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`A RAPID METHOD
`HIPPURIC
`
`FOR THE DETERMINATION
`ACID
`IN URINE.*
`
`OF
`
`(From
`
`AND W. W. SWANSON.
`BY F. B. KINGSBURY
`Department
`of Physiology,
`the Biochemical
`Laboratory,
`of Minnesota, Minneapolis.)
`
`University
`
`(Received
`
`for publication,
`
`June 20, 1921.)
`
`In making benzoate tests for renal efhciency we were confronted
`with the necessity for having a rapid and accurate method for the
`determination of hippuric acid in urine. The Folin-Flanders
`method which we have been using required more time than was
`thought necessary. By means of this method analyses could be
`made in 9 or 10 hours when necessary, but with
`the routine of
`teaching and other university work 24 hours were usually required.
`It was our object to devise a method which would conserve the
`accuracy of the Folin-Flanders
`(1) method, but one which could
`be completed within 2 or 3 hours and be as applicable for hospital
`routine work as are any of the other modern biochemical methods.
`A careful review of the more recent methods for the determina-
`tion of hippuric acid shows that at present there is only one method
`which fulfils the requirements of accuracy and simplicity.
`This
`is the method of Folin and Flanders. Two other methods which
`appeared at about the same time, Steenbock’s (2) and Hrynt-
`schak’s (3) methods meet the requirements of accuracy fairly well
`but are too tedious to compete with the Folin-Flanders method.
`Ito’s (4) method appearing 4 years later is more complicated than
`those mentioned above and does not represent an advance in this
`field. Steenbock’s and Hryntschak’s procedures depend upon
`the isolation and weighing of benzoic acid, which are accompanied
`by slight losses, more in the latter method than in the former, and
`are necessary only in those cases in which benzoic acid cannot be
`directly titrated, as for instance, in the presence of other titratable
`
`of
`is made to the Graduate School of the University
`*Acknowledgment
`Minnesota
`for the purchase of a portion
`of the chemicals used in this work.
`13
`
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`
`Determination
`
`of Hippuric Acid
`
`in the final extrac-
`Since there are no other acids present
`acids.
`method?
`titration
`tion and titration
`stages of the Folin-Flanders
`in this case is not only easier to accomplish but more accurate.
`Folin and Flanders proved
`that
`their method gave quantitative
`results with pure hippuric acid solutions which we have confirmed
`many
`times
`in the last
`few years.
`They did not compare
`their
`method, as applied
`to urine with any other procedure of analysis,
`nor as far as we can find, has any other
`investigator.
`They have
`assumed, however,
`that
`their method gives
`the most accurate
`re-
`sults of any method devised up to that time. We have proved in
`the experiments which are directly
`to follow
`that
`the Folin-
`Flanders method does correctly estimate the amount of hippuric
`acid that can be extracted directly
`from urine by means of ethyl
`acetate.
`
`I.- 0.561 gm. of pure sodium hippurate was dissolved
`in 100
`Experiment
`then
`cc. of water, 1 cc. of concentrated
`nitric acid added, and the mixture
`extracted with
`ten 50 cc. portions of ethyl acetate, shaking exactly 2 minutes
`each time.
`The aqueous mixture
`left was then
`filtered,
`the filtrate evap-
`orated
`to dryness over night on the steam bath with 10 cc. more of 5 per
`cent sodium hydroxide
`than
`that
`required
`for neutralization
`of the nitric
`acid present.
`The
`residue was then analyzed
`for any remaining
`hippuric
`acid by the Folin-Flanders
`method.
`The
`titration
`value was 0.07 cc.,
`which
`is the ordinary
`blank of the method.
`The hippuric
`acid was com-
`pletely extracted by this procedure.
`value of which was 13.58 cc.
`100 cc. of urine,
`the hippuric
`acid titration
`of one-tenth
`normal sodium ethylate were acidified with 2 cc. of concen-
`trated nitric
`acid and extracted with
`ten 50 cc. portions of ethyl acetate,
`shaking 2 minutes each
`time.
`The combined
`extracts were washed with
`two 200 cc. portions
`of the Folin-Flanders
`sodium
`chloride
`solution
`and
`then steam distilled
`until all of the ethyl acetate and approximately
`300 cc.
`of water had passed over.
`The aqueous solution of hippuric
`acid remain-
`ing in the distilling
`flask was quantitatively
`transferred
`to a casserole and
`analyzed
`according
`to the Folin-Flanders
`method.
`The
`titration
`value
`was 13.43 cc. of one-tenth
`normal sodium ethylate, agreeing with
`the value
`obtained
`directly
`as well as duplicates
`can usually be obtained
`by this
`method.
`
`Experimental Methods of Analysis.
`
`Our problem resolved itself into increasing the speed of the
`hydrolysis of hippuric acid either by acids or alkalies and the effec-
`tive oxidation of urinary pigments and other disturbing sub-
`stances. Without going into the details of many experiments
`
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`
`F. B. Kingsbury
`
`and W. W. Swanson
`
`15
`
`that by using 15 gm. of solid sodium
`carried out it may be stated
`the hippuric
`acid of 100 cc. of
`hydroxide
`in hydrolyzing
`for 30 minutes and subsequently
`acidi-
`urine at the boiling point
`fying, extracting,
`and titrating,
`results were obtained
`that were,
`in one experiment,
`22 per cent higher
`than
`the known
`titration
`value
`for
`this specimen of urine.
`It was also found
`that values
`from 10 to 33 per cent higher
`than
`those obtained by
`the Folin-
`Flanders method
`resulted when urine was boiled with
`an equal
`volume of a mixture of concentrated
`nitric and sulfuric
`acids
`for
`30 minutes
`in a proeess that gave 100 per cent recovery when applied
`to solutions
`of pure hippuric
`acid. Oxidation
`of the urine with
`alkaline potassium permanganate
`after the plan of Hryntschak
`was
`tried and yielded such promising
`results
`that
`the details of one
`typical
`experiment
`are given below:
`
`a
`
`sodium
`of solid
`7.5 gm.
`with
`boiled
`were
`cc. of urine
`8.-50
`Experiment
`in
`for
`30 minutes
`permanganate
`1.5 gm.
`of potassium
`and
`hydroxide
`condenser
`in
`the neck.
`fitting
`test-tube
`flask with
`a rather
`closely
`Kjeldahl
`nitric
`acid
`slowly
`poured
`was
`cooled
`and
`50 cc. of concentrated
`The
`flask
`mixture
`cleared
`up after
`boil-
`side of
`the
`condenser.
`The brown
`down
`the
`for 30 minutes,
`then
`cooled
`and
`ing a few minutes,
`but
`this was
`continued
`using
`comparative
`amounts
`extracted
`as
`in
`the Folin-Flanders
`procedure
`was
`16.72
`cc. of one-tenth
`of
`the
`various
`materials;
`The
`titration
`value
`cc.
`normal
`sodium
`ethylate;
`the
`regular
`Folin-Flanders
`method,
`16.95
`by
`97
`In a series
`of 12 analyses
`made
`in
`this way
`it was
`found
`that
`values
`from
`to 99 per
`cent
`of
`the Folio
`-Flanders
`figures
`cuuld
`always
`be obtained
`when
`these were
`as large
`as 15 c :., but with
`lower
`values
`the error
`was sometimes
`as
`large
`as 25 per
`cent.
`This was believed
`to be due
`to
`the
`action
`of
`the
`potassium
`permanganate
`on
`the benzoic
`acid present
`as it was always
`most
`pronounced
`in
`the urines
`which
`were
`the most
`dilute
`and
`therefore
`con-
`taining
`less of
`the
`other
`substances
`to
`combine
`with
`the
`permanganate.
`It was difficult
`to estimate
`the
`correct
`amount
`of potassium
`permanganate
`a
`to be added
`in each
`case and
`it
`frequently
`happened
`that
`1.5 gm. were
`greater
`amount
`than
`could
`be
`reduced
`beyond
`the manganate
`stage
`and 0.5
`gm. portions
`of sodium
`bisulfite
`had
`to be added
`to complete
`the
`reduction.
`It was also
`found
`that
`if
`this method
`were
`applied
`to a pure
`solution
`of hip-
`puric
`acid,
`allowing
`the potassium
`permanganate
`to act only
`2 or 3.minutes
`before
`reducing
`it with
`sodium
`bisulfite
`that
`it was
`impossible
`to
`obtain
`of
`more
`than
`95 per
`cent
`the
`theoretical
`amount.
`In Hryntschak’s
`method
`with
`the urine
`was boiled
`10 gm.
`of sodium
`hydroxide
`for 2.5 hours
`then
`gm. of potassium
`permanganate
`were
`added
`and
`the boiling
`was continued
`for 6 or 7 minutes.
`The
`excess
`of permanganate
`was
`removed
`bv
`adding
`about
`15 gm. of sodium
`bisulfite
`prior
`to acidification
`and extraction.
`He
`subjected
`benzoic
`acid
`to
`the
`same
`conditions
`and
`was
`able
`to
`recover
`
`10
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`
`16
`
`Determination
`
`of Hippuric Acid
`
`that
`from this
`and concluded
`98.24 and 98.17 per cent in two experiments
`This
`is con-
`potassium
`permanganate
`did not destroy any bensoic acid.
`trary
`to our findings using
`the more sensistive
`titration method.
`We were reluctant
`about giving up the use of potassium
`permanganate
`because the subsequbnt
`chloroform
`extracts were always practically
`color-
`less and remained
`so until
`the definite pink end-point
`of t: tration was
`reached.
`No decicledly yellow extracts such as are rather
`fret uent
`in the
`Folin-Flanders
`method were ever encountered.
`It was
`foun 1 by one of
`us that if a small quantity
`of magnesium oxide were present
`the sffect of the
`permanganate
`in decreasing
`the
`titration
`value was prevente 1. The de-
`tails of the method as we have adopted
`it follow:
`
`Description of the Method.
`
`50 cc. of urine are treated with 7.5 gm. of sodium hydroxide
`and 0.5 gm. of magnesium oxide in a 500 or 800 cc. KjeJ lahl flask.
`This mixture
`is boiled at such a rate as to bring its v )I lme down
`to approximately 25 cc. in the course of half an hour.
`it the end
`of this time, while still at the boiling temperature, 1 0 cc. of a 7
`per cent solution of potassium permanganate (a solut on approxi--
`mately saturated% at room temperature) is added, care Jeing taken
`to rinse down any that may remain on the neck of th ? flask with
`since no uncl anged per-
`the smallest possible amount of water
`manganate must be present when the acid is subsequc ltly added.
`The flask with its brown contents is twirled gently f( r a minute
`or two, cooled under the tap, a fairly closely fitting
`te t-tube con-
`denser placed in the neck and 30 cc. of concentrated nitric acid
`slowly poured in down the side of the condenser. T. e mixture,
`which rapidly clears up on the addition of the acid, is now gently
`boiled for 45 minutes (30 minutes are sufficient for accu -ate results,
`but a less colored, more easily titratable extract
`s obtained
`by boiling it 45’minutes) with a good current of w: ter flowing
`through the condenser, cooled under the tap, and the extraction
`with chloroform carried out approximately according t 1 the Folin-
`Flanders method. The condenser is rinsed down wit 1 25 cc. of
`water to remove any benzoic acid sublimed on the bo tom of the
`condenser, the contents of the flask are transferred tc/ a 500 cc.
`separatory funnel containing 25 gm. of solid ammonium sulfate.
`The flask is rinsed with 20 cc. of water which is poured into the
`separatory funnel. After dissolving the ammonium sulfate the
`benzoic acid is extracted successively with one 50 cc., one 35 cc.,
`
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`
`F. B. Kingsbury
`
`and W. W. Swanson
`
`17
`
`of the chloroform
`treatment
`that
`is reliable as far as its
`
`The
`chloroform.
`of neutral, well washed
`and two 25 cc. portions
`flask.
`the Kjeldahl
`first 2 porc,ions of chloroform
`are used to rinse
`funnel are washed
`The combined extracts
`in a second separatory
`once with 100 cc. of the Folin- Flanders
`salt solution
`(containing
`1.0 cc. of concentrated
`HCI
`in 2 liters of saturated NaCl solution)
`and drawn off through a dry filter paper into a dry Erlenmeyer
`flask.
`The separatory
`funnel
`from which
`the extract was drawn
`is rinsed
`with 20 cc. of chloroform.
`This is drawn off into a small beaker
`to
`which
`the wet
`filter paper had been transferred.
`The paper
`is
`rinsed with
`the chloroform
`and the latter
`is poured
`through a dry
`4
`filter
`into
`the main bulk of extract
`in the Erlenmeyer
`flask.
`drops of 1 per cent phenolphthalein
`in absolute alcohol are added
`and the benzoic acid solution
`titrated
`to a faint, but definite pink
`with
`tenth normal sodium ethylate.
`The preparation
`and stand-
`ardization
`of this alkali solution are adequately
`described
`in the
`original paper of Folin and Flanders.
`We have
`found
`that
`the following
`used in this method
`insures a product
`neutrality
`is concerned
`:
`is of the u. s. P. grade and contains about
`New chloroform, which
`0.75 per cent of ethyl alcohol, should be washed with an equal vol-
`ume of distilled water
`twice before being used
`for the extraction
`of benzoic acid. Chloroform
`which has been used in analysis and
`therefore
`contains
`sodium benzoate and alcohol
`is
`first
`filtered
`through
`a dry
`filter paper which
`removes a considerable
`part of
`the sodium benzoate
`in those determinations
`in which
`the titra-
`tion
`figure was
`fairly
`large.
`It
`is now washed
`successively
`with
`equal volumes of tap water, once; tap water
`containing 5 to 10 cc.
`of a saturated
`solution
`of NaOH,
`twice;
`tap water,
`twice;
`and
`in all. Since the accuracy
`of
`distilled water,
`once; six washings
`this method depends primarily
`upon the use of a sample of chloro-
`form which not only reacts neutral when
`tested, but which must
`remain neutral after being shaken with nitric acid, we have used
`the test which
`follows
`to determine
`this point:
`155 CC. of chloroform,
`the amount used in an analysis, washed
`as described above, are shaken with dilute nitric acid, washed with
`100 cc. of the Folin-Flanders
`salt solution,
`filtered
`through a dry
`paper, and
`titrated.
`The titration
`of this amount of chloroform
`suitable
`for use should not exceed 0.10 cc. of tenth normal sodium
`ethylate.
`
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`
`Determination
`
`of Hippuric Acid
`
`of this method or that of Folin and Flanders
`The application
`requires
`the removal of protein
`from
`the urine when
`this
`is present,
`as in nephritic urines.
`Directions
`for doing this have already been
`published,
`but perhaps may be repeated here.
`The albuminous
`urine
`is collected
`in 2 per cent nitric acid which
`was
`found by Raiziss and Dubin
`(5) to be effective
`in preventing
`the hydrolysis
`of hippuric
`acid.
`15 cc. of this dilute nitric acid
`are sufficient
`for a 3 hour nephritic
`urine.
`50 cc. of this urine,
`treated with 3 or 4 drops of 0.1 per cent methyl
`red solution
`in
`alcohol, are brought
`to the
`first definite
`yellow by the addition of
`approximately
`normal NaOH.
`The solution
`is then boiled, and
`during
`the boiling sufficient one-tenth normal HCl
`is added to pro-
`duce
`the
`first definite
`red color.
`This procedure
`removes
`the
`albumin nearly quantitatively
`so that
`there is no increase
`in the
`resulting
`titration,
`as has already been shown
`(6).
`The coagulum
`of albumin on the filter paper is washed
`twice with 50 cc. of boiling
`water.
`The combined washings
`and main bulk of
`filtrate
`are
`evaporated
`rapidly over a free flame in an 800 cc. Kjeldahl
`flask
`after being made slightly alkaline
`to methyl
`red by the addition
`of a small amount
`of dilute alkali.
`Bumping
`and
`frothing,
`should
`they occur, are checked by adding a glass pearl and a
`drop of caprylic
`alcohol.
`By
`supporting
`the
`funnel
`in
`the
`neck of the flask by means of a slice of a large cork stopper
`the
`filtration
`and evaporation
`are continued
`simultaneously.
`When
`the contents of the flask have been evaporated
`to approximately
`50 cc., 7.5 gm. of NaOH
`and 0.5 gm. of MgO are added and the
`analysis made according
`to the directions
`already given.
`spec-
`In Table
`I are given the comparative
`results with various
`imens of urine, normal and pathological,
`obtained by
`the new
`method and by that of Folin and Flanders.
`In a series of approxi-
`mately half of the determinations
`one of us used one method and the
`other,
`the other method.
`The results of neither of us were known
`to the other until all the determinations
`of this series had been made,
`when
`they were compared.
`No. 19 in Table I is a comparison
`of
`the two methods with 50 cc. aliquots of a pure sodium hippurate
`solution.
`The only modification
`in this case was
`the reduction
`of
`the permanganate with 0.5 gm. of sodium bisulfite as a substitute
`for the urinary
`constituents
`which ordinarily
`function
`in this man-
`ner, prior
`to the acid treatment.
`It
`is noted
`that
`the agreement
`
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`

`
`F. B. Kingsbury
`
`and W. W. Swanson
`
`19
`
`is good, as close in general as duplicates
`the two methods
`between
`can be made by the older method, and that duplicates
`by the new
`method, where
`they have been made show a very close agreement.
`TABLE
`I.
`
`Urine
`
`No.
`
`0.1 N Na ethylate.
`
`New method.
`
`F&n-Flanders
`
`method.
`
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`
`cc.
`4.70
`4.50
`
`28.95
`29.60
`13.55
`13.70
`33.10
`33.80
`0.95
`24.80
`26.75
`24.50
`14.20
`19 80
`8.65
`5.35
`8.70
`16.95
`8.80
`8.80
`12.35
`14.00
`
`14.25
`13.80
`14.15
`
`1
`
`2
`
`3
`
`4
`
`5
`6 P.*
`7 p.*
`8 P.*
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`
`19
`
`~
`
`cc.
`4.50
`4.35
`4.80
`4.50
`29.40
`29.50
`13.95
`
`33.65
`33.50
`1.05
`24.55
`26.60
`24.50
`14.20
`20.10
`8.50
`5.25
`8.65
`17.55
`8.55
`‘8.95
`12.56
`13.75
`13.55
`14.25
`
`designated
`
`by
`
`“P.
`
`“are
`
`pathological
`
`specimens.
`
`All others
`
`are
`
`*Urines
`normal.
`t50
`
`cc. of a sodium
`
`hippurate
`
`solution
`
`were
`
`used.
`
`have been made several days
`determinations
`duplicate
`A few
`apart with no evidence of loss of hippuric
`acid
`in acid urines at
`room temperature
`‘preserved with a small amount of a IO per cent
`solution of thymol
`in chloroform.
`
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`

`
`20
`
`Der;ermination
`
`of Hippuric Acid
`
`CONCLUSION.
`
`of hippuric
`the determination
`for
`rapid method
`An accurate,
`for comple-
`requires about 2 hours
`acid in urine
`is described which
`tion with normal urine and about 3 hours with urine containing
`albumin.
`
`BIBLIOGRAPHY.
`
`1912,
`
`xi, 257.
`
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` by guest on April 13, 2016
`
`331.
`921,
`
`xxviii,
`
`220.
`
`Chem.,
`J. Biol.
`F. F.,
`O., and Flanders,
`1. Folin,
`xi, 201.
`1912,
`Chem.,
`2. Steenbock,
`H.,
`J. Biol.
`xliii,
`315.
`1912,
`Z.,
`3. Hryntschak,
`T., Biochem.
`1916,
`xxxviii,
`2188.
`4.
`Ito,
`H.,
`J. Am. Chem.
`Sot.,
`H.,
`J. Biol.
`Chem.,
`5. Raiziss,
`G. W., and Dubin,
`6. Kingsbury,
`F. B., and Swanson,
`W. W., Arch.
`
`1915, xxi,
`Int. Med.,
`
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`
`A RAPID METHOD FOR THE
`DETERMINATION OF HIPPURIC ACID
`IN URINE
`F. B. Kingsbury and W. W. Swanson
`1921, 48:13-20.
`
`J. Biol. Chem.(cid:160)(cid:160)
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`Par Pharmaceutical, Inc. Ex. 1040
`Par v. Hyperion, IPR2015-01117
`Page 9 of 9
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`(cid:160)