FURTHER
`
`ON THE DETOXICATION
`STUDIES
`PHENYLACETIC
`ACID
`
`OF
`
`BY ANTHONY
`
`M.
`
`W. POWER,
`
`AND
`
`FRANCIS
`AMBROSE,
`CARL
`P. SHERWIN
`the Chemical Research Laboratory,
`Fordham University, New York)
`
`(From
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`(Received
`
`for
`
`publication,
`
`May
`
`4, 1933)
`
`In the previous work done on the detoxication of phenylacetic
`acid in t.his laboratory, we have never been able to account for
`more than half of it as the glutamine conjugate in the human
`urine. We ascribed this to either (a) conjugation with other
`substances besides glutamine, (b) an incomplete detoxication, or
`(c) the met.hods of analysis.
`
`Conjugation
`
`with Glucuronic
`
`Acid
`
`Quick (1) believed t,hat glucuronic acid may arise not from
`glucose itself, but from glycogen and sugar-forming amino acids
`In his experi-
`when glucuronic acid is needed for detoxicat,ion.
`ments depancreatized dogs were able to produce glucuronic acid
`the same as normal dogs, but under these conditions glucuronic
`acid is produced at the expense of glucose which would otherwise
`appear in the urine.
`The ratio between the conjugation of benzoic acid with glu-
`curonic acid and glycine in the case of animals was also deter-
`mined by Quick (2). The results show that
`the amount com-
`bined with glucuronic acid in these cases was by far larger than
`had previously been suspected.
`Brakefield (3) finds only traces of or no glucuronic acid after
`feeding benzoic acid to humans while Quick (4) under the same
`conditions reports 11 per cent conjugation. However, when
`phenylacetic acid is fed to dogs (a),
`it is detoxicated in a manner
`similar to benzoic acid.
`
`669
`
`
`Page 1 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`670
`
`Detoxication
`
`of Phenylacetic Acid
`
`Incomplete Detoxication
`subjects were
`fed phenylacetic
`acid; the urine was acidi-
`Human
`fied and extracted
`with
`various
`organic
`solvents,
`but no
`free
`phcnylacetic
`acid was
`found.
`to determine
`~~~e~irne~,t
`I-The
`object of our experiment, was
`to what extent,
`if any, glucuronic
`acid is employed by the human
`
`Rate of Glucuronic
`
`Acid
`
`TABLE
`Excretion
`of Phenylacetic
`
`I
`after
`
`Feeding
`Acid
`
`Varying
`
`Phenylacetic
`aid
`eliminated
`
`Amounts
`-
`
`8 Phenylacetio
`acid eliminated
`
`Date
`
`Date
`
`l-4
`4, 5
`
`Nov.
`“
`Dec.
`Jan.
`“
`“
`“
`“
`“
`“
`“
`“
`“
`‘I
`“
`“
`
`19, 23
`30, Dec.
`5-8,
`Jan.
`6, 7, 10
`11
`12
`13
`14
`16
`17
`18
`20
`21
`23
`25
`26
`
`gm.
`1
`2
`3
`4
`5
`5
`5
`5
`5
`5
`5
`6
`6
`6
`7
`7
`
`-
`
`93.0
`97.5
`98.0
`97.9
`97.0
`98.2
`98.5
`98.4
`98.2
`98.4
`98.4
`98.2
`98.6
`97.9
`98.9
`99.0
`
`i
`
`0.0 0.0
`
`1.0
`4.0
`4.9
`4.6
`3.5
`6.4
`7.1
`4.9
`4.9
`5.6
`5.7
`6.6
`5.7
`5.4
`
`Jan.
`“
`Feb.
`(‘
`“
`“
`“
`“
`“
`“
`“
`“
`“
`“
`“
`“
`
`27
`30
`1
`2
`3
`6
`8
`9
`10
`11
`13
`15
`16
`17
`23
`24
`
`CJm.
`7
`8
`8
`8
`10
`10
`10
`10
`10
`10
`8
`8
`7
`7
`6
`6
`
`99.1
`99.1
`98.4
`97.4
`98.8
`98.7
`99.5
`98.4
`98.9
`99.0
`98.5
`98.5
`98.5
`98.0
`98.7
`99.3
`
`4.5
`6.8
`5.7
`5.0
`5.3
`5.2
`5.3
`5.3
`5.2
`5.6
`5.6
`5.2
`5.1
`5.5
`6.5
`6.8
`
`of phenylacetic
`for detoxication
`subject
`take place.
`under which
`this might
`to human sub-
`fed
`acid was
`phenylacetic
`In these experiments
`in increasing daily doses in order
`to place
`jects as the sodium salt
`a continual
`and ever
`increasing
`strain on the defense mechanism
`of the body,
`in the hope
`that
`in this way we might overtax
`the
`
`the conditions
`
`acid and
`
`
`Page 2 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`Ambrose, Power, and Sherwin
`
`671
`
`to a
`to produce glutamine and force it to resort
`power of the body
`glucuronic
`acid detoxication.
`Table
`I shows
`the results obtained.
`Experiment
`,%-Small
`doses of phenylacetic
`acid, 3 gm ., were
`fed
`to thirty-four
`normal
`students
`and
`the urine was
`collected
`hourly over a period of 5 hours.
`It was
`thought
`that
`the small
`dose might be detoxicated
`by means of glucuronic
`acid and if so
`we wished
`to ascertain
`the rate at which
`this combination
`took
`place.
`It seemed quite possible
`to us
`that
`small amounts
`of
`phenylacetic
`acid excreted during
`the early stage of detoxication
`
`Glucuronic
`5 gm. were
`given
`
`Acid
`every
`
`Excretion
`8 hours,
`
`TABLE
`after
`a total
`
`II
`Acid
`Phenylacetic
`Feeding
`of 45 gm.
`in 72 hours.
`
`Time
`
`of
`
`feeding
`
`Phenylaoetio
`
`acid
`-
`
`9 a.m.
`5 p.m.
`12
`“
`9 a.m.
`5 p.m.
`12
`“
`9 a.m.
`5 p.m.
`12
`“
`
`Total
`
`gm.
`1.84
`4.74
`4.33
`2.68
`3.40
`3.89
`3.34
`4.36
`6.10
`
`Total................
`
`34.68
`
`I
`
`eliminated
`
`Combined
`
`wivii
`
`glucuronio
`
`gm.
`0.0
`0.807
`0.252
`0.602
`1.515
`0.384
`0.301
`1.566
`0.231
`
`5.658
`
`79.29
`cent
`per
`12.40 per
`
`per
`of
`cent
`
`acid
`phenylacetic
`total
`the
`of
`cent
`conjugated
`eliminated
`was
`total
`the
`of the
`total
`fed was conjugated
`with
`
`fed was
`with
`glucuronic
`
`recovered.
`glucuronic
`acid.
`
`16.00
`acid.
`
`large volumes of urine were
`when
`might have been overlooked,
`collected over a period of 24 hours.
`The results
`of the experi-
`ment, however, may be considered
`entirely negative
`as five sub-
`jects of the thirty-four
`showed
`less than 1 per cent of glucuronic
`acid conjugation
`at one time or another, and the others
`showed
`less or none at all.
`the
`in much
`was undert,aken
`experiment
`Experiment S-This
`same fashion, except, that
`the subject was
`fed 5 gm. of phenyl-
`acetic acid every 8 hours
`for a period of 72 hours, a total
`feeding of
`
`
`Page 3 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`672
`
`Detoxication
`
`of Phenylacetic Acid
`
`acid
`
`acid and phenylacetic
`45 gm., and the amount of glucuronic
`(See Table
`II.)
`determined at the end of each 8 hour period.
`Methods-Phenylacetylglutamine
`was prepared
`by
`the usual
`method
`(5).
`Total phenylacetic
`acid was determined
`by
`the
`method of Kingsbury
`and Swanson
`(6) as used by
`them
`for
`the
`determination
`of total benzoic acid, except
`that,
`instead of using
`quant.ities
`of urine as described
`in their method,
`the quantity
`varied
`inversely with
`the amount of phenylacetic
`acid ingested.
`Glucuronic
`acid was determined
`by
`its
`reducing
`power
`towards
`Somogyi’s
`reagent
`(7).
`acid plays a
`From
`this work
`it is clearly seen that glucuronic
`acid
`in the
`rather
`small
`role
`in the detoxication
`of phenylacetic
`human as compared with
`similar work
`on animals except when
`excessive
`amounts
`are
`ingested.
`Thus Quick
`(2) showed
`t.hat
`phenylacetic
`acid
`is excreted by
`the dog, conjugated with
`glu-
`curonic acid to the extent of about 34 per cent,
`the other 66 per
`cent appearing as the glycine conjugate.
`Table I shows
`that after
`repeated
`ingestions of phenylacetic
`acid at 24 hour
`intervals
`the
`body
`is still capable of furnishing
`glutamine
`for detoxication
`pur-
`poses and
`that
`the amount of glucuronic
`acid produced
`rarely
`exceeds a 5 per cent conjugation.
`However,
`the same or smaller doses, but ingested at 8 hour inter-
`vals,
`required
`considerable
`amounts of this substance,
`as Experi-
`ment 3 (Ta,ble
`II)
`shows,
`but we were unable
`to complete
`the
`series as the subject became nauseated on account, of t,he repeated
`ingestions.
`During
`the second period we obtained
`greater glu-
`curonic acid conjugation,
`indicating
`the body’s
`inability
`to synthe-
`size sufficient glutamine,
`thus
`resorting
`to glucuronic
`acid conju-
`gation.
`Phenylacetic
`acid retention was also observed.
`
`of Phenylacetylglutamine
`Methods of Analysis
`acid in the
`Folin and Flanders
`(8) have noticed
`that hippuric
`on a water
`urine
`is appreciably
`hydrolyzed
`while evaporating
`which
`is
`bath, so it is to be expected
`that phenylacetylglutamine,
`extent.
`quite unstable, would also be destroyed
`to a considerable
`In a series of four experiments,
`assuming
`that all the phenylacetic
`acid ingested should appear as a glutamine
`conjugate, an average
`of 51 per cent of phenylacetylglutamine
`was
`recovered
`from
`the
`urine.
`To check
`this by a recovery
`experiment,,
`16.4 gm. of
`
`
`Page 4 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`Ambrose, Power, and Sherwin
`
`in 2 liters of normal urine
`were dissolved
`phenylacetylglut,amine
`treated
`in the usual way
`(5). Only 47.4 per
`and the urine was
`cent of the original amount was
`recovered.
`Therefore,
`it seems
`t,hat the large amount of phenylacetylglutamine
`unaccounted
`for
`is not to be ascribed
`to any extensive
`conjugation with any other
`substances,
`but rather
`to t’he losses inherent
`in the evaporation,
`as
`the extraction
`itself
`seems
`to give quantitative
`removal
`of the
`phenylacetylglutamine
`once it is in the apparatus.
`In connection with
`this work we were able to get an approxi-
`mate estimate
`of the
`time
`required
`for
`the body
`to detoxicate
`phenylacetic
`acid.
`A total of 20 gm. (in four portions)
`was
`in-
`gested by two
`subjects, each taking 5 gm. in two doses 12 hours
`apart ; 12 hour volumes of the urine, each including both night and
`day specimens, were evaporated
`and extracted;
`and the
`first 12
`hour sample was compared with
`the following
`12 hour sample.
`It
`was
`found
`that
`in the urine excreted
`during
`the
`first 12 hours
`following
`ingestion
`of the acid, 53.8 per cent of the
`theoretical
`phenylacetylglutamine
`was
`recovered,
`and in the second 12 hour
`period only 1.4 per cent.
`Thus over 95 per cent of the phenyl-
`acetic acid is taken care of by the body during
`the first 12 hours
`and less than 5 per cent remains
`to be detoxicated
`in the second 12
`hours.
`Hence, whether
`we approach
`this detoxication
`problem
`from
`the glutamine
`conjugate or from
`that of glucuronic
`acid, the
`ratio of one to the other
`is very nearly
`the same, that
`is about 18: 1.
`In aqueous solution, pure phenylacetylglutamine
`shows a fairly
`strong
`acidity.
`Its hydrogen
`ion concentration,
`measured
`by
`indicators
`and checked approximately
`electrometrically,
`for 0.1 N
`solution
`is 6.9 X 10-3, corresponding
`to an ionization
`of about
`7 per cent, and for 0.01 N solution, 2.6 X 10-3, corresponding
`to an
`ionization of about 25 per cent.
`Its warm 0.1 N solution decom-
`poses
`the carbonates
`of alkalies and alkaline earths, also those of
`lead and copper.
`Its
`salts
`thus
`far studied’ are all soluble
`in
`water, making
`separation
`by precipitation
`thus
`far
`impossible.
`The
`insolubility
`of the barium
`salt
`in absolute alcohol
`(0.87 gm.
`per liter)
`is used to separate
`it from urea; the presence of 5 per cent
`of water
`in the alcohol raises
`its solubility
`over 400 per cent.
`in
`Decomposition
`by Evaporation-Two
`samples were dissolved
`in
`25 cc. of water
`in small Kjeldahl
`flasks which were
`immersed
`boiling water and a slow
`current
`of air passed
`through
`(over 2%
`
`
`Page 5 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`674
`
`Detoxication
`
`of Phenylacetic Acid
`
`and run
`hours),
`were as follows:
`
`into hydrochloric
`
`acid 0.5024 N.
`
`The
`
`results
`
`Substance
`
`om.
`0.1632
`0.1625
`
`Acid
`
`neutralized
`
`cc.
`0.26
`0.32
`
`Decomposition
`per cent
`21.1
`26.1
`
`that
`is based on t,he assumption
`decomposition
`This percentage
`the phenylacetylglutamine
`lost 1 molecule of ammonia and was
`converted
`into phenylacetylglutamic
`acid.
`A similar experiment
`run at 60” showed only one-fourth
`as much decomposition.
`with
`Decomposition
`of Phenylacetylglutamine
`on Evaporation
`in one
`Alkaline Solutions-Experiments
`were conducted as above;
`case 50 cc. of saturated
`barium hydroxide
`solution were
`taken and
`boiled off slowly during 1 hour
`into standard
`acid kept
`in an ice
`bath;
`in the other case N KOH
`solution was used.
`The results
`were as follows:
`
`Substance
`
`om.
`0.2002
`0.2015
`
`I
`
`Acid
`
`neutralized
`
`I
`
`Decomposition
`
`cc.
`(0.0894
`(0.1014N)
`
`N)
`
`8.80
`7.48
`
`per cent
`104.0
`99.5
`
`caused 71
`for 15 minutes
`solution
`Boiling with barium hydroxide
`per cent decomposition,
`and even in the cold there
`is a slight
`loss
`of ammonia on standing.
`by Means of Its Amino
`Determination
`of Phenylacetylglutamine
`Nitrogen-Small
`samples
`of phenylacetylglutamine
`(0.1266 gm.
`in each case) were boiled 2 hours under a reflux with 10 cc. of 62
`per cent sulfuric
`acid.
`The mixture was cooled, diluted, made
`alkaline with sodium hydroxide,
`and boiled until all the ammonia
`was
`liberated,
`then evaporated
`to dryness on the steam bath, and
`the residue dried
`in a vacuum
`oven overnight
`at 55-60”.
`The
`dry residue was ground up and extracted wit,h 95 per cent alcohol
`made faintly acid with
`sulfuric
`acid.
`This alcoholic solution was
`concentrated
`to small volume,
`run
`into
`the Van Slyke apparatus,
`and the amino nitrogen
`determined,
`after 10 minutes
`shaking.
`
`
`Page 6 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`Ambrose, Power, and Sherwin
`
`675
`
`1 cc. of Nz = 0.0118 gm. of phenylacetylglutamine.
`were as follows:
`
`The results
`
`N*
`cc.
`10.52
`10.12
`11.79
`11.70
`and
`
`pressure
`
`Yield
`per cent
`98.0
`94.4
`109.9
`109.0
`corrected
`for blank.
`
`* Normal
`
`temperature
`
`figures give an average yield of 102.8 per cent. Using
`These
`phenylacetylglutamine
`without
`previous hydrolysis,
`we observed
`on two
`samples 2.17 and 3.20 per cent decomposition,
`average
`2.69 per cent, after 10 minutes shaking.
`
`SUMMARY
`acid
`of phenylacetic
`incomplete
`conjugation
`1. The apparently
`in
`it
`is
`ingested
`by
`the human
`subject
`with
`glutamine when
`moderate doses may best be ascribed
`to the hydrolysis
`of phenyl-
`acetylglutamine
`when
`the urine containing
`it is evaporated
`on the
`water bath.
`in moder-
`acid ingested
`2. About 95 per cent of the phenylacetic
`glutamine,
`ate doses by
`the human
`subject
`is detoxicated with
`inges-
`acid. On continued
`and about 5 per cent with glucuronic
`tion the ratio
`is shifted
`in favor of the latter.
`3. Some properties
`of phenylacetylglutamine
`
`are enumerated.
`
`BIBLIOGRAPHY
`
`physiological
`
`chemistry,
`
`1. Quick,
`(1926).
`70,59,397
`Chem.,
`A. J., J. Biol.
`(1928).
`77,581
`Chem.,
`A. J., J. Biol.
`2. Quick,
`(1927).
`74, 783
`3. Brakefield,
`J. L.,
`J. Biol.
`Chem.,
`4. Quick,
`A. J., J. Biol.
`Chem.,
`92, 65
`(1931).
`5. Hawk,
`P. B.,
`and
`Bergeim,
`O.,
`Practical
`Philadelphia,
`9th
`edition,
`304
`(1926).
`J. Biol.
`Kingsbury,
`F. B., and Swanson,
`W. W.,
`Somogyi,
`J. BioZ. Chem.,
`70, 599
`(1926).
`M.,
`Folin,
`O., and Flanders,
`F. F., J. BioZ. Chem.,
`
`6.
`7.
`8.
`
`Chem.,
`
`48, 13 (1921).
`
`11,257
`
`(1912).
`
`
`Page 7 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on February 10, 2016
`
`FURTHER STUDIES ON THE
`DETOXICATION OF PHENYLACETIC
`ACID
`Anthony M. Ambrose, Francis W. Power and
`Carl P. Sherwin
`1933, 101:669-675.
`
`J. Biol. Chem.(cid:160)(cid:160)
`
`Access the most updated version of this article at
`
` http://www.jbc.org/content/101/3/669.citation
`
`
`
`Alerts:
`When this article is cited
`•(cid:160)
`
` When a correction for this article is posted
`•(cid:160)
`(cid:160)
`Click here
`alerts
`
` to choose from all of JBC's e-mail
`
`This article cites 0 references, 0 of which can be
`accessed free at
`http://www.jbc.org/content/101/3/669.citation.full.h
`
`tml#ref-list-1
`(cid:160)
`
`
`Page 8 of 8
`
`Owner Ex. 2027
`Par Pharm. v. Horizon
`IPR2015-01117,-01127
`
`(cid:160)
`