were applied to a silica gel plate. The plates were developed in n-butanol :acetone :acetic acid :28~, ammonia :water (45 :15:10:2 :28) .
Three dark blue spots were visualized on a green fluoresce
background under a ehortwave UV scanning lamp .
The spots were eluted and the
identity of each determined in preliminary experiments by : 1.
TLC using other systems (after acid hydrolysis and ether extraction) .
2.
Assay for glycine (6) .
3.
Assay for glucuronides (7) .
Free acids (pAA and pHHA) appeared at Rf ...0 .97, glycine conjugates at Rf . .. 0.79 and glucuronic acid conjugates Rf ... 0.53.
No indication of other
metabolites was seen . Each spot was marked with a pencil and scraped into a separate test tube . Three ml of 6N FECl was added to each tube and the tubes were heated for 1 hour in a boiling water bath to hydrolyze glycine and glucuronic acid conjugates .
Vol. 10, No. 21
Metabolism ad p-Anisic Acid
1257
After cooling, the contests of each tube were extracted three times with ml portions of ether.
3.0
The combined ether extracts were evaporated to dryness
under n streean of nitrogen.
$ach residue was taken up in 100 W1 ether,
Polloved by rinses with tro
50 ~ portions of ether . The combined ether
washings were spotted on a silica gel plate end developed in benzeae :diozane :
(90 :25 :4) . The spots (pAA at Rf ...0 .64 and pfBA at RP ...0 .51)
acetic acid
were visualised sad marked ns before end scraped into separate teat tubes. Tw ml .0lä AsOH wsa added to each tube .
After mi~dng end ceatrifugntion,
the optical density of each solution was measured, pAA at density of
247 mW (optical
0.49 was equivalent to 89 .4 ~[ piAA/ml ; optical density oP the 13" 5 NM pAA/ml) and pH9A at 279 m~ (optical
standard urine was equivale~ to density of 0.20 res equivalent to urine was equivalent to
29.7 ;+M pHBA/m1 ; optical density of standard
3.7 ~l pHBA/ml) .
Hlanàs (urine Prom untreated rats), standards (urine from untreated rats to which known qualities of piAA and püßA had been added), and acid hydrolyzed (12F HC1 and reflux Por 1 .5 haws) urine Pram treetefl animals were carried through the satire pnrocedure .
Recovery of pAA, pHBA end their conjugates
using the above procedures rna approximately 100 . Results Preliminary ezperimeats showed that ether extracts of acid hydrolyzed urine Prom pJyp-treated rata yielded only tro UV-absorbing spots after TLC in several systems .
In every case, the Rf oP one spot corresponded to that of
PAA end the Rf of the other corresponded to that oP pHBA .
The former gave a
negative response and the latter a positive response Then sprayed with a phenol-detecting reagent (8) .
When eluted rich .OlN NaOH, the spots yielded
iN abaorptioa spectra identical to the corresponding pure acids.
The remaiadsr
of the study was acco®pliahed based on the coaclusioa that the primary, end probably the only, product of oxidative p~AA metabolismi in the rat was pHBA . Table I s~mnmrized the analyses of pAA end its metabolites appearing is rat urine during the 24 hours after IP injection of pAA.
125 8
Metabolism od p-Anisic Acid
Vol . 10, No . 21
Table I Urinary Excretion of p-Anisic Acid and Metabolitesl Component
p-aniaic acid
p-hydro~rbenzoic acid
Form t
Percerxt of Injected Dose (mean t standard error)
Free acid
6 .0 t 0.4
Glycine conjugate
15 .5 t 1 .9
Glucuronide conjugate
58 .0 t 2 .4
Tote13
79 " 5 t 3 .9
Free acid
1 .4 t 0.2
Glycine conjugate
0 .6 t 0 .3
Glucuronide conjugate
4 .2 t 0.6
Tota13
6.2 ± 0.6
1 Urine collected under dry ice for 24 hours after IP injection of 100 mK p-eaisic acid to five male rats . Water, but no food, was freely available. Free acids, glycine conjugates, and glucuronide conjugates were separated by silica gel TLC of raw urine. Each spot was treated as described in methods . 3 Sum of free acid + glycine conjugate + glucuronide conjugate .
These account for 85 .7, of the administered dose of pAA . dose was excreted as same form of pAA. conjugated with glucuronic acid .
Almost 8cß of the
Most of this (5F~ of the dose) was
Another 15 .5
conjugate and the remainder was free pAA.
was excreted as the glycine
Of the relatively small proportion
of the dose metabolized to pHBA (6 .2~), most was excreted as the glucuronide. N~rly all the remainder was free pHBA . Discussion The rather large done of pAA (100 mg per rat) administered IP to rats in this study was metabolized and excreted in the urine almost completely within 24 hours.
of pAA.
The major metabolites ware the glucuronide and glycine conjugates Approximately 6;$ of the dose of pAA was converted to püBA .
Moat of
Vol . 10, No . 21
Metabolism of p-Anisíc Acid
this metabolite was excreted es the glucuronide .
1259
pHHA could form en ether
glucuronide through its phenol group, an ester glucuronide through its csrboxyl group, or a diglucuronide through both functional groups .
!Qo attempt
was made to determine the contribution of each of these to the total pHBA glucuronides . Since no toxic effects were noted in this study and since pAA was rapidly metabolized and excreted, ne toxicologic problem should be associated with pAA arising from hydrolysis of a single therapeutic dose of the experimental dr.:g p-methoMybenzoylscopolarrine . Acknowledgment - The authors thank Mr . Robert E . Su~vth for his technical assistance during this study . Aeferencea
47, 4t)3 (1y32) .
1.
A . .f, dUICK, J. Biol . Chem .
2.
H. G. BRAY, H . A. HIJMPFRiIS, W. V. PHORPE, K. WHITE and P . B . WOOD, Biochem . J . 59, 162 (1955) .
3.
J. AXEIIZOD, Bioc hem . J .
4.
H. G. BRAY, H. E. RYMAN and W . V. THORPE, Biochem . J .
5.
M . B . CR4MER, unp~ibliahed data .
6.
H. G . BRAY, R . C. CIAWES, W. V. THJRPE, K. ?+iHITE an~i F . H . WC`(~D,
7.
H. G. BRAY, B . G. HUNIPfBîIB, W. V. THDRPE, K. WHITE and P .
8.
H . G. i3RAY, W. V. Ti~RPE ard K. WHITE, Biochem . J .
63, cí34 (1956) . 41,
212 ~l. a+'?)
3lochem . 3 . 50, 5~3 (1y52) . Biochem . J . 52 , 412 (1r+52) .
B . WOOD,
15, 271 (195n) .
.
Pergamon Press
Mrrreanr .r.~ 0~ p-ANI3IC ACID BY TAE RAT Michael H. Cramer* and William R. Michael The Procter 8e Gamble Compaq, Miami Valley Laboratories P. 0. Box 39175, Cincinnati, Ohio 45239
(Received 28 June 1971; in final form 11 October 1971) Summar~r The rat metabolized and excreted in the urine over 85~, of a 100 mg IP dose of pAA within 24 hours . The only oxidation product of pAA metabolimm vas pHHA . Both pAA end pHBA were excreted primarily as conjugates . IN
1932,
wick reported that men excreted p-aniaic acid (pAA ;p-methoxy-benzoic
acid) as the glucuronide conjugate and the glycine conjugate in approximately equal proportions (1) .
Bray et el ., in
1955,
rabbits primarily as the glucuronide (2) . as the glycine conjugate ; less thaw as the free acid .
5`~
found that pAA vas excreted by
Most of the remai_^~pr vas excreted
of a single dose appeared in the urine
Administration of glycine concurrently with pAA increased
the proportion of the glycine conjugate in the urine. Axalrod has shorn that pAA could be 0-demethylated by enzymes in rabbit liver microsames (3) .
Qaick reported that dogs excreted p-hydro~grbenzoic acid
(pHHA), the product of pAA 0-demethylation, as the glucuronide while man ex crated the compound as the free acid and the glycine conjugate (1) .
Hrey et
al ., found that rabbits excreted pHBA primarily as the free acid but also as the glycine and glucuronide conjuga-tes ; a small fraction of a single dose was excreted as the ethereal sulfate (4) . Metabolism studies with p-methoxybenzoylscopolemiae, an experimental anticholinergic drug, have revealed that rat liver, akin and serum ere capable of hydrolyzing the drug to scopolamine end pAA (5) .
*Present address :
This study vas undertaken
University of Houston, College of Pharmacy, Hauston, Texas 77004.
1255
125 8
Metabolism ad p-Anisic Acid
Vol. 10, No. 21
to determine the fate of one of these metabolic products, pAA,
in the rat .
Materials and Methods pAA was purchased from Chemicals Procureme~ Labs, College Point, New York, and recrystallized three times from methyl ethyl ketone before use. pHHA, for use as n standard for analysis, was purchased from Metheson, Coleman and Hell, Cincinnati .
All thin-layer chromatography (TLC) was done using
silica gel F-254 precoated analytical glass plates with fluorescent indicator (Hrinkmana Instruments, Westbury, N. Y.) . An aqueous solution of pAA was prepared using a slight excess of NaOH . After the pH was adjusted to 7.0 with HC1, the solution was diluted with deionized water to contain 100 m8 PAA per ml .
Five male Sprague-Dawley rats
(Sprague-Dawley, Inc ., Madison, Wisconsin), 210-270 g were administered intraperitoneal (IP)
injections of 1 ml of the pAA solution (100 mg pAA per rat) .
Urine was collected under dry ice for 24 hours following injection.
Free
access to water, but no food, was permitted during the collection period.
All
urine samples were diluted to 50 ml i®nediately prior to use . Aliquots (25 ul) of diluted urine were applied to a silica gel plate. The plates were developed in n-butanol :acetone :acetic acid :28~, ammonia :water (45 :15:10:2 :28) .
Three dark blue spots were visualized on a green fluoresce
background under a ehortwave UV scanning lamp .
The spots were eluted and the
identity of each determined in preliminary experiments by : 1.
TLC using other systems (after acid hydrolysis and ether extraction) .
2.
Assay for glycine (6) .
3.
Assay for glucuronides (7) .
Free acids (pAA and pHHA) appeared at Rf ...0 .97, glycine conjugates at Rf . .. 0.79 and glucuronic acid conjugates Rf ... 0.53.
No indication of other
metabolites was seen . Each spot was marked with a pencil and scraped into a separate test tube . Three ml of 6N FECl was added to each tube and the tubes were heated for 1 hour in a boiling water bath to hydrolyze glycine and glucuronic acid conjugates .
Vol. 10, No. 21
Metabolism ad p-Anisic Acid
1257
After cooling, the contests of each tube were extracted three times with ml portions of ether.
3.0
The combined ether extracts were evaporated to dryness
under n streean of nitrogen.
$ach residue was taken up in 100 W1 ether,
Polloved by rinses with tro
50 ~ portions of ether . The combined ether
washings were spotted on a silica gel plate end developed in benzeae :diozane :
(90 :25 :4) . The spots (pAA at Rf ...0 .64 and pfBA at RP ...0 .51)
acetic acid
were visualised sad marked ns before end scraped into separate teat tubes. Tw ml .0lä AsOH wsa added to each tube .
After mi~dng end ceatrifugntion,
the optical density of each solution was measured, pAA at density of
247 mW (optical
0.49 was equivalent to 89 .4 ~[ piAA/ml ; optical density oP the 13" 5 NM pAA/ml) and pH9A at 279 m~ (optical
standard urine was equivale~ to density of 0.20 res equivalent to urine was equivalent to
29.7 ;+M pHBA/m1 ; optical density of standard
3.7 ~l pHBA/ml) .
Hlanàs (urine Prom untreated rats), standards (urine from untreated rats to which known qualities of piAA and püßA had been added), and acid hydrolyzed (12F HC1 and reflux Por 1 .5 haws) urine Pram treetefl animals were carried through the satire pnrocedure .
Recovery of pAA, pHBA end their conjugates
using the above procedures rna approximately 100 . Results Preliminary ezperimeats showed that ether extracts of acid hydrolyzed urine Prom pJyp-treated rata yielded only tro UV-absorbing spots after TLC in several systems .
In every case, the Rf oP one spot corresponded to that of
PAA end the Rf of the other corresponded to that oP pHBA .
The former gave a
negative response and the latter a positive response Then sprayed with a phenol-detecting reagent (8) .
When eluted rich .OlN NaOH, the spots yielded
iN abaorptioa spectra identical to the corresponding pure acids.
The remaiadsr
of the study was acco®pliahed based on the coaclusioa that the primary, end probably the only, product of oxidative p~AA metabolismi in the rat was pHBA . Table I s~mnmrized the analyses of pAA end its metabolites appearing is rat urine during the 24 hours after IP injection of pAA.
125 8
Metabolism od p-Anisic Acid
Vol . 10, No . 21
Table I Urinary Excretion of p-Anisic Acid and Metabolitesl Component
p-aniaic acid
p-hydro~rbenzoic acid
Form t
Percerxt of Injected Dose (mean t standard error)
Free acid
6 .0 t 0.4
Glycine conjugate
15 .5 t 1 .9
Glucuronide conjugate
58 .0 t 2 .4
Tote13
79 " 5 t 3 .9
Free acid
1 .4 t 0.2
Glycine conjugate
0 .6 t 0 .3
Glucuronide conjugate
4 .2 t 0.6
Tota13
6.2 ± 0.6
1 Urine collected under dry ice for 24 hours after IP injection of 100 mK p-eaisic acid to five male rats . Water, but no food, was freely available. Free acids, glycine conjugates, and glucuronide conjugates were separated by silica gel TLC of raw urine. Each spot was treated as described in methods . 3 Sum of free acid + glycine conjugate + glucuronide conjugate .
These account for 85 .7, of the administered dose of pAA . dose was excreted as same form of pAA. conjugated with glucuronic acid .
Almost 8cß of the
Most of this (5F~ of the dose) was
Another 15 .5
conjugate and the remainder was free pAA.
was excreted as the glycine
Of the relatively small proportion
of the dose metabolized to pHBA (6 .2~), most was excreted as the glucuronide. N~rly all the remainder was free pHBA . Discussion The rather large done of pAA (100 mg per rat) administered IP to rats in this study was metabolized and excreted in the urine almost completely within 24 hours.
of pAA.
The major metabolites ware the glucuronide and glycine conjugates Approximately 6;$ of the dose of pAA was converted to püBA .
Moat of
Vol . 10, No . 21
Metabolism of p-Anisíc Acid
this metabolite was excreted es the glucuronide .
1259
pHHA could form en ether
glucuronide through its phenol group, an ester glucuronide through its csrboxyl group, or a diglucuronide through both functional groups .
!Qo attempt
was made to determine the contribution of each of these to the total pHBA glucuronides . Since no toxic effects were noted in this study and since pAA was rapidly metabolized and excreted, ne toxicologic problem should be associated with pAA arising from hydrolysis of a single therapeutic dose of the experimental dr.:g p-methoMybenzoylscopolarrine . Acknowledgment - The authors thank Mr . Robert E . Su~vth for his technical assistance during this study . Aeferencea
47, 4t)3 (1y32) .
1.
A . .f, dUICK, J. Biol . Chem .
2.
H. G. BRAY, H . A. HIJMPFRiIS, W. V. PHORPE, K. WHITE and P . B . WOOD, Biochem . J . 59, 162 (1955) .
3.
J. AXEIIZOD, Bioc hem . J .
4.
H. G. BRAY, H. E. RYMAN and W . V. THORPE, Biochem . J .
5.
M . B . CR4MER, unp~ibliahed data .
6.
H. G . BRAY, R . C. CIAWES, W. V. THJRPE, K. ?+iHITE an~i F . H . WC`(~D,
7.
H. G. BRAY, B . G. HUNIPfBîIB, W. V. THDRPE, K. WHITE and P .
8.
H . G. i3RAY, W. V. Ti~RPE ard K. WHITE, Biochem . J .
63, cí34 (1956) . 41,
212 ~l. a+'?)
3lochem . 3 . 50, 5~3 (1y52) . Biochem . J . 52 , 412 (1r+52) .
B . WOOD,
15, 271 (195n) .
.