`
`Assay of the Human Liver Citric Acid Cycle Probe
`Phenylacetylglutamine and of Phenylacetate in Plasma
`by Gas Chromatography-Mass Spectrometry
`
`Dawei Yang,* Michel Beylot,t Kamlesh C. Agarwal,* Maxim V. Soloviev,* and Henri Brunengraber*· 1
`*Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106; and tiNSERM U197, Lyon, France
`
`Received February 11, 1993
`
`Phenylacetate, derived from phenylalanine, is con(cid:173)
`verted in human and primate liver to phenylacetylglu(cid:173)
`tamine. The latter has been used to assess the labeling
`pattern of liver citric acid cycle intermediates. We pres(cid:173)
`ent gas chromatographic-mass spectrometric assays of
`phenylacetylglutamine, phenylacetate, and phenylala(cid:173)
`nine in biological fluids. The compounds are deriva(cid:173)
`tized with dimethylformamide dimethyl acetal. Limits
`of detection are O.I nmol for phenylacetylglutamine
`and phenylacetate and 2 nmol for phenylalanine. Base(cid:173)
`line plasma concentrations of pheny I acetate and phenyl(cid:173)
`acetylglutamine are I and 3 #J.M, respectively. The 24-h
`urinary excretions of phenylacetate and phenylacetyl(cid:173)
`glutamine are about 4 #'mol and I mmol, respectively.
`Ingestion of phenylalanine (in the form of aspartame)
`by a human is followed by sequential increases in phe(cid:173)
`nylacetate and phenylacetylglutamine concentrations
`in plasma and urine. This assay opens the way to nonin(cid:173)
`vasive probing of the 13C-labeling pattern of liver citric
`acid cycle intermediates in humans.
`"'' 1993 Academic
`Press, Inc.
`
`Phenylacetylglutamine (PAGN) 2 is formed in the
`liver of primates and humans from the condensation of
`glutamine with phenylacetyl-CoA (1-3). The latter is
`formed from the activation of phenylacetate (PA), a
`side product of phenylalanine metabolism. PAGN is a
`normal constituent of human urine (4). It accumulates
`in the plasma of uremic patients (5,6). Large doses of
`P A are administered to children with hyperammonemia
`
`1 To whom correspondence should be addressed at: Department of
`Nutrition, Mt. Sinai Medical Center, Cleveland, OH 44106-4198.
`2 Abbreviations used: CI, chemical ionization; EI, electron ioniza(cid:173)
`tion; PA, phenylacetate; PAG, phenylacetylglutamate; PAGN, phe(cid:173)
`nylacetylglutamine.
`
`0003-2697/93 $5.00
`Copyright© 1993 by Academic Press, Inc.
`All rights of reproduction in any form reserved.
`
`resulting from inborn errors of the urea cycle (7). These
`children excrete nitrogen as PAG N instead of urea. P A
`is also used to treat other hyperammonemic conditions,
`such as those that occur in lysinuric protein intolerance
`(8), in propionic acidemia (9), during treatment of leu(cid:173)
`kemia (10), and in portal encephalopathy (11). Finally,
`P A is being tested as an inducer of tumor cell differen(cid:173)
`tiation (12).
`The pathway of PAGN production has been used re(cid:173)
`cently to set up the "non-invasive chemical biopsy of the
`human liver" (13,14). A 14C-substrate that labels liver
`citric acid cycle intermediates is administered intrave(cid:173)
`nously, and sodium PAis given orally. PAGN is isolated
`from urine, and the 14C-labeling pattern of its glutamine
`moiety is determined by sequential degradations to C02
`(15,16). This labeling pattern is assumed to reflect that
`ofliver citric acid cycle intermediates, in particular a-ke(cid:173)
`toglutarate. This technique yields useful information on
`the regulation of the citric acid cycle and gluconeogene(cid:173)
`sis in liver, in particular the ratio of activities (pyruvate
`dehydrogenase)/(pyruvate carboxylase) in the intact
`liver (13,14).
`The concentration of PAGN in normal urine and in
`the plasma of uremic patients has been assayed by
`HPLC (5,6). However, to the best of our review of the
`literature its physiological concentration in body fluids
`has not been directly assayed by isotope dilution gas
`chromatography-mass spectrometry (GC-MS). An iso(cid:173)
`tope dilution assay for PA, derivatized with pentafluoro(cid:173)
`propionic anhydride has been published (17). Using this
`assay, the concentration of PAGN in plasma and cere(cid:173)
`brospinal fluid of humans and monkeys was assayed as
`the difference between total PA and free PA (18). The
`former was measured after hydrolysis of PAGN to PA
`by HCl at 100°C.
`The physiological production of P AG N opens the pos(cid:173)
`sibility to conduct chemical biopsy investigations with
`
`277
`
`Par Pharmaceutical, Inc. Ex. 1024
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 1 of 6
`
`
`
`278
`
`YANG ET AL.
`
`13C-tracers and minimal or even zero doses of P A. The
`labeling pattern of PAGN might be assayable either by
`GC-MS or by NMR. As a first step in this endeavor, we
`set up sensitive assays of PA and PAGN in plasma and
`urine, using isotope dilution GC-MS. We determined
`baseline concentrations of these substrates in plasma
`and their modulation by a surcharge in phenylalanine or
`in PA.
`
`METHODS
`Materials
`Chemicals were obtained from Sigma-Aldrich. [2H 5 )(cid:173)
`Phenylalanine and [2H 5]phenylacetic acid were pur(cid:173)
`chased from Isotec. Methyl-8 (dimethylaminometh(cid:173)
`ylformamide dimethyl acetal) was from Pierce.
`Aspartame (N- L-a-aspartyl-L-pheny !alanine 1-methyl
`ester), in the form of sweetening pills (Equal), was pur(cid:173)
`chased from a local drugstore.
`Unlabeled and [2H 5]phenylacetyl chloride were pre(cid:173)
`pared by reacting unlabeled or [2H 5]phenylacetate with
`SOC12 • Pheny lacetylglutamine, [2H 5 ] pheny lacetylgluta(cid:173)
`mine, and phenylacetyl-C5N -amido] glutamine were syn(cid:173)
`thesized (19) by reacting unlabeled or [2H 5 ]phenylacetyl
`[15N-amido]glutamine.
`chloride with glutamine or
`Phenylacetylglutamate (PAG) was synthesized by
`reacting unlabeled phenylacetyl chloride with gluta(cid:173)
`mate. Purity of the synthesized PAGN and PAG was
`checked by HPLC on a 2.1 X 10-cm C18 column, devel(cid:173)
`oped with 8% acetonitrile in 0.05% H 3P04 (0.5 ml/min),
`using uv detection at 254 nm.
`
`Sample Preparation
`Standard curves of PA, PAGN, and phenylalanine
`were prepared in human plasma, dialyzed overnight
`against saline (to remove endogenous substrates). One
`set of 1-ml plasma samples was spiked with PA, phenyl(cid:173)
`alanine, and internal standards of [2H 5]PA (5 nmol) and
`2H 5 ]phenylalanine (100 nmol). A second set of 1-ml
`[
`plasma !'lamples was !'lpiked with P AGN and [2H 5)PAGN
`( 10 nmol). Both sets of samples were deproteinized with
`25 ~Ll saturated sulfosalicylic acid and centrifuged.
`The first set of supernatants were diluted two-fold
`with water, acidified to pH 1.5 with HCl, and loaded on a
`2-ml AG-1-50-H+ column, developed first with 3 ml
`water, then with 4 ml of NH 40H, 3M. The water ef(cid:173)
`fluent of the column, containing P A, was adjusted to pH
`2.0, saturated with NaCl and extracted 3 times with
`ethyl ether. The extract was dried over Na2S04 and
`evaporated. The ammonia effluent of the column, con(cid:173)
`taining phenylalanine, was evaporated. The two resi(cid:173)
`dues were reacted with 60 ~Ll of a mixture Methyl-8/ace(cid:173)
`tonitrile/methanol (3/2/1, v/v/v) at 100°C for 20 min.
`The second set of acidic supernantants was adjusted
`to pH 12 and incubated overnight at 75°C to convert
`
`PAGN to PAG. Then, the solutions were brought to pH
`1.5, saturated with NaCl, and extracted 3 times with
`ethyl ether. The extracts were dried over Na2S04 and
`evaporated and the residues reacted with Methyl-8 as
`above. In some experiments, trimethylsilyl derivatives
`of PAGN were prepared.
`
`Mass Spectrometric Assays
`The gas chromatograph (Hewlett-Packard 5890) was
`equipped with a 12-m HP-1 capillary column (12 m X
`0.2 mm i.d., 3-~Lm film thickness, Hewlett-Packard).
`Carrier gas was helium (1 ml/min), and column head
`pressure was 36 kPa. The injector port was at 290°C.
`The column temperature programs were: for PA, 95°C
`for 2 min, then increase by 5°C/min until 220°C; for
`PAGN, 140°C for 1 min, then increase by 15°C/min un(cid:173)
`til 250°C; for phenylalanine, 140 oc for 1 min, then in(cid:173)
`crease by l5°C/min until240°C. The column was inter(cid:173)
`faced with a HP-Engine mass spectrometer, operated
`under electron ionization (EI, 70 eV), or with ammonia
`(1 Torr) negative chemical ionization (CI), and the ion(cid:173)
`ization source temperature was 300°. All samples were
`injected twice. Nominal masses at m/z 150/155 (PA,
`El), 118/123 (PAGN, El) 279/284 and 262/267 (PAGN,
`Cl), and 235/240 (phenylalanine, Cl) were monitored
`with a dwell time of 100 ms per ion.
`A healthy male subject abstained from foods contain(cid:173)
`ing aspartame for 2 days. After an overnight fast, he
`ingested 20 mg/kg of aspartame (Nutrasweet, equiva(cid:173)
`lent to 70 #!mol phenylalanine/kg) dissolved in a glass of
`water. Heparinized blood and urine were collected be(cid:173)
`fore and at various times after aspartame ingestion.
`Other normal subjects abstained from aspartame for 2
`days, collected 24-h urine, and gave a blood sample after
`overnight fast for PA and PAGN assays.
`A 5-kg female maccaca mulatta rhesus monkey, anes(cid:173)
`thetized with halothane, fitted with arterial, venous,
`and urinary bladder cathethers, was infused intrave(cid:173)
`nously with PA (100 #Lmol bolus, followed by 100 #!mol/
`h) and [2- 13C]acetate (20 ~Lmol/kg X min) for 4 h. Arte(cid:173)
`rial blood and urine were collected at regular intervals.
`The 13C-enrichment of urinary PAGN was measured by
`monitoring mlz 262 to 266 of the CI mass spectrum.
`These enrichments are corrected for natural 13C-enrich(cid:173)
`ment by recurrent background corrections at each mass,
`based on the baseline unlabeled PAGN mass distribu(cid:173)
`tion.
`
`RESULTS
`Figure lA shows the EI mass spectrum of unlabeled
`PAGN derivatized with Methyl-8. The spectrum is
`identical to that obtained starting from PAG. The pro(cid:173)
`posed structure of this derivative is shown in Fig. 2. The
`major peak at mlz 118, shifting to 123 with [2H 6]PAGN,
`corresponds to C6H 5 -CH=CO (19). The loss of the
`
`Par Pharmaceutical, Inc. Ex. 1024
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 2 of 6
`
`
`
`ASSAY OF PHENYLACETATE AND PHENYLACETYLGLUTAMINE
`
`279
`
`100,---------r------------------------,
`
`...
`
`80
`
`" u c
`0
`~ 60
`~
`.~
`0
`4i a:
`
`40
`
`20
`
`0
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`40
`
`100
`
`80
`
`60·
`
`40
`
`20
`
`0
`
`" u c
`0 ,
`c
`:::1
`.0
`<(
`
`.~
`:S!
`" a:
`
`c
`:>
`
`" '"' c
`0 "
`~ ..
`,.
`·~
`4i a:
`
`Phenylocety(jlutomine
`
`..
`..
`11 J
`
`...
`
`...
`
`...
`
`230
`
`.. .
`
`80
`
`120
`
`160
`
`200
`
`240
`
`280
`
`m/z
`
`..
`
`Phenylocetate ...
`
`B
`
`..
`..
`
`..
`
`I
`
`~
`80
`
`....
`
`...
`
`J..
`
`...
`II
`120
`
`m/z
`
`160
`
`...
`
`c
`
`Phenylalanine
`
`••
`
`BO
`
`120
`
`160
`
`200
`
`240
`
`m/z
`
`FIG. 1. EI mass spectra of methyl-8 derivatives ofphenylacetylglu(cid:173)
`tamine (A) and phenylacetic acid (B) and CI mass spectrum of
`methyl-8R derivative of phenylalanine (C).
`
`amido nitrogen ofPAGN in the formation of this deriva(cid:173)
`tive was confirmed by using [15N-amido]PAGN. The
`spectrum of the latter showed no mass shift. The ammo(cid:173)
`nia CI mass spectrum of PAGN is characterized by two
`major ions at mlz 262 and 279, corresponding to the [M
`+ H]+ and [M + NH 4 ]+ ions (not shown). These clusters
`have normal isotopomeric profiles. Thus, they can be
`used to measure the total [13C]enrichment of PAGN
`generated in experiments with e3C]tracers.
`Figure 1B shows the EI mass spectrum of unlabeled
`PA, as its methyl ester. The peak at mlz 150, shifting to
`
`155 with [2H 5]PA, corresponds to the molecular ion.
`The base peaks at mlz 91 or96 show M-1 to M-5 masses
`and are not suitable for quantitation.
`Figure 1C shows the ammonia positive ion CI mass
`spectra of unlabeled phenylalanine, as the dimethylami(cid:173)
`nomethyl methyl ester. An advantage of the Methyl-S
`reagent is that it derivatizes in one step the carboxyl and
`the amino groups of aminoacids (20). A clean cluster at
`mlz 235, shifting to 240 with [2H 5]phenylalanine, corre(cid:173)
`sponds to the molecular ions of the two species. Under
`EI conditions, the main peak is at mlz 143, as reported
`in Ref. (20). However, the 143 peak does not shift with
`2H 5 ]phenylalanine. Thus, it is not suitable for quanti(cid:173)
`[
`tation of phenylalanine using the (2H 5]internal stan(cid:173)
`dard .
`Concentration standard curves for PAGN, PA, and
`phenylalanine in dialyzed human plasma are linear (Fig.
`3) and the limits of detection are 0.1 nmol for PA and
`PAGN and 2 nmol for phenylalanine .
`In seven normal adult subjects, the baseline plasma
`concentrations of PA and PAGN, after an overnight
`fast, were 1.22 ± 0.09 and 3.34 ± 0.31 (SE) JiM, respec(cid:173)
`tively. These are similar to concentrations reported by
`Karoum et al. (18) who assayed PAGN as the difference
`between total PA and free PA. Urinary excretion of PA
`and PAGN were 3.63 ± 0.45 JLmol/24 hand 1.08 ± 0.09
`mmol/24 h (n = 6), respectively. The 24-h urinary clear(cid:173)
`ance of PA and PAGN, expressed as percentage of the
`clearance of creatinine, were 2.8 ± 0.7 and 291 ± 37%,
`respectively. In one normal subject eating three meals
`per day, the plasma and urine concentrations of PAGN
`were measured 17 times over 46 consecutives h. The
`integrated plasma PAGN concentration was 3.4 JiM
`(range 2.5 to 6 JiM), and the integrated urinary clearance
`of PAGN was 216% of the creatinine clearance.
`Figure 4 shows the profiles of PAGN, PA, and phenyl(cid:173)
`alanine concentration in the plasma of a human subject
`after ingestion of 20 mg aspartame/kg (equivalent to 70
`JLmol of phenylalanine/kg). Concentrations peaked at
`20, 200, and 250 min for phenylalanine, PA, and PAGN,
`
`CH 2 - CH- COOCH 3
`I
`I
`CH 2
`N
`~/~
`o
`C
`C=O
`I
`CH 2
`I
`CaHs
`
`FIG. 2. Proposed structure of the phenylacetylglutamine deriva(cid:173)
`tive. Note that the amido nitrogen of the glutamine moiety has heen
`lost during the preparation of the derivative. An identical spectrum is
`obtained by derivatizing phenylacetylglutamate.
`
`Par Pharmaceutical, Inc. Ex. 1024
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 3 of 6
`
`
`
`280
`
`YANG ET AL.
`
`2.5
`
`2.0
`
`A
`
`•
`
`5
`
`10
`
`15
`
`20
`
`25
`
`Phenylocetyl<iJiutomine (nmol)
`
`B
`
`2
`
`J
`
`4
`
`5
`
`6
`
`Phenylocetote (nmol)
`
`c
`
`;n 1.5
`+
`~
`
`1.0
`
`2
`
`0.5
`
`1.2
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`;n
`+
`2
`::::::
`2
`
`1.4
`
`, .2
`
`1.0
`
`0.8
`
`-;;:;-
`+
`:::li
`::::::: 0.6
`:::li
`
`0.4
`
`0.2
`
`20 40 60 80 100 120 140
`Phenylalanine (nmol)
`
`FIG. 3. Standard curves of phenylacetylglutamine (A), phenylace(cid:173)
`tate (B), and phenylalanine (C) in human plasma. Human plasma
`was dialyzed against saline and spiked with various amounts of unla(cid:173)
`beled phenylacetylglutamine, phenylacetate, phenylalanine and the
`corresponding [2H,) internal standards.
`
`respectively. Urinary excretions of PA and PAGN fol(cid:173)
`lowed a similar pattern (Fig. 5). The profile of phenylala(cid:173)
`nine concentration in plasma was similar to that re(cid:173)
`ported by Gupta et al.
`(21) in humans ingesting
`aspartame.
`Figure 6 shows the plasma concentrations of P A and
`PAGN in an anesthetized monkey infused with 20 ~mol
`PA/kg X hand 20 ~mol [2- 13C]acetate/kg X min. By 4 h,
`concentrations of PA and PAGN almost leveled at 33
`and 24 ~M. respectively. From the 400 ~mol of PAin(cid:173)
`fused during the experiment, 2% was excreted as un-
`
`changed PA and 54% as P AG N. Figure 7 shows the M +
`1 and M + 2 13C-enrichment of urinary PAGN that be(cid:173)
`came labeled from infused [2- 13C]acetate. This labeling
`profile is similar to that of glutamate isolated from rat
`livers perfused with [2- 13C)acetate (22).
`
`DISCUSSION
`
`PAGN has been quantitated in normal urine and in
`uremic plasma by HPLC (5,6). To the best of our review
`of the literature, it has not been quantitated in normal
`plasma and urine using isotope dilution mass spectrome(cid:173)
`try. The preparation of the [2H 5 ]PAGN internal stan-
`
`25
`
`15
`
`i 20
`.. c ·e
`B
`" ;:;.
`..
`~ 10
`" 0
`~
`1
`ll.
`
`5
`
`0
`
`.3.0
`
`2.5
`
`i 2.0
`.!
`-! 1.5
`" 0 1-
`1
`ll.
`
`0.5
`
`0.0
`
`120
`
`110
`
`c
`
`! 100
`.I
`J 90
`
`80
`
`10
`
`80
`0
`
`100
`
`300
`200
`Time (min)
`
`400
`
`FIG. 4. Profile of phenylacetylglutamine (A), phenylacetate (B),
`and phenylalanine (C) in human plasma after ingestion of aspartame.
`An overnight fasted subject ingested 20 mg/kg of aspartame at zero
`time.
`
`Par Pharmaceutical, Inc. Ex. 1024
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 4 of 6
`
`
`
`A
`
`~L
`j
`
`B
`
`20
`
`18
`
`~ a.
`E 16
`0 "' ' 0
`E
`-..:!> 14
`z
`<:>
`~
`
`12
`
`10
`
`1.4
`
`1.2
`
`1 0
`
`.,
`a.
`E
`0
`"' ' 0
`
`E
`-.3.
`
`0..
`
`<( 0.8 J
`
`0.6 p
`
`0
`
`100
`
`200
`
`Time (m1n)
`
`~
`
`400
`
`300
`
`FIG. 5. Profile of phenylacetylglutamine (A) and phenylacetate
`(B) in human urine after ingestion of aspartame. Same experiment as
`in Fig. 4.
`
`dard is fairly simple, starting from commercial [2H 5]PA.
`The latter is also used for the isotope dilution assay
`ofPA.
`In early experiments, we prepared trimethylsilyl and
`tret-butyl-dimethylsilyl derivatives of PAGN and P A.
`However, the limits of detection were 10 nmol versus 0.1
`nmol with the derivatives obtained using Methyl-8.
`Also, the spectra were complex with overlapping clus(cid:173)
`ters. Finally, when using the silylated derivatives, the
`satellite peaks corresponding to naturally occuring
`heavy isotopes of carbon and silicon were relatively
`high. This would make future investigations on the 13C(cid:173)
`labeling pattern of PAGN less precise. In contrast, for
`the Methyl-8 derivative of PAGN (CI spectrum), theM
`+ 1 satellite in the mlz 279 cluster is 17% of theM peak.
`The profile of phenylalanine, PA and PAGN concen(cid:173)
`trations in human plasma, after ingestion of aspartame
`show classical precursor to product relationships, in
`that the three concentrations peak sequentially in the
`expected order.
`
`ASSAY OF PHENYLACETATE AND PHENYLACETYLGLUTAMINE
`
`281
`
`-
`
`___j"
`
`'-----
`
`The 24 hr urinary clearances of PA and PAGN are
`maximal values since they were calculated based on sin(cid:173)
`gle plasma concentrations after overnight fast. In one
`subject, the clearance of PAGN was calculated using the
`plasma PAGN concentration integrated over 46 h. It
`appears that PA and PAGN are handled by the human
`kidney in very different ways. The 24-h clearance ofPA
`amounts to at most 3% of the creatinine clearance.
`Thus, PA filtered in the glomerulus is almost entirely
`reabsorbed, presumably at the proximal convoluted tu(cid:173)
`bule which is the site of the reabsorption of carboxylic
`acids. In contrast, the clearance of PAGN is two to four
`times that of creatinine. Thus, in addition to glomerular
`filtration, P AG N is actively excreted by the nephron, as
`has been reported by Zimmerman et al. (5,6), using a
`HPLC assay.
`Magnusson et al. (13) and Ensenmo et al. (14) have
`administered gram amounts of PA to human subjects
`infused with [14C]tracers that label the citric acid cycle
`and the gluconeogenic pathways in liver. From the label(cid:173)
`ing pattern of urinary PAGN, they drew novel conclu(cid:173)
`sions on the regulation of these pathways in human
`
`25
`
`20
`
`:i'
`~
`" c
`E 15
`0
`"5
`c;.
`~ .. 10
`<J
`>. c ..
`.2
`"' Q.
`
`5
`
`0
`
`35
`
`30
`
`25
`
`:i'
`.,3.
`.!
`~ ..,
`~ 20
`i .c
`
`D..
`
`15
`
`10
`0
`
`50
`
`100
`150
`lime (min)
`
`200
`
`250
`
`FIG. 6. Profile of phenylacetylglutamine (A) and phenylacetate
`(B) in the plasma of a monkey infused with phenylacetate. An anes(cid:173)
`thetized monkey was infused with 20 ~mol phenylacetate/kg X h and
`20 ~mol [2- 13C]acetate/kg x min.
`
`Par Pharmaceutical, Inc. Ex. 1024
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 5 of 6
`
`
`
`282
`
`YANG ET AL.
`
`6
`
`5
`
`E 4
`w
`Q_
`:::t 3
`
`2
`
`M+1
`
`M+2
`
`50
`
`100
`
`150
`
`200
`
`250
`
`Time (m'1n)
`
`FIG. 7.
`13C-Enrichment of urinary phenylacetylglutamine in a
`monkey. Same experiment as in Fig. 6.
`
`liver. This technique of noninvasive chemical biopsy of
`the human liver could be extended to stable (13C]tracers
`with minimal or even zero load of P A. In humans, pro(cid:173)
`duction of PA from endogenous phenylalanine is suffi(cid:173)
`cient to supply enough PAGN (1 mmol/24 h) so that its
`total 13C-enrichment could be measured in urine and
`even plasma by our GC-MS technique. Further work is
`required to set up a technique to measure the distribu(cid:173)
`tion of rt 3CJ on each carbon of the glutamine moiety of
`PAGN.
`
`ACKNOWLEDGMENTS
`This work was supported by grants from the NIH (DK35543), the
`Nutrition Fund of the Cleveland Mt. Sinai Medical Center, and the
`North Atlantic Treaty Organization.
`
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`Par Pharmaceutical, Inc. Ex. 1024
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 6 of 6
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