0U§|tJ-‘)5Sfi.-'lJ=l.’32(J1-1U 19320.00
`DiaL:('.
`i\rl1"l.-\N}tt-:1! Man Dismwiiriru
`Copyrigltt '.‘C.- 2004 by '."].1e .-’\1I.1crIea1I Society for Pi.i.:iriu:rcol0gy tultl Experilnctital 'l'11t:rt=.peLttIt:s
`DMD 32.-‘:|lJ 1‘). 200:1
`
`I
`Vol. 32. No.
`liti|."ll]1l87
`Prh.=rr::! fir L-'.S..-t.
`
`NEW SECONDARY METABOLITES OF PHENYLBUTYFIATE IN HUMANS AND RATS
`
`Takhar Kasumov. Laura L. Brunengraber, Blandine Comte, Michelle A. Puchowicz,
`Kathryn Jobbins, Katherine Thomas, France David, Renee Kinman, Suzanne Wehrli,
`William Dahms, Douglas Kerr, Itzhak Nissim, AND Henri Brunengraber
`
`Departments of Nutrition {T.K.. L.L.B.. B.C., M1’-l.P., K.J.. K.T., F.D., R.K.. H3.) and Pediatrics {H.K.. W.D.. D.K.), Case Western
`Reserve University. Cleveland, Ohio; and Chiidren’s Hospitai (S. W.} and Department of Pediatrics (!.N.), University of
`Pennsylvania. Philadelphia. Pennsylvania
`
`[Received May 14. 2003; accepted September 2. 2003}
`
`This article is available online at http:i.idmd.aspetjouma|s.org
`
`ABSTRACT:
`
`Phenylbutyrate is used to treat inborn errors of ureagenesis, ma-
`lignancies, cystic fibrosis, and thalassemia. High-dose pl1eny|bu-
`tyrate therapy results in toxicity. the mechanism of which is unex-
`plained. The known metabolites of phenylbutyrate are phenylacetate,
`phenylacetylglutamine, and phenylbutyrylglutamine. These are ex-
`creted in urine, accounting for a variable fraction of the dose. We
`identified new metabolites of phenylbutyrate in urine of normal
`humans and in perfused rat livers. These metabolites result from
`interference between the metabolism of phenylbutyrate and that
`of carbohydrates and lipids. The new metabolites fall into two
`
`categories, glucuronides and phenylbutyrate {J-oxidation side
`products. Two questions are raised by these data. First, is the
`nitrogen-excreting potential of phenylbutyrate diminished by in-
`gestion oi carbohydrates or lipids? Second. does competition
`between the metabolism of phenylbutyrate, carbohydrates, and
`lipids alter the profile of phenylbutyrate metabolites? Finally, we
`synthesized glycerol esters of phenylbutyrate. These are par-
`tially bioavailable in rats and could be used to administer large
`doses of phenylbutyrate in a sodium-free, noncaustic form.
`
`Sodium phciiylbutyratc (PB'} is a highly ellcctix-‘c drug for the
`treatment of patients with hyperammonemirt resulting from inborn
`errors of urea synthesis (Bmshaw et al.. 1981, 2001; Brusilow. 199]).
`Tltesc paticitts excrete nitrogen as phonylacctylglutamint: (PACiN}
`tBatsl1aw et al.. 1981). the latter is also fonned when the patients are
`treated with [lhcnylacctate {PA}. H.owe\'cr, PB is prefcncd as a
`prodntg 01' PA because it does not have tl1e foul smell ol'the latter. ln
`ttddition, PB shows promise lot the treatment of cystic lihrosis he-
`caust: it it1L:l't.‘.t1St:S rr'rrns—I11c1nbmt1t: chloride contltictaticc (ltubcnslein
`and Zeitlin. 2000: Zeitlin et ul.. 2002). Also. PB is used in clinical
`trials for the treatment ofsick|c—cc|| anemia and thalasscmia because
`
`I994:
`induces the |'orn1ation ol' fetal hemoglobin (Dover et al._.
`it
`[loppc et aI._. I999). Lastly. PB is l.LfiCEl in clinical trials as a cytostatic
`antineoplustic a,«.zen1. because it inhibits histone deacetylases and po-
`tcntiatcs the effect of cytotoxic agents on tumors (Samid ct a|.,
`I997’:
`Gilbert er al__ 2[]f]l)_
`
`This work was stipported by the National Institutes of Health [Research Grants
`DK581 EB. GATQ4-Q5, and DKSSTB1. and Training Grant Dt<EI?3l 9} and the Cleve-
`land Mt. Sinai Health Care Foundation.
`' Abbreviations used are: PB, 4—phenylbutyrate; PAGN, phenylacetylglw
`tamine: PA. phenylacetatez PEIGN, 4-phenylbtrlyrylglutarninez PHEL 3-l"I)|'ClfCIXy--|'l--
`phenylbutyrate; FIG, 4—phenyl-trans-crotonate; PKB, -1-phehyl-3-ltetobutyrate;
`TMS.
`trimethylsilylj GC—MS. gas chromatography—mass spectrometry: SW.
`sweep width: TD. data points; COSY. correlation spectroscopy; HSCJC. hetero
`nuclear single-quantum coherence; AUG, area under the curve.
`
`Address correspondence to: Henri Brunengraber, Department of Nutrition,
`Room 2841 Case Western Reserve University, 11000 Cedar Rd. Cleveland OH
`44106—?139. Email: hxoB@cwnJ.edu
`
`The clinical cltcctiveiicss of PE in some of these situations is
`
`limited by occasionail incidences oftoxitzity at high doses t[Cardt.Icci et
`at, 2001; Gore et at. 2003). Concems [rave been raised by clinical
`investigators who treat patients with large closes of PB as a soditun
`salt. First. the total amount of PB and its knovvii metabolites excreted
`in urine (PA. PAGX) is less than the atlrninistered PB close. mine-
`tirncs as low as 50%. Some of the unknown metabolites might
`contribute to PB toxicity at hi gh doses. Second, the large sod itlm load
`of the treatment is potentially dangerous for patients with impaired
`cardiac andfor renal Function. Third. the eaustieity of sodium PB can
`result in csopliagal andjor gastric distress. even when it
`is adminis-
`tered as a powder suspentterl
`in water (this has been extensively
`debated till the lnteniet discttssion group Metal)-L at |ittp:n'|ists.l'raI1-
`kcn.dea‘mai|man;-‘Iistinfoimctab-L}. Neither the biochemical mecha-
`nismts) of PB toxicity nor the identity oi" the inissittg metabolites oi"
`PB is knonnt. Also, it is not clear whether the metabolism of PB ta
`modified fatty acid) interferes with, or is inthicnecd by. the metabo-
`lism of fatty acids and earboliydtates present in foodstuITs. Lastly. it
`is not known wlietltcr stimulatioit of lipolysis under stress conditions
`iiitcrfcrcs with PB metabolism.
`
`interest in PB metabolism was related to it being a
`Our original
`precursor til‘ FAG '. whiclt can be used as it noninvasive probe of the
`"iC- or DC-labeling patient of citric acid cycle intermediates in
`human liver tl\/lagnusson et al.. 1991; Yang et al._. 1996). As part of
`these itivestigaliotis. we recently identilied pllellyllI)tIl}'l')ilf_.§lllT.8.l'l‘l.ll'1E
`(PBGN) as a new metabolite of PB [Comte er al.. 2002). PBGN is
`prestiinatbly fonned from the reaction of l’B—C‘oA with glutaminc. by
`analogy with the fonnation of PAGN tiom the reaction between
`t’A—(.‘oA and glutaminc [Webster er al., I976}. In normal adult sub-
`
`10
`
`PAR PHARMACEUTICAL, INC.
`EX. 1015
`PAR PHARMACEUTICAL, INC.
`EX. 1015
`
`

`
`NEW PH ENYLBUTYFIATE METABOLITES
`
`11
`
`0
`
`°
`
`°><+ or
`
`C’
`
`o
`
`.
`
`0
`
`O
`
`.><
`
`1
`
`E20“.
`remix
`
`lINoOl-L
`
`0
`
`OH MB“: r \/
`‘j
`one
`0
`2
`
`0H 0
`
`0
`
`o
`
`D
`
`DD 0 OH l}
`3lNflBl34
`0H o
`3IHt_'|
`
`.t.s-|2,z,.t.4.4-1H5:-run
`
`in.
`
`nu;
`
`t-]~DlP{‘lTM
`
`OH
`
`HCl
`ton c
`
`0
`
`0
`
`OH
`
`|'hmrl-met-me
`
`”*‘E'"*
`
`0
`
`D
`
`R-PI-IB
`
`I-Fhenyl-2-|2-Jllfpropannl
`.S‘c'lre.=m.’.for- the .s'_t‘n.'!re.vi'.\' rgl'rrtitr:.F.1ele'tJ‘ aria’ t."emet'rnea’ .smm.tum!s'.
`
`FIG. 1.
`
`jccts who ingested a fairly low dose of PB (5 g/?5 kg). we found that
`the total excretion of PB + PA + PAGN + PBGN accounted for only
`half the ingested PB dosc (Comte et al.. 2002). The missing fraction
`of t|'tc dose may be disposed of either in urine as unknown metabo-
`lites, or in feces as unabsorbed PB and/or PB metabolites.
`In the present study. we report
`the irlcntificatinn of nclditiottnl
`metabolites of PB in humans. i.e.. 13- and S-3—hydroxy—4—phenylbu—
`tyrate (PHB), phenylacetonc, and l—pltenyl-2—propttno]. as well as PA
`and PB glueuronides. We studied the mechanism of PIIB fomiaiion
`lrutn PR in ])I:rrllht¢Ll ral livers 'dfI(l itlcntilit-:tl lvm mltliliurtal tm:labu—
`lites, 4-phenyl-ri'un.e-ctotonate (PIC) and 4-phcnyl-3-ketobutyrate
`(PKB}. Lastly, we prepared sotlium—tree esters of PB and investigated
`their bioavailability in rats. Glycerol-PB esters appear promising for
`the adtttiuistration of large amounts of PB without the corresponding
`sodium load.
`
`Materials and Methods
`
`Materials. All chemicals used in syntheses. general chenlicals. and solvents
`were obtained ficrn Sigma-Aldricli (St. Louis. MO). All organic solvents were
`dried and distilled inn1tedi.=tte1y before use. [:}l,]l"henylz1cetic acid 199%] and
`[31-l,_]ben2ene were prirchased ft'nl'I‘I lsntec lnc. tltetiamisbtirg. Oil). The derivati-
`7/ation agent N-Inethy1—N—(triIrtcthylsilylttritluoroncetamide was supplied by Rcgis
`Technologies, Inc. [Morton Grove. IL}. All nqtlootls solutions were made with
`water purified with tho Mi1li—Q system (l\r‘Ii1liporc Corporation, Bcdforrl. MA}.
`Preparation of lfnlubelett and neuterated Standards. .S‘—2—PhenylhutyryI
`chloride was prepared by reacting S-2-phcnylbutyric acid with SOCI3. and was
`vacunm—di.stillerl and stored at 4°(‘. as a 11.5 M solution in henrene. [31-l5]PH
`was prepared by aluniinurn chloride-catalyzed condensation of 'y—bulyrolnc—
`tone with [llildbenzene as previously described (Comte ct al., 1002).
`'y-Phe-
`nyl-rt'w.=s'—crotonic acid v\':.is prepared by at Fridel~Crafis reaction of benzene
`with ethyl
`‘y-bromo-ti‘w:.r-crotonate. followed by acid hydrolysis of ethyl
`‘y-phenylvrt'mr.u'—C1'cIEOI1£ItC t[.offlet ct al.. l9?t)). The imns configuration of the
`product was confirmed by '1] NMR. [III,]Phenylacctylglycitte was synthe-
`sized by reacting [ilisjphcnyleeetyl chloride with glycine. as described previ~
`
`I977».
`and Tanaka,
`(Ran-isdcll
`analog
`unlabeled
`the
`for
`ously
`[3II,]Phc1iylacetyl chloride was prepared by activation of [3[I-flphcnylacetic
`acid with freshly distilled dichlorornethyl methyl ether and used immediately
`tiller evaporation of’ excess dichlonomctliyl methyl ether and of the niclhyl
`chloroformate byproduct. The synthetic protocol for preparation ofl-‘l-Iii, PKB.
`[1I'I_;]P]I}3. phcnylacctotic. and I-phcnyl-2-[2-2H]pruput1ol is outlined in Fig. I.
`Erin’! at‘—P.‘wrg-I—3—ke.roilvtrg=rrrrc4 (2: Fig.
`I) was synthesized by a method
`adapted from Capozzi et al. t'l9‘J3‘r. The cotnmercial isopropylidcnc nialonate
`(2.2—(liu1elhy1-4.6-diketo—l.3-dioxane. also called Meldrnnfs acid: Aldrich
`Chemical Co.. Milwatiltec. WI} was reacted with phcnylacetyl chloride in the
`prcscncc of dry pyridine in anhydrous methylene chloride. Thc crudc pheny-
`Iacctylaterl Melt‘lr|1m‘s acid [compound I. Fig, I: was refluxed in absolute
`ethanol until evolution of CO3 ceased (about 3 h). After evaporation of the
`solvent. ethyl -l-phenyl-3-ketohutyrate was purified on a silica gel column.
`4-Pfreirtui-3—iretrJfJt.i.f1=rtt'
`t'l't.‘t(tl (PKB). A [(1% molar excess of I N NaOII
`solution was added slowly to ice-cooled ethyl -l-pheuyl-3--lcetobutyrate and
`stirred at room tcnipcmlurt: until the organic phase disappeared (approxinnatcly
`12 bl. The solution was acidified with I N I-lL‘l to pl-I 2 and extracted three
`times with 3 Volumes ufcthyl ether. J‘-\fl.ct drying uvcrNa3SO1. the solvenl was
`evaporated to give the white solid product t yield 94%. m.p. '.’t)‘C 1.
`'1! NMR
`(300 MHZ. 6. C'DC‘l3_t: keto. 3.33 (s. 2H. CI-IQCOO). 333 (5. 2H. C'H1Ph).
`‘LI ? 7.33 trn. 5|-I. Phtzenol.-1.82 ts. Ill. (Ill). I113 15. Hi, OI-ll: ketoienol =
`7.8:}. “C NMR [TS MHZ. 3. CDCI3): 48.36 {CI‘I2CO0)u 49.87 {CII2Ph}.
`E2141 {C-4 Phl.
`|2S.I'3 (C-3. C-5 Ph}. 129.50 (C-2. (#6 Phi. 133.14 ((.‘—ipso.
`P11). 169.35 (F00). 20l.5'r' (CO).
`l rcduction of4~phcnyl—
`The identity of the product was also confirmed by I
`3-ketobttryrate with NaBH,,. 2': extraction of R.S-PHB with ethyl acetate. and
`3} TMS derivatization and NtI_.—positive chemical
`ionization CiC—MS. The
`mass spectrum of the derivative was identical to that of PHB syntltcsized as
`described below. The 4—phe1iyl—3—ketobutyi'ic acid was stored at -Eit.l"(‘ to
`prevent decomposition. Just before use in liver pertitsion experiments. the acid
`was dissolved in water and titrated to p11 a—'— 140.
`32.3 3-h{wJr.«;2qi'-4-giiitwiiterrrtir-to acid (PHB). Ethyl 4~p11ci1yl—3~ketobutyrate
`(2.06 g. 10 mmol) was mixed with 10 ml of 1-130 and a calculated amount of
`N30]-l.*'l-{:0 was added dropwise with cooling to give (ISM solution of [0l'i'].
`
`

`
`12
`
`KASUMOV El AL.
`
`NaBH, t0.3i"g, 10 mmol} was added and the mixture stirred for 24 h. After the
`rcaction mixture was cooled to 0"C. the pH was brought to 7.0 with }ICl tool}
`and more than half of water removed by lyophilizer. The pll was brought to 2
`by I-ICI ti’: N} and the solution extracted 3 times with 2- volumes of ethyl ether.
`After drying the combined ether extract. the ether was evaporated giving a
`white solid product. Yield ?9%. Purity of product was assayed by GC—MS after
`derivatization with '1“MS.
`R-3-H:itu’ru.r_t--if-p}ieirt'.llJrr{t'm.'e [R-PI-TB] was prepared by reducing the cor-
`responding -"l-phenyl-.3-kctobutyric acid with Ll-chlorodiisopinocarnpheylbo
`ianc [I ““l-DIP-Cl] {Wang ct al.. 1999). The yield was 89% from PKB:
`enantiorneric excess was 927% [GC-MS of the methyl .8‘-2-phenylbutyryl de-
`rivative (see below). and purity was confirnted by NM R].
`4—l’he—
`R.S-3—.Hycl!'t).Y_lJ-4-p.Ili£:‘l‘I[l~'l,{'i?.3.3.4.4-:.H5J'ilJti{tJJ'cJi‘e
`rre.s—i-‘nyyenisi.
`ttyl-3-kctobutyiic acid €0.26?
`g. LS rninol) was suspended in 3 ml of EH30
`(99.9%; to which 0.36 ml of 40% or l\a0°H (3.5 l‘rJ.1‘r‘lOll in EH30 was slowly
`added at 0 --5°C. This ptoccduvc exchanges 'H for 2I'[ atoms on the tncthylciic
`groups adjacent to the carbonyl. The solution was stirred at room temperature
`overnight and then lyophilized. The residue was dissolved in 3 ml of EH30 and
`stirred for another 5 h at room temperature. The solution was cooled on ice.
`treated with Na]i3I[.._ ('63 mg. 1.5 ntrnoll. and stirred overnight at room
`temperature [Des Rosicrs et al., 1988}. After acidification to pH l
`to 2 with
`IIC1 (6 N}. this solution was saturated with NaCl and extracted three times with
`3 volumes of diethyl ether. Solvent evaporation yielded R.5'-[2l-[_.]Pl{B (yield
`74%. purity 99% by NMR. M5 isotopic enriclinient 95% by GC~MS of the
`TMS derivative). The free acid was titrated to 131-1 8 with .\la0}'l and stored
`frozen as a 0.5 M solution until use.
`Pill'é?l{l’.lfl£.'é.’f0H'é' was prepared by decarboxylating plienyllrtelobutyrztte in acid
`at I00°C. It was ncduccd to 1-phciiy1-2-[2-3I1]piupauoi with Nai33i1.,.
`Esters of Phenylbutyrate. Diliydroityacctone-di-P13. glycerol-tri-PB. ri-
`hose-tetra~Pl3_ glucose—p-enta—Pl3. and sorhitol—hexa—Pl3 were prepared by re-
`acting the polyohsugar with excess phenylhuryryl chloride in the presence of
`pyridine and catalytic amounts of .-‘V.N~diri1et|iy|a.nii11opy1'idine. Products were
`purified by flash column chromatography on silica. To prepare glycero|—mono—
`PB. isopiiopylidcnc glycerol was reacted with phcnylbutyryl chloride as above.
`and the isopropylidene group was removed by mild acidic hydrolysis in water.
`The structttrc and purity of all products were confirmed by 'H and “C NMR.
`The structure of‘ glycerol-mono-PB was confirmed by acerylation with acetic
`anliydride, followed by GC-MS analysis of the derivative.
`Sample Preparation. For the determination of the [tee acids concentration
`(PA. PHB. PKB, PIC. and PE) in pcrfitsatc. samples l0.l toll were spiked with
`0.1? ptrriol of [31-1,] PA.
`[ll-1_..]I’B_ and R,S—[°H,]I’I-lB before deproteinizatioii
`with 20 ptl of saturated sulfosalicylic acid. The slurrics were saturated with
`Na("l. acidified with one drop of 6 M IIC1. and extracted three times with 5 ml
`of diethyl ether. For the assay of conjugates of PI} and PA. 0.l—ml aliquots of
`final liver perfusttte or of human urine were spiked with internal standards and
`trcatod with [.0 ml MC] (6 ‘it at 90°C overnight to hydrolyze the conjugates.
`Ghtcuronides of PB and PA were identified by the amount ofthese compounds
`rclcascd after incubation of pcrfizsatc and urine samples with ,8-glucuronidasc
`in 0.2 M ammonitint acetate buffer. pH 5.0. o"vet'night at 17°C.
`Bilc samples were analyzed for Ercc and total {conjugated ~l~ Encc} PB and
`its nielabnlites. lit the first series ofassays. 0.05-ml samples of'l1i|e were spiked
`with intcrnal standalris. acidified to [:11 2 to 2.5, and extracted three tirrics with
`diethyl ether. In the second series of assays, samples spiked with internal
`standards were hydrolyzed with 0.3 ml of Nri0li to N} at 90°C.‘ for 3 h before
`acidification and extraction.
`
`Phenylztcetylglycine was extracted in acid and dcrivtttizcd with l‘l'leIhfID0l-"
`llCl. For the assay of phenylacctone and I-phen_v|-2-prnpanol. urine and
`perfusate samples were spiked with the structural analog l—phenyl—
`[°II5]ethanol and then treated with .\laB3II. to reduce phenylacetonc to mono-
`dcntcratcd I-phcny—2~propanol. The labeled and unlabeled l-phenyI—2~
`propziiiol were assayed as TMS derivatives.
`For the assays in urine. 0.]-rnl samples were spiked with (ll 5 p.mol
`[°H_.]l‘-‘HE. acidifieti to pH I to 2 with H{.‘l. samrated with Natl, and extracted
`three times with 3 ml of diethyl ether. The combined extracts were dried with
`Na3SO. and evaporated before reacting the residues with 70 pl of TMS at
`t‘i0"C for 20 min.
`For the cliiral assay of PHB enantiomers ("Powers et al.. 1994}. 0.]-ml
`samples were spiked with R.S-[3li5]Pl-IB. and either deproteinizcd with 50 to]
`
`to
`of saturated sulfosalicylic acid (if containing proteins} or acidified to pH 1
`2 with MC] (for uri.ucl.'1'hcI:t. thc slurrit-s or solutions were saturated with NaCl
`and extracted three times with 3 ml of diethyl ether. The combined extracts
`were dried with Na_-.804 and evaporated before reacting the residues with 0.15
`ml of l'l'lcthfl.t10lr'l‘l(.'l for l h at 65%,‘. to derivatize the carboxyl groups of the
`PHB enantioniers. After cooling.
`I ml of water was added to the |r1ixt:Ltre and
`the hydroxyacid methyl ester was extracted with diethyl ether (three times in
`3 ml}. After complete evaporation of the combined ether extract. S-2-
`phenylbutyryl chloride benzene solution {tJ.l
`rnl, 0,5 M) and 0.05 ml of
`aqueous 12 N NaOH were acldcd. Alter vorlcxing. the ruiittttrc was incubated
`for l h on a slow shaker at room temperature. The derivatives were extracted
`with ether [ three times in 3 1‘l'lll and 1 ml of water. The combined ether phase
`was dried with Na3SU,. and evaporated completely. The residue was dissolved
`in 0.] ml of ct.l:tyl acetate. and 1 5.1.1 was injected into the GE"-MS.
`(EC-l\*IS Methods. All of the metabolites. except pheiiylacetylglycine, were
`aiialyzed as their TMS derivatives on a I'Iewlctt-Packard 5890 gas chromato-
`graph equipped with a ZB-S capillary column :60 in X 0.25 l1'l1'l‘l i.d.. 0.5 I'I'll'l'l
`film thickness; Hewlett Packard. Palo Alto. CA) and coupled to a 5989.43 mass
`selective detector. Samples t0.2—J pill were injected with a split ratio 1:0 to
`50: l. The carrier gas was lteliurri ll ntlfininl and nominal initial pressure was
`20.01 psi. The injector port temperature was at 2i"0“'C'. the transfer line at
`305°C. the source tcnipcrature at 200°C and quadrupole at 150°C. The column
`temperature program Was: start at l00"C. hold for l min. increase by S°C‘!mi1i
`to 23(i“C. increase by .35”C.’tuin to 3l0“C. t’: min at 3l0”C. After automatic
`calibration. the mass spectrometer was operated under amtnonia-positive ion-
`ization mode. Appropriate ion sets were monitored with a dwell time of 25 to
`35 rrisfion. at ms: 225x233 {PA,’[°H,]PA_}. 2542259 {PB='[2H_..]PB). 325x330
`(P[lBi'[3ll5]Pll.Bl. and 252 (P'rC't. Note that PKB and pl]C1'l}'lfiCl:l.01']C had been
`reduced with NaB2H..
`to monodeuterated Ii’,S‘-3-hydroxy-4-phenylbutyrate
`(monitored at rm‘: .‘!-265330} and I-phenyl—2—pI'opanoJ (nionitored at mi’: '_’l'lll.«’
`Elf) andfor 2l"i'.f217i. Also. since I-plienyl-2-propanol was assayed with a
`stauidard of I"|3l'l€1‘|)"l[2ll<]Bll't3l10l.
`ions monitored for this assay were 200x209
`or 2170225.
`For the analysis of chiral PIIB derivatives. the GC iujoclor Icmpcralurc was
`set at 280°C. The column {DB-S, 60 in X 0.25 mm id. 0.5 nun film tliicknessz
`Hewlett Packard) program was rnodificd to the initial
`l:i0°C for 2 min,
`increased b_v
`lS"Cfrnin to 230°C. 25 rain at 230°C.
`increased by 35°C to
`290°C. and held 10 rnin. Ions monitored were l} 358 {M -I
`13.
`i.e.. M -I
`N]-1.1"} for analytes and 2) 363 {M + 18 + 5,
`i.e.. M + 5+NH.+I for
`["H5]I“HB. The mass spectrometer was operated under amrnonia- positive
`chemical ionization and was tuned autotiziatically.
`Phcnylacctylglycinc was analyzed as its methyl ester derivative using an
`OV-225 column ['29 m X 0 33! mm i.d..
`1
`tun film thickness: Quadrex
`Corporation. Woodbridge. CT). This column yielded better resolution of
`N—phenylact-tylglycine methyl ester with no peak tailing. Sarnplcs 012-] i.tll
`wcrc injected with a split ratio 20:1 . The carrier gas was hcliuni l constant flow:
`l.2 rnlrminj. The injector port temperature was at 220°C, the transfer line at
`240°C. the source tcmperaturc at 200°C. and quadrupole at 106°C. The column
`tempcrariire program was: start at 90°C. hold for I mitt. increase by |0'°C‘r'rnin
`to 240°C. 15 min at 240°C. After automatic calibration, the mass spectrometer
`was operated under ammonia-positive ion i7arinn mode { pressure adjtistcri to
`optimize peak areas]. Ions monitored wcrc H208 tM + I, i.c.. M + H“ and
`225 {M + 18, i.e., M + NH.’') forthe annlyte and 2] 215 IM + T + l. M +
`T + H '} and 2.32 (M + 7 + I8. M + ? + Nl—l_l" ) forN—[3tI,JP.1\-glyciiie with
`:1 dwell time of 25 I'nsi’ion.
`
`Areas under each chrornutogrztm were determined by interactive computer
`integration, and corrected for naturally occurring heavy isotopes and light
`isotopic irnpI.u‘itics in the synthesized labeled internal standards.
`NMR Spectroscopy. Proton NMR spectroscopy was performed at 400
`MHZ on a Brukcr Avancc (Bruker. Newark. DE} DMX -100 wide-bore spec-
`trometer nsitig a 5—rnm inverse probe. Fllll-S[t“t3t'Ig[l"I urine sutnples were ob-
`tained by Iyophilizing 5 ml ofurine to dryness. The residue was dissolved in
`0.5 ml ol"H,0_ and the solution was iiitzroduced into a 5—mm NMR tube. An
`external standard made of J. settled capillary containing a solution of triruelh—
`ylsilylpropionic acid in 31-I;O was introduced into the NMR tube and used as
`chemical shift reference. Standard acquisition conditions were as follows for
`one-dimensional spectra: 45° pulse. 8-s repetition time. water saturation during
`the relaxation delay. sweep width (SW: 6775 Hz, MK data points {TD}. and
`
`

`
`NEW PH EN YLBUTYFIATE METABOLITES
`
`13
`
`32 scans of dala collection. Two—dimensional correlation spectroscopy
`lCOSYl spectra Wl.'I't.' obtained with the following conditions for the sect‘.-itd
`dimension: SW 3500 H2. TD 2K. [6 scans. and For the lirst ditriensiou. 512
`iticretticnls of 2T3 _u.s zcrulillcd to IK. A uonshillcd siticbcll wiritlun. was
`applied in both dimensions, and rnagrtilude spectra were calculated. Two-
`dirnensional
`‘[L""C correlations via double insensitive nuclei enhanced by
`pOlt'll'lZzl.ll0rI transfer (HSQC'} were performed in the phuse—sensiti\te rnode
`(Tl’Pl;
`tirne-proportional receiver phase inercmentation) using gtaadients For
`coherence selection and carbon decoupling during acquisition. The following
`conditions were used in the second t1in'1enston'. SW 7:200 Ilz. TD 1K. 128
`scans. and in the first dimension: SW I2 kHz. 256 increments of 20.? _:.ts
`zerofillcrl to 512. A shifted sinehell window was applied in both dimensions.
`Pt-oton—decoupled ca1'ho11 spectra of the concentrated urine samples were
`obtained at l00.62 Mllz in a 5-mm dual probe. Acquisitiolt conditions were as
`follows: 20” pulse. repetition time [.3 s. SW 25 kHz. TD 64K. 40,000 scans.
`The free induction decays were zerolilled to 128K. and a Lorentz to tjauss
`tratisfc-I‘Jt1atioIt (LB = —I Hz. GB =-
`lll_‘.I was applied before Fotuier trans-
`lbrmation.
`Clinical Investigation. The protocol was reviewed and approved by the
`Institutional Review Board of University Hospitals of Cleveland. All subjects
`were free of any chronic or acute illness. Wotnen had a negative pregnancy test
`and were not breastfeeding. Seven subjects t_lh.ree men. four women: 3].? t
`5.0 years: I'll .3 I 3.4 cm: 79.5 1 5.9 kg} received detailed information on the
`purpose ofthc investigation and signed an itiforrncd consent form. Alter an
`overnight Fast. the sttbjects were admitted to the Clinical Research Center at
`7:30 AM. Tire); rennaiticd Fasting until
`the completion of the study. An
`intravenous line was installed in the forearm with a saline infusion (20 rt'i|:"|1l
`and a short blood satnpling catlicter was inset'ted into a superficial vein of the
`conttalatcral hand. The hand was placed in a heating box at 60"C' for sampling.
`of arterialized venous blood. At 8:00 AM, after baseline l)l0DCl and urine
`sampling. each subject ingested 0.3!‘: n1nio1i'kg(S g.«'?S kg} Na-l-‘Ii. This dose
`corresponds to II
`to l'?'% of‘ the doses commonly used in the treatment of
`patients. with inborn errors of urea synthesis (0.4 0.6 g - kg" - day‘ "'1. Water
`intake was adjusted to intluce 21 diuresis ol" at least l00 ml.n’30 min. Urine
`samples were collected at 30-min intervals for the iirst 3 h alter PB ingestion.
`and then every hour until 8 ll. Urine samples were quickly frozen and stored
`at —30'°C until analysis.
`Organ Perfusion Experiments. Livers from Fed male Sprague~Dawley rats
`kept on standard rat chow (200 230 g} were perfiased (Bruuengraber et 21]..
`l‘T.I'5] wilh recirculating K.tebs—Ringer~bicai'bonate buffer containing 4% ‘oo-
`vine serum albumin [fraction V. fatty acid poor; Intcrgcn. Purchase. NY} and
`10 niM glucose. The hile. duct was cannulated with PF.
`I0 tithing IBD
`Bioscicnccs, San Jose. CA! For bile collection. Throughoul the 2-h experiment.
`sodiultt taurocholate 1'38 _u.ntol.-'l1l was infused into the perfiusion reservoir to
`stituulatt: bile llow tRolJins and Brutictigraber. 1982}. After 30 min of equil-
`ibration, a calculated amount ofcither PB. R.5-Pl'il3. or PKB was added to the
`petfusatt: to set an initial cottcctitratiou uf5 mM. The perfusion continued until
`120 min. The pll of the perfusatc was monitored and kept at 1,3 to 7.4 by
`adding 0.3 \r[ Na0H. Samples. of bile and perfusate were collected at regular
`intervals. For the assay of PKB, perfitsate samples I2 rnll were treated immev
`diately with u.?. ml of 0.1 M NaB”I-[4 in 0.1 rnM N210!-I to convert unstable
`PKI3 to stable titonodeuterated R..5‘~Pl'|I3. Bile samples were collected every 30
`min. At the end of the experiment. the livers were quick—I‘rozen with aluminum
`tongs prccoolcd in liquid nitrogen.
`Rat in Viva lixperintents‘. We tested the hioavailzthility oltwu T-‘B esters as
`tricans to deliver large amounts of PB without the corresponding sodium
`at
`load. 0ver1iight—F2:sted rats [330 -400 g] were divided into six groups (SJ! rats
`per group} for the testing of llztrec diiTercnt PB preparations: Na-PB. glycerol-
`mono—l’l:l. glyct-:rol—tri—l’B.
`ribose—tetra—PB. glucose-peuta—I’B. and sorbitol—
`licxa-PB. Each rat received one stomach gavagc of the sodium salt or ester in
`an amount that delivered 2.15 rnmol PBi'l-Lg. The weiglzted dose for each rat was
`mixed with 3 Llll of Tween and atltuiiiislcrctl lo the rats through a slotttacli
`gavage needle.
`Whole blood samples H00 -200 poll wen: taken at ‘-5. I5. 30. I50. [.30. 240.
`330. "I20. and 480 min from a small incision in a tail vein. Blood was collected
`in heparinized microcapillary tubes and centrifuged. The plasma ['50 100 toll
`was transferred to an l-lppendorf nine and quick frozen.
`
`Results
`
`Human Study. In our previous study, we had identified PBG.\‘ in
`the plasma and urine ofnorrnal adults who had ingested a small dose
`of PB. We now report the data of additional analyses conducted on the
`some samples of human urine. First. we subjected to .\'MR analysis
`two samples of urine produced by each subject before and 2 h after
`ingestion of PB. The NMR spectrum of the second sample, but not of
`the lirst. was highly suggestive of the presence of a pt'ot‘luct of
`hydroxylation ofthc side chain of PB. In the COSY spcctrtuu ofthc
`lyophilized urine dissolved in D30 (alter PB ingestion), we identified
`a proton at 4.25 ppm cottplcd with two Cl-155. The first CH2 has
`protons at 2.8? and 2.70 ppm: the sccortd has nearly itlcitlical protons
`at 2.4 ppm. A chemical shift at 4.25 ppm is likely corresponding to a
`proton coupled to the OH group. Therefore,
`the COSY spectrum
`revealed the presence of a metabolite having —Cll3—CIlOll—Cll:—
`moiety. In the HSQC‘ spcctntm tltcsc proton signals correlated with
`the following carbons: CH at 7'0 ppm overlapping with other CH
`carboltyclratc carbons, CH, z1t44,6 ppm and CH3 at 42.5 ppm. Lastly.
`in the aromatic region. signals at I29. 6. 128.7. and 126.6 ppm corre-
`sponded to a monostthstirutcd phcnyl group having the same intensity
`pcr carbon as the signals of the -CH2—CH0H~Cl-12- group. We
`therefore concluded that ,8-ltydroxy-PB (Pl-1B] was present in the
`urine of patients treated with PB. PllB would be a very likely
`metabolite since it would be formed via partial ‘B-oxidation of PB to
`l’HB—CoA (presumably the S-t.‘.[1El[lllt'J[Ilt3l'), which would be hydro-
`lyzed to fret: PHB.
`'l'o ftLrtht:t'contit1n the identity of the urinary metabolite detected by
`N.\:lR. we synthesized unlabeled and R.S[3I-l_.]Pl--LB. The di-TMS
`tlt.'rivntivt: of syitlltctic R.S—PH.B was analyzed by GC—MS itt parallel
`with an extract of htttnan Ltrinc lallcr PB itigcstion) that had been
`reacted with TMS. In the sample derived from urine, we found a peak
`at the same retention time and with the same mass spectra (electron
`itirtixnlitirt antl l\ll‘l3-|"l(lNlll\-‘L’. u:l’|t:|nit:ul
`ltiltixztlititt) as Iln: .\'lHIItl'dI‘il
`til‘
`R.S—PHB. In addition. thc NMR spectrum of synthetic PHB had the
`same chemical shifts as the material identified in human urine. This
`
`Confimled the identity of PllB in human urine but did not yield
`information about its chiralily. Thc chiralily of excreted Pll.B yields
`infoniiation on the mechanism of its fonnation (see below).
`For the cltrotnatograpltic separation of Pl-[B cnantiomcts from the
`synthetic raocmate. we tried various chiral hydroxyl dcrivatization
`reagents before selecting the combination of I) methylation of the
`catboxyl group. and 2) reaction of the ltydroxyl group with S-2-
`pltcnylbutyryl chloride (Powers ct al..l994}. The cxpcctccl derivatives
`of R— and S-PHB were well separated. and their order ofclutton was
`confimicd using, a sample of R-PllB that we had synthesized. R-Pl-1B
`clutes altcad of the S—c|ct-ivativc (Fig. 2).
`Chiral GC—MS analysis of the hutnan urine samples (after PB
`ingestion) revealed that PHB is present as an cnantiomeric Inixttlre
`with 10% R-PllB and 90% S-PI-lB (Fig. 2). Figure 3 shows the time
`profile ul'(R+S')-PIlB excretion it1 urine after an oral holtts ol'Na-PB.
`The excretion of (R+S)—PHB peaked at 120 to 240 min. Eight hours
`after ingestion of PB.
`the cumttlative excretion of IjR+S)—PHB
`(1.35 : 0.13 mrnol) amounted to 4.4 I 0.56% of the PB dose.
`Small amounts of phenylacetone and 1-plienyl-2-propattol were
`idctttificd in tltt: urine samples (Table 1). Treatment ofurint: samples
`with ;‘3—glttcurottidase increased their PB —l— PA content. The total
`amount of PB + PA released by i3—g|ttcttronidasc amottntcd to 2.4 t
`0.3% ofthe PB dose.
`
`Perl‘used Rat Liver Study. The metabolism of PB was studied in
`perfused rat livcrs by the addition of 5 mM PB to the recirculating
`pcrfitsatc. The tune profile of the PB concentration was curvilinear
`
`

`
`KASUMOV EF AL.
`
`TABLE I
`
`ilk.’ rflfltl
`tli'e(.‘nvt'.’1:\- (JP-3 um." i:r.\' mc'.fah0lirc.\ in i"mm.r1n' m'ir:r:.’ (1: — 1,! a_'f.'fr.'i‘
`i:i_<.3¢'.§'.':'m: :J,|l':".l.3I‘J mu-:r).l'.r'.l'rg A-'u—PB a::u';_=:'riu'ur.'ri(::i rJ__f'}".l? I-i:r.*i'u.’Ja':i'r'.rt<:.\' by mr .l'r'\-wt‘
`(H ‘- 5) pt*r'.I'it.i‘e‘u' n'.".'.’.' 5 ffllltzlt PB
`-3-.. ol'1“B Uptake
`
`Ml.‘.l:|l1x.tlitt‘
`
`FTEL‘ FR
`PB--[5-glueumnitlc
`Free PA
`PA-glycine
`PA-I3-glileututiide
`PAGN“
`1"BCr1\"
`P1113
`PKJ3
`PIC
`Pltenylaeetone
`|—1"]1en}'l—2-propanol
`Total bile rncl
`Tulul
`
`Humziris
`0.9? L 0.33
`I29 ; 0.33
`0.26 “' 0.06
`
`|.l1 L 0.19
`32.6 '
`1.9
`21.5 .' 2.-l
`-l-.a'l- “' 0.36
`
`0.] 1 + 0.00?
`0.01 1- 0.001
`
`62,4 '_ ?..l
`
`Rats
`
`21.52 L 4.32
`7.64 "' 0.87
`7.68 + 0.84
`5.25 L 0.88
`
`l5.T1 “' 1.63
`4.54 1*.‘ 0.29
`5.15 i 0.59
`3.6? *7 0.23
`Trace amounts
`2.9? L 0.tSl
`74.12 L 5.58
`
`14
`
`10
`
`in
`
`3 2 1 a
`
`Abundancex10‘
`
`El PB-giucur.
`
`O (51.3)-PHB
`
`0 [SJ-PI-IB
`
`* PA
`*3 PA-glycine
`0 PA-gsucur.
`I8IF'lB
`‘' PKB
`
`12' Phenylac.
`
`" Prt:vluIisl5' itlctltilictl [rll:lEIl}lIIl[C.H' {(‘nIIlle :4! ml . 2003!.
`
`E.
`
`5.
`.
`
`EE
`
`§-
`
`E E3-
`
`9
`g
`
`39.64
`
`sass
`
`39.12
`
`39.76
`
`39.90
`
`39.94
`
`Tlme (rninj
`
`Flt}. 2.
`
`r._1fR'— z.'u:.I' 5'—.PH3 é'x(.-i‘e.*."eta’ ii: om: .\'ar.Iipi'e' qflrirriruti
`-f.‘?.I.='r'£(:l CC."-fl-f3 ::.\.\'q|'
`rr.-‘lite a_,f.'rer om! .irt'g(-'.'.‘."."nH q,f'I‘J.36 trirarot-"fig .-’\"a—PB (bow n-ace).
`The surtiple w:1:~: spiked with R..'i' [31-l5]I'1-l13.
`ll1e t::'Ia1'|tio1't1t;r profile nf \\'hicl1 is
`shown by the thin trace.
`
`0.3
`
`0.2
`
`[L1
`
`
`
`
`
`Ilmnl{R5}.PH!
`
`-00
`
`II
`
`N
`
`120
`
`180
`
`240
`
`SW 360
`
`$20
`
`400
`
`'I'in'II- [min]
`
`Flt}. 3. Tr'me wmwe af'r'R-S}—PI-1B urinar_\r excretion in normal hunmrts ul'tt.'r
`oral ingestion of 0.36 Inmol-’k1__L Na—I’B (ruean * S.E_M.: H = 7].
`
`/IA). Plotting of the data under semilogaritlunie coordinates
`(Fig.
`yielded a linear relationship compatible with a first order kinetic
`process for PR 1]p[F