003 |-3§l98,'9M2902-0l47$03.00f0
`PEDIATRIC RESEARCH
`Copyright @ I9‘}| International Pediatric Research Foundation. Inc.
`
`|99|
`Vol. 29, N0. 2,
`Printed in l';'..S'./I.
`
`Phenylacetylglutamine May Replace Urea as a
`Vehicle for Waste Nitrogen Excretion‘
`
`Department Q,"Pediatrics. The Johns Hopkmt Um'vei'rity .5‘olrooJ' of.‘.»Ica‘icr'ne. Bot'tr'm0r£, Maryland 21205
`
`SAUL W. BRUSILUW
`
`the amino
`ABSTRACT. Phenylacetylglutamine (PAC),
`acid acetylation product of phenylacetate (or plienyllJutyr-
`ate 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
`phenylaeetate 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. Treatment
`with relatively high dose phenylacctate or phenylbutyrate
`(0.5—l].6 gfkgfd) resulted in normal daytime levels of glu-
`tamine. These data suggest that PAG may replace urea as
`a waste nitrogen product when phenylbutyrnte is adminis-
`tered at a dose that yields PAG nitrogen excretion equal
`to 40-44% of a low nitrogen intake. (Pediarr Res 29: I47-
`l5{|', 1991)
`
`Abbreviations
`
`PAG, phenylaeetylglutarnine
`
`The physiologic problem faced by a patient with inborn errors
`of urea synthesis is excretion of waste nitrogen,
`."_e_ 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
`rcquirenicnts for urea synthesis and thereby be helpful (especially
`in patients with significant residual ureagenic 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 benzoate.
`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,/Ll)
`and sodium phcnylacctate (0.25 gfkgfd), 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.
`Correspondence and reprint reqtlests: Dr. Saul Brusilow, The Johns Hopkins
`HI"IS]1llfll.F3l'l! 301,600 N. Wolfe Street. Baltimore. MD 2| 205.
`Supported by the National Institutes of Health Grants No. HD l1l34. HD
`16358, and RR 0005?, The [J.S. Food and Drug Administration Grant no. FD-R-
`000l98. The T.!\. and MA. O‘Malley Foundation. and the Kettering Family
`Foundation.
`' Presented in part at the Annual Meeting of the American Pediatric Society.
`Washington. D.C.. May 2-5. I939.
`
`hippuratc nitrogen and,/or PAG 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 estimate the requirement for
`hippurate and/or PAC} nitrogen synthesis and excretion,
`it
`is
`necessary to know urine 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/'d) of 6.5-7.5 (40.6 46.9 g of protein/d) 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 adniinistration of 18 g of
`sodium phenylacetate should result in the excretion of 3.23 g of
`PAG nitrogen, an amount that would completely replace 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) receiving a diet
`of0.2 gfkg/d of nitrogen/d { 1.25 gfkg/cl of protein) excrete 0.094
`g of urea nitrogenfkg/d, 4?'% of dietary nitrogen. To excrete
`0.094 g/kg/d of PAG nitrogen would require 0.524 gfkgxd of
`sodium phenylacetate. This represents a 36% improvement in
`nitrogen excretion as compared to the combination of sodium
`benzoate and sodium phcnylacctatc, each at a dose of 0.25 gfkgf
`d, which would result in the excretion of 0.069 g/kgfd of nitrogen
`(0.025 g as hippuratc nitrogen and 0.045 g as PAG nitrogen}.
`These theoretical considerations suggest that, on a molar basis,
`phenylacetate (mol wt. 158) is twice as ctfcetive as benzoatc
`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
`lrom phenylbutyric acid,
`secreted as a defensive weapon by the stinkpot turtle (8)]. There-
`fore, sodium phenylbutyrate (mol wt, 186), which is known to
`be ,6-UXiCli7.3d in vivo to phenylacetate (9), may serve as 21 pro-
`drug for phenylacetate.
`
`MATERIALS AND METHODS
`
`Three studies were performed. In the first, the stoiehiometry
`l)etween oral sodium phenylacetate or sodium phenylbutyrate
`administration and PAG excretion was studied in a 7‘/2-yr-old,
`27.2-kg boy with carbamyl phosphate synthctasc 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. 1012
`
`LUPIN
`
`EX. 1012
`
`

`
`148
`
`BRUSILOW
`
`Table 1. Urinary excretion ofPAG during three 3—d periods during which 7'/2—y—otd boy with carbamyi phosphate syntherase
`deficiency was treated with sodium .sat’t.r ofphenyiacetate and phenyibtttyrate {g;'3 d)
`Period l
`Period 1]
`Period III
`
`gt 3 d
`Predicted FAG excretion (mmol)
`Measured FAG excretion (mmol)
`Measured FAG X 100
`Predicted PAG
`r=A(;-N X I00‘
`Dietary N
`* Also shown is a calculation of the percentage of dietary nitrogen excreted as PAL} nitrogen (Pr-'tG—N}. _
`
`__
`
`Na phenylacetatc
`30
`190
`15"!
`83%
`
`38.1%
`
`Na phcnyibutyrate
`36
`I93
`I74
`90%
`
`42%
`
`Na phenylbutyrate
`42
`225
`181
`80%
`
`44%
`
`Table 2. Partition of urinary nitrogen in patient
`described in Tab.-’e I
`Period I
`Period 11
`(3 d)
`(3 d}
`
`Period Ill
`(3 (1)
`
`Total N (g)
`Urea N (g)
`NH: N (g)
`
`8.96
`1.05
`0.36
`
`9.6?
`135
`0.30
`
`9.39
`0.94
`0.29
`
`phenylacetate, 12 g (64.5 mmol) of sodium phenylbutyratc, and
`14 g (75.2 mmol) of sodium phenylbutyrate. His daily diet during
`the three periods consisted of It g of natural protein, 1 1 g of an
`essential amino acid mixture (nitrogen density 12%), and 4.5 g
`(25.'t' 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
`phenylacetate, phenylbutyrate, and PAG were measured in pa-
`tients with various urea cycle disorders receiving varying dosages
`of sodium phenylbutyrate.
`In the third study, the diurnal variation in plasma levels of
`glutaminc, phenylacetate, phenylbutyratc, PAG, and ammo-
`nium was studied in five patients with deficiencies of carbamyl
`phosphate synthetasc or ornithine transearbamylase, four of
`whom were treated with phenylaccate or phenyibutyratc.
`Plasma levels of phenylacetatc and PAG were measured by
`reverse phase HPLJC (Waters, Milford, MA) after precipitation
`with methanol. The technique includes isocratic elution using
`the mobile phase of 0.005 M phosphoric acid in 10% methanol
`at a [low rate of 1.2 mL/min with spectrophotometric detection
`at 2 18 nm. Urine levels were similarly measured after appropriate
`dilution. The detection limits in plasma and urine for phenyl-
`
`butyratc, phenylacetate, and PAC: were 0.05, 0.03, and 0.02
`1‘nmol;’L, respectively.
`Phcnylbutyrate 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 phenylacetyl chloride and glu-
`tarnine ( I0}. Plasma amino acids were measured by automated
`colu Inn chromatography (tnodcl 6300; Beckman, Palo Alto, CA).
`Urinary creatinine was measured by the Jaffc reaction after
`absorption and elution from Lloyds reagent (11). Plasma am-
`monium was measured by visible spectrophotometry using the
`indophenol reaction after separatiott of the ammonium ion by a
`batch cation exchange technique (12). Urine glucuronides were
`measured using the naptltaresorcinol reagent (13). 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 stoichiomctry between phenylacetate or
`phenylhutyratc administration and urinary excretion of PAG.
`The amount of PAG cxcrctod was a function of phenylaoetate
`or phenylbutyrate dose; between 80 and 90% of the predicted
`amount of PAG synthesized is excreted. That these may be
`minimum excretion values is suggested by the coefiicient 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. Phenylacetate, phenylbutyrate, 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. Overntghtfasting piasma ieveis ofpnenyibatyrate, phenytacetare. and FAG in 10 patients receiving various doses of
`sodium phenyibiayrate*
`
`Enzyme
`deficiency
`OTC
`A8
`A3
`CPS
`OTC
`OTC
`OTC
`OTC
`CPS
`OTC
`
`Age
`(y)
`I3
`5
`4
`9
`8
`8
`2
`2
`I
`7
`
`Sex
`F
`M
`M
`M
`F
`F
`M
`M
`M
`M
`
`Protein intake
`(gfkg/d)
`I .0
`I .2
`1.5
`0.91
`[.0
`[.0
`[.01
`1.141
`1.01
`1.31
`
`Phenylbutyrate
`(gfkg./d)
`0.306
`0420
`0.440
`0.490
`0.5 30
`0.530
`0.565
`0.590
`0.600
`0.650
`
`Plasmd immol}
`EA
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`0.‘t'5
`ND
`
`FAG
`0.42
`0.04
`0.26
`009
`0.06
`019
`0.09
`0.08
`0.29
`0.10
`
`1253
`ND
`ND
`[.21
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`
`“eB_. plienylbutyrate: 95A, phcnylaeetatc; OTC, ornithine transcarbamylase; AS, argininosuccinic acid synthetase; CPS, earbamyl phosphate
`synthetase; ND. not detectable.
`1 Protein intake consisted of approximately equal amounts of natural protein and an essential amino acid mixtune.
`
`

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`Pl'lENYLACl:L'l'YL(_iLUTAMINE REPLACES UREA
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`Time (hl after Fasting Glutamine (El
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`1. Plasma glutamine levels exprcsfl as a percent of overnight
`Fig.
`fasting level {ll 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 transcarbamylase; CPS,
`carbamyl phosphate synthctasc; Nafifit, sodium phcnylacctatc; Na¢B,
`sodium phenylbutyrare.
`
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`CPS
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`(JTC
`OTC
`UTC
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`Age (y)
`4
`3’
`7
`9
`12
`48
`
`Sex
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`F
`M
`M
`F
`F
`F
`
`Treatment
`
`_
`
`(g/kg)
`Naas, 0.49:)
`Natal}, 0.565
`Naofl, 0.600
`NaoI\, 0. 500
`None
`None
`
`nitrogen, and ammonium nitrogen in each of the 3-d periods,
`during which he received between [1 and 12 g ofdietary nitrogen
`and 13.5 g('l'!.l mmol) of eitrulline.
`To evaluate whether phenylaoetate, phenylbutyratc, or PAG
`accumulate, overnight fasting plasma levels were measured in 10
`patients receiving oral sodium phenylbutyrate at doses varying
`from 0.306 to 0.65 g,/kg/d (Table 3). With only two exceptions,
`overnight fasting plasma levels of phenylbutyrate and phenylac—
`etate were below the limits ofdetectability. 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 transcarbamylase.
`Plasma glutaminc lcvcls returned to normal during the day in
`each treated patient regardless of the overnight fasting glutamine
`levels, which were 0.67, 1.31, 1.26, and l.20 mmol/‘L (normal,
`0.596 i 0.66 mmol,"I.). The plasma glutamine level remained
`unchanged at high levels (>1 mmol/L) in the two untreated
`patients.
`In patients receiving sodium phcnylbutyrate, the mean (it
`SD) diurnal plasma levels of phenylacetatc, phenylbutyrate, and
`PAG {excluding overnight fasting values described earlier) were
`0.37 i 0.3, 0.17 i 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 phcnylacctatc 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 mrnolfI., re-
`spectively. Throughout the day, the mean plasma ammonium
`level For the four treated patients was 25.5 1 3.3 urnoly’L, range
`20-34 (upper limit ofnormal, -<30).
`
`as shown in Table [ demonstrates both that phenylbutyrite
`appears to be completely oxidized to phenylacetate and that
`phenylacetate is completely, or nearly so, conjugated with glu-
`tamine.
`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 PAC} nitrogen
`excretion and dietary nitrogen. At doses of sodium phenylbutyr—
`ate of'0.44l and 0.515 g/kg/d, 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 PAC 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-d period varied
`from 0.94 g (33 mmol urca) to 1.715 g (62.5 mmol urea), all of
`which can be accounted for by the normal metabolic fate of the
`supplementary dietary citrullinc in each period (711 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 glutaminc levels increase
`before the onset of symptomatic hyperamrnonemia (1?"). Figure
`1 suggests that phenylacetate 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 significant accumulation of
`drugs or their reaction products.
`Our data support the hypothesis that high doses of phenyIac-
`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
`syntliesis, which will increase or decrease in proportion to nitro-
`gen intake, PAG nitrogen synthesis is a function of the 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 (tag. 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 phenylacetate or phcnylbutyrate. For example,
`a nutritionally stable 6—y—old boy with ornithinc lranscarbamyl-
`ase deficiency receiving an essential amino acid diet developed
`alopecia, periorbita] edema, and hypoproteinemia shordy alter
`phenylbutyrate was substituted for benzoate [unpublished obser-
`vations). His nutritional deficiencies promptly resolvcd when
`protein was added to his diet.
`Whether phenylacctatc or phcnylbutyratc 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 glutaminc conjugation { 18, 19],
`pheny|aoe1ylCoA ligase and acyl-CoA:L-gl utaminc N—acyl—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.
`
`Acknowlcdgmrmfr. 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.
`
`DISCUSSION
`
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`[2,
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`
`14.
`
`IS.
`
`I6.
`
`1'}.
`
`l3.
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`I9.