0031-3998/91/2902-0147$03.00/0
`PEDIATRIC RESEARCH
`1991 International Pediatric Research Foundation, Inc.
`Copyright
`
`VoL 29, No.2, 1991
`Primed in U.S.A.
`
`Phenylacetylglutamine May Replace Urea as a
`Vehicle for Waste Nitrogen Excretion1
`
`Department of Pediatrics, The Johns Hopkins University School of Medicine. Baltimore, Mary/and 21205
`
`SAUL W. BRUS!LOW
`
`ABSTRACT. Phenylacetylglutamine (PAG), the amino
`acid acetylation product of phenylacetate (or phenylbutyr(cid:173)
`ate after /:1-oxidation) was evaluated as a waste nitrogen
`product in patients with inborn errors of urea synthesis. A
`boy with .carbamyl phosphate synthetase deficiency receiv(cid:173)
`ing a low nitrogen intake excreted 80-90% of administered
`phenylacetate or phenylbutyrate as PAG. 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
`dietaty nitrogen. With few exceptions, neither phenylace(cid:173)
`tate nor phenylbutyrate accumulated in plasma. Treatment
`with relatively high dose phenylacetate or phenylbutyrate
`(0.5-0.6 g/kgfd) resulted in normal daytime levels of glu(cid:173)
`tamine. These data suggest that PAG may replace urea as
`a waste nitrogen product when phenylbutyrate is adminis(cid:173)
`tered at a dose that yields PAG nitrogen excretion equal
`to 40-44% of a low nitrogen intake. (Pediatr Res 29: 147-
`150, 1991)
`
`Abbreviations
`PAG, phenylacetylglutamine
`
`The physiologic problem faced by a patient with inborn errors
`of urea synthesis is excretion of waste nitrogen, i.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
`requirements 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 nitrogen-containing 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 of amino 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 g/kg/d)
`and sodium phenylacetate (0.25 g/kg/d), 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 ll, 1990: accepted September 19, 1990.
`Correspondence and reprint requests: Dr. Saul Brusilow, The Johns Hopkins
`Hospital, Park 301,600 N, Wolfe Street, Baltimore, MD 21205.
`Supported by the National Institutes of Health Grants No. HD ll 134, HD
`26358, and RR 00052, The U.S. Food and Drug Administration Grant no. FD-R-
`000198, The T.A. and M.A. 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, 1989.
`
`hippurate 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 PAG 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 ± 0.3 g/d 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
`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
`of 0.2 g/kg/ d of nitrogen/ d ( 1.25 g/kg/d of protein) excrete 0.094
`g of urea nitrogen/kg/d, 47% of dietary nitrogen. To excrete
`0.094 gjkg/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 phenylacetate, each at a dose of0.25 g/kg/
`d, which would result in the excretion of0.069 g/kg/d of nitrogen
`(0.025 gas hippurate nitrogen and 0.045 gas PAG nitrogen).
`These theoretical considerations suggest that, on a molar basis,
`phenylacetate (mol wt, 158) is twice as effective as benzoate
`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(cid:173)
`fore, sodium phenylbutyrate (mol wt, 186), wnich is known to
`be 1'3-oxidized in vivo to phenylacetate (9), may serve as a pro(cid:173)
`drug for phenylacetate.
`
`MATERIALS AND METHODS
`Three studies were performed. In the first, the stoichiometry
`between oral sodium phenylacetate or sodium phenylbutyrate
`administration and PAG excretion was studied in a 7 112-yr-old,
`27.2-kg boy with carbamyl phosphate synthetase deficiency.
`During three 3-d periods (each separated by a 24-h transition
`period), he respectively received 10 g (63.3 mmol) of sodium
`147
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`

`148
`Table 1. Urinary excretion of PAG during three 3-d periods
`was treated with sodium salts
`Period I
`
`BRUSILOW
`
`Period II
`
`Period III
`
`Na
`
`g/3 d
`Predicted PAG excretion (mmol)
`Measured PAG excretion (mmol)
`Measured PAG x 100
`Predicted PAG
`PAG-N X 100*
`D1etary N
`*Also shown is a calculation of the percentage of dietary nitrogen excreted as PAG nitrogen (PAG-N).
`
`30
`190
`!57
`83%
`
`38.1%
`
`36
`193
`174
`90%
`
`42%
`
`42
`225
`181
`80%
`
`44%
`
`Table 2. Partition of urinary nitrogen in patient
`described in Table 1
`Period II
`Period I
`(3d)
`(3d)
`9.67
`8.96
`1.75
`1.05
`0.30
`0.36
`
`Period III
`(3d)
`9.89
`0.94
`0.29
`
`Total N (g)
`Urea N (g)
`NH4+N
`
`phenylacetate, 12 g (64.5 mmol) of sodium phenylbutyrate, and
`14 g (75.2 mmol) of sodium phenylbutyrate. His daily diet during
`the three periods consisted of 11 g of natural protein, 11 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(cid:173)
`lated to be 3.84 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 P AG were measured in pa(cid:173)
`tients with various urea cycle disorders receiving varying dosages
`of sodium phenylbutyrate.
`In the third study, the diurnal variation in plasma levels of
`glutamine, phenylacetate, phenylbutyrate, PAG, and ammo(cid:173)
`nium was studied in five patients with deficiencies of carbamyl
`phosphate synthetase or ornithine transcarbamylase, four of
`whom were treated with phenylaceate or phenylbutyrate.
`Plasma levels of phenylacetate and PAG were measured by
`reverse phase HPLC (Waters, Milford, MA) after precipitation
`with methanol. The technique includes isocratic elution using
`the mobile phase of0.005 M phosphoric acid in 10% methanol
`at a flow rate of 1.2 mL/min with spectrophotometric detection
`at 218 nm. Urine levels were similarly measured after appropriate
`dilution. The detection limits in plasma and urine for phenyl-
`
`Table 3. Overnight fasting plasma levels ofphenylbutyrate,
`sodium
`
`butyrate, phenylacetate, and PAG were 0.05, 0.03, and 0.02
`mmol/L, respectively.
`Phenylbutyrate 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(cid:173)
`tamine ( 1 0). Plasma amino acids were measured by automated
`column chromatography (model6300; Beckman, Palo Alto, CA).
`Urinary creatinine was measured by the Jaffe reaction after
`absorption and elution from Lloyds reagent ( 11 ). Plasma am(cid:173)
`monium was measured by visible spectrophotometry using the
`indophenol reaction after separation of the ammonium ion by a
`batch cation exchange technique (12). Urine glucuronides were
`measured using the naptharesorcinol reagent ( 13). Urinary nitro(cid:173)
`gen was measured by the Kjeldahl method previously described
`(14) and urinary urea and ammonium were measured as de(cid:173)
`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 phenylacetate or
`phenylbutyrate administration and urinary excretion of PAG.
`The amount of PAG excreted was a function of phenylacetate
`or phenylbutyrate dose; between 80 and 90% of the predicted
`amount of P AG synthesized is excreted. That these may be
`minimum excretion values is suggested by the coefficient of
`variation of the creatinine 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
`
`and PAG in 10 patients receiving various doses of
`
`Enzyme
`
`Age
`
`Protein intake
`
`Phenylbutyrate
`
`Plasma (mmol)
`PAG
`IZIA
`IZIB
`Sex
`0.42
`ND
`ND
`0.306
`LO
`13
`OTC
`F
`s
`0.04
`ND
`ND
`0.420
`1.2
`M
`AS
`0.26
`ND
`1.21
`0.440
`1.5
`M
`4
`AS
`0.09
`ND
`ND
`0.490
`0.9t
`M
`9
`CPS
`0.06
`ND
`ND
`0.530
`1.0
`8
`OTC
`F
`0.19
`ND
`ND
`0.530
`1.0
`OTC
`8
`F
`0.09
`ND
`ND
`0.565
`M
`LOt
`2
`OTC
`0.08
`ND
`ND
`0.590
`1.14t
`M
`OTC
`2
`0.29
`0.75
`ND
`0.600
`LOt
`M
`CPS
`I
`0.10
`ND
`ND
`0.650
`M
`7
`OTC
`I
`* 0B, phenylbutyrate; 0A, phenylacetate; OTC, ornithine transcarbamylase; AS, argininosuccinic acid synthetase; CPS, carbamyl phosphate
`synthetase; ND, not detectable.
`t Protein intake consisted of approximately equal amounts of natural protein and an essential amino acid mixture.
`
`Par Pharmaceutical, Inc. Ex. 1022
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`Page 2 of 4
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`

`

`PHENYLACETYLGLUTAMINE REPLACES UREA
`
`149
`
`(j)
`>
`(j)
`_J
`
`,25 ~
`01
`~ 100
`'"'
`(f)
`(I)
`LL
`
`••
`
`••
`
`• •
`
`•
`•
`
`•"o
`
`0
`t;
`
`nO
`
`0
`
`v
`
`t;
`
`v
`
`6
`
`0
`
`0
`
`ct> 0
`v
`ts9 6
`
`t;
`
`0
`
`0
`
`0
`
`75
`
`50
`
`25
`
`~ 0
`
`(J)
`c
`E
`:;
`G
`
`(I)
`
`(I)
`E
`(f)
`(I)
`
`0::
`
`0
`
`0
`
`10
`
`20
`
`30
`
`Time (h) after Fasting Glutamine (~)
`
`l. Plasma glutamine levels expressed as a percent of overnight
`Fig.
`fasting level (IJ, 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 synthetase; NapA, sodium phenylacetate; Na¢B,
`sodium phenylbutyrate.
`
`Fasting
`
`Enzyme
`
`Treatment
`
`0 1.30
`/':, 0.67
`\1 1.26
`<) 1.21
`• 1.03
`• 1.11
`
`OTC
`CPS
`OTC
`OTC
`OTC
`OTC
`
`4
`7
`7
`9
`12
`48
`
`Sex
`
`F
`M
`M
`F
`F
`F
`
`NaoB, 0.490
`NaoB, 0.565
`NaoB, 0.600
`NaoA, 0.500
`None
`None
`
`nitrogen, and ammonium nitrogen in each of the 3-d periods,
`during which he received between 11 and 12 g of dietary nitrogen
`and 13.5 g (77.1 mmol) of citrulline.
`To evaluate whether phenylacetate, phenylbutyrate, 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 gjkg/d (Table 3). With only two exceptions,
`overnight fasting plasma levels of phenylbutyrate and phenylac(cid:173)
`etate were below the limits of detectability. Plasma levels ofPAG
`were below 0.5 mmoljL.
`Figure 1 shows the diurnal variation of plasma glutamine level
`in two untreated females with ornithine transcarbamylase defi(cid:173)
`ciency and four treated patients with a deficiency of either
`carbamyl phosphate synthetase or ornithine transcarbamylase.
`Plasma glutamine levels 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 1.20 mmol/L (normal,
`0.596 ± 0.66 mmol/L). The plasma glutamine level remained
`unchanged at high levels (> 1 mmoljL) in the two untreated
`patients.
`In patients receiving sodium phenylbutyrate, the mean (± 1
`SD) diurnal plasma levels of phenylacetate, phenylbutyrate, and
`PAG (excluding overnight fasting values described earlier) were
`0.37 ± 0.3, 0.17 ± 0.25, and 1.42 ± 0.91 mmol/L, respectively.
`For the patient who received only sodium phenylacetate, the
`mean (±SD) diurnal plasma levels of phenylacetate and PAG
`were (excluding overnight fasting levels) 0.88 ± 0.49 and 0.79 ±
`0.48 mmol/L, respectively. Excluding overnight fasting values,
`the range of plasma levels of phenylacetate, phenylbutyrate, and
`PAG were 0.026-1.87, 0-0.872, and 0.093-3.15 mmol/L, re(cid:173)
`spectively. Throughout the day, the mean plasma ammonium
`level for the four treated patients was 25.5 ± 3.3 p:moljL, range
`20-34 (upper limit of normal, <30).
`
`DISCUSSION
`Examination of the stoichiometry between sodium phenylac(cid:173)
`etate or phenylbutyrate administration and the excretion ofPAG
`
`as shown in Table 1 demonstrates both that phenylbutyrate
`appears to be completely oxidized to phenylacetate and that
`phenylacetate is completely, or nearly so, conjugated with glu(cid:173)
`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 PAG nitrogen
`excretion and dietary nitrogen. At doses of sodium phenylbutyr(cid:173)
`ate of0.441 and 0.515 g/kg/d, 1.62 and 2.88 g/d ofPAG nitrogen
`were excreted representing at least 42 and 44% of dietary nitro(cid:173)
`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-d period varied
`from 0.94 g (33 mmol urea) to 1.75 g (62.5 mmol urea), all of
`which can be accounted for by the normal metabolic fate of the
`supplementary dietary citrulline in each period (77.1 mmol).
`It has been apparent for a number of years that hyperammo(cid:173)
`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 ( 1 7). Figure
`1 suggests that phenylacetate or phenylbutyrate are effective in
`maintaining normal nitrogen homeostasis as manifested by
`maintenance of plasma glutamine levels at normal or near nor(cid:173)
`mal levels during the day without significant accumulation of
`drugs or their reaction products.
`Our data support the hypothesis that high doses of phenylac(cid:173)
`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(cid:173)
`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 (e.g. premature
`or full-term infants and patients on marginal nitrogen intakes) it
`may be possible to induce negative nitrogen balance by admin(cid:173)
`istering high-dose phenylacetate or phenylbutyrate. For example,
`a nutritionally stable 6-y-old boy with ornithine transcarbamyl(cid:173)
`ase deficiency receiving an essential amino acid diet developed
`alopecia, periorbital edema, and hypoproteinemia shortly after
`phenylbutyrate was substituted for benzoate (unpublished obser(cid:173)
`vations). His nutritional deficiencies promptly resolved when
`protein was added to his diet.
`Whether phenylacetate or phenylbutyrate 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),
`phenylacetyl CoA ligase and acyl-CoAL-glutamine N-acyl-trans(cid:173)
`ferase, it is not certain that either organ alone will have the
`requisite activity or, in the case of chronic renal disease, whether
`PAG accumulation may limit the usefulness of these drugs.
`
`Acknowledgments. The author thanks Ellen Gordes and
`Evelyn Bull for their excellent technical assistance and also
`thanks the staff of the Pediatric Clinical Research Center for
`nursing support.
`
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`
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`150
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