003l-3998/86/20l H I 17502.00/0
`PEDIAI‘RIC RESEARCH
`Copyright in I986 International Pediatric Research Foundation. Inc.
`
`Vol. 20. No. l I. 1980
`Printed in USA
`
`Waste Nitrogen Excretion Via Amino Acid
`Acylation: Benzoate and Phenylacetate in
`Lysinuric Protein Intolerance
`
`OLLI SIMELL. ILKKA SIPILA, JUKKA RAJANTIE. DAVID L. VALLE. AND
`SAUL W. BRUSILOW
`
`Children is Hospital. Universin och/sinki. SF—00290 Helsinki. Finland: and Deparlmc'nt ochdiatrics. The
`Johns Hopkins Univarsity School ofMedicine. Baltimore. Marv/and 2I205
`
`ABSTRACT. Benzoate and phenylacetate improve prog-
`nosis in inherited urea cyclc enzyme deficiencies by increas-
`ing waste nitrogen excretion as amino acid acylation prod-
`ucts. We studied metabolic changes caused by these sub-
`stances and their pharmacokinetics in a biochemically
`different urea cycle disorder, lysinuric protein intolerance
`(LPI), under strictly standardized induction of hyperam-
`monemia. Five patients with LPI received an intravenous
`infusion of 6.6 mmol/ltg L-alanine alone and separately
`with 2.0 mmol/kg of benzoate or phenylacetate in 90 min.
`Blood for ammonia, serum urea and creatinine, plasma
`benzoate. hippurate, phenylacetate. phenylacetylgluta-
`mine, and amino acids was obtained at 0, 120, 180, and
`270 min. Urine was collected in four consecutive 6-h pe-
`riods. Alanine caused hyperammonemia: maximum in—
`crease l07, 28—411 uM (geometric mean, 95% confidence
`interval); ammonia increments were nearly identical after
`alanine + benzoate (60, 17—213 12M) and alanine + phen-
`ylacetate (79, 13-467 uM) (NS). Mean plasma benzoate
`was 6.0 mM when extrapolated to the end of alanine +
`benzoate infusions; phenylacetate was 4.9 mM at the end
`of alanine + phenylacetate. Transient toxicity (dizziness,
`nausea, vomiting) occurred in four patients at the end of
`combined infusions, and we suggest upper therapeutic
`plasma concentrations of 4.5 mM for benzoate and 3.5
`mM for phenylacetate. Benzoate and phenylacetate then
`decreased following first-order kinetics with tms of 273
`and 254 min, respectively. Maximal plasma hippurate
`(0.24, 0.l4—0.40 mM) was lower than maximal phenylac-
`etylglutamine (0.48, 0.22-l.06 mM, p = 0.008). Orotic
`acid excretion was 5.62, l.84—l7.l4 nmol/kg per h after
`alanine, but only l.07, 0.04—25.62 umoI/kg per h after
`alanine + benzoate (p < 0.l5l ) and 2.74, 0.01-16.25umol/
`kg per h after alanine + phenylacetate (p < 0.0l6). Urea
`excretions were in the same range after all loads. Urinary
`hippurate nitrogen after alanine + benzoate and phenyl—
`acetylglutamine nitrogen after alanine + phenylacetate ac-
`counted for an average of 12 and 22 of that in urea in the
`first 6 h. 0f the benzoate and phenylacetate given, 65 and
`51% were excreted in 24 h as hippurate and phenylacetyl-
`
`less than 3.5% appeared un-
`glutamine, respectively;
`changed in urine. (Pediatr Res 20: 1117—1 l2], 1986)
`
`Abbreviations
`
`LP], lysinuric protein intolerance
`iv, intravenous
`
`In 1914, Lewis ( l ) showed that benzoate modifies waste nitro
`gen excretion by decreasing production of urea via excretion of
`waste nitrogen as the acylation product of glycine with benzoate.
`ie. hippurate. A few years later (2) phenylacetate was noted to
`be another efficient acylating agent, combining with glutaminc
`to form phenylacetylglutaminc. Excretion of these nitrogenous
`products decreases the requirement for urea synthesis. Despite
`these observations, these alternative ways of waste nitrogen ex-
`cretion were not used clinically until the encouraging results in
`the treatment of inborn errors of the urea cycle were reported
`(3—9).
`In life-threatening hyperammonemia measurements of the
`effects and metabolism of benzoate and phenylacetate are diffi-
`CUlt if not impossible. We thus selected to test these substances
`in LPI, a disease with typical clinical picture (10, II), we"-
`characterized hyperammonemia. and defective transport of the
`diamino acids at the basolateral membrane of the intestinal (12-
`14), renal (15. 16), and probably liver (l7,
`l8) epithelial cells.
`We gave the patients an iv alanine load, which leaves healthy
`subjects unaffected but causes moderate hyperammonemia in
`the patients, either alone or together with benzoate or phenyl-
`acetate. The results indicate that hippurate and phenylacetylglu-
`tamine nitrogen excretions accounted for over 10 and 20%.
`respectively. of the amount of nitrogen exeretcd in urea in the
`first 6 h. Neither one of these substances was able to abolish
`alanine-induced hyperammonemia in these patients, although
`orotic aciduria diminished.
`
`MATERIALS AND METHODS
`
`Rcccivcd December l0. I985: accepted June 6. I986.
`Address for correspondence and reprint requests Dr. Olli Simell. Children's
`Hospital. UI'IIVCl'Slly of Hclsrnkt. 00290 Helsinki. Finland.
`This study was supported by the Finnish Academy, by the Sigrid Jusélius
`Foundation and Foundation for Pediatric Research. Finland. and by Grants ROI-
`IlD-l 1134. MOI—RR—OOOSZ. and KO7-NS~0034Z from the National Institutes of
`Health. by the Randi Klavan Mcmonal Fund. and by the Kittcring Family
`Foundation. 0.5. was a Visiting Scientist at
`the McGill University-Montreal
`Children‘s Hospital. sponsored by the Medical Research Council ofCanada. during
`preparation of the manuscript. D.L.V. was an investigat0r in the Heward Hughes
`Medical Institute.
`
`I: a
`Patients. Five Finnish children (see the legend of Fig.
`sister-brother pair. otherwise no consanguinity between the pa-
`tients) with a mean age (range) of 8.8 (2.8—l2.6) yr, weight for
`age of —l.6 [—0.9-(-3.2)] SD and height for age of —2.4 [—1.3-
`(—4. I )] SD and homozygous for LPl were studied after informed
`consent and acceptance of the study by the ethics committee of
`the hospital.
`The patients were of normal intelligence, had hepatospleno-
`megaly, hypotrophic muscles, osteoporosis. and aversion to pro-
`tein-rich food. They cxcrctcd massive amounts oflysinc and less
`Ill7
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 1 of 5
`
`

`

`lll8
`
`500
`
`SIMELL ET AL.
`
`400
`
`(Pmolll)
`
`BLOODAMMONIA
`
`300
`
`200
`
`100
`
`
`
`I. Blood ammonia responses to 6.6 mmol/kg loads of alanine infused iv in 90 min either alone (ALA) or together with 2.0 mmol/kg of
`Fig.
`benzoate (ALA + BA) or phenylacetate (ALA + PAA) to live Finnish patients with lysinuric protein intolerance. For identification, the patients are
`named from A to E; A is the sister of B.
`
`TIME (h)
`
`of arginine and omithine. All except one patient with recent
`diagnosis had been on citrulline supplementation. 0.5 mmol/g
`of daily dietary protein. Even on this regimen the protein intake
`had remained low (12, 19). The citrullinc supplementation was
`stopped 48 h before the infusion studies.
`The infusions were started after an Overnight fast at 0900 h,
`and no food was a110wed during the first 6 h. L—Alanine, 6.6
`mmol/kg, was given iv as a 5% aqueous solution in 90 min,
`followed by 0.9% saline. 200 ml/m2 of body surface area per h
`for additional 4.5 h. This load causes a rapid increase in serum
`urea in healthy controls but blood ammonia and urinary orotic
`acid excretion remain unchanged ()9). A similar infusion of
`alanine was given to each patient together with 2.0 mmol/kg of
`sodium benzoate. and on a separate day, with 2.0 mmol/kg of
`sodium phenylacetate.
`Blood and urine samples. Blood for ammonia, serum urea and
`creatinine. plasma benzoatc, hippurate, phenylacetate, phenyl-
`acetylglutamine. and amino acids was drawn at 0. 90. 120, 180.
`and 270 min. Urine was collected in four consecutive 6-h periods.
`Blood samples were immediately placed on ice and processed for
`measurement or storage at —20° C. Urine was collected on ice
`and frozen at the end of each collection.
`Measurements. Blood ammonia was measured with an am-
`monia specific electrode (Orion Research Inc., Cambridge, MA)
`(20). Plasma and urinary benzoic acid, hippun'c acid, phenyla-
`cetic acid. and phenylacetylglutamine were measured by reverse
`phase liquid chromatography with the use of a Waters C”
`column, with a 20% methanol solution in 0.0l M acetate buffer.
`pH 3. as an eluant. Plasma and urinary amino acids were
`quantitated with a Beckman 121 M Amino Acid Analyzer using
`lithium buffers and norleucine as internal standard.
`Statistical analysis. Values are expressed, if not otherwise
`stated, as geometric means with 95% confidence limits. For
`statistical evaluation. Mann—Whitney’s U test was used.
`Urinary values are expressed normalized to body weight. Re-
`sults normalized to creatinine excretion gave little new infor-
`mation and were omitted.
`
`RESULTS
`
`Clinical symptoms. The youngest patient was pale and floppy
`for l h after alanine load, but others talented alanine-induced
`hyperammonemia without symptoms. Four patients, including
`the youngest one. had symptoms at the end of loads containing
`
`benzoate or phenylacetate. Three complained of dizziness and
`two vomited at the end of alanine + benzoate loads, and three
`were pale and dizzy after alanine + phenylacetate. The symptoms
`disappeared in 45—60 min.
`Blood ammonia. The fasting blood ammonia concentration
`was normal (70 uM) in l l of the IS measurements, and elevated
`in four (Fig.
`l). The three hyperammonemic values at the
`beginning of the alanine + phenylacetate infusions were in
`patients having just arrived at the hospital in an overnight train;
`their ammonia increments were well within the range of the
`other subjects. Ammonia values usually peaked at 120 min. The
`geometric mean of peak increments was slightly higher after
`alanine ( I07, 28-4ll uM) than after alanine + benzoate (60, I7—
`213 pM, NS) and alanine + phenylacetate (79, 13-467 aM, NS).
`The concentrations returned to basal values in all except one
`subject by 270 min.
`Serum urea and creatinine. The increase in the geometric
`mean values of serum urea after alanine (from 4.5 to 5.l mM)
`and alanine + benzoate (from 6.6 to 8.3 mM) loads was caused
`by rising values in one patient. The values were stable in the
`others. Urea increased in this patient also after alanine + phen-
`ylacetate load.
`Serum creatinine fluctuated slightly and randomly in the in-
`dividual patients. The log mean values remained stable after
`alanine,
`increased after alaninae + benzoatc, and decreased
`slightly after alanine + phenylacetate.
`Plasma benzoate and hippurate. Plasma benzoate peaked 2 h
`afler the start of the infusion and decreased linearly with a mean
`t”; of 273 min (Fig. 2). By extrapolation, benzoate concentration
`at the end ofinfusion (at 90 min) was 6.01, 5.17-6.98 mM.
`Plasma hippurate levels peaked 120 min after start of the
`infusions (0.24, 0.14—0.40 mM) and remained nearly stable for
`the next 3 h (Fig. 2), implying that synthesis from benzoatc,
`distribution between plasma and other compartments and elim—
`ination were balanced, despite continuous decrease in plasma
`benzoate concentration. At all time points, the hippurate con-
`centrations were less than 10% those of benzoate.
`Peak
`Plasma phenylacetate and phenylacetylglutamine.
`plasma concentration of phenylacetate was at 120 min afler the
`alanine + phenylacetate infusion (Fig. 3) and was significantly
`(p = 0.008) be10w the concentration of plasma benzoate reached
`after the alanine + benzoate load. The disappearance curve of
`phenylacetate from plasma was linear with a t”, of 254 min,
`similar to that of benzoate. By extrapolation, phenylacetate
`concentration was 4.77, 3.71-6.l 1 mM at the end of the infusion.
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 2 of 5
`
`

`

`BENZOATE AND PHENYLACETATE IN HYPERAMMONEMIA
`
`1119
`
`Plasma phenylaeetylglutamine values were stable in 180- and
`270-min samples in two subjects but still increasing in three at
`270 min (Fig. 3). Mean value thus peaked at 270 min; concen-
`tration peaks (0.48, 0.22—l .06 mM) were above those of hippur-
`ate after alanine + benzoate (p = 0.008) (Figs. 2 and 3). Phen-
`ylacetylglutaminc/phenylaeetate percentages (linear means i
`
`SD), 7 i 2, 12 t 4, and 19 t 10 at the three measurement points
`(Fig 3), were clearly above the hippurate/benzoatc percentages
`after alanine + benzoate infusions (Fig. 2).
`Plasma amino acids Plasma alanine concentrations at the
`four measurement times and peak increments (5.86, 3.56—9.67;
`6.39, 299—1365 and 4.66,
`i.05-20.7O mM) differed insignifi-
`cantly after the three loads. Infusion of alanine caused an initial
`rise in plasma glycine concentration in all patients, whereas
`glycine increased in only three patients after alanine + benzoate
`and in four patients after alanine + phenylaeetate. The peak
`increments in plasma glutamine + glutamie acid were 1.08, 0.25—
`4.70; 0.84, 0.26—2.7l; and 0.22, 0.16—2.99 mM (NS) after the
`three loads. respectively. Plasma proline concentrations increased
`and plasma citrulline rose slightly after alanine and alanine
`benzoate loads but decreased after alanine + phenylaeetate.
`Arginine, omithine, and lysine remained unchanged. Plasma
`concentrations of several other amino acids were slightly higher
`at 120 min as compared with the fasting value but responses
`were similar after all three loads.
`Orotic acid excretion. The patients excreted l0 times more
`orotic acid during the first 6-h collection after the alanine load
`(5.62,
`l.84-—l3.l4 uM/kg per h) (Fig. 4) than they do in their
`daily urine (17). Addition of benzoate or phenylaeetate to the
`infusion decreased this value to 19% (1.07, 0.04—25.6 umol/kg
`per h, p = 0.151) and 49% (2.74, 0.014625 umol/kg per h, p
`= 0.016) respectively, of alanine alone.
`Urea excretion. Twenty-four-h urea excretion was 8.36, 4.51-
`I5.49 mmol/kg afteralanine, as compared with 9.30, 6. l2—l4. l 3
`mmol/kg after alanine + benzoate (NS) and 7.99, 422-1515
`mmol/kg after alanine + phenylaeetate (NS) with highest values
`in the first two 6-h collections (data not shown).
`
`
`
`PLASMABENZOICACID(mmol/I)
`
`
`
`
`
`HIPPURICACID(mmol/l)
`PLASMA
`
`TIME (h)
`
`ALA + BA
`
`.3
`
`°‘—‘°\.
`
`//;>;(
`
`
`
`Fig. 2. Plasma benzoate and hippuratc aflcr alanine + benzoate loads
`in patients with lysinuric protein intolerance. For details see legend to
`Figure l.
`
`1.0
`
`ALA+PAA
`
`w E
`
`2
`<
`
`
`
`s
`(8"
`2.1:.
`w>'5
`(l—
`E
`48E
`a.<v
`.J
`>-
`Z
`Lu
`.
`
`I6
`
`ALA + PAA
`
`o
`-
`
`3
`(9
`2h:
`ml”\
`03
`<
`.155
`1,5
`Z
`W
`I
`a
`
`
`
`TIME (h)
`
`Fig. 3. Plasma phenylaeetate and phenylaeetylglutamine concentratiOns after alanine + phenylaeetate loads in patients with lysinuric protein
`intolerance. For details see legend to Figure I.
`
`Do
`
`.at
`
`anO
`
`U)
`
`(pmol/kg/h) &
`
`0'6
`
`12-18
`6—12
`
`18-24
`
`
`
`OROTICACIDEXCRETION
`
`ALA + BA
`
`ALA + PAA
`
`
`
`12'18
`18-24
`
`6-12
`COLLECTION HOURS
`
`
`
`0'6
`
`12-18
`6—12
`
`18-24
`
`Fig 4. Urinary orotic acid cxcrctions after alanine. alanine + benzoate. and alanine + phenylaeetate infusions in patients with lysinuric protein
`intolerance. Urine was collected for four consecutive 6-h periods. For details of the infusions see legend to Figure I.
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 3 of 5
`
`

`

`[120
`
`SIMELL ET AL.
`
`Benzoate and hippurate excretion. Less than 2% of the admin-
`istered dose of benzoate appeared unchanged in the urine in 24
`h. Hippurate excretion peaked in the first 6-h collection, de-
`creased steadily and then was in the range of basal excretion in
`the last collection (Fig. 5). The combined amount of benzoate
`and hippurate excreted in 24 h (1.33. 0.71-2.52 mmol/kg)
`accounted for 67. 35—126% of the benzoate dose given.
`Phenylacetate and phenylacelylglulamine excretion. The
`amount of phenylacetate excreted unchanged in 24 h was 61, 9—
`425 umol/kg or 3, 0.4—2 1% of the dose infused. Excretion peaked
`in the first collection period. and approached zero values in the
`third and fourth collections.
`For unknown reasons, measured urinary phenylacetylgluta-
`mine excretion in one patient markedly exceeded the infused
`dose of phenylacetate (Fig. 5). When her data are deleted from
`the results. 809. 297—2200 umol or 40, 15—110% of infused
`phenylacetate was excreted as phenylacetylglutamine in 24 h.
`The major part of phenylacetylglutamine excretion occurred
`within 12 h but small amounts continued to be excreted even in
`the last collection.
`Molar excretions of the acylation products (hippurate and
`phenylacetylglutamine) by the patient group were almost iden-
`tical (Fig. 5) but “hippurate excretors” and “phenylacetylgluta-
`minc excretors” differed. Further. excretions of the acylation
`products did not correlate with orotic acid excretions, or peak or
`mean blood ammonic levels in individual patients.
`Amino acid excretion. Mean 24-h alanine excretion was less
`than 2% of the administered dose and identical after the three
`
`loads. Excretion of alanine and other amino acids, including
`ornithine. arginine. and lysine was increased in the first collec-
`tion, but returned to basal rates (data not shown).
`Relative nitrogen excretions. The major pan of urinary nitro-
`gen output occurred as urea, but excretion as hippurate after
`alanine + benzoate and as phenylacetylglutamine after alanine
`+ phenylacetate formed 11.5 and 22.1% of the urea nitrogen in
`the first collection period, respectively (Table I). The excretion
`of nitrogen in other measured constituents of the urine, including
`orotic acid, was small after all loads.
`
`DISCUSSION
`
`Toxic dosage or plasma concentrations associated with acute
`toxicity after intravenous benzoate or phenylacetate are currently
`poorly known. The clinical responses (dizziness. nausea. vomit-
`ing) in our patients after the infusions of alanine + benzoate and
`alanine + phenylacetate suggest that the 2.0 mmol/kg dose is
`likely to cause symptoms in the majority of subjects. However,
`simultaneous hyperammonemia may have influenced the clini-
`cal symptoms A dose between 1.5—1.8 mmol/kg, given iv in 90
`min during a hyperammonemic crisis is probably safe, and the
`plasma concentrations reached should perhaps not exceed 4.0-
`4.5 mM for benzoate or 3.0—3.5 mM for phenylacetate (calcu-
`lated by extrapolating the mean plasma curves of these substances
`after the loads to the assumed peak concentration at the end of
`the infusion, minus 30%). The calculated iv dose is within the
`range now safely used in patients with urea cycle enzyme defi-
`ciencies (8). Plasma concentrations measured in such patients
`imply that somewhat higher peak phenylacetate values (exceed-
`ing 4 mM) are acceptable.
`The t. ,2 values in plasma for both benzoate and phenylacetate
`were short and almost identical, suggesting fast metabolism and
`distribution into tissues. Our results clearly indicate that the
`main metabolic pathway of these substances is via conjugation
`to hippurate and phenylacetylglutamine, respectively. Compari-
`son of plasma benzoate and hippurate concentrations after the
`alanine + benzoate infusions (Fig. 2) suggests that benzoate
`concentration was not rate-limiting for acylation and lower con-
`centrations might be as efficient for hippurate production with—
`out causing side effects. Plasma phenylacetate values after the
`alanine + phenylacetate infusions also remained high for a long
`period, and excretion of the acylation product continued for even
`longer.
`The 6.6 mmol/kg dose of L—alanine iv has been experimentally
`found suitable for induction of mild hyperammonemia in LPl
`(10). In theory, addition of 2.0 mmol/kg of benzoate or phenyl-
`acetate to the load optimally leads to stoichiometric eliminatiOn
`of 2 and 4 mmol/kg of nitrogen as hippurate and phenylacetyl-
`glutamine, respectively. Because lower doses of alanine are tol-
`erated without hyperammonemia in LPl, we assumed that these
`benzoate and phenylacetate doses, despite of the apparent non-
`
`0
`
`ALA+ PAA
`
`(mmol/kglh)
`
`EXCRETION
`PHENYLACETYLGLUTAMINE
`
`HIPPURlCACIDEXCRETION
`
`(mmol/kg/h)
`
`0-6
`
`12-18
`5-12
`18-24
`COLLECTION HOURS
`
`0-6
`
`6-12
`
`12-18
`18-24
`
`Fig. 5. Urinary hippurate excretion after alanine + benzoate loads
`and phenylacetylglutamine excretion after alanine + phenylacetate loads
`in patients with lysinun'c protein intolerance. For details see the legend
`to Figure l.
`
`Table l. Nitrogen excretion (pmol/kg body wt per II. or percent ofnitrogen excreted in urea) in five patients with lysinuric protein
`intolerance in thefirst 6-h collections afteriloads ofalanine, alanine + benzoate, and alanine + phenylacetate"
`Load
`
`Alanine '
`784'. 364-1970
`
`_ %
`100
`
`Alanine + benzoate
`l 100'. 400—3020
`126; 29—542
`
`
`N excreted in
`Urea
`Hippurate
`Phenylacetylgluta-
`mine
`Orotic acid
`Alanine
`Glycine
`Glutaminc + glutamic
`acid
`10.4; 1.3-85.0
`1.]
`8.7; 6.5-1 LG
`Lysine
`14.6;72.3~792.S
`1.1
`8.6; 10.8-26.1
`Creatininc
`" Values are log means; 95% confidence limits. Percentages are calculated for the means.
`
`I 1.2; 3.7-34.3
`11.8; 0.8-1672
`3.6;0.3—55.9
`18.7: 3.5-101.l
`
`1.4
`1.5
`0.5
`2.4
`
`2.1; 0.1-51.2
`16.2; 2.0—129.2
`1.8: 0.1—43.3
`23.9; 1.3—92.5
`
`% _r _£lanine + phenylacetate
`100
`84 I; 396-3020
`11.5
`1.6; 0.2—11.1
`I86: 28—1200
`
`0.2
`1.5
`0.2
`1.3
`
`1.0
`1.3
`
`5.5; 0.0—32.5
`14.2; 1.9-104.4
`1.9; l.5—|9.9
`4.5; 0.5—38.1
`
`14.4; 4.0—51.1
`17.4: 8.4—35.9
`
`%
`100
`0.2
`22.]
`
`0.7
`1.7
`0.2
`0.5
`
`1.7
`2.1
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 4 of 5
`
`

`

`BENZOATE AND PHENYLACETATE IN HYPERAMMONEMIA
`
`ll2l
`
`stoichiometry. would prevent the induced hyperammonemia.
`lnstead, in this study. blood ammonia values and urinary orotic
`acid excretion were only moderately diminished by addition of
`benzoate or phenylacetate to the alanine load. suggesting that
`hyperammonemia. earbamyl phosphate. and. subsequently. or-
`otic acid production occur rapidly after alanine infusion and are
`not efficiently abolished by simultaneous infusion of the acylat-
`ing agents. Induction of hyperammonemia by protein intake (5—
`9. 2|) probably leads to more even distribution of nitrogen in
`metabolic compartments and, consequently, may be associated
`with better responses to treatment with the acylating agents.
`Interestingly. correlation of orotic acid excretion with blood
`ammonia was good in these experiments as in previous studies
`(22—23).
`Our findings of the excretion rates of hippurate and phenyl-
`acetylglutamine after the loads differ somewhat from earlier
`findings in healthy subjects. Lewis's volunteer, receiving to g of
`sodium benzoate orally. excreted 23% of urinary total nitrogen
`as hippurate. but an equimolar decrease occurred in urea +
`ammonia nitrogen excretions. We did not measure total nitrogen
`output in our patients. but hippurate nitrogen excretion was l2%
`of urea nitrogen excreted in the first 6 h after the load. We also
`found that 54% of the single phenylacetate dose was excreted as
`phenylacctylglutamine in 24 h after the load. Earlier Ambrose er
`a]. (24). after having administered 5—7 g/day of phenylacetate to
`a healthy man. found 98% of the dose in urine as phenylacetyl-
`glutamine. In another study (25). 91% of the 85 mg/kg dose was
`excreted in the urine as phenylacetylglutamine.
`In patients with urea cycle enzyme deficiency who have re-
`ceived benzoate or phenylacetate treatment with proper meas-
`urement of nitrogen excretion (3—5. 7—9). urea excretion re-
`mained unchanged even though the total nitrogen excretion
`increased by approximately the amount accountable for hippur-
`ate or phenylacetylglutamine. just as in our experiments. The
`cause for the discrepancy in the urea excretions after administra-
`tion of benzoate or phenylacetate in healthy subjects and in
`patients with urea cycle failure remains open. However. it sug-
`gests that urea excretion in urea cycle diseases (often with a close
`to zero enzyme activity of the cycle in irilrri) cannot be easily
`changed and represents nitrogen coming from another pool than
`the one affected in similar experiments in healthy subjects (8. 9).
`After a single dose of benzoate or phenylacetate with alanine,
`the acylation products formed 12 and 22% of the urea nitrogen
`in the urine immediately after the dose. During prolonged ad-
`ministration the percentages are probably higher (3—5. 7—9). The
`fast disappearance from plasma of both benzoate and phenylac-
`etate Suggests that frequent oral doses or continuous infusion are
`optimal ways for their administration.
`In summary. this study shows that waste nitrogen excretion as
`hippurate or phenylacetylglutamine can be induced in patients
`with LPI by infusion of benzoate or phenylacetate. Neither one
`ofthese substances. when added to an iv alanine infusion. is able
`to abolish the resulting hyperammonemia and orotic aciduria in
`the patients. In hyperammonemic crisis. addition of benzoate.
`phenylacetate. or both to the iv ornithine or arginine or oral or
`iv citrulline treatment of LP] (IO.
`I I) may prove helpful.just as
`they are effective in hyperammonemic urea cycle enzyme defi-
`ciencies (3—9).
`
`Ackn0wledgmems. The skillful technical assistance of Ms. Mar-
`jatta Viikari, Ellen H. Gordes, and Evelyn Bull is acknowledged.
`The authors thank Huguette Rizziéro and Lynne Prevost (McGill
`University-Montreal Children's Hospital Research Institute) for
`preparing the typeseript
`
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`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
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