003 I-399 8}‘) l/2902-0 l4'i'$03.00/0
`PEDIATRIC RESEARCH
`Copyrigln @ I9‘)! International Pediatric Research Foundation. Inc.
`
`Vol. 29, N0. 2, I99!
`Printed in L-'..S'/I.
`
`Phenylacetylglutamine May Replace Urea as a
`Vehicle for Waste Nitrogen Excretion‘
`
`Department qfPad£arricr. The Johru Ho,nkm.r Umverriry brlicol t:y".'.=lrdrcinr. Baltimore, Maryland 2:205
`
`SAUL W. BRUSILOW
`
`the amino
`ABSTRACT. Phenylacetylglutamine {l‘AG),
`acid acetylation product of phenylacetale (or phenylbutyr-
`are after ,6-oxidation) was evaluated as a waste nitrogen
`product in patients with inborn errors of urea synthesis. A
`boy with carbamyl phosphate synthetase deficiency receiv-
`ing a low nitrogen intake excreted 80-90% of administered
`phenylacetate or phenylbutyrate as FAG. The amount of
`PAG nitrogen excreted varied from 38-44% of his dietary
`nitrogen, similar to the relationship between urea nitrogen
`and dietary nitrogen found in normal subjects receiving low
`dietary nitrogen. With few exceptions, neither phenylace-
`tate nor phenylbutyrate accumulated in plasma. 'I‘reat.ment
`with relatively high dose phenylacctate or phenylbutyrate
`(0.54].6 g/kgfd) resulted in normal daytime levels of glu-
`tamine. These data suggest that FAG may replace urea as
`a waste nitrogen product when phenylbutyrate is adminis-
`tered at a dose that yields PAG nitrogen excretion equal
`to 4044% of a low nitrogen intake. (Pedtarr Res 29: l4'7—
`1 50, l 991 )
`
`Abbreviations
`
`PAG, phenylacetylglutamine
`
`The physiologic problem faced by a patient with inborn errors
`of urea synthesis is excretion of waste nitrogen,
`tie. dietary
`nitrogen not used for net protein synthesis or excreted in other
`ways (stool, skin, etc). Treatment of such patients by modifying
`the quantity and quality of nitrogen intake may reduce the
`requirements for urea synthesis and thereby be helpful (especially
`in patients with significant residual urcagcnic capacity). Dietary
`therapy alone has been unsuccessful in severely affected patients
`(1. 2)-
`That other nitrogemcontaining compounds may substitute for
`urea nitrogen may be adduced from the report by Lewis (3), who
`described a stoichiometric relationship between the decrease in
`urine urea nitrogen and appearance of urine hippurate nitrogen
`in a normal subject given sodium bcnzoatc.
`The use ofamino acid acylation pathways has been successfully
`exploited in empiric studies of patients with inborn errors of urea
`synthesis (4, 5). Treatment with sodium benzoate (0.25 gfkg/L1)
`and sodium phcnylacctatc (0.25 g/kgXd), respectively, activate
`the synthesis and excretion of hippurate and PAG, both of‘which
`may serve as waste nitrogen products. The degree to which
`Received May 11, 1990: accepted September I9, I990.
`Corrcsponrlencc and reprint rnqllesls: Dr. Saul Bruxilnw, The Johns Hopkins
`Hruipilal. Park 30] , 600 N. Wolfe Street. Baltimore. MD 2| 205.
`Supported by the National lnslitulrs of Health Grants No. HD l1l3-l. HD
`26358, and RR 0005?, The [l.S. Food and Drug Administration Grant no. FD-R-
`tltltlltltl, The TA. and MA. O‘MaIIey Foundation. and the Kettering Family
`Foundation.
`‘Pr:-._~:4~.n1cd in pan at the Annual Meeting ofihe American Pediatric Society.
`Washington D.C.. May 2-5. I939.
`
`hippuratc nitrogen and/or FAG nitrogen can substitute for urea
`nitrogen in patients receiving low nitrogen intakes has not been
`studied.
`We propose to examine the hypothesis that PAG nitrogen
`alone can replace urea nitrogen as a vehicle for waste nitrogen
`synthesis and excretion in patients on low protein intakes.
`Theoretical considerations. To cstimatc the requirement for
`hippuratc and/or PAG nitrogen synthesis and excretion,
`it
`is
`necessary to know urinc urea nitrogen excretion in normal
`subjects as a function of dietary nitrogen.
`Although there are many studies of the effect of variations of
`dietary nitrogen intake or urine nitrogen excretion, there are,
`curiously. very few such studies where urine urea nitrogen has
`been measured in normal subjects receiving varying nitrogen
`intakes.
`Calloway and Margan (6) reported that on dietary nitrogen
`intakes (g/cl) of 6.5-7.5 (40.6 46.9 g of protcinfd) normal adult
`males excreted 3.16 i 0.3 gfd of‘ urea nitrogen, approximately
`47% of their dietary nitrogen. Assuming complete conversion to
`its amino acid conjugate,
`the oral administration of 18 g of
`Sodium phenylacetate should result in the excretion of 3.23 g of
`PAC} nitrogen, an anmunt that would completely rcplacc urea
`nitrogen as a vehicle for waste nitrogen excretion in subjects
`receiving low protein intakes.
`There appear to be no studies of normal children receiving
`varying nitrogen intakes in whom urine urea nitrogen excretion
`was measured. However, it is possible to calculate from a report
`of Waterlow (7) that children (6-24 mo of age) rocciving a diet
`of0.2 g/kg/d of nitrogen/d ( 1.25 g/kg/cl ofproiein) excrete 0.094
`g of urea nitrogen/kg/d, 47% of dietary nitrogen. To cxcrctc
`0.094 3/kg/d of PAG nitrogen would require 0.524 g/kg/d of
`sodium phenylacetate. This represents a 36% improvement in
`nitrogen excretion as compared to the combination of sodium
`benzoate and sodium phcnylacctalc, each at a close of 0.25 gfkgf
`d, which would result in the excretion of0.069 g/kg./d of nitrogen
`(0.025 g as hippuratc nitrogen and 0.045 g as PAG nitrogen).
`These theoretical oonsiderations suggest that, on a molar basis,
`phenylacetate (mol wt. 158) is twice as effective as bcnzoatc
`because PAG contains two nitrogen atoms as compared to the
`one nitrogen atom of hippurate. Phenylacetate, however has a
`disadvantage as consequence of its offensive odor [it is one of
`several phenylalkanoic acids, apart from phenylbutyric acid,
`secreted as a defensive weapon by the stinkpot turtle (8)]. There-
`fore, sodium phenylbutyrate (mol wt. 186), which is known to
`be ti-oxidized in viva to phcnylacetate (9), may serve as a pro-
`drug for phenylacetate.
`
`MATERIALS AN D METHODS
`
`Three studies were performed. In the first, the stoichiometry
`between oral sodium phenylacelate or sodium phenylbutyrate
`administration and PAG excretion was studied in a 7‘/2-yr-old,
`27.2-kg boy with carbamyl phosphate synrhctasc deficiency.
`During three 3-d periods (each separated by a 24-h transition
`period), he respectively received £0 g (63.3 mmol) of sodium
`147
`
`LUPIN EX. 1011
`LUPIN EX. 1011
`
`1 of 4
`
`

`

`148
`
`BRUSILOW
`
`Table 1. Urinary excretion ofP/{G during three 3—d periods during which 79‘:-}LoId boy with earbamyl phosphate s}=mheta.s'e
`deficiency was {retried with sodium .S‘t:l.i!.\“ ofphenyiacemte and phenylb_:rryrate {g/3 62')
`Period 1]
`Period III
`Period l
`Na phcnylbmyrare
`Na phcnylbutyrate
`Na phcnylaeetalt:
`36
`42
`I93
`225
`l?4
`l 8 l
`90%
`30%
`
`gr 3 Li
`Predicted FAG excretion (mmol)
`Measured PAC] excretion (mmol)
`Measured FAG X 100
`Predicted FAG
`PAG—N
`_
`Dietary N x I00
`* Also shown is a calculation of the percentage of dietary nitrogen excreted as PAU nitrogen [PAG~N}.
`
`30
`190
`15?
`83%
`38.1%
`
`42%
`
`44%
`
`Table 2. Partition ofttrirtary nitrogen in patient
`described in Tobie]
`Period l
`Period ll
`(3 Cl)
`(3 Cl)
`8.96
`9.67
`L05
`|.I"§
`0.36
`0.30
`
`Period II]
`(3 d)
`9.89
`0.94
`0.29
`
`Total N (g)
`Um-I N (8)
`NHK N (g)
`
`phcnylacclate, 12 g (64.5 mmol) of sodium phenylbutyrate, and
`14 g (75.2 mmol) ofsodium phenylbutyrate. His daily diet during
`the three periods consisted of ll g of natural protein, 1 l g of an
`essential amino acid mixture (nitrogen density 12%), and 4.5 g
`(25.7 mmol) of citrulline. The total nitrogen intake was calcu-
`lated to be 3.34 g, which included 0.4 g of nitrogen in the gelatin
`capsules containing the drugs and the one third of administered
`citrulline nitrogen that enters the free amino acid pool. Total
`urinary nitrogen, urea nitrogen. and ammonium nitrogen were
`measured in each period.
`In the second study, the overnight fasting plasma levels of
`phenylacetatc, phcnylbutyralc, and FAG were measured in pa-
`tients with various urea cycle disorders receiving varying dosages
`of sodium phenylbutyratc.
`In the third study, the diurnal variation in plasma levels of
`glutamine, phenylacetate, phenylhulyratc, PAG, and ammo-
`nium was studied in five patients with deficiencies of carbamyl
`phosphate synthetase or Ornithine trattscarbamylasc, four of
`whom were treated with phenylaeeate or phenylbutyratc.
`Plasma levels Of phenylacetate and PAG were measured by
`reverse phase HPJJC (Waters, Milford, MA) alter precipitation
`with methanol. The technique includes isocrafic clution using
`the mobile phase of 0.005 M phosphoric acid in 10% methanol
`at a [low rate Of 1.2 mL/min with spectrophotomctric detection
`at 2 18 nm. Urine levels were similarly measured after appropriate
`dilution. The detection limits in plasma and urine for phenyl-
`
`butyratc. phcnylncetate, and FAG were 0.05, 0.03, and 0.02
`mmol/L, respectively.
`Phenylbutyrale levels in plasma were also similarly measured
`except for the mobile phase, which consisted Of 0.005 mol/L
`phosphoric acid in 40% methanol. PAG {for use as an external
`standard) was synthesized from phcuylacetyl chloride and glu-
`tamine (I0). Plasma amino acids were measured by automated
`oolu mn chromatography (model 6300; Beckman. Palo Alto, CA).
`Urinary creatinine was measured by the Jaffe reaction after
`absorption and elution from Lloyds reagent (11). Plasma am-
`monium was measured by visible spectrophotometry using the
`indophcnol reaction after separation of the ammonium ion by a
`batch cation exchange technique (12). Urine glucuronides were
`measured using the naptl1anesorcinolreagent(l3). Urinary nitro-
`gen was measured by the Kjeldahl method previously described
`(14) and urinary urea and ammonium were measured as de-
`scribed by Chaney and Marbach (15).
`These studies were approved by The Johns Hopkins Joint
`Committee on Clinical Investigation.
`
`RESULTS
`
`Table 1 compares the stoichiometry between phcnylaeetatc or
`phenylbutyratc administration and urinary excretion of PAG.
`The amount of PAG excreted was a function of phenylacetate
`or phenylbutyratc close; between 80 and 90% of the predicted
`amount of PAG synthesized is excreted. That these may be
`minimum excretion values is suggested by the coefllcient Of
`variation of the creatininc excretion over the 9 d, which was
`14%. Table 1 also demonstrates that when PAG excretion is
`expressed as PAG nitrogen, it accounts for at least 38-44% of
`dietary nitrogen intake. Phertylacetate, phcnylbutyrate, or total
`glucuronide excretion in the urine did not exceed 1% of the
`administered drug in any period.
`Table 2 shows the excretion of total urinary nitrogen, urea
`
`Table 3. Overnight fasting piasma levels afphenylbutyrarte, phenylacetare. and FAG in 10 patiems receiving various doses of
`sodium p!2enylbn.tyrote*
`
`Enzyme
`deficiency
`OTC
`AS
`As
`CPS
`OTC
`OTC
`OTC
`OTC
`CPS
`OTC
`
`Age
`(y)
`13
`5
`4
`
`I
`7
`
`-
`
`Protein intake
`(gfkgfL1)
`I .0
`I .2
`1.5
`0.91
`t .0
`t .0
`l .07
`1 . 141
`1 .01
`1.31
`
`Phenylbutyrate
`(gfkg/Ll)
`0.306
`0.420
`0 .440
`0490
`0.5 30
`0530
`0.565
`0590
`0.600
`0.650
`
`dB
`ND
`ND
`[.2 l
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`
`Pl
`
`l
`'
`asmd (mmo )
`EEA
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`035
`ND
`
`FAG
`0.4-2
`0.04
`0.26
`0.09
`0.06
`0. 19
`0.09
`0.08
`0.29
`0. I0
`
`“.913. phenylbutyrate: wk, phenylacctatc; OTC, ornithine lranscarbamylase; AS, argininosnccinic acid synthelase: CPS, carbarnyl pltospltatc
`synthetase; ND. not detectable.
`1' Protein intake consisted of approximately equal amounts of natural protein and an essential amino acid mixture.
`
`2 of 4
`
`

`

`PH ENYLr\CE'l'YL(_iLUTA MINE REPLACES UREA
`
`£49
`
`as shown in Table [ demonstrates both that phen ylbutymtc
`appears to be Completely oxidized to phenylacetate and that
`phenylacetate is completely, or nearly so, conjugated with glu-
`tarnine.
`That complete conjugation of the drugs occurs may be further
`adduced by the insignificant amount of unchanged drugs or their
`esters in urine and by the lack of accumulation in overnight
`fasting plasma {Table 2).
`Table 1 also shows the relationship between PAG nitrogen
`excretion and dietary nitrogen. At doses of sodium phenylbutyr—
`ate of 0.441 and 0.515 g/kg/cl, 1.62 and 2.38 g/d OFPAG nitrogen
`were excreted representing at least 42 and 44% of dietary nitro-
`gen. When compared to the relationship between urea nitrogen
`excretion in normal adults or children receiving low nitrogen
`intakes, it appears that PAG nitrogen may serve as a replacement
`vehicle for waste nitrogen synthesis and excretion in children
`with little or no ability to synthesize urea.
`That this patient synthesized little or no urea may be inferred
`by comparing urinary urea excretion (Table 2} with citrulline
`intake. Urinary urea nitrogen excretion in each 3~Cl period varied
`from 0.94 g (33 mmol urca) to 1.75 g (62.5 mmol urea), all of
`which can be accounted for by the non-nal metabolic fate of the
`supplementary dietary citmllinc in each period (77.1 mmol).
`It has been apparent for a number of years that hyperammo-
`nemia in patients with inborn errors of urea synthesis is always
`associated with high plasma glutamine levels (1, 16). It also has
`been shown in such patients that plasma glutamine levels increase
`before the onset of symptomatic hyperammonemia (17). Figure
`I suggests that phenylacelate or phenylbutyratc are effective in
`maintaining normal nitrogen homeostasis as manifested by
`maintenance of plasma glutamine levels at normal or near nor-
`mal levels during the day without significztnt accumulation of
`drugs or their reaction products.
`Our data support the hypothesis that high doses ol" phenylao-
`etate or phenylbutyrate will result in the synthesis and excretion
`of PAG nitrogen similar to the amount of urea nitrogen that is
`excreted in normal subjects on a low—protein diet. Unlike urea
`synthesis, which will increase or decrease in proportion to nitro-
`gen intake, PAG nitrogen synthesis is a function ofthe dose of
`phenylacetate or phenylbutyrate. Therefore, the appropriate dose
`will be a function of dietary nitrogen and nitrogen retention.
`Under Circumstances of avid nitrogen retention (sag. premature
`or full-term infants and patients on marginal nitrogen intakes) it
`may be possible to induce negative nitrogen balance by admin-
`istering high-dose phenylaeetate or phenylbutyrate. For example,
`a nutritionally stable 6—y—old boy with ornithinc transcarbamyl-
`ase deficiency receiving an essential amino acid diet developed
`alopocia, periorbita] edema, and hypoproteinernia shortly alier
`phenylbutyratc was substituted for benzoate (unpublished obser-
`vations). His nutritional deficiencies promptly resolved when
`protein was added to his diet.
`Whether phenylacetatc or phcnylbutyrate may be helpful in
`the management of other nitrogen accumulation diseases, such
`as hepatic encephalopathy or chronic renal disease, remains to
`be tested. Although both the liver and the kidney have the
`requisite enzyme activity for glutamine conjugation { 18, 19],
`phenylaoelyl Con ligase and acyl-CoA:l.-gl utamine N—acy1-trans-
`ferase, it is not certain that either organ alone will have the
`requisite activity or, in the case ofchronic renal disease, whether
`PAG accumulation may limit the usefulness of these drugs.
`
`Acknowledgments. The author thanks Ellen Gordcs and
`Evelyn Bull for their excellent
`technical assistance and also
`thanks the staff of the Pediatric Clinical Research Center for
`nursing support.
`
`
`
`
`
`PlasmaGutamine.%FastingLevel
`
`‘I0
`
`20
`
`Time lhl after Fasting Glutamine {E}
`
`1. Plasma glutarnine levels exprcsfl as a percent of overnight
`Fig.
`fasting level (I1 100%) measured at time 0 (between 0730 and 0930) in
`five patients with inborn errors of urea synthesis. Drugs were given in
`three to four divided doses. OTC. ornithine trar-scarbamylase; CPS,
`carbamyl phosphate synthctase; Nan.-X, sodium phcnylacctatc; Na¢B,
`snrlium phcnylbutyrare.
`
`Treatment
`(gfks)
`NanB, 0.490
`Nash, 0.565
`Naoli, 0.600
`NMSA, 0. 500
`None
`None
`
`Sex
`
`F
`M
`
`MFFF
`
`Age (y)
`4
`7
`7
`9
`l 2
`48
`
`Fasting
`glutamine
`O [.30
`A0.67
`V1.26
`01.21
`I L03
`A ].1l
`
`Firwyrtlti
`deficiency
`OTC
`CPS
`OTC
`OTC
`OTC
`UTC
`
`nitrogen, and ammonium nitrogen in each of tire 3—d periods,
`during which he received between [1 and I2 gofdietary nitrogen
`and 13.5 g(T'a'.] mmol) ofcitrullinc.
`To evaluate whether pherrylaoetate, phenylbutyratc, or PAG
`accumulate, overnight fasting plasma levels were measured in 10
`patients receiving oral sodium phenylbutymte at doses varying
`from 0.306 to 0.65 g/kg/d (Table 3). With only two exceptions,
`overnight fasting plasma levels of phenylburyratc and phenylac-
`ctate were below the limits ofdetcctability. Plasma levels of PAG
`were below 0.5 mmol/L.
`Figure 1 shows the diurnal variation of plasma glutamine level
`in two untreated females with ornithine transcarbamylase defi-
`ciency and four treated patients with a deficiency of either
`carbamyl phosphate synthetase or ornithine transcarbamylasc.
`Plasma glutaminc levels returned to normal during the day in
`each treated patient regardless of the overnight fasting glutamine
`levels, which were 0.67, l.3l, 1.26, and L20 mmol/L (normal,
`0.596 t 0.66 mmol/L). The plasma glutamine level remained
`unchanged at high levels (>1 mmol/L) in the two untreated
`patients.
`In patients receiving sodium pherlylhutyrate, the mean (:1
`SD) diurnal plasma levels of phenylacetatc, phenylbutyrate, and
`PAG (excluding overnight fasting values described earlier) were
`0.37 i 0.3, 0.l7 : 0.25, and 1.42 i 0.91 mmol/L, respectively.
`For the patient who received only sodium phenylacetate, the
`mean (iSD) diurnal plasma levels of phenylacctate and PAG
`were (excluding overnight fasting levels) 0.88 1 0.49 and 0.79 i
`0.48 mmol/L, respectively. Excluding overnight fasting values,
`the range of plasma levels of phcnylacctatc, phcnylbutyrate, and
`PAG were 0.026—l.87, 0-0872, and 0.093—3.l5 mmol/L, re-
`spectively. Throughout the day, the mean plasma ammonium
`level For the four treated patients was 25.5 1 3.3 umol/’L, range
`20-34 (upper limit ofnormal, <30).
`
`DISCUSSION
`
`REFERENCES
`
`Examination of the stoichiometry between sodium phenylac-
`etate or phenylbutyrate administration and the excretion of PAG
`
`l, Snih VF, 1976 Hereditary urea cycle disorders. In: Grisolia S, Baguena R,
`Major R (eds) The Urea Cycle. John Wiley. New York, pp 367-414
`
`3 of 4
`
`

`

`150
`
`BRUSILOW
`
`2. Brustiow SW. Bauthaw ML, Wubcr Ll 19'J£i The 115: of kctcracids in inborn
`errors of urea synthesis. In: Winick M (ed) The Nu1I'itinl'ta| Management u!‘
`Genetic Disorders. John Wiley, New York, pp 6'L75
`. bcwi-5 HE [914 Studios in the synthesis of hippuric acid in the animal
`organisms. l[_ The synthesis and rate of ctirninalimi of liippurir: acid after
`benzoatoingestion in man. J Biol Chem I8:22$—-23]
`. Br-usilow SW, Tinker '1", Bat.shnwML I980 Amino acid acy|ation:a mechanism
`of nitrogen t'.xt:1‘:.'titJn in inborn crrors of urea synthesis. Scicnoc 20':':i559A
`661
`. P-rusilow SW. Horwich A [989 Urea cycle enzymes. In: Sc-river C, Bcaudet A,
`Sly W, Valle D Icds) The Metabolic Basis of Inherited Disease, 6:11 Ed.
`Mcfiraw-Hill, New York. pp 629—664
`. Calloway DII, Margan S 19?1 Variation in endogenous nitrogen excretion and
`dietary nitrogen utilimtion as determinants ofhnman protein requirements.
`JNu1r l0|:2D5—2l6
`, Ware-rlow JC I963 The partition of nitrogen in the urine by malttuurished
`Jamaican infants. Am J Clin Nutr l2:235-.2-ID
`. Eisner T. Conner WE. Hicks K. Dodge KR, Rosenberg I-II, Jonu TII, Cohen
`M, Meinwalcl J I977 Stink ofstinkpol turfleideiitiflcd: plicnylalkanoic acids.
`Science l96:I 347- I349
`. Knoopl-7 1905 Der abbau arommischerfettsaurctic1'korper.BeitrChcm Phystot
`Path 6:105-I62
`. Thierfelcler H, Sherwin CP 1915 Phenylacerylgjutamin und seirt Bildung in
`
`II.
`
`ll
`
`13.
`
`14.
`
`merischlichen Korpen nztch Eingabe V011 Phenyicimegsaure Hoppe-Saylnr
`Zcit Physiul Chem 94: I49
`Van Pilsum JF 1959 Determination of creatinine and related gunnidiurn
`compounds. Methods Biochem Anal 'l':l93—2Sl
`Brusitow SW, Batshaw ML, Wat-ser LI 1982 Neonatal hyneramrnoncmiac
`coma, Adv Pediatr 29;69— I 03
`Nir I
`I954 Determination of glncuronit‘: acid by napiltaresorcinol. Anal
`Biochcm 8:20-23
`Brusilow SW, Tinkur J. Balsliaw ML L980 Amino acid acylation: ii mccltanisrn
`of nitrogen excretion in inbnrn errors of urea synthesis. Science 2t]?:IS59-
`66I
`, Chaney AL, Marbacii EP I97] Modified reagents tor determination of urea
`and ammonia. Clin Chem 3:130-I32
`. Levin B I9'Il Hereditary metabolic disorders of the urea cycle. Adv Clin Chem
`I-$65-I63
`. Eatsltaw ML, Walser M, Brusilciw SW I980 Plasma alpha-ketoglutarate in urea
`cycle enzymopathies and its role as a harbinger of hyperzmmunernic Corns.
`Pediatr Res ‘)?:I316—l3t9
`. Webster LT. Siddiqui VA, Lucas SV. Strong IM, Mieynl .IJ t9‘.‘6Iden1il'1cation
`of separate acyl-COA: glycine and acyl-Cofit: Ldglutarnine Nitcyl-transfcmsc
`activities 111 mitochondrial fractions from liver of rhesus monkey and man.
`J Biol Chem 2513352-335H
`. Moldavc K, Meister A 1957 Synthesis of phenyiacctylglutamine by human
`tissue. I Biol Chem 229:463—476
`
`4 of 4
`
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