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`
`PROTEIN
`
`METABOLISM
`BLOOD
`AND
`
`FROM
`TISSUE
`
`THE STANDPOINT
`ANALYSIS.
`
`OF
`
`THIRD
`
`PAPER.
`
`FURTHER
`ENCE
`
`ABSORPTION
`TO
`THE
`BEHAVIOR
`AND
`TO
`THE
`
`EXPERIMENTS
`OF
`CREATINE
`FORMATION
`
`WITH
`
`ESPECIAL
`AND
`CREATININE
`OF UREA.
`
`REFER-
`
`BY OTTO
`
`FOLIN
`
`AND W. DENIS.
`
`(From
`
`the Biochemical
`
`Laboratory
`
`of Harvard
`
`Medical
`
`School,
`
`Boston.)
`
`(Received
`
`for
`
`publication,
`
`June
`
`1, 1912.)
`
`are
`products
`digestion
`the nitrogenous
`that
`The hypothesis
`the
`resynthesized
`into albuminous materials while passing
`through
`mucous membrane of the intestine and therefore
`cannot be detected
`in the blood was originally
`advanced
`to explain
`the absence of
`peptones
`in the blood during digestion.
`The subsequent
`discovery
`that protein digestion normally
`proceeds
`further
`than
`to the pep-
`tone stage and results
`in the formation
`of amino-acids
`suggested
`another explanationof
`the earlier negative
`results.
`Asthe attempts
`to find the amino-acids
`in the blood were no more successful
`than
`had been the earlier attempts
`to find the peptones,
`the hypothesis
`of the immediate
`resynthesis
`of the digestion products
`into albumin-
`ous substances
`was
`revived.
`Kutscher
`adopted
`it? and Abder-
`halden has become more and more positive
`that
`it represents
`the
`only adequate explanation
`of the negative
`results obtained by him
`and his coworkers
`in their search
`for peptones and amino-acids
`in the blood.
`Since the resynthesis
`hypothesis
`is based on nega-
`tive
`findings alone it has no value after
`the presence
`in the blood
`of amino-acids
`absorbed
`from
`the
`intestine
`has been positively
`demonstrated.
`In our
`first paper2 we showed
`that
`it is possible
`by means of our new analytical method9
`to trace urea, glycocoll
`and pancreatic
`amino-acid mixtures
`not only
`into
`the blood, but
`
`f. physiol.
`xxxiv,
`Chem.,
`1 Zeitschr.
`p. 87, 1912.
`* This
`Journal,
`xi,
`xi, pp. 493-536,
`1912.
`3 Ibid.,
`
`p. 529,
`
`1902.
`
`141
`
`001
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`
`142
`
`.
`
`Urea
`
`Formation
`
`the increases
`tissues of the body. Moreover,
`also into the general
`nit’rogen of the blood and muscles which we
`in the non-protein
`have obtained
`in absorption
`experiments
`are large enough
`to
`account
`for practically
`all the nitrogenous material absorbed
`from
`the
`intestine.
`We do not claim
`to account
`exactly
`or quanti-
`tatively
`for all that has been absorbed.
`It
`is entirely
`possible
`that different
`tissues absorb different amounts
`from
`the blood, and
`we have
`therefore
`omitted
`all calculations
`tending
`to show
`that
`all the absorbed mat,erial
`is present
`in the animal
`in non-protein
`form.
`The result’s obtained
`indicate,
`however,
`that practically
`all the absorbed nitrogen
`can be accounted
`for, so that
`for
`the
`present at least
`the hypothesis
`of immediate
`protein
`synthesis
`in
`the walls of the
`intestine must be regarded
`as superfluousand
`untenable.
`of
`idea served only as an explanation
`regeneration
`The protein
`the failure
`to find the absorbed amino-acids;
`it was anything but an
`explanation
`of the formation
`of those amino-acids
`in the intestine;
`it was
`inconsistent
`with
`t’he rapid urea elimination
`after protein
`feeding, and it necessarily
`presupposed
`that
`in chemical
`composi-
`tion the serum proteins are veryvariable,
`whereas, on the contrary,
`they appear
`to remain decidedly uniform
`in composition.
`As an explanation
`of the failure
`to find peptones or amino-acids
`in the blood when
`they should be there
`in unusual amount,s,
`the
`immediate deaminization
`hypothesis
`therefore
`seemed more plausi-
`ble than the protein
`regenerat,ion doctrine, and was usually accepted
`as a working
`hypothesis
`by t,hose who could not accept
`the older
`t’heory of protein synthesis.
`content of the portal blood, and
`The supposedly
`high ammonia
`the results
`report’ed by Cohnheim4 on the production
`of ammonia
`and volatile bases accompanying
`the disappearance
`of peptone
`and amino-acids
`from
`the digestive
`tract of fishes (when
`isolated
`and suspended
`in blood or salt solutions)
`pointed certainly
`in the
`direction
`of deaminizat.ion.
`The experiments
`of Jacobi5 and of
`Lang6 on
`the deaminization
`of amino-acids
`by
`tissue extracts
`
`lix,
`Chem.,
`paper
`Cohnheim
`
`lxi,
`1909;
`p. 239,
`reports
`only
`
`lxxvi,
`p. 181, 1909;
`the
`finding
`of ammonia
`
`p.
`
`physiol.
`f.
`4 Zeitschr.
`In his
`last
`293, 1912.
`and
`no amino-acids.
`5 Zeitschl.
`j. physiol.
`6 Ho.jmeister’s
`Hritrtige,
`
`p. 149,
`xxx.
`Chem.,
`v, p. 321, 1904.
`
`1900.
`
`002
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`
`Otto
`
`Folin
`
`and W. Denis
`
`143
`
`direct evidence of t,he presence of deaminizing
`to furnish
`seemed
`in the intestine
`and
`liver, a,nd helped
`therefore mat,e-
`ferments
`rially
`to strengthen
`the theory of immediate deaminization
`as the
`characteristic
`feature of intermediary
`protein
`cat!abolism.
`Folin’s
`theory of protein metabolism
`is based on
`the same conception.
`Folin did not commit himself
`to the localiza,t,ion of the deamini-
`zation process
`in the intestine and liver.
`though he believed
`that
`those organs were
`chiefly
`responsible
`for
`the ammonia and urea
`formations.
`to explain
`failed of course
`theory
`The immediate deaminization
`why
`the non-prot,ein
`nitrogen did not increase
`in the blood during
`active absorption.
`The adherents
`of that
`theory had to content
`themselves with
`the “calculations”
`of Bergma.nn and Langstein’
`and others
`that
`the speed of the blood was so grea.t as to remove
`the urea practically
`as fast as it was
`formed.
`In such an appli-
`cation of these calculations
`it is tacit,ly a.ssumed tha.t the excretory
`capacity
`of
`the k’dneys
`is quite as efficient as
`the absorptive
`capacity of the intestine,
`for if such were not, t,he case, the volume
`and speed of the blood stream
`in the mesenteric
`and portal circu-
`lation could not prevent a temporary
`accumdlat,ion
`of t,he absorbed
`digestion products
`in the blood.
`t,hat the deami-
`indicated
`The results
`reported
`in our first paper
`is by no means
`nizing power ascribed
`to the int.estine and liver
`adequate
`to prevent
`the accumulation
`of amino-acids
`in the blood;
`and in our second papers we showed
`that no specialized deamini-
`zation process
`is located
`in the intestine, and that
`t,he ammonia
`in
`the portal blood is very small in amount and represents
`the absorp-
`tion of ammonia produced
`in the
`intestinal
`lumen,
`chiefly by
`putrefactive
`bacteria.
`in so
`two pa,pers are surprising
`in our first
`The results
`reported
`far as they
`failed
`to reveal any urea formation
`at all except when
`ammonia was present
`in the material used
`for absorption.
`This
`result might be regarded as inconsistent
`with
`the fact shown
`by
`ordinary
`feeding experiments
`that
`the urea elimination
`is rapidly
`increased when protein or amino-acids
`are fed to men or to animals.
`The
`inconsistency
`is, however, more a,pparent
`than
`real.
`The
`“rapid
`urea elimination”
`after
`the intake of nit.rogenous
`food
`is
`
`7 Hofmeistey’s
`6 This
`Jownal,
`
`p. 27, 1904.
`vi,
`Beitriige,
`xi, p. 161, 1912.
`
`003
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`
`I44
`
`Urea
`
`Formation
`
`we
`experiments
`in our absorption
`by hours, whereas
`measured
`the experiments
`and did not carry
`were dealing with minutes,
`for the urea dur-
`beyond one hour.
`The negative
`results obtained
`ing the first stages of absorption
`prove
`that
`the urea
`formation
`is not
`localized
`in the
`intestine
`and
`liver
`for
`the purpose
`of
`holding back all amino-acids which are not needed for the rebuild-
`ing of body
`tissues, because
`if such were
`the case the increase
`in
`the urea contents
`of the blood should begin practically
`at once,
`and should be much greater
`than
`the accumulation
`of amino-acid
`nitrogen.
`On the other hand, unless we could show
`that
`the urea
`formation
`does take place later
`there would
`remain a discrepancy
`between our findings and the facts established by ordinary
`feeding
`experiments.
`In this paper we wish
`to report
`results which
`seem
`to clear up in a measure
`the formation
`of urea from amino-acids.
`Before
`taking
`up the consideration
`of experiments
`specifically
`planned
`to throw
`light on the urea formation we wish
`to record a
`few additional
`absorption
`experiments with
`substances which are
`not adapted
`for the study of the urea production.
`
`repre-
`which
`details
`certain
`here
`to state
`we wish
`of brevity
`sake
`the
`For
`paper
`in
`this
`recorded
`the
`experiments
`in all
`followed
`sent
`the
`procedure
`alone,
`ether
`we have
`used
`(1) F or anesthetics
`described.
`unless
`otherwise
`sulphate,
`or
`of morphine
`with
`a subcutaneous
`injection
`or ether
`together
`with
`the substance
`inves-
`(the
`latter
`injected,
`together
`ether
`with
`chloretone
`tigated,
`into
`the
`intestine).
`After
`the
`first
`few minutes
`the ether was admin-
`istered
`by means
`of a tracheal
`cannula.
`(2) As soon
`as
`the
`animals
`were
`unconscious
`we
`laid
`bare
`the
`common
`carotids
`to have
`them
`ready
`when
`wanted
`and
`inserted
`a tracheal
`cannula
`in
`the usual manner.
`(3) The
`first
`sample
`of blood
`was
`then
`taken
`from
`one of
`the
`femoral
`arteries
`by means
`of a 2 or 5 cc. pipette,
`as described
`in
`this
`journal,9
`the
`gracilis
`muscle
`of
`the
`opened
`leg was dissected
`out,
`and
`5 grams
`of
`it was
`immediately
`cut
`fine with
`scissors
`and
`immersed
`in pure methyl
`alcohol.
`(4) The abdomen
`of
`the animal
`was
`then
`opened
`and
`ligatures
`applied
`to
`the
`small
`intestine,
`one
`just
`below
`the
`stomach,
`the
`other
`just
`above
`caecum.
`(6) Unless
`the
`otherwise
`stated
`the
`kidneys
`were
`not
`ligatured
`disturbed.
`(6) The
`or
`50
`substance
`under
`investigation,
`dissolved
`in
`from
`to 100 grams
`of water,
`was
`then
`injected
`into
`the
`intestine
`by means
`of a large
`syringe
`and
`small
`a hypodermic
`needle
`and
`the abdomen
`promptly
`closed
`by means
`of artery
`clips.
`(7) The
`animal
`was
`kept
`warm
`by means
`of an electric
`stove
`below
`the
`holding
`frame.
`(8) The
`alcoholic
`blood
`and
`tissue
`extracts
`were
`pre-
`pared
`as described.lO
`(9)
`The
`analytical
`results
`are
`calculated
`in
`terms
`of
`
`9 This
`lo Ibid.,
`
`xi, p. 527,
`Journal,
`xi, p. 528, 1912.
`
`1912.
`
`004
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`

`
`Otto
`
`Folin
`
`and W. Denis
`
`145
`
`(10)
`out
`
`The difference
`tissue.
`or
`of blood
`per 100 grams
`of nitrogen
`milligrams
`at
`the
`intestine
`by washing
`of nitrogen
`obtained
`the
`amount
`between
`the
`contained
`in
`of nitrogen
`of
`the
`experiment
`and
`the
`amount
`the
`end
`absorbed.
`A small
`product
`is given
`as
`the
`amount
`of substance
`injected
`the
`to keep
`animals
`be
`involved
`in
`this
`procedure,
`but
`in order
`may
`error
`the
`intestine
`before
`as possible
`we did
`not
`want
`to wash
`out
`as normal
`the experiment.
`We have washed
`out
`the
`intestine
`at
`the
`begin-
`beginning
`ning and have
`found
`only
`a few
`(30
`to 40) milligrams
`of nitrogen,
`so the error
`is certainly
`of
`very
`little
`significance.
`(11) Unless
`otherwise
`stated,
`no
`account
`has been
`taken
`of
`the
`absorption
`of water
`from
`the
`intestine
`or of
`urine
`elimination.
`In nearly
`all of our
`experiments
`there
`was
`practically
`no elimination
`of urine
`as the bladder
`remained
`collapsed,
`also
`there
`was very
`little
`water
`absorption,
`for
`the
`intestine
`remained
`full.
`
`ABSORPTION
`
`OF
`
`ASPARAGINE.
`
`feeding,
`Last
`3163 grams).
`(weight,
`1. Cat 41
`EXPERIMENT
`hours before
`the operation.
`Anesthetic,
`ether and
`twenty-four
`After
`ligaturing
`the blood supply of both kidneys
`10
`morphine.
`grams of Kahlbaum’s
`asparagine
`dissolved
`in about 100 cc. of
`water were
`injected
`into the ligatured
`intestine.
`Asparagine
`is not
`very soluble and the absorption was allowed
`to continue
`for
`two
`hours.
`The following
`analytical
`results were obtained.
`
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`
`2154
`. 1054
`43
`
`49
`
`69
`
`52
`
`62
`
`70
`
`83
`
`62
`200
`
`235
`
`.
`
`.
`
`I.
`II.
`
`III.
`
`IV.
`
`V.
`
`VI.
`
`VII.
`
`VIII.
`
`. . . . .
`.
`.
`blood..
`vein,
`
`.
`
`.
`
`.
`
`. .
`. . . . . . _
`twenty-two
`
`vein,
`
`twenty-four
`
`.
`min-
`
`min-
`
`injected..
`nitrogen
`Asparagine
`absorbed..
`nitrogen
`Asparagine
`control
`nitrogen,
`Non-protein
`jugular
`nitrogen,
`Non-protein
`utes
`afterinjection...............................
`Non-protein
`nit.rogen,
`portal
`utesaftertheinjection...........................
`artery,
`Non-protein
`nitrogen,
`carotid
`.
`minutes
`after
`the
`injection..
`artery,
`Non-protein
`nitrogen,
`carotid
`injection..
`the
`and
`twenty
`minutes
`after
`vein,
`one
`Non-protein
`nitrogen,
`portal
`.
`the
`injection..
`twenty-three
`minutes
`after
`one hundred
`Non-prot,ein
`nitrogen,
`mesenteric
`vein,
`injection.
`.
`and
`twenty-nine
`minutes
`after
`the
`Non-protein
`nitrogen,
`inferior
`vena
`cava,
`one hundred
`and
`thirty-two
`minutes
`after
`the
`injection..
`injection
`Non-protein
`nitrogen
`in muscle
`before
`the
`Non-protein
`nitrogen
`in muscle
`one
`hundred
`and
`thirty-seven
`minutes
`after
`the
`injection..
`
`forty-seven
`
`one
`
`hundred
`
`hundred
`
`and
`
`005
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`
`146
`
`Urea
`
`Formation
`
`to make urea
`it is useless
`injections
`the case of asparagine
`In
`determinations
`because
`the imide group
`is not sufficiently
`stable
`to resist
`the treat*ment employed
`for decomposing
`urea.
`The figures
`recorded
`in the above experiments
`are not entirely
`satisfactory.
`There is an unmistakable
`increase
`in the non-protein
`nitrogen of the blood and in the muscle extract.
`Quantitatively
`the figures are perhaps
`rather
`smaller
`than
`they should be on the
`basis of the asparagine absorption.
`The explanationof
`thenitrogen
`deficit
`is the fact
`that asparagine
`is precipitated
`together with
`the
`albuminous materials by the methyl alcohol used as a precipitant.
`The same difficulty
`is met with even with
`such a soluble product
`as urea, unless
`the precipitated
`protein materials
`are washed with
`alcohol.
`The loss of non-protein
`ment recorded below.
`EXPERIMENT
`30 mgm.
`blood containing
`2. To slaughter-house
`of non-protein
`nitrogen per 100 cc. was added (1) urea, (2) glyco-
`~011, (3) asparagine,
`in the proportion
`of (a) 100, (b) 50 mgm. of
`nitrogen
`per 100 cc. of blood.
`After precipitating
`the blood
`in
`the usual manner
`(but without
`washing
`the coagulum) with nine
`volumes of pure methyl alcohol and a little zinc chloride,
`the fol-
`lowing
`figures were obtained
`from
`the filtrates:
`
`is shown
`
`in the experi-
`
`nitrogen
`
`involved
`
`.
`nitrogen..
`1 a Urea
`nitrogen...............................
`1 b Urea
`2 a Glycocoll
`nitrogen.
`2 b Glycocoll
`nitrogen.
`3 a Asparagine
`nitrogen..
`3 b Asparagine
`nitrogen.
`_____
`
`_.
`
`. .
`
`ADDED
`w7.
`100
`50
`100
`50
`100
`50
`
`RECOVERED
`mg.
`82
`36
`82
`36
`44
`36
`
`to the extent of washing
`procedure
`the analytical
`Ry varying
`fresh portions of methyl
`albuminous material with
`the precipitated
`alcohol
`the urea and glycocoll added to the blood can be recovered
`almost quantitatively,
`but with
`asparagine
`as with
`creatine
`or
`creatinine
`the loss remains
`large.
`Thus when adding 100 mgm. of
`asparagine nitrogen we
`recovered
`only 33, and when adding 50
`we recovered 21.
`
`006
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`
`Otto Folin
`
`and W. Denis
`
`147
`
`the values
`in the case of asparagine,
`In all cases, but particularly
`therefore
`nitrogen of the blood must
`obtained
`for the non-protein
`be regarded as minimum
`values so far as the increase due to the
`absorbed
`substance
`is concerned.
`
`ABSORPTION
`
`OF TYROSINE.
`
`In view of the insolubility and low nitrogen content of tyrosine
`it seemed practically hopeless to try
`to trace its transportation
`from the digestive tract to the blood and tissues by means of nitro-
`gen determinations. The negative results which have heretofore
`been obtained by means of Millon’s reagent’1 also indicate that
`tyrosine is absorbed very slowly indeed. Millon’s reaction is how-
`ever not a particularly delicate one for the detection of tyrosine.
`One part in 10,000 seems to be about the limit for an unmistakable
`test on the basis of that reaction. Afar more delicate color reaction
`for tyrosine is one recently discovered by Folin and Macallum on
`the basis of a certain phosphotungstic phosphomolybdic compound,
`the exact composition of which we do not know, but the practical
`preparation of which is described elsewhere.12 By means of this
`reagent we obtain an unmistakable blue color with solutions con-
`taining 1 part of tyrosine in 1 million parts of water. Here we
`had, therefore, a method by means of which it should be an easy
`matter to trace tyrosine in the body.
`3. Cat 48 (weight, 2530 grams). Last feeding,
`EXPERIMENT
`twenty-four hours before the operation. Anesthetic, ether and
`morphine. Two grams of tyrosine dissolved in about 125 cc. of
`warm 1 per cent sodium carbonate solution was used for the injec-
`tion. More
`tyrosine could not be used for want of a suitable
`solvent. The usual nitrogen determinations were made on the
`blood and on muscle extracts only to determine whether any
`increase in the non-protein nitrogen occurs in the absence of active
`absorption.
`
`in control blood..
`nitrogen
`I. Non-protein
`Ureanitrogen....................................
`II. Non-protein
`nitrogen,
`carotid
`artery,
`minutes after the injection.
`. . .
`Urea nitrogen....................................
`
`This Journal,
`I1 H. C. Bradley:
`xi, p. 10, 1912.
`12 See the next number of this Journal.
`
`.
`
`Milligrams.
`39
`20
`
`eighty-four
`. .
`
`. .
`
`40
`20
`
`007
`
`Harvest Trading Group - Ex. 1120
`
`

`
`148
`
`Urea Formation
`
`III.
`
`IV.
`
`V.
`
`20 cc. of
`tyrosine we evaporated
`for
`test
`the calorimetric
`For
`first about
`in the usual manner, and added
`the alcoholic
`filtrates
`1 cc. of the phosphotungstic-molybdenum
`reagent, 2 or 3 cc. of
`water, and then solid sodium carbonate
`in excess (as much as would
`dissolve).
`The control
`extracts
`of blood and muscle gave an
`extremely
`faint
`reaction, while all the other
`tests both in the blood
`and in the second muscle extract were so strong as to uny istak-
`ably prove
`the presence of the tyrosine.
`It seemed hardly worth
`while
`to make
`the test quantitative
`because of the small amounts
`involved,
`but
`the reaction
`is decided enough
`to be available
`for
`quantitative
`purposes.
`
`ABSORPTION
`
`OF CREATININE.
`
`to the albumi-
`like asparagine adheres so tenaciously
`Creatinine
`nous precipitate
`produced
`on adding alcohol
`to blood
`that all of
`it cannot be recovered.
`The absorption
`and general distribution
`of creatinine
`is however
`easily demonstrated
`by means of the non-
`protein nitrogen determinations.
`The calorimetric
`estimation
`of
`creatinine
`in the alcoholic blood and tissue extracts proved at first
`rather
`puzzling.
`Although
`our nitrogen
`determinations
`showed
`that our extracts
`contained enough of the absorbed
`creatinine
`to
`make a calorimetric
`determination
`feasible, we failed to get even a
`sure qualitative
`test with
`the alkaline pierate
`reagent.
`The nega-
`tive results could have been due to the transformation
`of creatinine
`into creatine, although
`this seemed unlikely
`from what we know
`concerning
`the metabolism
`of these
`two products.
`Roiling
`the
`
`Milligrams.
`and
`
`one hundred
`.
`
`one
`vein,
`the
`injection..
`
`hundred
`
`and
`
`artery,
`carotid
`nitrogen,
`Non-protein
`the
`injection.
`minutes
`after
`eighty
`nitrogen.....................................
`Urea
`nitrogen,
`portal
`Non-protein
`minutes
`after
`eighty-three
`Ureanitrogen.....................................
`Non-protein
`nitrogen,
`mesenteric
`and
`eighty-six
`minutes
`after
`Urea
`nitrogen.....................................
`Non-protein
`nitrogen
`in muscle
`Ureanitrogen.....................................
`Non-protein
`nitrogen
`in muscle
`minutes
`after
`the
`injection..
`Urea
`nitrogen.....................................
`
`one
`vein,
`injection..
`
`hundred
`
`the
`
`before
`
`the
`
`injection
`
`one hundred
`
`and ninety
`
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`
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`
` by guest on October 19, 2015
`
`39
`19
`
`39
`20
`
`39
`19
`180
`26
`
`180
`26
`
`008
`
`Harvest Trading Group - Ex. 1120
`
`

`
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`
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`
` by guest on October 19, 2015
`
`Otto Folin and W. Denis
`
`‘49
`
`failed
`residues
`the dry
`acid or heating
`extracts with hydrochloric
`The
`true explanation
`however
`to yield
`the creatinine
`reaction.
`was
`found on adding creatinine directly
`to alcoholic blood extracts.
`The negative
`results obtained are due to the presence
`insuch extracts
`of some inhibiting
`substance.
`Fortunately
`that substance
`is solu-
`ble in ether and in chloroform.
`The fact
`that muscle and blood
`contain substances which
`interfere with
`the calorimetric
`determina-
`tion of creatinine doubtless
`invalidates
`at least some of the creatine
`and creatinine
`determinations
`recorded
`in
`the
`literature.
`The
`creatinine determinations
`of Noel PatonI
`in goose flesh, for exam-
`ple, were made under conditions which precluded his finding any
`creatinine which might have been present
`in his muscle extracts.
`To test
`for creatinine or to determine
`it we have therefore worked
`out
`the
`following
`procedure:
`To
`the alcoholic
`blood or
`tissue
`extract
`in an evaporating
`dish add 0.1 or 0.2 gram of picric acid
`Wash
`the
`(in methyl alcohol solution)
`and evaporate
`to dryness.
`residue several
`times with
`chloroform
`saturated with
`picric acid
`Then add a little water
`and,
`finally, once with pure chloroform.
`and alkali
`in the usual manner and make up to a suitable volume
`(usually 25 or 50 cc.).
`
`creatinine
`pure
`determinations
`As a standard
`potassium
`the
`half-normal
`rather
`more
`are
`by Folin.
`Creatinine
`keeps
`solutions
`originally
`solutions,
`and
`since
`creatinine
`acid
`in
`tenth-normal
`the
`chief
`reason
`for
`using
`condition,
`easily
`be prepared
`One
`gram
`of
`creatinine
`longer
`holds.
`mate
`as a standard
`acid
`can be used
`for
`several
`hundred
`in a liter
`of
`tenth-normal
`tions
`by
`taking
`1 cc. and making
`the
`reaction
`in a 50 cc. volumetric
`
`for such creatinine
`satisfactory
`than
`recommended
`hydrochloric
`in pure
`no
`
`solutions
`bichromate
`indefinitely
`can now
`bichro-
`the
`dissolved
`determina
`flask.
`
`4. Cat 42 (weight, 2353 grams). Three days after
`EXPERIMENT
`the last feeding the animal was anesthetized (ether and morphine),
`the right kidney was removed, the ureter of the left kidney was
`ligatured, and 6 grams of creatinine together with about 100 of
`water were introduced into the ligatured intestine.
`
`Creatinine
`Creatinine
`Non-protein
`Urea
`
`injected..
`nitrogen
`absorbed
`nitrogen
`in control
`nitrogen
`nitrogen.......................................l$
`
`I.
`
`in eighty
`blood
`
`minutes.
`
`Milligrams.
`2172
`822
`31
`
`I3 Joum.
`
`of Physiol.
`
`xxxix.
`
`p. 488, 1010.
`
`009
`
`Harvest Trading Group - Ex. 1120
`
`

`
`150
`
`Urea
`
`Formation
`
`artery,
`
`nine minutes
`
`Milligranzs.
`
`II
`
`III.
`
`IV.
`
`V.
`
`VI.
`
`VII.
`
`this
`The calorimetric creatinine determinations shattered in
`experiment
`for reasons which we have already described. We
`had kept some of the samples of blood, however, and later made
`calorimetric determinations on blood samples IV, V, and VI.
`The calorimetric values obtained gave us 51, 44 and 70 mgm.,
`respectively, per 100 cc. of blood for the creatinine nitrogen. The
`corresponding direct nitrogen determinations, after subtracting the
`normal value 31, are 65, 47 and 73.
`5. Cat 44 (weight, 2743 grams). For two weeks
`EXPERIMENT
`prior to the experiment this animal had been kept on a low nitrogen
`diet consisting of rice (25 grams) and cream (50 cc., 15 per cent
`fat). For anesthetic we used ether and morphine. Before inject-
`ing the creatinine solution the bladder was emptied by means of
`a syringe.
`
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`
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`
` by guest on October 19, 2015
`
`t3l
`18
`
`79
`18
`
`91
`17
`
`78
`17
`
`104
`17
`
`91
`18
`216
`26
`
`250
`26
`147
`19
`
`181
`16
`
`carotid
`nitrogen,
`Non-protein
`aftertheinjection................................
`Ureanitrogen....................................
`Non-protein
`nitrogen,
`portal
`aftertheinjection................................
`Ureanitrogen....................................
`Non-protein
`nitrogen,
`portal
`aftertheinjection................................
`Ureanitrogen....................................
`Non-protein
`nitrogen,
`carotid
`utes
`after
`the
`injection..
`Ureanitrogen....................................
`vein,
`Non-protein,
`mesenteric
`after
`the
`injection................................
`Ureanitrogen....................................
`Non-protein
`nitrogen,
`carotid
`.
`after
`the
`injection.
`Ureanitrogen....................................
`Non-protein
`nitrogen
`in muscle
`Urea
`nitrogen..
`.
`Non-protein
`nitrogen
`in muscle
`theinjection.....................................
`Ureanitrogen....................................
`Non-protein
`nitrogen
`in kidney
`Ureanitrogen....................................
`Non-protein
`nitrogen
`in kidney
`theinjection.....................................
`Ureanitrogen....................................
`
`vein,
`
`fourteen
`
`minutes
`
`vein,
`
`fifty-one
`
`minutes
`
`.
`
`artery,
`
`fifty-four
`
`min-
`
`sixty-seven
`
`minutes
`
`artery,
`
`eighty
`
`minutes
`.
`
`before
`
`the
`
`injection.
`
`eighty
`
`minutes
`
`after
`
`before
`
`the
`
`injection.
`
`eighty
`
`minutes
`
`after
`
`.
`
`010
`
`Harvest Trading Group - Ex. 1120
`
`

`
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`
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`
` by guest on October 19, 2015
`
`Otto Folin and W. Denis
`
`151
`
`Milligrams.
`.
`2172
`and
`
`922
`30
`20
`
`45
`21
`
`.
`
`12
`
`(15)
`
`blood.
`.
`one hundred
`vein,
`the
`injection.
`
`determina-
`
`vein,
`mesenteric
`minutes
`
`one hun-
`after
`the
`
`56
`21
`
`.
`
`.
`
`colosimetric
`
`determina-
`
`vein,
`after
`
`hundred
`one
`the
`injection.
`
`calorimetric
`
`determina-
`
`artery,
`iliac
`after
`minutes
`calorimetric
`
`one hundred
`the
`injeCtion.
`determina-
`
`before
`
`the
`
`injec-
`
`25
`
`(26)
`
`(22)
`
`42
`21
`
`9
`
`45
`
`12
`
`(15)
`
`.
`iniected..
`nitrogen
`Creatinine
`in one hundred
`absorbed
`nitrogen
`Creatinine
`minutes..
`. . . .
`twenty-three
`nitrogen
`in control
`Non-protein
`Urea
`nitrogen..
`portal
`Non-protein
`nitrogen,
`after
`and
`twenty
`minutes
`Urea
`nitrogen...............................
`Creatinine
`nitrogen,
`calorimetric
`tion.........................................
`Non-protein
`nitrogen,
`and
`dred
`twenty-three
`injection....................................
`Urea
`nitrogen..
`Creatinine
`nitrogen,
`tion.......................................
`iliac
`Non-protein
`nitrogen,
`and
`twenty-five
`minutes
`Ureanitrogen...............................
`Creatinine
`nitrogen,
`tion.......................................
`Non-protein
`nitrogen,
`and
`twenty-seven
`Creatinine
`nitrogen,
`tion.......................................
`Non-protein
`nitrogen
`in muscle
`tion.......................................
`Urea
`nitrogen...............................
`test.
`Creatinine
`nitrogen,
`calorimetric
`Non-protein
`nitrogen
`in muscle
`one hundred
`twenty-seven
`minutes
`after
`the
`injection.
`Ureanitrogen...............................
`Creatinine
`nitrogen,
`tion.......................................
`
`I.
`
`II.
`
`III.
`
`IV.
`
`V.
`
`130
`16
`trace
`
`180
`16
`
`and
`
`.
`
`54
`
`(50)
`
`calorimetric
`
`determina-
`
`in Experi-
`recorded
`in the results
`difference
`The most striking
`ments 4 and 5 is the different distribution
`of the absorbed
`creat-
`inine.
`In
`the former
`there
`is a greater accumulation
`in the blood,
`and in the latter
`the muscles have taken up a greater proportion.
`It
`is not possible at
`the present
`time
`to definitely
`explainthis
`variation.
`The
`two experiments
`are not strictly
`similar.
`The
`absorption
`periods are
`longer
`in Experiment
`5, and it
`is quite
`likely
`that
`the speed of the absorption
`diminishes
`as the absorbed
`product accumulates
`in the blood, and that
`the muscles
`therefore
`
`011
`
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`
`

`
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`
` by guest on October 19, 2015
`
`I.52
`
`Urea
`
`Formation
`
`Cat 42 had been fed on a
`Further,
`absorb a larger proportion.
`to the fasting, while Cat 44 (Experi-
`protein-rich
`diet preliminary
`ment 5) had been kept on a low nitrogen
`diet which,
`especially
`in a naturally
`high protein
`animal
`like
`the cat, might
`leave the
`muscles unusually
`low
`in non-protein
`nitrogen
`and
`thereby
`lead
`to a greater avidity
`for
`the absorbed products.
`The non-protein
`nitrogen per 100 grams of control muscle
`in Cat 42 is 214 mgm.,
`as against only 130 mgm.
`in Cat 44.
`In Cat 42 the elimination
`of urine had been prevented,
`but
`this has
`little
`to do with
`the
`result,
`for in the bladder of Cat 44 we obtained only a small quan-
`tity of urine secreted during
`the experiment and it contained only
`a few milligrams of creatinine.
`During
`the last few years numerous
`interesting
`and suggestive
`papers have appeared on creatine and
`creatinine,
`on the transformation
`of the one into
`the other, and
`vice versa, as well as on their complete destruction within
`the body.
`The results of our absorption
`experiments
`seem to throw
`some new
`light
`from a different
`angle on the problems
`thus
`raised.
`The
`hypothetical
`transformation
`of creatinine
`into creatine
`in the mus-
`cles, as suggested
`by Mellanby,14 or by means of ferments,
`as
`claimed by Gottlieb andStangassinger,15
`isnot
`founded on convinc-
`ing experiments
`and is certainly
`not supported
`by the determjna-
`tions
`recorded above.
`Considering
`the nature of our experiments
`and the somewhat
`preliminary
`character
`of the analytical
`tech-
`nique,
`the calorimetric
`values obtained
`for
`the creatinine
`in the
`blood as well as in the muscle correspond
`surprisingly
`well with
`the
`total
`increase
`in the non-protein
`nitrogen.
`opposite
`in brackets
`To make
`this point clear we have
`inserted
`the creatinine
`nitrogen
`figures
`the corresponding
`figures derived
`from
`the total non-protein
`nitrogen determinations.
`the
`According
`to the experiments
`of Gottlieb and Stangassinger
`tissues of the animal body contain not only
`ferments
`capable of
`transforming
`creatinine
`into creatine but also ferments
`capable of
`decomposing
`creatine and creatinine.
`The figures which we have
`obtained
`for the urea in the above experiments
`are however entirely
`negative
`so far as the
`‘Ldestruction”
`of creatinine
`is concerned.
`And since our results were obtained
`from experiments with
`living
`animals we are inclined
`to insist
`that
`the results obtained by Gott-
`
`14 Journ. of Physiol.,
`I5 Zeitschr.
`f. physiol.
`
`1908.
`p. 447,
`xli,
`Iii, p. 1, 1908;
`Chem.,
`
`lvii, p. 131, 1908.
`
`012
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`
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`
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`
` by guest on October 19, 2015
`
`Otto
`
`Folin
`
`and W. Denis
`
`I53
`
`in their autolysis and perfusion experiments
`lieb and Stangassinger
`have no bearing on the processes of animal metabolism.
`More-
`over,
`the analytical
`results of Gottlieb and Stangassinger
`are more
`or less open to suspicion
`in view of the circumstance
`that
`they did
`not discover and did not take
`into account
`the fact that blood and
`tissues
`contain
`substances
`which
`interfere with
`the calorimetric
`creatinine determinations.
`Their conclusion
`that
`the figures
`they
`obtained
`represented maximum
`values because of other substances
`giving
`the same reaction
`is certainly
`untenable.
`To be sure Van
`Hoogenhuyze
`and Verploegh have verified
`their
`result@
`but Mel-
`lanby on the other hand was unable
`to do so. At all events our
`results
`lend no support
`to the theory
`that
`the animal body contains
`special
`ferments
`for
`the destruction
`of creatinine.
`
`ABSORPTION
`
`OF CREATINE.
`
`In the study of the creatine we have made use of the following
`method:
`
`in a
`contained
`extract
`tissue
`or
`blood
`alcoholic
`the
`To 10 to 20 cc. of
`acid
`dissolved
`in me