"Pharmaceutical Sciences, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Zheng, Jiaojiao"@en . "2008-10-11T21:09:22Z"@en . "1993"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Valproic acid (VPA) is an anticonvulsant agent widely used in the treatment of several types of epileptic seizures. The drug is unique within its therapeutic class, in terms of its mechanism of action, its chemical structure, as well as its extensive biotransformation into at\r\nleast 16 different metabolites. The interest in VPA metabolites has\r\nbeen stimulated by the potential of VPA to produce severe\r\nhepatotoxicity. Metabolites 4-ene VPA and 2,4-diene VPA are thought to be responsible for the rare but fatal hepatotoxicity associated with VPA. Thus, methodology to study the pharmacokinetics of VPA metabolitesis important to an evaluation of the role that metabolites may play during VPA therapy.\r\nStable isotope techniques and gas chromatography mass spectrometry (GCMS) have been used in several areas of research on VPA. For example the application of a stable isotope labelled analog as a \"pulse dose\" in antiepileptic drug studies allows the elimination kinetics of the drug to be determined without discontinuing therapy and risking the exacerbation of seizures. In the present study, [21-10PA and [13C4] VPAwere evaluated as to their applicability to pharmacokinetic studies of\r\nVPA. Pharmacokinetic parameters of VPA, [21-161VPA were measured in a\r\nhealthy human volunteer. Potential isotope effects of [2H6JVPA and\r\n[13C4] VPA were studied based on the urine recovery ratio or AUC ratio of VPA and its metabolites to their isotope labelled analogs. No apparent isotope effect was found in the metabolism of [13C4]VPA, which makes [13C4]VPA qualified for use in a \"pulse dose\" manner. Upon [2H6JVPA administration, a large isotope effect was observed in the metabolic formation of [24]5-0H VPA and [2H3]2-PGA, and a small isotope effect was apparent for the formation of [2H6](E)-2,4-diene VPA. Based on the latter observation it was proposed that the formation of 2,4-diene VPA might occur partly from 3-ene VPA. (E)- and (Z)-3-ene VPA were synthesized to test this proposal.\r\nThe use of stable isotope labelled analogs as internal standards can minimize the variance arising from extraction of VPA metabolites due to a slight pH change or incompleteness of derivatization due to time and temperature. In order to obtain optimal analytical results, eight deuterium labelled VPA metabolites were synthesized as internal standards, which included [2H7]VPA, [2H7]2-ene VPA, [2H7]3-keto VPA,[2H7]3-0H VPA, [2H7]4-ene VPA, [2H7]4-0H VPA, [2H7]4-keto VPA, and[2H7]5-0H VPA. These internal standards were applied to the analysis ofVPA, [13C4]VPA and their metabolites in serum and urine samples collected from two nonpregnant sheep following single dose\r\nadministration of VPA:[13C4]VPA (50:50). The elimination half-life of VPA in the sheep was estimated to be approximately 2.5-5 hours.\r\nGCMS conditions for both electron ionization (EI) and negative chemical ionization (NCI) were optimized to obtain the best resolution and sensitivity for VPA metabolites. A single temperature program with a run time of 47 min was established for NCI analysis of PFB derivatives of VPA, [2H6]VPA and their metabolites. Two temperature programs were investigated for the EI analysis of t-BDMS derivatives. One run time of 35 min was used for VPA unsaturated metabolites, while a run time of 20 min was used for the more polar metabolites of VPA.\r\nAll the urine and serum samples were analyzed with both El and NCI techniques. PFB derivatives of VPA metabolites analyzed by the NCI technique gave higher sensitivity and better resolution than t-BDMS derivatives of VPA metabolites analyzed by El methods. All urine samples were hydrolyzed with glucuronidase and with NaOH solution. No difference was observed between the results obtained with the different hydrolysis methods, which indicated that there was little or no fl-glucuronidase-resistant conjugate present in the urine samples of this human volunteer after urine samples were kept at -20 \u00B0C for about two months. The conjugated fractions were measured, more than 90% of VPAand its unsaturated metabolites were excreted into urine in the form of their glucuronic conjugates. Metabolites 3-0H, 4-0H and 5-0H VPA were excreted partly as glucuronides, while 3-keto, 4-keto VPA, 2-PSA and 2-PGA were excreted mostly as the free metabolites.\r\nThe present investigation reaffirmed the importance of GCMS assay techniques to studies of VPA pharmacokinetics and disposition. Stableisotope labelled internal standards improved the accuracy and precision of VPA metabolite analysis by GCMS. [13C4JVPA was ideal for \"pulsedose\" VPA studies of pharmacokinetics, in which techniques it was demonstrated that the pharmacokinetic parameters of VPA metabolites will be obtained for the first time in pediatric patients."@en . "https://circle.library.ubc.ca/rest/handle/2429/2582?expand=metadata"@en . "4962066 bytes"@en . "application/pdf"@en . "METABOLISM AND PHARMACOKINETIC STUDIES OF VALPROIC ACID USING STABLEISOTOPE TECHNIQUESbyJIAOJIAO ZHENGB.Sc. (Chem.) Zhejiang Normal University, 1983M.Sc. (Chem.) Shanghai Institute of Materia Medica, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESFaculty of Pharmaceutical Sciences(Division of Pharmaceutical Chemistry)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJanuary 1993\u00C2\u00A9 JIAOJIAO ZHENG, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of phad-rri.The University of British ColumbiaVancouver, CanadaDate^24 , g3DE-6 (2/88)ABSTRACTValproic acid (VPA) is an anticonvulsant agent widely used in thetreatment of several types of epileptic seizures. The drug is uniquewithin its therapeutic class, in terms of its mechanism of action, itschemical structure, as well as its extensive biotransformation into atleast 16 different metabolites. The interest in VPA metabolites hasbeen stimulated by the potential of VPA to produce severehepatotoxicity. Metabolites 4-ene VPA and 2,4-diene VPA are thought tobe responsible for the rare but fatal hepatotoxicity associated withVPA. Thus, methodology to study the pharmacokinetics of VPA metabolitesis important to an evaluation of the role that metabolites may playduring VPA therapy.Stable isotope techniques and gas chromatography mass spectrometry(GCMS) have been used in several areas of research on VPA. For examplethe application of a stable isotope labelled analog as a \"pulse dose\" inantiepileptic drug studies allows the elimination kinetics of the drugto be determined without discontinuing therapy and risking theexacerbation of seizures. In the present study, [21-10PA and [13C4]VPAwere evaluated as to their applicability to pharmacokinetic studies ofVPA. Pharmacokinetic parameters of VPA, [21-161VPA were measured in ahealthy human volunteer. Potential isotope effects of [2H6JVPA and[13C4]VPA were studied based on the urine recovery ratio or AUC ratio ofVPA and its metabolites to their isotope labelled analogs. No apparentisotope effect was found in the metabolism of [13C4]VPA, which makes[13C4]VPA qualified for use in a \"pulse dose\" manner. Upon [2H6JVPAi iadministration, a large isotope effect was observed in the metabolicformation of [ 24]5-0H VPA and [ 2H3]2-PGA, and a small isotope effectwas apparent for the formation of [ 2H6](E)-2,4-diene VPA. Based on thelatter observation it was proposed that the formation of 2,4-diene VPAmight occur partly from 3-ene VPA. (E)- and (Z)-3-ene VPA weresynthesized to test this proposal.The use of stable isotope labelled analogs as internal standardscan minimize the variance arising from extraction of VPA metabolites dueto a slight pH change or incompleteness of derivatization due to timeand temperature. In order to obtain optimal analytical results, eightdeuterium labelled VPA metabolites were synthesized as internalstandards, which included [ 2H7]VPA, [ 2H7]2-ene VPA, [ 2H7]3-keto VPA,[ 2H7]3-0H VPA, [ 2H7]4-ene VPA, [ 2H7]4-0H VPA, [ 2H7]4-keto VPA, and[ 2H7]5-0H VPA. These internal standards were applied to the analysis ofVPA, [ 13C4]VPA and their metabolites in serum and urine samplescollected from two nonpregnant sheep following single doseadministration of VPA:[ 13C4]VPA (50:50).^The elimination half-life ofVPA in the sheep was estimated to be approximately 2.5-5 hours.GCMS conditions for both electron ionization (EI) and negativechemical ionization (NCI) were optimized to obtain the best resolutionand sensitivity for VPA metabolites. A single temperature program witha run time of 47 min was established for NCI analysis of PFB derivativesof VPA, [ 2H6]VPA and their metabolites. Two temperature programs wereinvestigated for the EI analysis of t-BDMS derivatives. One run time ofiii35 min was used for VPA unsaturated metabolites, while a run time of 20min was used for the more polar metabolites of VPA.All the urine and serum samples were analyzed with both El and NCItechniques. PFB derivatives of VPA metabolites analyzed by the NCItechnique gave higher sensitivity and better resolution than t-BDMSderivatives of VPA metabolites analyzed by El methods. All urinesamples were hydrolyzed with glucuronidase and with NaOH solution. Nodifference was observed between the results obtained with the differenthydrolysis methods, which indicated that there was little or no fl-glucuronidase-resistant conjugate present in the urine samples of thishuman volunteer after urine samples were kept at -20 \u00C2\u00B0C for about twomonths. The conjugated fractions were measured, more than 90% of VPAand its unsaturated metabolites were excreted into urine in the form oftheir glucuronic conjugates. Metabolites 3-0H, 4-0H and 5-0H VPA wereexcreted partly as glucuronides, while 3-keto, 4-keto VPA, 2-PSA and 2-PGA were excreted mostly as the free metabolites.The present investigation reaffirmed the importance of GCMS assaytechniques to studies of VPA pharmacokinetics and disposition. Stableisotope labelled internal standards improved the accuracy and precisionof VPA metabolite analysis by GCMS. [ 13C4JVPA was ideal for \"pulsedose\" VPA studies of pharmacokinetics, in which techniques it wasdemonstrated that the pharmacokinetic parameters of VPA metabolites willbe obtained for the first time in pediatric patients.ivTABLE OF CONTENTSABSTRACT^ iiLIST OF TABLES^ ixLIST OF FIGURES xiiLIST OF SCHEMES^ xivLIST OF ABBREVIATIONS xvACKNOWLEDGEMENT^ xviii1. Introduction 11.1 Overview of VPA 11.2 Mechanism of action 21.2.1 GABAergic hypothesis of VPA 21.2.2 VPA potentiates the postsynaptic response to GABA 41.2.3 VPA action on neuronal membrane 41.3 Metabolism 51.3.1 Conjugated metabolites of VPA 61.3.2 fl-Oxidation pathway of VPA administration 91.3.3 w-Oxidation and (w-1) oxidation 101.4 Pharmacokinetics and pharmacodynamics 111.5 Toxicity 141.6 Chemical^derivatization and analysis 171.7 Stable isotope techniques 181.8 Specific objectives 242. Experimental 262.1 Chemicals and instrumentation 26v2.1.1 Chemicals and reagents 262.1.2 VPA metabolites and internal^standards 272.1.3 Nuclear magnetic resonance spectrometry 272.1.4 Centrifuges 282.1.5 Packed column gas chromatography - mass spectrometry 282.1.6 Capillary column gas chromatography - mass spectrometry 292.1.7 Mass selective detector (MSD) 302.2 Chemistry 302.2.1 Synthesis of [ 2H7]VPA 302.2.2 Synthesis of [ 2H7]4-ene VPA 312.2.3 Esterification of [ 2H7]4-ene VPA 322.2.4 Synthesis of [ 2H7]4-keto VPA 332.2.5 Synthesis of [ 2H7]4-0H VPA 342.2.6 Synthesis of [ 2H7]5-0H VPA 352.2.7 Synthesis of [ 2H7]3-keto VPA 362.2.7a Synthesis of ethyl 3-keto pentanoate 362.2.7b Alkylation of ethyl 3-keto-pentanoate with [ 2H7]bromopropane 372.2.8 Synthesis of [ 2H7]3-0H VPA 382.2.9 Synthesis of [ 2H7]2-ene VPA 392.2.10 Synthesis of (E)-3-ene VPA 402.2.11 Synthesis of (Z)-3-ene VPA 412.3 Pharmacokinetic studies 422.3.1 Pharmacokinetic study with [ 2H6]VPA 422.3.2 Pharmacokinetic study with [ 13C4]VPA 432.3.2a Human study 432.3.2b Animal^study 432.4 Metabolic studies of (E)- and^(Z)-3-ene VPA 44vivi i2.4.1^Study design^ 442.4.2^Metabolism of (Z)- and (E)-3-ene VPA^ 442.5^Calibration curves^ 442.6^Extraction and derivatization^ 482.7^Calculation and data evaluation 522.7.1^Pharmacokinetic parameters^ 522.7.2^Isotope effects^ 522.7.3^Conjugated fraction of VPA and its metabolites in urinesamples^ 522.7.4^Evaluation of data^ 533.^Results and discussion^ 543.1^Synthesis of deuterium labelled internal standards^543.1.1^Synthesis of [2H7]VPA^ 543.1.2^Synthesis of [21-17]4-ene VPA 593.1.3^Synthesis of [ 2H7]4-keto VPA^ 633.1.4^Synthesis of [2H7]4-0H VPA 663.1.5^Synthesis of [ 2H7]5-0H VPA^ 693.1.6^Synthesis of [ 2H7]3-keto VPA 723.1.6a Synthesis of ethyl 3-keto pentanoate 723.1.6b Alkylation of ethyl 3-keto pentanoate with [2H7]bromopropane 753.1.7 Synthesis of [2H7]3-0H VPA 793.1.8^Synthesis of (E)-[ 2H7]2-ene VPA^ 823.1.9^Stereoselective syntheses of (E)- and (Z)-3-ene VPA^863.2^Optimizing GCMS conditions for the analysis of VPA metabolitesin El (t-BDMS derivatives) and NCI (PFB derivatives) modes^92viii3.3 Pharmacokinetics of [ 2H6]VPA and its metabolites in ahealthy volunteer 973.4 Isotope effects of [ 2H6]VPA metabolism 1063.5 Isotope effects with respect to [ 13C4]VPA metabolism 1113.6 Urinary recoveries of VPA and its metabolites 1143.7 Conjugated fraction of VPA and its metabolites in urinesamples 1193.8 Comparison of analyzing and hydrolyzing methods 1233.9 A pharmacokinetic study of VPA in sheep using [ 13C4]VPA 1253.10 Metabolic studies of (Z)-and (E)-3-ene VPA 1324. Summary and conclusions 1355. References 138ixLIST OF TABLESTable 1: Stock solution concentrations (ug/mL) used fer thepreparation of calibration curves for VPA, [916]VPAand their metabolites.^ 45Table 2: Stock solution concentrations (ug/mL) used f9K thepreparation of calibration curves for VPA, [1C4] VPAand their metabolites.^ 47Table 3: Mass to charge (m/z) for the internal standards (*), VPA,and VPA metabolites that were used for ion monitoringin the NCI (PFB derivatives) and El (t-BDMS derivatives)mode.^ 51Table 4: List of the negative ions monitored and retention timesfor the PFB derivatives of VPA, [LHOPA, theirmetabolites and internal standards (I.S.) for theNCI analysis mode.^ 93Table 5: Positive ions monitored and the retention times of thet-BDMS derivatives of VPA, [LHOPA, their unsaturatedmetabolites, and the internal standards (I.S.) in the Elanalysis mode.^ 95Table 6: Positive ions monitored and the retention times of thet-BDMS derivatives of VPA, [LHOPA, their keto andhydroxyl metabolites and internal standards in the Elanalysis mode.^ 96Table 7: Linearity of calibration curves for quantitative assaysof VPA, VPA metabolites and their [LH7]-labelled analogueswhich were isolated from urine saples of a human volunteeradministered with 700 mg of VPA:[917PPA (50:50) every 12hours for two and half days.^ 98Table 8: Linearity of calibration curves for quantitative assaysof VPA, VPA metabolites and their [917]-labelled analoguesin serum total, serum free and saliva samples of a humanvolunteer administered with 700 mg of VPA:rH7PPA (50:50)every 12 hours for two and half days.^ 99Table 9: Pharmacokinetic parameters of VPA(I) and [2H6]VPA(II)measured by NCI technique in serum and saliva samples ofone subject under steady state conditions (5 oral dosesof 700 mg of VPA : [9161VPA).^ 103Table 10: Pharmacokinetic Parameters of deuterium labelled andunlabeled metabolites of VPA measured in a healthyvolunteer under steady state conditions; all data werebased on NCI results.^ 105Table 11: Area under curve (AUC) ratios of VPA, VPA metabolites totheir deuterium labelled analogs over 12 hours after thefinal dose in the serum samples of a,healthy volunteeradministered 5 doses of 700 mg VPA:[ 2 H6 ]VPA (50:50); allvalues were based on NCI results.Table 12: Steady state urinary recovery molar ratio of VPA aqd itsmetabolites to their deuterium labelled analogs, rHOPAand metabolites in a healthy human volunteer administered5 doses of 700 mg of VPA:[ H6]VPA (50:50), based on 12hour urine collected following the final dose*.^109Table 13: Metabolic equivalence of [ 13C4]VPA and VPA based 911 meanTIC peak area ratio of VPA metabolites to their [ 1\u00C2\u00B0C4]-labelled analogs (13 serum samples and urine samplecollected 3-9 hr after the dose from a healthy humanvolunteer administered a single dose of 700 mg of VPA:[\"C4]VPA were analyzed by NCI techniques).^113Table 14: Steady state urinary recoveries of VPA, [ 2H6]VPA andmetabolites (free plus conjugated) in the urine collected12 hr following the final dose , hydrolyzed with NaOHsolution, and analyzed by NCI GCMS.^ 115Table 15: Steady state urinary recoveries of VPA, [ 2H6]VPA andmetabolites (free plus conjugated) in a urine samplecollected for 12 hours following the final dose,hydrolyzed with glucuronidase and analyzed by NCI GCMS.^116Table 16: Steady state urinary recoveries of VPA, [ 2H6]VPA andmetabolites (free plus conjugated, in the urine collected12 hours following the final dose , hydrolyzed with NaOHsolution, and analyzed by EI GCMS.^ 117Table 17: Steady state urinary recoveries of VPA, [ 24]VPA andmetabolites (free plus conjugated) in the urine collected12 hours following the final dose*, hydrolyzed withglucuronidase, and analyzed by EI GCMS.^ 118x108Table 18: Conjugated fraction (%) of VPA and its metabolites inurine samples collected for 12 hours after final dosemeasured by different hydrolysis and assay methods.Table 19: P-values of paired t-test over different hydrolysis andanalysis methods.^ 122Table 20: Correlation coefficients (r 2 , n=11) between concentrationsof VPA urine metabolites measured by different hydrolysis(base or enzyme) and analysis (NCI or EI) methods. Urinesamples were collected from a human volunteer in themultiple dose study (700 mg of VPA : [ H6]VPA (50:50)every 12 hr for 2.5 days).^ 124Table 21: The retention time and m/z values of the (M-57) 4\"diagnostic ions of VPA and its metabolites isolated fromserum sample ,of sheep dosed with single i.v. dose ofT'1 g of VPACOPA (50:50).^ 127Table 22: Linearity of calibration curves for the quantitativeanalysis of VPA, VPA metabolites and their C-13 labelledanalogues isolated from urine samples 9f sheep dosed i.v.with a single dose of 1000 mg of VPA:[ C4]VPA (50:50).^130Table 23: Pharmacokinetic parameter,for VPA(I), [ 13C4]VPA(II),(E)-2-ene VPA (III) and [\"C4](E)-2-ene VPA (IV) measuredin two sheep dosed i.v. with a single 1000 mg dose ofVPA:[ i3C4]VPA (50:50); based on serum samples whichwere analyzed by EI GCMS.^ 131Table 24: Retention time and peak area of monitored ions m/z 199and 197 which represent parent drug 3-ene VPA and itsdiene metabolites. 134xi121LIST OF FIGURESFig. 1.^Proposed metabolic pathways of VPA in humans. The brokenline indicates a likely metabolic route not yet confirmed.The compounds in brackets have also not been confirmed(Kassahun et al., 1989).^ 7Fig. 2:^The structure of 1-0-acyl-P-linked VPA glucuronide.^8Fig. 3:^GCMS mass spectra of the methyl esters of [2H7]/PA(top) and VPA (bottom).^ 57Fig. 4:^1 H NMR spectrum of [2H7]VPA. 58Fig. 5:^GCMS mass spectra of the methyl esters of [2H7]4-ene VPA(top) and 4-ene VPA (bottom).^ 61Fig. 6:^IH NMR spectrum of [ 2H7]4-ene VPA. 62Fig. 7:^GCMS mass spectra of ethyl esters of [2H7]4-keto VPA(top) and 4-keto VPA (bottom).^ 65Fig. 8:^GCMS mass spectra of ethyl esters of [2H7]4-0H VPA (top)and 4-0H VPA (bottom).^ 68Fig. 9:^GCMS mass spectra of the ethyl esters of [2H7]5-0H VPA(top) and 5-0H VPA (bottom). 71Fig. 10: GCMS mass spectra of ethyl 3-keto-pentanoate.^73Fig. 11: 1 H NMR spectrum of ethyl 3-keto-pentanoate. 74Fig. 12: GCMS mass spectra of the ethyl esters of [2H7]3-ketoVPA (top) and 3-keto VPA (bottom).^ 77Fig. 13: 111 NMR spectrum of ethyl ester of [2H7]3-keto VPA.^78Fig. 14: GCMS mass spectra of the ethyl esters of [2H7]3-0H VPA(top) and 3-0H VPA (bottom).^ 81Fig. 15: GCMS mass spectra of the ethyl esters of (E)-[2H7]2-eneVPA (top) and (E)-2-ene VPA (bottom).^ 84Fig. 16: IH NMR spectrum of the ethyl ester of [ 2H7]2-ene VPA.^85Fig. 17: GCMS mass spectra of the methyl esters of (E)- (top) and(Z)-3-ene VPA (bottom).^ 89Fig. 18: 1 H NMR spectrum of (E)-3-ene VPA.^ 90Fig. 19: IH NMR spectrum of (Z)-3-ene VPA. 91xiiFig. 20: Elimination curves of VPA and [21-16]VPA in serum total(top), serum free (middle) and saliva (bottom) whichwere measured with NCI techniques. 101Fig. 21: Time course (12 hr) of labelled and unlabeleda-oxidation metabolites of VPA.^ 104Fig. 22: SIM chromatograms of,[21-17PPA (top, internal standard),VPA (middle), and [-\"COPA (bottom).^ 128LIST OF SCHEMESScheme 1: Sample handling procedure for serum (total and free) andsaliva samples.^ 49Scheme 2: Sample handling procedure for urine samples.^50Scheme 3: Synthesis of [ 2H7]VPA.^ 56Scheme 4: Synthesis of [ 2H7]4-ene VPA. 60Scheme 5: Synthesis of [ 2H7]4-keto VPA.^ 64Scheme 6: Synthesis of [ 2H7]4-0H VPA. 67Scheme 7: Synthesis of [ 2H7]5-0H VPA.^ 70Scheme 8: Synthesis of [ 2H7]3-keto VPA. 76Scheme 9: Synthesis of [ 2H7]3-0H VPA.^ 80Scheme 10: Synthesis of (E)-[ 2H7]2-ene VPA. 83Scheme 11: Synthesis of (E)-3-ene VPA.^ 88x ivLIST OF ABBREVIATIONSAUG^Area Under the CurveBDZ Benzodiazepinesbp^Boiling Pointn-BuLi n-ButyllithiumCL^Clearancecm CentimeterCNS^Central Nervous SystemCSF Cerebral Spinal Fluidd^DoubletDBU 1,8-Diazabicyclo[5,4,0]undec-7-eneE^TransEl Electron ImpactEt0H^EthanoleV Electron VoltGABA^Gamma Aminobutyric AcidGABA-T Gamma Aminobutyric Acid TransaminaseGAD^Glutaric Acid DecarboxylaseGCMS Gas Chromatography Mass Spectrometryh^HourHMPA HexamethylphosphoramideHz^Hertzi.d. Internal DiameterIDMS^Isotope Dilution Mass SpectrometryI.S. Internal Standardi.v.^IntravenousXVxviJ^Coupling Constant in HertzKE Elimination Rate Constantkg^KilogramL LitreLiICA^Lithium n-IsopropylcyclohexylamineLDA Lithium Diisopropylamidem^MultipletM MolarityM+^Molecular IonMe0H Methanolmg^Milligram2-MGA 2-Methylglutaric AcidMHz^MegahertzMIDAS Michigan Interactive Data analysis Systemmin^MinutemL Millilitermmoles^MillimolesMSD Mass Spectrometry DetectorMW^Molecular Weightm/z Mass/chargeNaOH^Sodium hydroxideNCI Negative Chemical IonizationNMR^Nuclear Magnetic ResonancePFB Pentafluorobenzyl2-PGA^2-Propylglutaric Acid2-PSA 2-Propylsuccinic Acidq^QuartetxviiSIM^Selective Ion MonitoringSSA-DH Succinic Semialdehyde Dehydrogenaset^Tripletti/2 Half Lifet-BDMS^tertiary-ButyldimethylsilylTMS TrimethylsilylTHF^TetrahydrofuranTIC Total Ion ChromatogramU^Unitug MicrogramuL^MicrolitreVd Volume of Distributionw^WideZ CisDEDICATIONxixTo Xudong and my parentsxviiiACKNOWLEDGEMENTI sincerely thank Dr. Frank S. Abbott for his generous support andexcellent supervision throughout this program. I am grateful to thecommittee members Dr. James Orr, Dr. Stelvio Bandiera, Dr. JamesAxelson, and Dr. Kathleen MacLeod for their effort and helpfulsuggestions. Special thanks go to Mr. R. Burton for his valuableassistance in GCMS and computer work; Mr. D. Yu for his kind help inGCMS analysis of sheep samples. I very much appreciate the assistancefrom my lab mates, Dr. R. Lee, Dr. K. Kassahun, Mr. A. Borel, Ms. S.Panesar, Mr. J. Palaty and Ms. S. Gopaul.1. Introduction 1.1 Overview of VPAValproic acid (VPA) is a relatively new antiepileptic compoundwhose pharmacological properties were discovered in 1963 (Meunier etal., 1963). Since its first clinical use in France in 1964 (Carraz,1964), valproate or VPA has rapidly established itself worldwide as amajor antiepileptic drug against several types of seizures. In 1983,VPA was marketed as a syrup and gelatin capsule under the trade nameDepakeneR . It was soon recognized as a highly effective first line drugagainst the primary generalized tonic-clonic, and myoclonic seizures.The drug is unique within its therapeutic class, in terms of both itsmechanism of action and its chemical structure, and as a consequence, ithas been the focus of much basic and applied research (Gram et a7.,1985; Chapman et al., 1982).In recent years, interest in VPA metabolites has been stimulated bythe potential of VPA to produce severe hepatotoxicity (Bohan et al.,1987; Dickinson et al., 1985; Kuhara et al., 1985; Zimmerman et al.,1982). The risk of fatal hepatic dysfunction has been assessed at 1 in37000 in patients receiving VPA as monotherapy, and as high as 1 in 500in children younger than 2 years of age who are receiving VPA incombination with other anticonvulsants (Dreifuss et al., 1987).1Although several studies of the pharmacokinetics of VPA and itsmetabolites in adults have been completed, no pharmacokinetic study ofVPA metabolites has been reported for children, the age group mostsusceptible to severe hepatotoxicity. Developing methodology that couldbe applied to a study of the pharmacokinetics of VPA metabolites inpediatric patients is one of the objectives of the present study.1.2 Mechanism of ActionThe mechanism of action of VPA is not clear.^There are threeproposed mechanisms of action:^1) VPA increases brain 7-aminobutyricacid (GABA) (Feriello et a7., 1983); 2) VPA potentiates thepostsynaptic response to GABA (Macdonald and Bergey, 1979) and 3) VPAexerts a direct membrane effect (Slater and Johnson, 1978). A briefdescription for each hypothesis is presented as follows.1.2.1 GABAergic hypothesis of VPAVPA is able to antagonize seizures induced by GABA antagonists,bicuculline and picrotoxin (Frey and Loscher, 1976; Worms and Lloyd,1981) and seizures induced by inhibitors of GABA synthesis, 3-mercaptopropionic acid, isoniazid and allyl-l-glycine (Dren et al.,1979). Research indicated that GABA levels in the whole brain ofrodents are elevated within 15-60 min of administration of VPA(Schechter et al., 1978; Perry and Hansen, 1978) and these remainelevated for 3-8 hours (Nau and Loscher, 1982). Godin (1969) reportedthat VPA inhibits in vitro GABA - transaminase (GABA-T), the enzyme inthe first step of GABA degradation. Harry et 0.(1975) also presentedtheir finding that VPA is a more potent inhibitor of in vitro succinic2semialdehyde dehydrogenase (SSA-DH), the next enzyme in the GABAdegradative pathway. Conversely, the activity of regional (Phillips andFowler, 1982) and whole brain (Loscher, 1981) glutamic aciddecarboxylase (GAD), the GABA synthesizing enzyme, is increased afterVPA administration. The inhibited activities of GABA degradationenzymes and the increased GAD activity all result in increased GABAlevels.NH2-CH2-CH2-CH2-COOHStructure of GABAIn the neuron, GABA is contained in the synaptosomes of nerveterminals as well as in the neuronal metabolic pool of soma and glialcells.^However, only the synaptosomal fraction is involved inneurotransmission.^Therefore, unless a clear distinction is madebetween the two pools, it is impossible to establish the effectivenessof increased levels of whole brain GABA in increasing GABA-mediatedinhibition (Balazs et al., 1970).Drugs which increase GABA function and elevate convulsantthresholds act as anticonvulsants. The fact that GABA levels in thebrain increase after VPA administration has been the basis for the abovehypothesis concerning the mechanism of action of this drug. However,the available evidence is still insufficient to make a final assessmentof the validity of this hypothesis.31.2.2 VPA potentiates the postsynaptic response to GABAVPA has been shown to potentiate GABA-mediated postsynapticinhibition^in^vitro,^which^is^similar^to^the^anticonvulsantbenzodiazepines (BDZ) and barbiturates (MacDonald, 1986). Thepotentiation of GABA by VPA was also observed in the rat corticalneurons in the substantia nigra (Kerwin et al., 1980). However, theconcentration of VPA initially used to potentiate GABA response washigher than that seen in vivo (Harrison and Simmonds, 1982) and when theconcentration was reduced to reflect serum levels, the results ofpotentiation could not be repeated. On the basis of in vitro bindingstudies, Ticku and Davis (1981) suggested that VPA action may be exertedat the picrotoxin binding site of the GABA receptor-chloride ionophorecomplex in the postsynaptic membrane. However, upon furtherinvestigation using a tritiated analogue, no evidence of binding tobrain membranes was found (Morre et al., 1984).1.2.3 VPA action on Neuronal MembraneWhen VPA is at concentrations 15 to 50 times higher than clinicallevels, an increase in membrane conductance to e has been observed inthe Aplysia neuron, a powerful hyperpolarizing mechanism (Slater andJohnson, 1978). Valproate at \"therapeutic\" cerebral spinal fluid (CSF)levels limits the depolarization-induced sustained repetitive firing toa few action potentials (McClean and MacDonald, 1986) through a blockageof voltage-sensitive Na+ influx. Similarly, in studying hippocampalslices, Franceschetti et al (1986) found that VPA markedly depressed45frequency potentiation and paired pulse facilitation.^VPA alsosuppressed spontaneous epileptiform activity and prolonged the afterdischarge elicited by antidromic stimulation (Franceschetti et al.,1986).From the discussion above, VPA was shown to possess direct membraneeffects at clinically obtainable levels. However, the relationshipbetween these direct membrane effects in these test systems and VPA'santiconvulsant effect, although plausible, remain unknown.Although there is considerable evidence to support each hypothesis,for the mechanism of action of VPA, none of the hypotheses cansatisfactorily explain all of its anticonvulsant activity. It istherefore, probable that VPA acts through more than one mechanism inproviding its broad anticonvulsant effects.1.3 MetabolismThe structure of VPA (1) - a branched chain fatty acid, differsdramatically from the substituted heterocyclic ring structure which iscommon to other anticonvulsants.CH3-CH2-CH2CH-COONCH3 - CH2 - CH2( 1 )Despite it structural simplicity, the metabolic fate of VPA iscomplex because of its branched chain structure. This short-chain fattyacid is metabolized in the body by a combination of mitochondrial,microsomal and cytosolic enzymes to produce at least sixteen knownmetabolites (Gugler et al., 1980; Acheampong et al., 1983, 1985;Kassahun et al., 1989; Rettie et al., 1987). In mammals, the fate ofVPA is mainly hepatic metabolism since only 1-3% of the dose is excretedunchanged in the urine (Gugler et al., 1980; Bailer et al., 1985). Themajor metabolic pathways of VPA include conjugation of VPA withglucuronic acid, a-oxidation, w-oxidation and (w-1)-oxidation. A seriesof unsaturated, hydroxyl and glucuronic acid conjugated metabolites areformed. Figure 1 summarizes the metabolic pathways of VPA in human(Kassahun et al., 1989).1.3.1 Conjugated metabolites of VPADirect conjugation^of valproate with glucuronic^acid^isquantitatively the most important route of valproate biotransformation.Metabolism of valproate is dose-dependent, and, at least in humans andrats, glucuronidation accounts for progressively more of a dose as thedose or blood concentration increases (Dickinson et al., 1979; Grannemanet al., 1984). In humans, glucuronidation of VPA varies between 20-70%of recovered dose (Abbott et al., 1986; Chapman et al., 1982).Dickinson et al. (1989) reported VPA glucuronide conjugation accountsfor 59.3 + 25.6 % of VPA dose, based on a study in 24 epileptic patients600HVPA00H3-ene VPAN 1 //51:7\00H(E,E)-2,3'-diene VPA4'-keto-2-ene VPAOH3-0H VPA00H4-keto VPA1Z-INHOOC2-PSA 74,4'-diene VPA^4-ene VPA^(E)-2.4-diene VPA^3-keto-4-ene VPA00-OWVPA glucuronidezCIOH^ 00H5-0H VPA^ 4-OH VPA00HHOOC2-PGA/...\/COOHCOON2- PMA(E)-2-ene VPA^(Z)-2-ene VPA13-keto VPAFig. 1: Proposed metabolic pathways of VPA in humans. The broken lineindicates a likely metabolic route not yet confirmed. The compounds inbrackets have also not been confirmed (Kassahun et al., 1989).under steady state conditions. The corresponding conjugate, 1-0-acyl-fl-linked ester glucuronide (Figure 2), is excreted into urine. Thisconjugate is also present at high concentrations in the bile of ratsgiven VPA (Dickinson et al., 1979), and is consistent with the findingthat VPA undergoes enterohepatic recycling in rats (Dickinson et a7.,1985a).8CH3-CH2-CH2CH-COCH3-CH2-CH2Fig. 2: The structure of 1-0-acyl-8-linked VPA glucuronide.It should be noted that glucuronidation also represents animportant pathway of biotransformation for primary metabolites of VPAthat have been formed by initial oxidative processes (Rettenmeier etal., 1985; 1986a; 1986b). In the case of hydroxylated VPA metabolites,both ether and ester glucuronides can result.Other minor conjugation routes of VPA metabolism exist. Carnitineconjugates (Bohan et e., 1984) were found in the urine of pediatricpatients receiving prolonged administration of the drug. VPA glycineconjugates have been identified in rat urine, together with somewhatgreater quantities of glycine conjugates of unsaturated VPA metabolites(Granneman et al., 1984). The existence of a VPA conjugate withcoenzyme A as a metabolic intermediate in liver tissue was proposed bythe evidence from animal studies (Thurston et al., 1983; 1985), eventhough rigorous structural characterization of this conjugate has notbeen reported. A novel metabolite of VPA, 5-(N-acetylcystein-S-y1)3-eneVPA identified in rat and human urine has been reported by Kassahun etal (1991). In their studies, 5-(glutathion-S-y1)-3-ene VPA was detectedin rat bile following the administration of either (E)-2,4-diene or 4-ene VPA, and it was assumed that GSH reacted in vivo with an activatedform of the diene, namely with the CoA ester.1.3.2 B -Oxidation pathway of VPA administrationThe second major route of VPA metabolism is 0-oxidation.Metabolites generated via this pathway are 2-ene VPA, 3-0H VPA and 3-keto VPA (Granneman et al., 1984). It is noteworthy that the structuresof these three metabolites are formally analogous to the sequentialintermediates of fatty acid 10-oxidation (Prickett and Baillie, 1984).The available evidence indicates that VPA and endogenous lipids competefor the enzymes of 0-oxidation (Bjorge and Baillie, 1985). In fact, VPAand its metabolites are thought to serve as competitive inhibitors offatty acid ig-oxidation, which causes the rare but fatal hepatotoxicity.Recently, stable isotope labelling techniques have been employed toinvestigate the fl-oxidation pathway of VPA metabolism in the rat. Thefindings indicated that have demonstrated that 3-0H VPA is not as wasoriginally suspected an exclusive product of 0-oxidation, but has a dualorigin in vivo being derived largely by direct, cytochrome P450-9dependent hydroxylation of the parent drug (Rettenmeier et al., 1987).This study also confirmed an earlier observation (Nau and Zierer, 1982)that 3-keto-VPA appeared to be formed mainly by oxidation of 2-ene VPArather than derived from 3-0H VPA.Recent studies on mitochondrial metabolism of VPA (Li et al., 1991,Bjorge and Baillie, 1991) identified 3-keto VPA together with threeunsaturated metabolites, viz. (E)-2-ene VPA, 3-ene VPA and (E,E)-2,3'diene VPA after incubating VPA with freshly isolated rat livermitochondria. The 3-ene VPA and (E,E)-2,3'-diene VPA were subsequentlyshown to be metabolites of 2-ene VPA. All three unsaturated metaboliteswere shown to serve as precursors of 3-keto VPA when incubated withmitochondrial preparations (Bjorge and Baillie, 1991). Metabolite 3-0HVPA was not detected as a metabolite of VPA in this in vitro system.However, trace amounts of 3-0H-VPA CoA were detected by HPLC. It wasconcluded that crotonase catalyzes the hydration of 2-ene-VPA CoA to 3-OH-VPA CoA, and that oxidation of the latter species to thecorresponding 3-keto metabolite is mediated by a novel NAD + -dependent 3-hydroxyacyl-CoA dehydrogenase (Li et al., 1991).1.3.3 w -Oxidation and (w - 1) OxidationProducts of w-oxidation of VPA are 5-0H VPA, 2-propylglutaric acid(2-PGA) and 2-propylmalonic acid (2-PMA), while (w-1) oxidation resultsin 4-0H VPA, 4-keto VPA and 2-propylsuccinic acid (2-PSA) (Granneman etal., 1984).10As in the case of its endogenous counterparts, VPA undergoeshydroxylation at the 4 and 5 position by the action of cytochrome P-450enzymes (Prickett and Baillie, 1984), mainly in liver tissue but also inother organs. A possible mechanism to account for the formation of 4-0Hand 5-0H VPA was proposed. A carbon-centered VPA free radical wasformed via hydrogen atom abstraction from position 4 or 5 by theperferryl oxygen of the heme prosthetic group, then followed byrecombination of carbon radical-perferric hydroxide radical pairs toyield the isomeric alcohols (Rettie et al., 1987). 4-Keto VPA is formedfrom 4-0H VPA (Granneman et al., 1984), and was first identified as ahuman metabolite of VPA in a stable isotope \"pulse dose\" experiment(Acheampong et al., 1983).2-PGA and 2-PSA are believed to arise from further oxidation of 5-OH and 4-0H VPA, respectively (Granneman et al., 1984).1.4 Pharmacokinetics and PharmacodynamicsVPA can be administered by intravenous (I.V.), oral and rectalroutes. Among them, the oral route is by far the most widely used.Despite differences in the population (healthy volunteer versusepileptic patients) and formulation (oral solution, immediate releasetablet, enteric-coated tablet), the absolute bioavailability of sodiumvalproate was consistently found to be close to unity (Levy and Shen,1989). The various forms of oral VPA essentially differ only in therate of absorption. The peak plasma levels are usually attained within110.5-2 hours after administration of an oral solution or coated tablet ofVPA.VPA is highly bound (90%) to human plasma albumin at therapeuticconcentrations (Levy and Lai, 1982). This property tends to keep mostof the drug within the vascular compartment. A value of 0.1-0.4 L/Kg ofVd (volume of distribution) is an indication that the distribution ofVPA is limited to the circulation and rapidly exchangeable extracellularwater (Gugler and Von Unruh, 1980).The clearance of VPA is independent of liver blood flow but ishighly dependent on the free fraction. It has been recognized that theblood level-dose relationship for VPA is highly variable betweenpatients. VPA is eliminated almost exclusively by hepatic metabolism(>96% of administered dose, Levy and Shen, 1989). The reported plasma(or metabolic) clearance in healthy volunteers is in the range of 6-11mL/Kg/h (Levy and Shen, 1989). Children younger than five, 5 to 10 and10 to 15 years of age have been reported to have mean VPA clearances of48.3, 39.1 and 24.8 mL/Kg/h respectively (Dodson and Tasch, 1981). Theclearance of VPA was found to increase (14.4-16.5 ml/Kg/h) in adults onpolytherapy, and is thought to be the result of hepatic enzyme induction(Schappel et al., 1980; Hoffman et al., 1981).The elimination half-life of VPA in plasma ranges from 8-16 hoursin adult epileptics (Gugler and Von Unruh, 1980; Bowdle et al., 1980)and 3-12 hr in children (Cloyd et al., 1983).12Besides aging and coadministration of other drugs, pregnancy alsoaffects the clearance of VPA. It was reported (Plasse et al., 1979)that the blood level-to-dose ratio began to decline in the latter partof the second trimester in a pregnant woman and continued through theearly part of the third trimester, finally reaching nadir within 3 weeksof delivery. Following parturition, VPA levels rose rapidly andregained pre-pregnancy values within 2-3 weeks. A similar experience infive pregnant patients was cited by Philbert and Dam (1982). Part ofthe reason for the apparent increase in clearance during late gestationis a decrease in maternal serum protein binding of VPA as a result ofelevated nonesterified fatty acids and hypoalbuminemia (Nau and Krauer,1986).VPA exhibits several distinct pharmacodynamic characteristics ascompared with traditional antiepileptic drugs. The anticonvulsanteffect of VPA has been shown repeatedly to correlate poorly with thesteady-state serum VPA concentration (Chadwick, 1984; Minns et al.,1982). Also, a striking dissociation between serum VPA concentrationand the time course of antiepileptic response has been demonstrated inpatients (Rowan, 1979a; 1979b;) and in several experimental models ofepilepsy (Lockard and Levy, 1976; Pellegrini et al., 1978; Walter etal., 1980).Notably, maximal anticonvulsant effect is usually not observedduring initial drug therapy until sometime after the attainment ofsteady-state serum VPA concentrations. In addition, followingdiscontinuation of VPA administration, seizure control persists long13after the intact drug has been cleared from the systemic circulation(Lockard and Levy, 1976; Harding et al., 1978). Since VPA isextensively metabolized by the liver, one explanation for the abovephenomena is that one or more of its metabolites may contributesignificantly to the antiepileptic action of the drug and that thesemetabolites are eliminated more slowly than the parent drug.In fact, the unsaturated metabolites 2-ene VPA, 3-ene VPA, 4-eneVPA (Loscher, 1981; Loscher et al., 1985) and 2,3'-diene (Abbott et al.,1988) were found to have significant anticonvulsant activity in rodentmodels.1.5 ToxicityAdverse reactions to VPA may be divided into physiological or dose-related side effects, metabolic effects, and unusual or rare drugreactions. Idiosyncratic reactions and effects other than those due tothe pharmacology of the drug may be mediated by the formation of unusualor novel metabolites. Altered target organ responses may also resultfrom genetic abnormalities. Finally, teratogenicity represents anadverse drug reaction which, in the case of valproate, appears to bedose-related (Jaeger-Roman et al., 1986; Diliberti, et al., 1984).Among the idiosyncratic side effects of VPA, hepatotoxicity drawsthe most attention. A comprehensive, retrospective analysis of thecases reported in the United States from 1978 to 1984 has provideddefinitive information on the primary risk for fatal liver failure with14valproate treatment (Dreifuss et al., 1987). According to this study,patients at risk are children two years old or younger who receivevalproic acid as part of anticonvulsant polypharmacy and who also haveother medical problems besides severe epilepsy, e.g., mentalretardation, developmental delay, and metabolic disorder. The incidenceof hepatotoxicity found is as high as 1 in 500 in pediatric patientswith multi- therapy. Outside of this group, the overall risk of fatalhepatic dysfunction with valproate (1 in 12,000 with polytherapy versus1 in 45,000 with monotherapy) is still higher among patients receivingmultiple anticonvulsants. In addition, no cases of hepatic failure wereidentified among those persons over the age of 10 who were administeredvalproate monotherapy (Dreifuss et al., 1987).The most common histopathological feature of VPA-induced liverinjury is microvesicular steatosis, similar to that produced by thetoxic metabolites of hypoglycin A and by 4-pentenoic acid (Rettie etal., 1988) which is a potent inhibitor of mitochondrial fatty acidmetabolism (Corredor et al., 1967). Evidence has been obtained fromstudies in vivo (Mortensen, 1980; Mortensen et al., 1980; Kesterson etal., 1984) and in vitro (Thurston et al., 1983; Bjorge & Baillie, 1985;Thurston et al., 1985) that VPA inhibits the fi-oxidation of endogenousfatty acids.Studies in animals have indicated that several metabolitescontribute to the toxic effects of VPA. The 4-ene VPA and 2,4-diene VPAmetabolites are hepatotoxic in rats (Kesterson et al., 1984) and arethought to be responsible for the rare but fatal hepatotoxicity1516associated with VPA. These metabolites may cause damage to livermitochondria, inhibit fatty acid a-oxidation activity and causeaccumulation of hepatic lipids (Zimmerman et al., 1982; Rettenmeier etal., 1986a). Additional studies with 4-ene VPA served to reinforce theview that this terminal olefin, like 4-pentenoic acid, acts as amechanism-based irreversible inhibitor of enzymes of the fatty acid /3-oxidation complex, and then induces the microvesicular steatosis in vivo(Rettenmeier et al., 1985, 1986a). In fact, 4-ene VPA was shown to bethe most toxic metabolite of VPA in the rat (Kingsley et al., 1983).Since the formation of 4-ene VPA is mediated by cytochrome P450, theprevalence of 4-ene VPA may be increased by the presence of enzymeinducers (Rettie et al., 1987). This may in part be the explanation whypatients on polytherapy are at considerably greater risk of developingfatal hepatic dysfunction than patients receiving valproate monotherapy.Kassahun et al. (1990) reported the discovery of GSH 3-ene VPA inrat bile following the administration of either (E)-2,4-diene VPA or 4-ene VPA. Since 2,4-diene is considered to be a mitochondrial oxidationproduct of 4-ene VPA CoA (Rettenmeier et al., 1985), it is conceivablethen that, by virtue of its electrophilic nature, (E)-2,4-diene VPA CoAmay bind to a nucleophilic site of a mitochondrial enzyme. This wouldaccount for the potent inhibition of mitochondrial a-oxidation of fattyacids observed for (E)-2,4-diene VPA in rats (Kesterson et al., 1984).An alternate mechanism for the hepatotoxicity of the reactive (E)-2,4-diene VPA CoA ester, according to Kassahun's study could be thelocalized depletion of GSH in mitochondria.Since fl-oxidation of VPA would require that the drug be in the formof its CoA thioester (Bjorge and Baillie, 1991; Li et al., 1991), itappears likely that the effects of VPA on the hepatic lipid metabolismare mediated in part by the ability of the drug to sequester limitedpools of CoASH, the obligatory cofactor for fl-oxidation.1.6 Chemical Derivatization and AnalysisGas chromatography mass spectrometry (GCMS) is widely used for theanalysis of VPA and its metabolites. The reported GCMS methods includeelectron impact (El) of the t-butyldimethylsilyl (t-BDMS) andtrimethylsilyl (TMS) derivatives (Abbott, et al., 1986; Nau et al.,1981; Rettenmeier et al., 1986b; 1989; Tatsuhara et al., 1987) andnegative chemical ionization (NCI) of pentafluorobenzyl (PFB)derivatives (Abbott et al., 1987; Kassahun et al., 1989). A completeGCMS assay using El techniques for VPA and its metabolites was reportedby Abbott et al. (1986). With the El method, t-BDMS derivatives of VPAand its metabolites have advantages over TMS derivatives except for thatof 3-0H VPA. When derivatized with t-BDMS, VPA and its metabolites haveincreased sensitivity in El mode because of the intense (M-57)4-fragments formed in contrast to the less intense (M-15)1\" fragment fromTMS derivatives. While very good sensitivity was obtained for the t-BDMS derivatized VPA unsaturated metabolites, problems arise in theanalysis of the keto and hydroxy metabolites which can yield eithermono- or di- derivatives depending upon the derivatization conditionsused. The 3-0H VPA does not derivatize readily under the conditionssuitable for derivatizing other metabolites with t-BDMS.17NCI GCMS is a very sensitive and specific assay method for theanalysis of valproic acid metabolites. The PFB derivatives produceabundant [M-181] - ions except for 3-keto VPA which gave an [M-181-0O2] -ion. However, when a combination of PFB and TMS (for hydroxy and 3-ketomoiety) derivatization was used, 3-keto VPA formed an [M-181] - ion whichwas practically the only ion present in the mass spectrum. Hydroxymetabolites derivatized by this method gave significant improvement inboth peak shape and sensitivity of detection (Kassahun et al., 1989,1990). The PFB derivatives (NCI mode) proved to be 30-50 times moresensitive than the t-BDMS derivatives under the El mode (Kassahun etal., 1989).1.7 Stable Isotope TechniquesStable isotope techniques have been used in different areas of VPAstudies including steady-state kinetics (Acheampong et al., 1984),bioavailability studies (Strong et al., 1975), drug interaction studies(Von Unruh et al., 1980), drug metabolism studies (Acheampong et al.,1983), and mechanistic studies of drug metabolism (Rettie et al., 1988;Rettenmeier et al., 1987).The identification of VPA metabolites is hindered by the chemicallability of some of the metabolites as well as the structural similarityof VPA to endogenous compounds. The use of stable-isotope labelleddrugs has been widely applied to facilitate identification of drugmetabolites (McMahon et al., 1973; Pohl et al., 1975). Usually an18equimolar mixture of unlabeled and stable isotope-labelled drug isadministered and biological fluids are examined by mass spectrometry forcharacteristic ion-doublets indicative of drug-derived metabolicproducts.A stable isotope technique was used in this lab (Acheampong et al.,1983) to search for new metabolites of VPA in human serum and urine.GCMS proved useful to clarify the identity of compounds described eitheras endogenous compounds or as VPA metabolites. In this study, a pulsedose of di-[(3,3,3-2H3)-propy1]-acetic acid ( [2H6}VPA, (2) ) wasadministered to a human volunteer at steady state serum concentrationsof unlabeled VPA. The location of the deuterium on the terminal carbonsprovided ion doublets in the mass spectra of GC metabolite peaks withmass differences of either three or six amu.C2H3-CH2-CH2 \CH-COOHr2u ru ru 1\u00E2\u0080\u0098- H3-112-'-'12(2)Stable isotope technique can also determine changes in the kineticparameters of a drug in patients on multiple dose therapy (Sullivan etal., 1975; Kapetanovic et al., 1980). To determine kinetic parametersof an anticonvulsant in patients on multiple dosing or multi-therapy, a'pulse' dose of stable isotope-labelled drug offers a convenient1920technique. Application of this method to antiepileptic drug studiesallows the elimination kinetics of the drug to be determined withoutdiscontinuing therapy and risking the exacerbation of seizures. Thelabelled drug elimination phase can be followed for 3-4 half-livesduring subsequent uninterrupted multiple dosing of unlabeled drug. Thistechnique was applied first by Von Unruh et al. (1980) using di-[(2,3'-2H2) propyl]-acetic acid to study the elimination kinetics of VPA understeady state conditions in patients on combined antiepileptic drugtherapy. Similar methodology was applied by Acheampong et al. in whicha human volunteer was given a single dose of di-[(3,3,3- 2H3)-propy1]-acetic acid (Acheampong et al., 1984). In the latter case, an isotopeeffect was observed for metabolites in the co-oxidation pathway.A stable isotope-labelled drug which shows a significant biologicalisotope effect is not appropriate for use in pharmacokinetic studies,particularly when metabolites are to be measured. Thus the position ofthe label in the drug should be optimal for minimizing isotope effectson metabolic reactions. In the present study, potential isotope effectsin the in vivo metabolism of [ 13C4]VPA and [ 2H6]VPA will beinvestigated. If no significant isotope effect is found in [ 13C4]VPA asexpected, [ 13C4]VPA will be used in pharmacokinetic studies of VPA andits metabolites in pediatric patients. Hence, pharmacokineticparameters for VPA metabolites will be determined for the first time inchildren.Stable isotope methodology, besides providing the identification ofnew metabolites and determining elimination kinetics under steady stateconditions, also represents a powerful technique for studies on theorigins of drug metabolites and for the elucidation of complex metabolicinter-relationships in vivo.VPA or its metabolites are thought to serve as competitiveinhibitors of fatty acid fl-oxidation, which causes the rare but fatalhepatotoxicity. It is known that VPA is metabolized to three products(2-ene VPA, 3-0H VPA and 3-keto VPA) whose structures are formallyanalogous to the sequential intermediates of fatty acid fl-oxidation(Prickett & Baillie, 1984). Rettenmeier and coworkers (1987) attemptedto determine whether these three metabolites share a common metabolicorigin in vivo and represent products of fl-oxidation activity usingstable isotope techniques. In that study, the metabolism of 2-[2H1]VPA(3) and 3,3-[ 2H2]VPA (4) was compared to unlabeled VPA. When 2-[2H1]VPAwas administered to rats, the metabolite 3-0H VPA exhibited partiallabeling (60%). This result demonstrated clearly that the 3-0H VPAformed in that experiment was not produced exclusively via fl-oxidation.When [3,3- 2H2]VPA was given to rats, the deuterium content of 3-keto-VPAwas very close to that of 2-ene VPA, which suggested that 3-keto-VPA wasformed solely via 2-ene VPA.CH3CH2CN\C2H-COOHCH3 CH2 C2N\CH-COOHCH3C H2CH 2^ CH3 C H2CH2212 - [21.11]VPA (3)^3-[2H2]VPA (4)Since kinetic isotope effects can be associated with the formationof some metabolites, stable isotope techniques are useful in mechanisticstudies of drug metabolism. Rettie and his coworkers (1988) used thistechnique in studying the formation of 4-ene VPA. The metabolite 4-eneVPA was shown to be the most toxic metabolite of VPA in rat hepatocytesin culture (Kingsley et al., 1983) and to be considerably more potent asan inducer of steatosis in young rats than was the parent drug(Kesterson et al., 1984).Based on the supposition that a carbon-centered free radical,localized at either C-4 or C-5 is formed before eliminating hydrogen toyield 4-ene VPA (Rettie et al., 1987), the mechanism of the denaturationreaction to form 4-ene was studied by inter- and intra- moleculardeuterium isotope effect experiments using deuterium substitutions ofVPA at either C-4 or C-5 (Compounds 5, 6, 7, 2, Rettie et al., 1988).22CH3C 2H2CH2CH-COON ;CH3CH2CH24-[ 2H2]VPA (5)CH3C2H2CH2CH-COONru r2u ru /%-113k, \"2L124,4'-[ 2H4]VPA (6)C 2H3CH2CH \CH-COON ;CH3CH2CH25-[ 2H3]VPA (7)C2H3CH2CH \CH-COONC2H3 CH2CHru ru I\"3'-\"2'-\"25,5'-[ 2H6]VPA (2)A large primary kinetic isotope effect was obtained for theformation of 4-0H VPA from 4-[ 2H2]VPA and for the formation of 5-0H VPAfrom 542H3]VPA. Conversely, some fl-secondary kinetic isotope effectscould be observed for the formation of 4-OH-VPA from 5-[2H3]VPA and forthe formation of 5-0H VPA from 4-[2H2]VPA. After calculating theisotope effects of 4-ene VPA, 4-0H VPA and 5-0H VPA, the authors foundthat the formation of 4-ene VPA and 4-0H from hexadeuterated VPA 2showed a small or no isotope effect, while high isotope effects wereobserved for 4-ene VPA and 4-0H VPA formed from compound 6. It was thenconcluded that removal of a hydrogen atom from the subterminal C-4position of VPA to form a carbon-centered free radical at C-4 is ratelimiting in the formation of both 4-ene and 4-0H, and the influencewhich the second hydrogen/deuterium exerts to form 4-ene on the overallkinetics of the reaction is small. Therefore, it is a carbon-centeredfree radical, localized at C-4 instead of C-5 that is formed beforeeliminating hydrogen to yield 4-ene VPA (Rettie et al., 1988).Another application of stable isotopes is also called isotopedilution mass spectrometry (IDMS). IDMS methods for organic analytesinvolve spiking a sample with a labelled version of the analyte as aninternal standard, processing the sample, and then measuring the ratioof unlabeled to labelled analyte by using GCMS. IDMS has been thetechnique of choice for definitive methods at the National Institute ofStandards and Technology, since it does not depend on sample recovery,shows high precision, and can be tested for bias and unknowninterferences.23When stable-isotope labelled VPA and its metabolites were used asinternal standards, calibration curves with high correlationcoefficients and good reproducibilities were obtained (Abbott et al.,1986; Kassahun et al., 1990; Von Unruh et al., 1980). The variance inextraction of VPA metabolites due to a slight pH change orincompleteness of derivatization due to time and temperature can beminimized by using stable isotope-labelled analogs as internalstandards.^In GCMS analysis, especially under CI conditions, thespectra depend on source pressure and temperature.^More accuratemeasurements can be made with a stable isotope-labelled internalstandard since the physicochemical properties of the internal standardclosely approximate that of the analyte.1.8 Specific objectives1. To investigate potential isotope effects of the metabolism of[ 2H6]VPA and [ 13C4]VPA in a healthy human volunteer.2. To determine the pharmacokinetics of [ 2H6]VPA, and itsmetabolites in a healthy volunteer, and compare the pharmacokineticbehavior of [ 2H6]VPA and its metabolites with those of unlabeledanalogs.3.^The applicability of [ 13C4]VPA for pulse dose studies of thepharmacokinetics of VPA and metabolites requires the development of a2425precise and sensitive assay technique.^This project will focus onevaluating and improving GCMS methods for the assay of labelled andunlabeled VPA metabolites.4. As part of the assay development, urine, serum and salivasamples are to be analyzed using different ionization methods (NCI andEl) to compare the sensitivities and reproducibilities of these two GCMSmethods. The conjugated fraction of VPA and its metabolites in urineare to be measured by hydrolyzing conjugates with alkali and withglucuronidase, in order to examine if any glucuronidase resistantconjugates exist.5. To synthesize stable isotope-labelled internal standards inorder to facilitate the quantitative analysis of VPA, [ 13C4]VPA andtheir metabolites by GCMS. The labelled metabolites to be synthesizedare:[2117]VPA;^ [2117]2-ene VPA;[2H713-keto VPA;^[2H7]3-OH VPA;[2117]4-ene VPA; [21-17]5-0H VPA;[21-17]4-01-1 VPA;^[2H7]4-keto VPA6. To perform preliminary study of the use of stable isotope labelledinternal standards in a pharmacokinetics assay of [13C4] VPA and itsmetabolites in nonpregnant sheep.2. Experimental2.1 Chemicals and Instrumentation2.1.1 Chemicals and ReagentsChemicals were reagent grade and were obtained from the followingsources:Aldrich Chemical Co. (Milwaukee, WI):Butyllithium (1.6 M in hexane), 1,8-diazabicyclo[5,4,0] undec-7-ene(DBU), DiazaldR, diisopropylamine, diisopropylethylamine,hexamethylphosphoramide, methanesulfonyl chloride, triethylamine, (E)-2-pentenoic acid, bromopropane, lithium aluminum hydride, propionylchloride, 4-pentenoic acid, tetrahydrofuran, isopropylcyclohexylamine.MSD Isotopes (Montreal, Canada):1,2,3,1'-[ 13C4]-2-propylpentanoic acid, [ 2H7] propyl bromide.BDH Chemicals (Toronto, Ontario):Benzene, chloroform, ether, hydrochloric acid, magnesium sulfate,sodium hydroxide, sodium sulfate, sulfuric acid, hexane.Caledon Laboratories Ltd. (Georgetown, Ontario):Dichloromethane, ethanol, ethyl acetate.26ICN Pharmaceuticals Inc. (Plainview, NY):Di-n-propylacetic acid (Valproic acid)2.1.2 VPA metabolites and internal standardsThe synthesis of the following VPA metabolites for use asanalytical standards has been described elsewhere: 3-ene VPA(stereochemistry undetermined), 4-ene VPA, 3-0H VPA, 4-0H VPA, 5-0H VPA,3-keto VPA, 4-keto VPA, 2-PSA and 2-PGA (Acheampong et al., 1983);(E,E)-2,3'-diene VPA (Acheampong et al., 1985); (E)-2,4-diene VPA (Leeet al., 1989).The internal standards used for the assay of VPA, [2H6]VPA andtheir metabolites were [2H3]2-ene VPA, synthesized by Abbott et al.(1986); [2H3]3-keto VPA, a kind gift from Dr. T.A. Baillie (Universityof Washington, School of Pharmacy, Seattle, WA); di-n-butylacetic acid;and 2-methylglutaric acid (2-MGA). In both the El and NCI modes, di-n-butylacetic acid was used as the internal standard for VPA and [2H6]VPA;[2H3]3-keto VPA for 3-keto VPA and [24]3-keto VPA; 2-MGA for 2-PSA, 2-PGA and their deuterated analogs; [ 2H3]2-ene VPA for all othermetabolites.2.1.3 Nuclear Magnetic Resonance SpectrometryHigh field 1H NMR spectra were obtained on the Bruker WH-400 andVarian XL-300 spectrometers in the Department of Chemistry, Universityof British Columbia NMR facility. Spectra were acquired in CD3C1 withtetramethylsilane as an internal standard. Carbon-13 NMR spectra were27obtained on the Varian XL-300 spectrometer in the Department ofChemistry.2.1.4 CentrifugesUnbound drugs were separated from serum samples by the use ofultrafiltration. Protein becomes selectively partitioned into afraction of the sample volume (retentate), while free ligand passesessentially unhindered through the membrane along with solvent into theultrafiltrate.^The physical separation does not change free ligandvolume or concentration.^CentrifreeTM micropartition system (Amicon,W.R. Grace & Co, Danvers, MA) and Beckman centrifuge, model J2-21,equipped with a 45\u00C2\u00B0 rotor were used to achieve this purpose. Serumsamples were centrifuged at a speed of 3500 rpm for 30 minutes.2.1.5 Packed Column Gas Chromatography - Mass SpectrometryGCMS analysis of synthesized compounds was performed on a HewlettPackard 5700A gas chromatograph interfaced to a Varian MAT-111 massspectrometer using a variable slit separator. Electron impact (EI) datawere recorded in scanning mode with a range of 15 - 750 mass units every5 seconds, and the data were processed using a Packard Bell computer(IBM AT clone) and a program developed in our laboratory. Total ioncurrent (TIC) plots were based on scanning range of m/z 50 - 500.The instrument was operated with an emission current of 300 uA,ionization of 70 eV, and source pressure of 5 x 10 -6 Torr. The column28(1.8 m x 2 mm i.d.) was packed with 3% Dexsil 300 on 100 - 120 meshSupelcoport. The oven temperature program was 50 - 300 \u00C2\u00B0C at 16 \u00C2\u00B0C/min.Injection port temperature was 250 \u00C2\u00B0C and the separator temperature setat 250 \u00C2\u00B0C. The carrier gas, with a flow of 25 ml/min, was helium.2.1.6 Capillary Column Gas Chromatography - Mass SpectrometryThe quantitative and qualitative analyses of VPA and itsmetabolites were performed on a Hewlett Packard 5987A GCMS having anopen-split interface. Data recording and processing were managed with aHP-1000 on-line computer. El techniques were used to analyze t-BDMSderivatives with an electron ionization energy of 70 eV. Operatingconditions were: source temperature 200 \u00C2\u00B0C, transfer line temperature240 \u00C2\u00B0C, injection port temperature 240 \u00C2\u00B0C and helium flow-rate 1 ml/min.The capillary column was an OV-1701 bonded phase, 25 m x 0.32 mm i.d.,with a film thickness of 0.25 um (Quadrex Scientific, New Haven, CT,U.S.A.). Two temperature programs were utilized for the GCMS, EIanalysis. The temperature program used for VPA and its unsaturatedmetabolites was initiated at 50 \u00C2\u00B0C, programmed at 30 \u00C2\u00B0C/min to 110 \u00C2\u00B0C,held for 18 min before being increased to 260 \u00C2\u00B0C at rate of 8 \u00C2\u00B0C/min.The temperature program used for the remaining polar metabolites of VPAwas initiated at 50 \u00C2\u00B0C, programmed to 100 \u00C2\u00B0C at 30 \u00C2\u00B0C/min, thenincreased to 250 \u00C2\u00B0C at rate of 8 \u00C2\u00B0C/min.In the negative ion chemical ionization (NICI) mode, ultra-highpurity methane was used as the reagent gas. The capillary column usedwas DB-1 (25m x 0.25mm, with a film thickness of 0.25 um, J&W29Scientific, Rancho Cordora, CA, U.S.A.). The source pressure was 0.8 -1.2 Torr, ionization energy 200 eV, and the emission current 250 uA.The instrument was programmed for SIM as well. The initial oventemperature was 50 \u00C2\u00B0C, programmed to 140 \u00C2\u00B0C, held at 140 \u00C2\u00B0C for 20 minbefore being increased to 260 \u00C2\u00B0C at 5 \u00C2\u00B0C/min.2.1.7 Mass Selective Detector (MSD) All samples obtained from the VPA study in sheep were extracted,derivatized with t-BDMS and analyzed with a HP 5890 GC interfaced to aHP 5971A MSD using EI mode. Data recording and processing were managedwith a HP VECTRA R 486 data system. Operation conditions were: injectiontemperature 250 \u00C2\u00B0C, ion source temperature 180 \u00C2\u00B0C, GCMS interfacetemperature 280 \u00C2\u00B0C, and helium flow-rate 1 ml/min. The capillary columnused was DB-1701 (30m x 0.25mm, with a film thickness of 0.25 um, J&WScientific, Rancho Cordora, CA, U.S.A.). The initial oven temperaturewas 80 \u00C2\u00B0C, programmed to 100 \u00C2\u00B0C at rate of 10 \u00C2\u00B0C/min, then to 130 \u00C2\u00B0C at2 \u00C2\u00B0C/min, and finally to 260 \u00C2\u00B0C at 30 \u00C2\u00B0C/min and held for 8 minutes.2.2 Chemistry:2.2.1 Synthesis of (2H71VPAIn a dry 250 ml 3-neck flask equipped with a mechanical stirrer,drying tube, dropping funnel, and reflux condenser, n-butyllithium inhexane (30 mL of 1.6 M, 48 mmol) was added dropwise to a solution ofdiisopropylamine (6.8 mL, 48 mmol distilled over CaH2) in 40 mL of THE30(distilled over L1A1H4) at 0 \u00C2\u00B0C under N2 atmosphere and stirred foranother 30 minutes. Distilled valeric acid (2.45 mL, 23 mmol) in 10 mLof THF was added dropwise so that the temperature of the reaction neverexceeded 5 \u00C2\u00B0C. The solution at first became milky, but when 5 mL ofHMPA was added the solution became clear. The reaction was allowed toproceed for 30 min before [2H7] bromopropane (18 mmol) was added to themixture. The ice/water bath was then removed and the mixture stirred atroom temperature for 3 h. The reaction was again cooled in ice andquenched with 15% HC1 (cooled) until a pH of 1 was attained. Themixture was extracted with diethyl ether (50 mL x 3), the ethereal layerwashed with 10% HC1 and water, and then dried over anhydrous Na2SO4.The solvent was removed by flash evaporation and the residue wasfractionally distilled (110-113 \u00C2\u00B0C/10 mm Hg) to afford 1.2 g of pure[2H7] VPA (yield 44%). A small portion of the sample was derivatizedwith diazomethane (Levitt, 1973) for GCMS analysis. GCMS mass spectrumof the methyl ester of [ 2H7]VPA, m/z(%): 89(100), 123(30), 117(28),61(23), 134(5), 106(5).1H NMR of [2H7]VPA (CDC13): 6 0.93 (t, 3H, CH3), 1.37 (m, 2H,CH3CH2), 1.46-1.62 (m, 2H, CH2CH), 2.39 (dd, 1H, CHCOOH), 8.0 (w, 1H,COOH).2.2.2 Synthesis of [2H7]4-ene VPAIn a dry 500 mL 3-necked flask equipped with a dropping funnel,condenser, mechanical stirrer, and drying tube, 90 mL of n-butyllithium(0.144 mol) in hexane was added dropwise to a solution of31diisopropylamine (20.2 mL, 0.144 mol) in 120 mL of THE (distilled overLiA1H4) in an ice/water bath under N2 atmosphere.^The mixture wasstirred for 30 min.^4-Pentenoic acid (6.75 mL, 0.066 mol) was addeddropwise over a period of 15 min, followed by 15 mL of HMPA (0.066 mol).The reaction was then allowed to proceed for 30 min. [ 2H7] Bromopropane(6.2 g, 0.06 mol) was added and the ice/water bath removed. The mixturewas stirred at room temperature for 2.5 hours before being quenched with15% HC1 solution. The aqueous layer was extracted 3 times with etherand the combined ether extracts dried over anhydrous Na2SO4. Thesolvent was removed by flash evaporation. A yield of 5.4 gram (77%) of[ 2H7]4-ene VPA was obtained by fractional distillation (110-120 \u00C2\u00B0C/10 mmHg). A small portion of the sample was derivatized with diazomethane(Levitt, 1973) for GCMS analysis.GCMS mass spectrum of the methyl ester of 4-ene [ 2H7]VPA, m/z(%):113(100), 59(67), 104(58), 115(37), 132(11), 163(M + , 2).1 H NMR of 4-ene [ 2H7]VPA (CDC13): (5 2.28 (m, 1H, CH2CH), 2.38-2.43(m, 2H, CH2CH), 5.03 (dd, 1H, CH2=CHCH2, J=9 Hz, cis), 5.08 (dd, 1H,CH2=CHCH2, J=15 Hz, trans), 5.78 (m, 1H, CH2=CH), 8.5 (w, 1H, COOH).2.2.3 Esterification of (2H7]4-ene VPAIn a 100 mL round-bottomed flask equipped with Dean-Starkapparatus, drying tube, and a reflux condenser, [ 2H7]4-ene VPA (3.7 g,0.027 mol), ethanol (6 mL), concentrated sulfuric acid (0.5 mL), and 12mL of benzene were refluxed at 90 \u00C2\u00B0C for 46 hours. The organic layer32was consecutively washed with saturated NaHCO3 solution and water untilthe wash was neutral, then dried over anhydrous Na2SO4. A yield of 3.75g of ethyl [2H7]4-ene VPA was obtained by fractional distillation (80\u00C2\u00B0C/10 mm Hg, 85% yield).GCMS mass spectrum of the ethyl ester of [2H7]4-ene VPA, m/z(%):104(100), 127(88), 59(86), 99(73), 143(17), 189(e, 1).2.2.4 Synthesis of [2H734-keto VPABenzoquinone (1.32 g) and PdC12 ( 0.033 g) were added to a mixtureof 38.5 mL of dimethyformamide and 5.5 mL of water in a 50 mL round-bottomed flask equipped with a reflux condenser. To this stirredmixture, ethyl [2H7]4-ene VPA (1.87 g, 0.013 mol) was then addeddropwise with a syringe over a period of 10 min and the reaction wasallowed to proceed at room temperature for 22 h. The product was thenpoured into 20 mL of cold 10% HC1 solution and extracted 3 times withether (80 mL x 3). The combined organic layer was washed three timeswith 20 mL of 10% NaOH solution, once with saturated NaC1 solution, andwas then dried over anhydrous Na2SO4. The solvent was removed by flashevaporation and the residue was fractionally distilled to afford 1.18 gof pure ethyl [ 2H7]4-keto VPA ( 110-115 \u00C2\u00B0C/10mm Hg, 60% yield).To 0.54 mL of ethyl [2H7]4-keto VPA in a 25 ml round-bottomedflask, 1.55 g of NaOH in 6 mL of H20 and 5 mL of CH3OH was added andheated at 50 \u00C2\u00B0C for 4 h.^Using an oil bath at 120\u00C2\u00B0C, methanol andethanol were distilled off at atmospheric pressure.^The remaining33aqueous solution was washed with ether (3 x 20 mL) to extract anyunhydrolyzed ethyl ester. The remaining aqueous solution was thenacidified to pH 2 with diluted HC1 and extracted with ether three times(3 x 40 mL).^The combined ethereal layers were dried over anhydrousNa2SO4.^The ether was evaporated under flash evaporation and theresidue was further dried under vacuum (0.5 mm Hg) for 3 hours. [ 2H7]4-keto VPA (0.43 g) was obtained (93% yield). A small portion of thesample was derivatized with diazomethane (Levitt, 1973) for GCMSanalysis.GCMS mass spectrum of the methyl ester of [ 2H714-keto VPA, m/z(%):43(100), 122(88), 87(40), 89(33) 75(28), 103(20), 148(17), 164(2),179(M+ ,1).2.2.5 Synthesis of [2H7]4-0H VPATo a well stirred mixture of 170 mg of NaBH4 (4.47 mmol) in 8 mL ofethanol, 0.64 g (3.3 mmol) of ethyl [ 2H7]4-keto VPA was added dropwiseover a period of 10 min. The reaction was allowed to proceed for 60 minat room temperature, and then quenched with saturated NH4C1 solution.The aqueous solution was extracted 3 times (3 x 20 mL) with ether andthe combined ethereal layers were dried over anhydrous Na2SO4.^Thesolvent was removed under flash evaporation.^The residue wasfractionalized by distillation (115-118 \u00C2\u00B0C / 8 mm Hg ) and 170 mg (yield26%) of ethyl [ 2H7]4-0H VPA was then obtained.34GCMS mass spectrum of ethyl [2H7]4-0H VPA, m/z(%): 101(100),43(45), 134(8), 116(5), 151(2).Ethyl [2H7]4-0H VPA (170 mg), 0.75 mL methanol and 0.3 g of NaOH in3 mL of H20 were heated at 50 \u00C2\u00B0C for 2 hours. After being cooled, thesolution was washed with ether to remove any unhydrolyzed ester.2.2.6 Synthesis of [2H7] 5-0H VPATo 0.75 g of ethyl [2H7]4-ene VPA (4.1 mmol) in dry THF in a 50 mLthree neck round-bottomed flask cooled to 4 \u00C2\u00B0C with ice. Under N2, 1.5mL of borane - THE was added dropwise. After the mixture was stirred atroom temperature for 30 min, the flask was immersed into an ice/waterbath and 100 gl of water was added to destroy excess hydride. To thiswas added after 5 min, 4 mL of 1.5 N NaOH followed with 0.5 mL of 30 %H202. The reaction temperature was maintained at 40-50 \u00C2\u00B0C for 1 h. Thereaction was then quenched by pouring into 20 mL of ice/water.^Thesolution was extracted 3 times with 30 mL of ether.^The combinedethereal layer was washed with water followed by saturated NaC1 solutionand then dried over anhydrous Na2SO4.^The solvent was removed underflash evaporation.^The residue was purified by flash columnchromatography (Rettenmeier et al., 1985) using a mobile phase of 5%methanol in ethyl acetate. A yield of 150 mg (19%) of ethyl [2H7]5-0HVPA was obtained.GCMS mass spectrum of the ethyl ester of [2H7]5-0H VPA, m/z(%):101(100), 57(28), 115(12), 150(7), 196(M+, 1).35Ethyl [ 2H7]5-0H VPA (150 mg) was hydrolyzed by being heated with 1mL of methanol and 0.35 g of NaOH in 4 mL of H2O at 60 \u00C2\u00B0C for 4 hours.After cooled, the solution was washed with ether to remove anyunhydrolyzed ester.2.2.7 Synthesis of [2H733-keto VPA 2.2.7a. Synthesis of ethyl 3-keto pentanoateIn a 1000 mL 3-necked flask equipped with a dropping funnel andmechanical stirrer, isopropylcyclohexylamine (40 mL) and THE (250 mL)under N2 atmosphere were cooled to 0 \u00C2\u00B0C with ice. N-butyllithium (150mL, 0.24 mol, 1.6 M in Hexane) was added dropwise over a period of 10min. The mixture was stirred for another 20 min before immersing theflask into a dry ice/acetone bath (-78 \u00C2\u00B0C). Ethyl acetate (11 mL, 0.12mol) dried over Na2SO4 was then added dropwise over a period of 10 minfollowed by propionyl chloride (9.3 mL). The reaction was allowed toproceed for an additional 15 min before being quenched by adding 4 N HC1slowly to a pH of 2. The product was extracted 3 times (100 mL x 3)with ether. The ethereal layer was washed with NaHCO3 solution and thendried over anhydrous Na2SO4. The solvent was removed under vacuum usingflash evaporation. The residue was fractionally distilled to afford 9.5g of ethyl 3-keto pentanoate (70 \u00C2\u00B0C/5 mm Hg, yield 55%).GCMS mass spectrum of ethyl 3-keto pentanoate, m/z(%): 57(100),29(95), 43(39), 98(14), 115(12.5), 144(1%1+ ,12).36300 MHz 1H-NMR of ethyl 3-keto pentanoate (CDC13): 6 1.1(t, 3H,CH3-CH2), 1.3(t, 3H, CH3-CH20), 2.6(q, 2H, CH3CH2C0), 3.45(s, 2H,COCH2CO2), 4.2(q, 2H, CH20).2.2.76 Alkylation of ethyl 3 - keto -pentanoate with [2H7] bromopropaneSodium metal (1.4 g, 0.06 mol) cut into small pieces was placed ina 50 mL 3-necked flask equipped with a dropping funnel filled withethanol (20 mL) and protected from moisture with drying tubes. A wettowel was kept in readiness to control the vigor of the subsequentreaction. Absolute ethanol (10 mL) dried over Mg was added to thesodium producing a vigorous reaction. As the reaction subsides, morealcohol was introduced to maintain vigorous, but controllable refluxing.In this manner, most of the Na reacted rapidly. Finally the remainderof ethanol was added and the mixture refluxed by a heating mantle untilthe Na had reacted completely. Ethyl 3-keto pentanoate (8.6 g, 0.06mol) was added dropwise over a period of 10 min, and the mixture wasstirred and refluxed for another 20 min. [ 2H7] Bromopropane (7 g, 0.054mol) was then introduced dropwise, and the reaction allowed to refluxfor 7 hours. The solution was filtered to remove the sodium bromideafter cooling. The filtered NaBr was washed with ethanol. The ethanolsolutions were combined and the ethanol was evaporated by flashevaporation. Water was added to the residue and extracted with ether 4times (4 x 50 mL). The combined organic layer was dried over anhydrousNa2504 over night. The ether was removed under flash evaporation and37the residue fractionally distilled to yield 8.2 gram of ethyl [ 2H7]3-keto VPA (75-80 \u00C2\u00B0C /3 mm Hg, 78% yield).GCMS of ethyl [ 2H7]3-keto VPA, m/z(%): 57(100), 103(42), 137(18),145(9), 164(3), 193(M+ ,1).1 11 NMR of ethyl [ 2H7]3-keto VPA (CDC13): 6 1.15(t, 3H, CH3CH2),1.26(t, 3H, CH3CH20), 2.55(m, 2H, CH3CH2), 3.42(d, 1H, COCHCO2), 4.18(q,2H, CH3CH2O).Ethyl [ 2H7]3-keto VPA (60 mg) was hydrolyzed by stirring with 1 mLof 3N NaOH and 0.4 mL of methanol at room temperature for two hours.Unhydrolyzed ester was extracted with hexane.2.2.8 Synthesis of [2H7] 3-0H VPAIn a dry 100 mL flask, ethyl [ 2H7]3-keto VPA (5 g, 0.026 mol) wasadded dropwise to the well mixed NaBH4 (1.425 g 0.0375 mol) in 60 mL ofethanol. The reaction was then allowed to proceed with stirring for 1hour before being quenched with saturated NH4C1. The solution wasextracted 5 times (5 x 50 mL) with ether, and the ethereal layer driedover anhydrous Na2SO4. The solvent was removed under flash evaporationand the residue further dried under vacuum (0.5 mm Hg). The reactionwas found to be quantitative with 4.75 g (94% yield) of ethyl [ 2H7]3-0HVPA being obtained.38GCMS of the ethyl ester of [2H7] 3-0H VPA, m/z(%): 103(100),75(52), 120(37),137(30), 166(25), 150(17), 196(M+, 1).Ethyl [2H7]3-0H VPA (45 mg) was hydrolyzed by refluxing with lmL 3NNaOH and 0.6 mL methanol at 50 \u00C2\u00B0C for two hours. Unreacted ester wasextracted with 3 mL of hexane. The aqueous solution was then acidifiedwith 4 N HC1 solution to pH of 2 and extracted with ether (5mL x 3).The ethereal solution was then evaporated under flash evaporation toafford [2H7]3-0H VPA.2.2.9 Synthesis of [2H7] 2-ene VPAIn a 50 mL flask equipped with a mechanical stirrer, ethyl [2H7]3-OH VPA (1.5g, 7.7 mmol), triethylamine (1.34 mL, 9.5 mmol) anddichloromethane (15 mL) were cooled to 0 \u00C2\u00B0C in an ice bath.Methanesulfonyl chloride (0.83 mL, 9.9 mmol) in dichloromethane wascooled to 0 \u00C2\u00B0C and added dropwise to the stirred mixture. The mixturewas stirred for a further 60 min at room temperature. A few mL of etherwere added to the mixture to precipitate the salt. The mixture was thenfiltered and the solvents removed by flash evaporation. The residue wasreconstituted in 20 mL of dry THE, a solution of DBU (1,8-diazabicyclo[5.4.0] undec-7-ene, 1.43 mL, 9.5 mmol) was added and the contentsgently refluxed for 3 hours. The reaction was quenched with water andthe aqueous layer extracted 3 times (3 x 30 mL) with ether. The organiclayer was washed first with 1M HC1, then with 1M NaOH solution andfinally dried over anhydrous Na2SO4.39The product was purified by flash column chromatography(Rettenmeier et al., 1985) using a mobile phase of 2-5% ethyl acetate inhexane. A yield of 260 mg of ethyl [ 2H7]2-ene VPA was obtained (19%yield). The product was confirmed with IH NMR to be mostly E isomerwith a small portion of Z isomer.GCMS of ethyl [ 2H7]2-ene VPA, m/z(%): 115(100), 57(95), 132(87),177(M+ , 82), 97(42), 104(40), 148(37).1 11 NMR of ethyl [ 2H7]2-ene VPA (CDC13): 1.06(t, 3H, CH3CH2), 1.3(t,3H, CH3CH2O), 2.2(m, 2H, CH3CH2), 4.2(q, 2H, CH3CH20), 6.73(t, 1H,CH=C).Ethyl [ 2H7]2-ene VPA (50 mg) was hydrolyzed by heating with 1 mL of3N NaOH and 0.3 mL of methanol at 80 \u00C2\u00B0C for 20 hours.2.2.10 Synthesis of (E)- 3-ene VPAA flame-dried 250 mL three-necked flask equipped with a graduatedseparatory funnel and mechanical stirrer was flushed with N2 andimmersed in an ice-water bath. Diisopropylamine (5.6 mL, 0.04 mol) and40 mL of THE were placed in the flask, 25 mL of n-butyllithium (1.6 M inhexane, 0.04 mol) was added dropwise from the funnel. The mixture wasallowed to stir for 20 min. The flask was then immersed in a dryice/acetone bath (-78 \u00C2\u00B0C), HMPA (7.9 mL, 0.044 mol) was added and themixture was stirred for 30 min. Ethyl (Z)-2-pentenoate (5.7 mL, 0.039mol) was added to the mixture dropwise over a period of 15 min, followed4041by dropwise addition of bromopropane (4.4 mL, 0.048 mol).^Afterstirring for 30 min, the reaction was quenched with 15% HC1 solution toa pH of 2. The aqueous layer was extracted twice with 20 mL of ether.The combined organic layer was washed with saturated NaHCO3 solution,with water, and then dried over anhydrous Na2SO4. The ester was thenhydrolyzed by heating with 8 mL of 3N NaOH solution and 2 mL of methanolat 50 \u00C2\u00B0C for 2 hours. Unhydrolyzed ethyl ester was extracted withhexane, and ethanol and methanol were distilled under atmosphericpressure. The aqueous solution was then acidified with 4N HC1 solutionto a pH of 1-2, and extracted three times (3 x 50 ml) with ether. Etherwas removed under flash evaporation. The residue was fractionallydistilled to afford 1 g of pure (E)-3-ene VPA (85 \u00C2\u00B0C/1 mm Hg, yield18%).GCMS of the methyl ester of (E)-3-ene VPA, m/z(%): 55(100), 97(25),113(18), 127(7), 156(M+,2).1H NMR of (E)-3-ene VPA (CDC13): 6 0.9(t, 3H, CH3CH2), 1.34(m, 2H,CH3CH2), 1.53(m, 1H, CH2CH), 1.75(m, 1H, CH2CH), 1.70(d, 3H, CH3C=),2.98(dd, 1H, CHC00), 5.44(dd, 1H, J=15Hz, =CHCH), 5.6(dt, 1H, J=15Hz,CH3CH=).2.2.11 Synthesis of (Z) -3 -ene VPA(Z)-3-ene VPA was synthesized by the same method as described forthe synthesis of (E)-3-ene VPA, but with the starting material beingethyl (E)-2-pentenoate.GCMS of the methyl ester of (Z)-3-ene VPA, m/z(%): 55(100), 97(25),113(18), 127(7), 156(M+ ,3).1 H NMR of (Z)-3-ene VPA (CD30D): S 0.93(t, 3H, CH3CH2), 1.34(m, 2H,CH3CH2), 1.48(m, 1H, CH2CH), 1.72(m, 1H, CH2CH), 1.66(d, 3H, CH3C=),3.35(dd, 1H, CHC00), 5.36(dd, 1H, J=11Hz, =CHCH), 5.6(dt, 1H, J=11Hz,CH3CH=).2.3 Pharmacokinetic studies2.3.1 Pharmacokinetic study with [2H6]VPAA healthy male volunteer (my research supervisor) participated in amultiple-dose study of the pharmacokinetics of [ 2H6]VPA. A 700 mg doseconsisting of VPA:[ 2N6]VRA (50:50) was given orally to the volunteerevery 12 hours for a period of two and half days. Following the fifthand final dose, blood samples were withdrawn at 0, 0.5, 1, 1.5, 2, 2.5,3, 5, 7, 9, 12, 24, 48, 96, 168, 240 and 336 h. Blood samples wereallowed to clot and then centrifuged to provide serum samples, whichwere transferred to sterile vacutainers and stored at -20\u00C2\u00B0C untilanalyzed. Saliva samples were taken at selected times convenient withthe taking of blood samples. Saliva production was stimulated with a 5%citric acid solution rinse of the mouth. Following the first dose,urine samples were collected in 12 hour blocks for 2 days and then in 2or 3 day blocks for another 8 days. Total urine volume and pH valueswere recorded and homogeneous aliquots were saved. Saliva and urine4243samples were also stored at -20\u00C2\u00B0C.^All samples were analyzedquantitatively by GCMS (HP 5987A) using El analysis of the t-BDMSderivatives and NCI analysis of the PFB derivatives. Dibutylaceticacid, 2-methylglutaric acid and the stable isotope labelled metabolites,[2H3]2-ene VPA and [2H3]3-keto VPA were used as internal standards.2.3.2 Pharmacokinetic study with [13C4]VPA2.3.2a Human study:A healthy human volunteer ( my research supervisor, 70 Kg) wasgiven a single oral dose consisting of 700 mg of VPA:[13C4]VPA (50:50).Blood samples were collected 0, 0.5, 1, 1.5, 2, 2.5, 3, 5, 7, 9, 12, 24,48 and 72 h after the single dose and allowed to clot. The samples werecentrifuged to obtain serum samples. Urine samples were collected atconvenient blocks for three days after the dose. Total urine volume andpH values were recorded and homogeneous aliquots were saved. Allsamples were stored at -20\u00C2\u00B0C until analyzed by GCMS using NCItechniques.2.3.2b Animal study:Two nonpregnant sheep (60 and 73.6 Kg respectively) were eachadministered i.v. a single dose of 1000 mg of VPA : [ 13C4]VPA (50:50).Blood^samples^were collected^at^5 min^before^injection, 2, 6, 10, 15,20,^30,^45,^60 min,^1.5,^2,^2.5,^3,^3.5,^4,^5,^6,^8,^10, 12, 24, 36, 48,72 h after the single dose. All the samples were allowed to clot beforecentrifuging to collect serum samples. Urine samples were collectedevery half an hour, then in 1 hour, 2 hour, 12 hour, or 24 hour blocks.Bile samples were taken at selected time 0-30 min, 60-90 min, 2.5-3.5 h,5-5.5 h, 7-7.5 h, 11-11.5 h, 24-24.5 h, 36-36.5 h, 48-48.5 h, and 72-72.5 h after administration. The volume of urine and bile samples wererecorded, and the pH values of urine samples were measured. All sampleswere stored at -20 \u00C2\u00B0C until the time of analysis.2.4 Metabolic studies of (E)- and (Z)-3-ene VPA2.4.1 Study designThe metabolic studies of (Z)-3-ene VPA and (E)-3-ene VPA wereperformed in rats. The animals were kept in a restraint cage equippedwith a funnel and a glass container to collect the urine.2.4.2 Metabolism of (Z) - and (E) - 3 - ene VPA(Z)- and (E)-3-ene VPA (150 mg/kg dose) were separatelyadministrated i.p. to two rats (adult male Wistar rats weighing 270 and308 g).^A blood sample from each rat was taken 2 h after the dose,allowed to clot, and centrifuged to obtain a serum sample.^Urinesamples were collected every 24 hours for two days. Samples were storedat -20 \u00C2\u00B0C until derivatized and analyzed by GCMS using EI techniques.2.5 Calibration CurvesTo analyze VPA, [ 2H6]VPA and their metabolites in the steady-statestudy, calibration curves having different concentration ranges were44prepared for urine, serum total, and serum free (saliva) samplesrespectively. Table 1 summarizes the concentrations of stock solutionsof VPA and VPA metabolites used in the preparation of the differentcalibration curves.Table 1: Stock solution concentrations (ug/mL) used for the preparationof calibration curves for VPA, rHOVPA and their metabolites.COMPOUNDS URINE SERUM TOTAL SERUM FREE(SALIVA)VPA 509.2 124.4 12.44-ene VPA 1.5 1.5 0.15(E+Z)-3-ene VPA 0.985 1.97 0.197(Z)-2-ene VPA 4.188(E)-2-ene VPA 103.6 20.43 2.043(E)-2,4-diene VPA 23 1.02 0.102(E,E)-2,3'-diene VPA 49.2 5.03 0.5033-keto VPA 300 10 14-keto VPA 40.8 1.04 0.1043-0H VPA 28.3 0.98 0.0984-01-1 VPA 52.8 1.908 0.19085-0H VPA 53.4 0.95 0.0952-PSA 19.7 0.124 0.01242-PGA 99.6 1.004 0.100445Calibration curves for VPA, [ 2H6]VPA and their metabolites weremade from samples prepared by the following dilution of the stocksolution described in Table 1.Amount of stock solution(%) * Control or water** (%)100 080 2060 4040 6020 800 100*total amount: 1 mL for EI, 0.25 mL for NCI.**water was used as a control for serum free.A second set of calibration curves was prepared to analyze thesamples from the pharmacokinetic study in sheep which were administereda single dose of VPA:[ 13C4]VPA (50:50). Table 2 summarizes theconcentrations of stock solutions used for the dilution of VPA and VPAmetabolites for the calibration standard samples.46Table 2: Stock solution concentc4tions (ug/mL) used for the preparationof calibration curves for VPA, [\"C4] VPA and their metabolites.COMPOUNDS^ Concentration (ug/mL)VPA^ 204-ene VPA^ 2(E+Z)-3-ene VPA^ 4(Z)-2-ene VPA 20(E)-2-ene VPA^ 20(E)-2,4-diene VPA 4(E,E)-2,3'-diene VPA^ 83-keto VPA^ 44-keto VPA 23-0H VPA^ 44-011 VPA 45-011 VPA^ 22-PSA 22-PGA^ 247Calibration curves for VPA, [ 13C4]VPA and their metabolites weremade from samples prepared by the following dilution of the stocksolution described in Table 2.Amount of stock solution(%) * Control or water** (%)100 050 5025 7512.5 87.56.25 93.751.56 98.44482.5 Extraction and DerivatizationExtraction and derivatization procedures for the biological samplesare summarized in Schemes 1 and 2.Serum (total or free) or Saliva sample (0.25 mL for NCI, 1 mL for El)+ internal standards/Adjust to pH=2 with 4N HC1iExtract twice with lmL ethyl acetate(centrifuge to optimize separation)iDry with Na2SO4, reduce volume to 200 ul with N2I /Add 10 ul PFBB (30% in ethyl acetate)^50 ul of MTBSTFAand 10 ul diisopropylethylamine,,^i I60 'C for 1 h^ 60 \u00C2\u00B0C for 1 hinject 1 ul^ inject 1 ul(NCI assay) (El assay)Scheme 1:^Sample handling procedure for serum (total and -free) andsaliva samples.491Urine sample (0.25 mL for NCI, 1 mL for EI)+ internal standardsiHydrolyzing conjugates *Adjust to pH=2 with 4N HC1Extract with 1 mL ethyl acetate1Dry with Na2SO4, reduce volume to 200 ul with N2I^ IAdd 10 ul PFBB (30% in ethyl acetate)^50 ul of MTBSTFAand 10 ul diisopropylethylamine\u00E2\u0080\u009E i60 uC for 1 h^ 60 \u00C2\u00B0C for 1 hinject 1 ul(NCI assay)iinject 1 ul(EI assay)Scheme 2: Sample handling procedure for urine samples.*The glucuronide conjugates in urine were hydrolyzed using twotechniques:^(i). enzymatic hydrolysis by fl-glucuronidase at 37uC for24 h (ii). alkaline (NaOH solution, pH=12) hydrolysis at 60 \u00C2\u00B0C for 1 h.All samples were run in selected ion monitoring (SIM) mode. Table3 summarizes the ions of derivatized VPA and metabolites that weremonitored for the EI or NCI ionization techniques.50Table 3: Mass to charge (m/z) for the internal standards (*), VPA, andVPA metabolites that were used for ion monitoring in the NCI (PFBderivatives) and El (t-BDMS derivatives) mode.COMPOUNDS^NCI^El(M-181)-^(M-57)+Dibutyl acetic acid*^171^229VPA^ 143 201(E)-[2H3] 2-ene VPA*^144^2024-ene VPA^141 199(E+Z)-3-ene VPA^141^199(Z)-2-ene VPA 141 199(E)-2-ene VPA^141^199(E)-2,4-diene VPA^139 197(E,E)-2,3'-diene VPA^139^1974-keto VPA^157 2153-0H VPA 231^2174-0H VPA^ 231 2175-0H VPA 231 (monoderiv)^331 (dideriv){2H3}3-keto VPA*^232 (dideriv)^332 (dideriv)160 (monoderiv)^218 (monoderiv)3-keto VPA^229 (dideriv)^329 (dideriv)157 (monoderiv)^215 (monoderiv)2-MGA*^ 325^3172-PSA 339 3312-PGA^ 353^345512.7 Calculation and Data Evaluation2.7.1 Pharmacokinetic parametersThe pharmacokinetic parameters, area under the curve (AUC), half-life (t1/2), elimination rate constant (KE), distribution volume (Vd),and clearance (CL) of [ 2H6]VPA and some of its metabolites werecalculated using the equations of Gibaldi and Perrier (1982) andcompared with those of VPA and its metabolites.2.7.2 Isotope effectsIsotope effects of [ 2H6]VPA were measured from a) serum data basedon the AUC ratio of [ 2H0]VPA and its metabolites to [ 2H6]VPA and itsdeuterated metabolites, and b) urine data determined from the recoveryratio of [ 2H0]VPA and its metabolites to [ 2H6]VPA and its metabolites inurine samples collected for 12 hours after the final dose.Isotope effects of [ 13C4]VPA was measured from serum data based onthe concentration ratio of VPA and its metabolites to [ 13C4]VPA and itsmetabolites.2.7.3 Conjugated fraction of VPA and its metabolites in urine samplesThe glucuronide conjugates in urine were hydrolyzed using twotechniques, enzymatic hydrolysis by fl-glucuronidase at 37 \u00C2\u00B0C for 24 hoursand alkaline (NaOH solution, pH=12) hydrolysis at 60 \u00C2\u00B0C for 1 hour. The52efficacy of the two hydrolyzing methods and whether any fl-glucuronidase-resistant conjugates exist or not were determined by comparing theresults of the two hydrolysis methods. The conjugated fractions weredetermined from the concentration differences of free drug andmetabolites in hydrolyzed and unhydrolyzed urine samples respectively.2.7.4 Evaluation of DataA MIDAS based computer program containing methodological statisticswas used to evaluate the two analytical methods, NCI and El, and tocompare the efficiencies of the two hydrolysis methods, fl-glucuronidaseand treatment with alkali (NaOH solution, pH=12).533. Results and Discussion3.1 Synthesis of deuterium labelled internal standardsStable isotope-labelled analogs provide optimal internal standardsfor GCMS assays. By using stable isotope-labelled analogs as internalstandards, the lowest variance factors due to sample manipulation andinstrumental errors was produced (Claeys et al., 1977). Ideally thelabelled internal standard should have a mass difference of at least 3mass units from the analyte to minimize interference from naturalisotopes and thus avoid correction for mass overlap. While [2H6]VPA issuitable for VPA analysis ( Au = 6), it is not a good internal standardfor [13C4]VPA ( Au = 2). Based on the above principle, [2H7]VPA shouldprovide an ideal internal standard for both [13C4]VPA and VPA analysisby GCMS. The previously reported assays (Abbott et al., 1986;Acheampong et al., 1983; Kassahun et al., 1989) for VPA metabolites fromthis laboratory could also be improved by the availability of adequatestable isotope labelled internal standards of the metabolites. Thus,syntheses were carried out to produce several deuterium labelled VPAmetabolites.3.1.1 Synthesis of [2H7]VPA[2H7]VPA was synthesized by a method for the alkylation of a-metalated aliphatic acids (Pfeffer et al., 1972). The procedure isoutlined in scheme 3. Distilled pentanoic acid was converted into thea-anion by means of two equivalents of lithium diisopropylamide intetrahydrofuran (THF)-hexane solution. Hexamethylphosphoramide (HMPA)54was added to the mixture to make the reaction more specific andefficient, due to its chemical and physical properties. First, themetalated straight-chain acids are insoluble in THF, but readilydissolve in the highly dipolar solvent, HMPA; second, becauseorganolithium compounds are known to associate to higher molecularweight aggregates, the degree of polymerization (n=2-6) varying withsolvent and structure (Mallan and Bebb, 1969), the aggregation state isdisrupted by complexation with polar solvents in the formation of asolvent-separated ion pair of higher reactivity than the originalaggregate. A strong dipolar solvent like HMPA should accordingly bemore efficient in association with metalated carboxylates than THF byforming a complex of higher reactivity, presumably a solvent-separatedion pair (Pfeffer et al., 1972). Pfeffer et al. (1972) also illustratedby comparative experiments that HMPA, in addition to solubilisingdianions, also accelerates the alkylation rates, and quantitativeconversion was observed in HMPA solution when 1.5 equivalents of alkylbromide was added.This method was first used to synthesize VPA in this lab byAcheampong (1985). The same method was applied in synthesizing[ 2H7]VPA, the only difference being the use of [ 2H7]propyl bromide.Since [ 2H7]propyl bromide is very expensive, we could not afford toredistill it before use, hence, the yield is less than the reference(91%, Lee, 1987). The final product was confirmed to be [ 2H7]VPA bycomparing its GCMS mass spectrum and NMR spectrum with unlabeled VPA.The mass spectrum of the methyl ester derivative of [ 2H7]VPA which wasprepared by derivatizing with diazomethane (Levitt, 1973) revealed55fragment ions at m/z 136(M-C2H5)+, 123(M-C3H6)+, 117(M-C32H6)+, and89(C2H2CH2COOCH3)+, corresponding to VPA fragments at m/z 129(M-C2H5)+,116(M-C3H6)+, and 87(CH2CH2COOCH3)+ respectively (Figure 3). The 1H NMRof the synthesized product also confirmed the [2H7]VPA structure (Figure4).CH3-CH2-CH2-CH2-COOH^valeric acid/ LDA/THFCH3-CH2-CH2-CH--000-^ dianion[2H7] propyl bromideCH3-CH2-CH29 /C2H3-C2H2-C412CH-COOH [2H7]VPA56Scheme 3: Synthesis of [2H7]VPA.87(CH2CH2C0OCH3CH3 -CH2 -CH211699106 134Al 69(C 2 H2CH2COOCH3 +CH3 -CH2 -CH2CH -COOCH3C 2H3-C 2H2 -C 2H2(M-C32[16 )4-^(M-C3H6)117 123611117,,^I, 111 11151, I, ...111 a^ sti^ a, I18CH -COOCH 3C H3 -CH2 -CH2( M - C3 H 6 )1829IMIIMIN\u00E2\u0096\u00A0111111\u00E2\u0096\u00A0\u00E2\u0080\u00A2\u00E2\u0096\u00A0111157(M - C2H5 ) 4-12969M+15943Fig. 3.^GCMS mass spectra of the methyl esters of [ 2H7]VPA (top) andVPA (bottom).578.5 2.5 5.0^4.59992.8^6.5 6.8^5.5 3.9^2.54.0^3.5 2.8^1.5^1.2CH3-CH2-CH2CH-COOHC2H3-C 2 H2-C2 H2CE3-CH2iCH3CH2-CH// CH3-CH2CH3CH2-C1^J 'LACH-COOHFig. 4:^111 NMR spectrum of [2H7]VPA.3.1.2 Synthesis of [2H7]4-ene VPA4-Ene VPA can usually be prepared via its ethyl ester, by reactionof ethyl valerate and allyl bromide according to the general method forthe alkylation of esters at the a-carbon atom (Cregge, et al., 1973).However, this method was not suitable for introducing the [2H7]deuterium labelled side chain. It turns out the a-metalation method wasalso applied by Pfeffer (1972) to olefinic acids. For example, 10-undecenoic and (Z)-9-octadecenoic acids, were converted to their aanions and then alkylated in good yields to the a-branched chainunsaturated acids. The isolated double bonds in these acids were foundto be unaltered positionally or geometrically in the formation of theirdianions by lithium diisopropylamide. An attempt to synthesize [2H7]4-ene VPA using this method was performed by starting first with thesynthesis of unlabeled 4-ene VPA. Good yields were obtained for 4-eneVPA, and thus [2H7] 4-ene VPA was synthesized in this study startingfrom 4-pentenoic acid (Scheme 4) by Pfeffer's method.[2H7]4-ene^VPA^was^successfully^purified^by^fractionaldistillation. The GCMS mass spectrum of the methyl ester of [2H7]4-eneVPA was compared with that of the methyl ester of 4-ene VPA (Figure 5).Fragments of [2117]4-ene VPA occurred at m/z 163 (e), 129 (M-C22H5)+,113(M-C32H7)+, and 104(M-COOCH3)+ and correspond to those of 4-ene VPAat m/z 156 (Mt), 127 (M-C2H5)+, 113(M-C3H7)+, and 97(M-COOCH3)+. 1H-NMR(Figure 6) confirmed the presence of the terminal double bond: 8 5.78(m, 1H, CH2=CH), 5.12 (d, 1H, J=17 Hz, proton at C-5 trans to proton atC-4), 5.08 (d, 1H, J=11 Hz, proton at C-5 cis to proton at C-4).59CH2=CH-CH2-CH2-000HLDA/THFCH2=CH-CH2-CH - -000 -[ 2H7] propyl bromide4-pentenoic aciddianion60CH2=CH-CH2/C 2H3-C 2H2-C 2H2CH-COON [ 2H7]4-ene VPAScheme 4: Synthesis of [ 2H7]4-ene VPA.61Fig. 5: GCMS mass spectra of the methyl esters of 4-ene [ 2H7PPA (top)and 4-ene VPA (bottom).CH2=CHFig. 6:^1H NMR spectrum of [2H7]4-ene VPA.3.1.3 Synthesis of [2H7]4-keto VPAEthyl [2H7]4-ene VPA was oxidized by benzoquinone to ethyl [2H714-keto VPA using palladium chloride as catalyst. This procedure is well-known as the Wacker process, and is one of the most important industrialprocesses employing transition metal catalysts (Smidt et al., 1959).There are several methods which can selectively oxidize terminal doublebonds to methyl ketones; of them, the palladium(II) chloride-catalyzedoxidation of terminal olefins seems to be one of the best (Tsuji, 1984).Since the oxidation proceeds under mild conditions, various functionalgroups, such as esters, carboxylic acids, aldehydes, are unchangedduring the reaction (Tsuji, 1984). The conversion of ethyl [2H714-eneVPA to ethyl [2H7]4-keto VPA was quantitatively completed in 22 hours.Scheme 5 summarizes the synthesis procedure of [2H7]4-keto VPA.The GCMS mass spectrum of ethyl [2H7]4-keto VPA (Figure 7) revealeda molecular ion (m/z 193) and other fragments, m/z 148 (M-OCH2CH3)+, 136(M-CH3COCH2)+, and 108 (C2H3C2H2C2H2CH2C00)+, which correspond to ethyl4-keto VPA fragments, m/z 186 (M+), 141 (M-OCH2CH3)+, 129 (M-CH3COCH2)+,and 101 (CH3CH2CH2CH2C00)+.63CH2=CH-CH2 \, /C 2H3-C 2H2 -C 42Ethyl [ 2H7]4-ene VPACH -CO0C2H5/c2Hr c2H 2 _cr_9 l_1 2CH -CO0C2H5 Ethyl [ 2H7]4-keto VPACH3-C O-CH2 \CH2=CH-CH2 64\CH-COONC 2H3 - C 2H2 -C492//[2H7]4-ene VPAEtOH, H2SO4BenzeneIBenzoquinonePdC12Na0H/Me0HCH3 -CO -CH2\9 /c2H3 _c2H 2 _u_H2CH-COOH^ [2H7]4-keto VPAScheme 5: Synthesis of [ 2H7]4-keto VPA.65Fig. 7: GCMS mass spectra of ethyl esters of (2H7]4-keto VPA (top) and4-keto VPA (bottom).3.1.4 Synthesis of 1-2H7,14-0H VPA The synthesis of [ 2H7]4-0H VPA was accomplished via its ethyl esterby reducing the ethyl ester of [ 2H7]4-keto VPA with NaBH4. The reactionwas quantitatively completed within 60 min at room temperature, whenexcess NaBH4 was used. The steps in the synthesis are summarized inScheme 6. The GCMS mass spectrum of ethyl [ 2H7]4-0H VPA was comparedwith that of unlabeled ethyl 4-0H VPA (Figure 8). The fragments ofethyl [ 2H7]4-0H VPA at m/z 150 (M-C2HSO), 134, 116 and 101 correspond toethyl 4-0H VPA at m/z 143, 127, 113 and 110.6667CH3 -CO -CH2CH -CO0C2H5^Ethyl [ 2H7]4-keto VPAC 2H3-C 2H2-C 2 H2NaBH4/EtOHOHCH3-CH-CH 2CH -CO0C2H5C 2 H3-C 2 H2-C 2 H2two isomers ofethyl [ 2H7]4-0H VPANaOH/MeOHOHCH3 -CH-CH2CH-COOH^two isomers ofC 2 H3-C 2H2-C 2 H2^ [ 2H7]4-0H VPAScheme 6: Synthesis of [ 2H7]4-0H VPA.Fig. 8: GCMS mass spectra of ethyl esters of [2H7]4-0H VPA (top) and 4-OH VPA (bottom).683.1.5 Synthesis of [2H7] 5-0H VPAThe ethyl ester of [ 2H7]5-0H VPA was prepared from ethyl [ 2 H7]4 -eneVPA by the same method that Rettenmeier et a/.(1985) used to synthesize5-0H VPA. Scheme 7 summarizes the synthesis procedure. Ethyl [ 2H7]4-ene VPA was hydroborated into the corresponding trialkylborane, whichwas then oxidized with alkaline hydrogen peroxide to provide ethyl[ 2H7}5-0H VPA. The GCMS mass spectrum (Figure 9) of ethyl [ 2 H7]5-0H VPAindicates the molecular ion at m/z 195 and other fragments at m/z 164(M-CH3OH) + , 150 (M-C2H5OH) + , 115, and 101, corresponding to fragments ofethyl 5-0H VPA at m/z: 157 (M-CH3OH) + , 143 (M-C2H5OH) + , 113, and 100.69CH2=CH-CH2\CH-COOC2H5 ethyl [ 2H7]4-ene VPA709 /c2H3 _c2H2 _c41 2H3 B- BH3-CH2-CH2-CH2C 2 H3-C 2 H2 -C 2 H2\/ CH-COOCH2CH3/trialkylborane3H202/NaOHHOCH2 - CH2 - CH2C 2H3-C 2H2-C 2 H 2\/CH-COOCH2CH3^ethyl [ 2H7]5-OH VPANaOH/MeOHHOCH2-CH2-CH2C 2H3-C 2H2-C 2 H 2\/CH-COON^ [2H7]5-0H VPAScheme 7: Synthesis of [ 2H7]5-0H VPA.Fig. 9: GCMS mass spectra of the ethyl esters of [2H7]5-0H VPA (top)and 5-0H VPA (bottom).713.1.6 Synthesis of [2H7]3-keto VPAEthyl 3-keto VPA is usually synthesized by alkylating the enolateester of valeric acid with propionyl chloride, a very well establishedmethod for the synthesis 3-keto VPA (Acheampong, 1982). However,[ 2 H7]pentanoic acid would have to be prepared first if this method wereapplied in the synthesis of [ 2H7]3-keto VPA. This adds to increasedcosts and a lower yield based on the availability of [ 2H7]bromopropane.It is optimal to introduce the [ 2H7]propyl side chain in the final stepof the synthesis for considerations of expense and efficacy.Accordingly, we designed a synthesis of ethyl 3-keto pentanoate, whichcould then be alkylated with [ 2H7]bromopropane to produce the desiredproduct.3.1.6a Synthesis of ethyl 3-keto pentanoate:Ethyl 3-keto pentanoate was synthesized according to the methodpublished by Micheal et a/.(1971) for the preparation of fl-keto esters.Scheme 8 summarizes the procedure. Ethyl acetate was converted into thecorresponding lithium enolate ester by reaction with lithium N-isopropylcyclohexylamide (LiICA). The enolate ester was then condensedwith propionyl chloride to form the desired fl-keto ester withoutsignificant self-condensation. However, attack of the enolate ester atthe ketone carbonyl of the product could lead to decreased yields. Thispossibility was minimized by using an extra equivalent of the generatingbase, LiICA, thereby converting the product into the relatively inert fi-keto enolate ester. The product gave only one peak in the TIC upon GCMS72analysis and proved to be ethyl 3-keto pentanoate from the GCMS massspectrum and from IH NMR, after purification by fractional distillation.GCMS mass spectrum (Figure 10) of the product shows a molecular ion ofethyl 3-keto-pentanoate at m/z 144, and other fragments m/z 115(M-C2H5)+, 98(M-C2H5OH)+, 87(M-CH3CH2C0)+, 69(M-CH3CH2COOH)+,57(CH3CH2C0)+. 111 NMR (Figure 11) also confirmed the structure of theproduct to be ethyl 3-keto-pentanoate.7357(CH3CH2C0 )4-CH3 -02-C-CH2-COOC 2H5(M-C21-150H)+69(M-CH3CH2CO) F 98 (M-C2F15)1^+r^.11,^ir 2943181.,m+.144Fig. 10: GCMS mass spectra of ethyl 3-keto-pentanoate.CH3CH2CO0IICH3 -CH2-C-CH2 - COOC2H5^lit I 1 I 1 TtitioillitijilfilliiiTi l 1 11^til\u00E2\u0080\u0094IM-1\u00E2\u0080\u0094VilFITI1^111\u00E2\u0080\u0094$11-1111111111 l l I 11 l 1 11 i 11\u00E2\u0096\u00A011111119 1^\u00E2\u0080\u00A2^8^7^6 5^M 3^2Fig. 11: 1 H NMR spectrum of ethyl 3-keto-pentanoate.CH3CH2Oj3.1.66 Alkylation of ethyl 3-keto pentanoate with [2H7]bromopropaneEthyl 3-keto-pentanoate was converted into the corresponding sodiumfl-keto enolate ester with sodium ethoxide which was prepared by addingabsolute alcohol to freshly cut Na metal. The enolate was thenalkylated with [2H7]bromopropane to afford a high yield of ethyl [2H7]3-keto VPA (Scheme 8). [2H7]3-keto VPA was purified by fractionaldistillation. Figure 12 shows the mass spectra of ethyl [2H7]3-keto VPAand ethyl 3-keto VPA. The molecular ion (m/z 193) and other fragmentsof [2H7]ethyl 3-keto VPA at m/z 164 (M-C2H5)+, 145 (M-C32H6)+, 137 (M-CH2CH2C0)+, 103 (C2H2CH2C00C2H5)+, correspond to those of ethyl 3-ketoVPA at m/z 186 (e), 157 (M-C2H5)+, 144 (M-C3H6)+, 130 (M-CH2CH2C0)+,101 (CH2CH2C00C2H5)+. IH NMR (Figure 13) of ethyl [2H7]3-keto VPAconfirmed the structure and purity of product.75\/CH3-CH2-00\C 2H3-C 2H2-C 2 H2 /CH-COOH^ [2H7]3-keto VPACH3-CO0C2H5EtONa[ 2H7]propyl bromideCH3 -CH2 -COCH -CO0C2H5C 2 H3 - C 2 H2 - C 2 H2ethyl acetateethyl 3-keto pentanoateethyl [ 2H7]3-keto VPA76LiICA/THFCH3CH2C0C1CH3-CH2-CO-CH2-CO0C2H5NaOH/MeOHScheme 8: Synthesis of [ 2H7]3-keto VPA.(CH2CH2C00C2H5)-1-MilCH3 -CH2 -CO57m\u00C3\u00B7193(H-CH2CH2C0)-1-137 (M-C324)+145^(M-C2H5)-1-116^Ii 164CH3-CH2 -COCH -COOC2H5/C2H3-C2H2 -C42(C2H2CH2C00C2H5)-1-1032975Si45I 185729CH-CO0C2H5CH3-CH2-CH2(M-CH2CH2C0 ) -1-(M-C3H6)-1-144(M-C2H5)4-157187130743 1158377Fig. 12: GCMS mass spectra of the ethyl esters of [2H7]3-keto VPA (top)and 3-keto VPA (bottom).Fig. 13: 1 H NMR spectrum of ethyl ester of [ 2H7]3-keto VPA.CH20^I)4.0^3.5PPM3.0 2. 5CH3-CH2-00C2H3-C2H2-C2H2C H 3 C H 0CH-CO0C2H5G.5^G0 5.0^4.5 2.^5^.^.5^0.05. 5CH3CH2C01^,^,3.1.7 Synthesis of 1-2H733-0H VPA The ethyl ester of [ 2H7]3-0H VPA was synthesized by reducing theethyl ester of [ 2H7]3-keto VPA with NaBH4, the same method that was usedin the synthesis of [ 2H7]4-0H VPA. Scheme 9 presents the syntheticprocedure.The product was confirmed to be ethyl [ 2H7]3-0H VPA by comparisonof its GCMS mass spectrum with that of ethyl 3-0H VPA (Figure 14). Thefragments of ethyl [ 2H7]3-0H VPA at m/z: 166 (M-C2H5) + , 150 (M-0C2H5) + ,137 (M-CH3CH2CHO) + , 120 (CH3CH2COCHC3 2H7) + , 103 (C 2H2CH2CO2C2H5) + ,correspond to the fragments of ethyl 3-0H VPA at m/z: 159 (M-C2H5) + , 143(M-0C2H5) + , 130 (M-CH3CH2CHO) + , 113 (CH3CH2COCHC3H7) + , and 101(CH2CH2CO2C2H5) 1\".79\Na0H/Me0HOH1CH3-CH2-CHCH-COOHc2H3_c2H2 2 /-C H2Two isomers of[2H7]3-OH VPA80CH3-CH2-CO \^CH-CO0C2H5^ ethyl CH713-keto VPAc2H3_c2H2_c2H2 /NaBH4/Et0HOH1CH3-CH2-CH\^ Two isomers of^C2H3-C2H2-C2H2 / CH -CO0C2H5^ethyl [ 2H7P-OH VPAScheme 9: Synthesis of [2H7]3-0H VPAFig. 14: GCMS mass spectra of the ethyl esters of [2H7}3-OH VPA (top)and 3-0H VPA (bottom).813.1.8 Synthesis of (E)-[ 2H7]2-ene VPAEthyl (E)-[ 2H7]2-ene VPA was synthesized from ethyl [ 2H7]3-0H VPAaccording to the procedure in scheme 10, and based on the method of Leeet al., (1989) to synthesize (E)-2-ene VPA. The formation of the mesylester of [ 2H7]3-0H VPA was completed within one hour aftermethanesulfonyl chloride was added. Nucleophilic elimination of themesyl group by 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) afforded ethyl[ 2H7]2-ene VPA.^The separation and purification of product wasperformed by column chromatography.^The main impurity was unreactedethyl [ 2H7]3-0H VPA, which combined with the stationary phase tightlybecause of the hydroxyl group. The impurity eluted only when using ahighly polar solvent, therefore, it was easy to separate the relativelynon-polar ethyl [ 2H7]2-ene VPA from it. Moreover, being an a,$-unsaturated ester, ethyl [ 2H7]2-ene VPA was visible under UV light,which facilitated monitoring the separation. The solvent was removedunder flash evaporation from the fraction shown to contain the 2-eneVPA, and the residue was dried under vacuum. The 1 H NMR (Figure 16)confirmed the product to be ethyl [ 2H7](E) 2-ene VPA, with a smallportion of (Z) isomer, which agrees with Lee's result (1989). Figure 15shows the mass spectra of labelled and unlabeled ethyl (E)-2-ene VPA.The fragments of ethyl [ 2H7]2-ene VPA at m/z 177 (e), 148 (M-C2H5) + ,132 (M-0C2H5) + , 115 (CH2=C(C 2H2)C00C2H5) + , and 97 (CH3CH=CHC(C0)=C 2 H2) + ,correspond to those of ethyl 2-ene VPA at m/z 170 (e), 141 (M-C2H5) + ,125(M-0C2H5) + , 113(CH2=C(CH2)C00C2H5) + , and 95(CH3CH=CHC(C0)=CH2) + .82OHCH3-CH2-CHC2H3-C2H2-C2H2CH -CO0C2H5 ethyl [ 2H7D-OH VPAMsC1Et3N, CH2C12OSO2C1CH3-CH2-CH^ mesyl ester of ethylC-CO0C2H5^ [2H7D-OH VPAC2H3-C2H2-C2H2DBU/THFCH3-CH2-CHC-COOC2H59 /C2H3-C2H2-CLH2ethyl (E)-[2H7]2-ene VPANa0H/Me0HCH3-CH2-CHC-COOH9 /c2H3_c2H2_cm2(E)-[2H7]2-ene VPAScheme 10: Synthesis of (E)-[ 2H7]2-ene VPA.83Fig. 15: GCMS mass spectra of the ethyl esters of (E)-42H7]2-ene VPA(top) and (E)-2-ene VPA (bottom).84Fig. 16: IH NMR spectrum of the ethyl ester of [2H7]2-ene VPA.3.1.9 Stereoselective syntheses of (E)- and (Z)-3-ene VPA(E)-3-ene VPA was synthesized as outlined in scheme 11, by a methodbased on general procedures described by Herrmann et al. (1973).According to the research of Kende and Toder (1982), alkylation of ethyl(Z)-2-alkenoate yields stereospecifically ethyl (E)-3-alkenoate. Thea,fl-unsaturated ester ethyl (Z)-2-pentenoate was converted into thelithium enolate by the non-nucleophilic form of lithium diisopropylamide(LDA) which is a 1:1 complex of LDA and HMPA. Besides being employed asbase, LDA might also act as a nucleophile and conjugatively add to theunsaturated ester at a rate competitive with proton abstraction. Afterapplying HMPA to modify LDA, no Micheal addition to (Z)-2-pentenoate wasobserved. The lithium enolate was then alkylated by bromopropane toafford ethyl (E)-3-ene VPA. Ethyl (E)-3-ene VPA was hydrolyzed withbase and the free acid purified by fractional distillation. The productwas confirmed by GCMS and 1H-NMR to be pure (E)-3-ene VPA. GCMS massspectrum (Figure 17) of 3-ene VPA after methylation affords ions at m/z156, 127, 113, 97, 55, corresponding to the molecular ion (M +) andfragments (M-C2H5) + , (M-C3H7) + , (M-COOCH3) +, and (CH3CH=CHCH2) +respectively.The NMR spectrum (Figure 18) confirmed the stereoselectivity ofthis reaction. The proton at C-4 has a chemical shift value at 5.6 ppm,and is split into a 16 peak multiplet with coupling constants of 15 Hz(with the proton at C-3), 6.5 Hz (with CH3), and 1 Hz (with the protonat C-2); the proton at C-3 has a chemical shift value of 5.43 ppm, and86is split into a 16 peak multiplet with coupling constants of 15 Hz (withthe proton at C-4), 8.6 Hz (with the proton at C-2) and 2 Hz (with CH3).(Z)-3-ene VPA was synthesized by the same method as (E)-3-ene VPAexcept the starting material was ethyl (E)-2-pentenoate. The GCMS massspectrum (Figure 17) of the methyl ester of (Z)-3-ene VPA was identicalwith that of the methyl ester of (E)-3-ene VPA. However, 1 H NMR candistinguish between these two isomers. Figure 19 shows the NMR spectrumfor (Z)-3-ene VPA. The signal for the vinylic proton at C-4 appears at5.6 ppm, and occurs as a 16 peak multiplet with coupling constants of 11Hz (with the proton at C-3), 6.5 Hz (with CH3), and 1 Hz (with theproton at C-2); the vinylic proton at C-3 shows a signal at 5.36 ppm,and is split into a 16 peak multiplet with coupling constants of 11 Hz(with the proton at C-4), 9.5 Hz (with the proton at C-2), and 2 Hz(with CH3 adjacent to the C-4).The purity of both (E)- and (Z)-3-ene VPA were checked by GCMS andby NMR, before they were used in the metabolism studies.87CH3-CH2^CO0C2H5/ LDA/HMPA, THF(Z)-ethyl 2-pentenoate88^CH3^H\ /C=C/ \^H^CHCO0C2H5ILilithium enol ate1^bromopropaneCH3^H\ /C=C/ \H^CHCO0C2H5/CH3CH2CH2ethyl (E)-3-ene VPAI^Na0H/Me0HCH3^H\ /C=C/ \H^CHCOOH/CH3CH2CH2(E)-3-ene VPAScheme 11: Synthesis of (E)-3-ene VPA.Fig. 17: GCMS mass spectra of the methyl esters of (E)- (top) and (Z)-3-ene VPA (bottom).89CH3CH=r-CH_CHCOOHCH3CH2^CH3 ^H/C=C/^H^CHCOOHCH3CH2CH22.5^7.5 5.5^G. fiFig. 18: IH NMR spectrum of (E)-3-ene VPA.L.5.8^\u00E2\u0080\u00A2^5.6^5.4^5.2-CHCHCOOH111111T 1114^ till41-1-1 ir^ITT-Fig. 19: 1H NMR spectrum of (Z)-3-ene VPA.3.2 Optimizing GCMS conditions for the analysis of VPA metabolites inEI (t-BDMS derivatives) and NCI (PFB derivatives) modesWhen a mixture of VPA and stable isotope labelled VPA isadministered to a subject, the number of metabolite peaks to bequantitated by GCMS will double.^Adequate resolution of metabolitepeaks becomes even more critical.^Under a constant carrier gas flowrate (1 ml/min), oven temperature is certainly the most important factorfor good resolution when using either EI or NCI techniques.By performing several experiments with different oven temperatureprograms, we obtained relatively satisfying results for both the EI andNCI methods. Table 4 lists the retention times of the PFB derivativesof VPA, [ 2H6]VPA and their metabolites analyzed by NCI technique, with aGC oven temperature initiated at 50 \u00C2\u00B0C, programmed to 140 \u00C2\u00B0C at30\u00C2\u00B0C/min, held for 20 min, and then increased to 260 \u00C2\u00B0C at 8 \u00C2\u00B0C/min.The PFB derivatives of VPA, [ 2H6]VPA and their metabolites havegood chromatographic properties and give sharp and symmetric peaks evenduring a relatively long GC run time. All of the derivatizedmetabolites gave an abundant ion at [M-181] - corresponding to the lossof the PFB moiety. These ions were monitored in NCI SIM mode for thequantitation of metabolites.9293Table^4:^List^of the^negative, ions monitored^and^retention^times^forthe^PFB^derivatives^of^VPA,^[9-16]VPA,^their^metabolites^and^internalstandards^(I.S.)^for the NCI analysis mode.COMPOUNDS NEGATIVE ION MONITORED(m/z)tR(min)Dibutyl^acetic acid^(I.S.) 171 27.0[2H3](Z)-2-ene VPA^(I.S.) 144 15.35[ 2H3](E)-2-ene VPA^(I.S.) 144 18.50[2H3]3-keto VPA (I.S.) 232 32.312-MGA^(I.S.) 325** 43.23VPA 143 14.67[ 2H6]VPA 149 14.464-ene VPA 141 14.34[24]4-ene VPA 146 14.143-ene VPA 141 14.92[ 2H03-ene VPA 147 14.71(Z)-2-ene VPA 141 15.44[24](Z)-2-ene VPA 147 15.21(E)-2-ene VPA 141 18.62[ 2H6](E)-2-ene VPA 147 18.332,4-diene VPA 139 19.93[ 2H5]2,4-diene VPA 144 19.21(E,Z)-2,3'-diene VPA 139 18.93[24](E,Z)-2,3'-diene VPA 145 18.60(E,E)-2,3'-diene VPA 139 22.43[241(E,E)-2,3'-diene VPA 145 22.034-keto VPA 157 25.03[ 2H04-keto VPA 160 24.873-0H VPA 231 29.69[24]3-0H VPA 237 29.534-0H VPA 231 29.30, 30.36*[ 2H614-0H VPA 237 29.14, 30.21*5-0H VPA 231 33.28[ 2H5]5-0H VPA 236 33.193-keto VPA 229 32.36[ 2H6]3-keto VPA 235 32.262-PSA 339** 41.76[2H3]2-PSA 342** 41.712-PGA 353** 43.43[2H3]2-PGA 356** 43.38* .isomers;** di-derivativesGood resolution of all peaks was obtained by using the GC conditiondescribed above. The 4-ene VPA peak which is frequently overlapped byhigh concentrations of VPA was well separated from VPA with retentiontime difference of about 0.34 min. (E,Z)-2,3'-diene VPA, which isusually missed by EI analysis of the t-BDMS derivative, was detected andwell separated from (E)-2,4-diene VPA with a retention time differenceof up to 1 minute.Two different temperature programs were investigated for t-BDMSderivatives of VPA and metabolites when analyzed by GCMS in EI mode.Table 5 summarizes retention times of the t-BDMS derivatives of theunsaturated metabolites of VPA and [ 2H6]VPA with the GC temperatureinitiated at 50 \u00C2\u00B0C, programmed at 30 \u00C2\u00B0C/min to 110 \u00C2\u00B0C, held for 18 minand then increased to 260 \u00C2\u00B0C at a rate of 10 \u00C2\u00B0C/min. All unsaturatedmetabolites were eluted by 26 min and gave good resolution and peakshapes by this temperature program. However, the run time was too longfor the keto and hydroxyl metabolites and these did not produce sharpand symmetric peak shapes because of their high polarity. Thus, anothertemperature program was set to analyze polar metabolites. Table 6 showsretention times of t-BDMS derivatives of VPA, [ 2H6]VPA and their ketoand hydroxyl metabolites with the GC temperature initiated at 50 \u00C2\u00B0C,programmed to 100 \u00C2\u00B0C at 30 \u00C2\u00B0C/min, then increased to 250 \u00C2\u00B0C at a rate of8 \u00C2\u00B0C/min. Total run time was less than 20 min.94Table 5: Positive ion monitored and the retention times of the t-BDMSderivatives of VPA, [915]VPA, their unsaturated metabolites, and theinternal standards (I.S.) in the El analysis mode.COMPOUNDS ION MONITORED tR(m/z) (min)[2H3](Z)-2-ene VPA^(I.S.) 202 16.79[2H3](E)-2-ene VPA (I.S.) 202 20.41VPA 201 15.71[2H6]VPA 207 15.664-ene VPA 199 15.68[24]4-ene VPA 204 15.533-ene VPA 199 15.88[2H5]3-ene VPA 205 15.62(Z)-2-ene VPA 199 16.86[2H5](Z)-2-ene VPA 205 16.61(E)-2-ene VPA 199 20.59[2H6](E)-2-ene VPA 205 20.192,4-diene VPA 197 22.88[2H5]2,4-diene VPA 202 22.71(E,E)-2,3'-diene VPA 197 24.91[24](E,E)-2,3'-diene VPA 203 24.719596Table 6:^Positive ion^monitored and the retention times of the t-BDMSderivatives^of VPA,^[ H6]VPA,^their^keto^and^hydroxyl^metabolites^andinternal^standards in the EI analysis mode.COMPOUNDS ION MONITORED(m/z)tR(min)Dibutyl^acetic acid^(I.S.) 229 11.02[ 2H3](E)-2-ene VPA^(I.S.) 202 9.45[ 2H3]3-keto VPA (I.S.) 218 11.33VPA 201 8.55[ 2H6JVPA 207 8.484-keto VPA 215 11.96[ 2H6]4-keto VPA 221 11.893-OH VPA 217 11.50, 11.83 *[ 24]3-0H VPA 223 11.43, 11.77*4-OH VPA 217 11.37, 11.94*[ 24]4-0H VPA 223 11.30, 11.90*5-OH VPA 331** 16.25[ 24]5-0H VPA 336** 16.213-keto VPA 332** 15.85[ 24]3-keto VPA 335** 15.832-MGA^(I.S.) 317** 16.162-PSA 331** 16.39[ 2H3]2-PSA 334** 16.362-PGA 345** 17.70[ 2H3]2-PGA 348** 17.67* isomers,^** di-derivatives973.3^Pharmacokinetics of [2H6]VPA and its metabolites in a healthyvolunteerA 700 mg dose consisting of VPA:[24]VPA (50:50) was given orallyto a healthy human volunteer every 12 hours for a period of two and halfdays to perform a multiple-dose study of the pharmacokinetics of VPA and[2H6]VPA. Following the final dose, blood samples were withdrawn atcertain times and serum samples were then obtained by centrifugation.Saliva samples were taken at selected times convenient with the takingof blood samples after stimulation with a 5% citric acid solution rinseof the mouth. Following the first dose, urine samples were collected in12 hour blocks for 2 days and then in 2 or 3 day blocks for another 8days.All samples were analyzed quantitatively by GCMS (HP 5987A) usingEl analysis of the t-BDMS derivatives and NCI analysis of the PFBderivatives with the GC condition we discussed in section 3.2.. Urineor serum samples (0.25 mL) were extracted and derivatized with PFB andthen TMS to be made ready for NCI analysis, while 1 mL of urine or serumwas required to be derivatized with t-BDMS for El analysis.Dibutylacetic acid was used as an internal standard to analyze VPA and[2H6JVPA, 2-methylglutaric acid for 2-PSA and 2-PGA, [2H3]3-keto VPA for3-keto VPA and [2H6]3-keto VPA, and [2H312-ene VPA for the rest of VPAmetabolites and their deuterium labeled analogs. Calibration curveswith different standard concentrations were made for urine, serum total,serum free and saliva respectively, and run for both PFB and t-BDMSderivatives. Table 7 and 8 summarize the coefficients of determinationfor calibration curves of VPA metabolites.Good linearity of calibration curves was obtained for most of theunsaturated VPA metabolites and 3-keto VPA, since [ 2H3]2-ene VPA and[ 2H3]3-keto VPA were used as internal standards. However, the linearitycould be improved for 4-keto and hydroxyl metabolites, if adequatestable isotope labelled internal standards were used for thosemetabolites.Table 7: Linearity of calibratiorl curves for quantitative assays ofVPA, VPA metabolites and their [ H7] labelled analogues which wereisolated frpm urine samples of a human volunteer administered with 700mg of VPA:[917]VPA (50:50) every 12 hours for two and half days.r2Metabolites NCI EIVPA 0.986 0.9844-ene 0.999 0.9973-ene 0.997 0.997(Z)-2-ene 0.999 0.999(E)-2-ene 1.000 0.996(E)-2,4-diene 0.998 0.992(E,Z)-2,3'-diene 0.971 N.D.(E,E)-2,3'-diene 0.993 0.9804-keto 0.987 0.9873-keto 0.998 0.9983-0H 0.996 0.9874-0H 0.984 0.9835-0H 0.990 0.9782-PSA 0.990 0.9992-PGA 0.997 0.9989899Table 8: Linearity (r2) of calibratipn curves for quantitative assaysof VPA, VPA metabolites and their [917] labelled analogues in serumtotal, serum free and saliva samples of a human volunteer administeredwith 700 mg of VPA:VH7NPA (50:50) every 12 hours for two and halfdays.Serum Total Serum Free & SalivaMetabolites NCI El NCI ElVPA 0.999 0.997 0.998 0.9994-ene 0.995 0.997 0.992 0.9933-ene 0.996 N.D. 0.992 0.991(Z)-2-ene 0.999 1.000 1.000 0.998(E)-2-ene 1.000 1.000 1.000 0.999(E)-2,4-diene 0.991 0.999 0.984 0.993E,Z 2,3'-diene 0.994 N.D. 0.992 N.D.E,E 2,3'-diene 0.994 0.998 0.992 0.9944-keto 0.997 0.953 0.961 0.9943-keto 0.998 0.999 0.998 0.9993-0H 0.995 0.960 0.992 N.D.4-0H 0.962 0.906 N.D. N.D.5-0H 0.971 N.D. 0.995 N.D.2-PSA N.D. 0.998 N.D. N.D.2-PGA 0.994 0.996 0.876 0.985Pharmacokinetic parameters of VPA and its metabolites were obtainedbased on the serum and saliva data. The elimination rate constants (KE)were obtained from the slopes of the log serum or saliva concentrationvs time plot using statistical linear regression program. Total bodyclearance (CL) and volume of distribution (Vd) were obtained from thefollowing equation where area under the curve (AUC) over 12 hours afterthe final dose was calculated using a computer program.DoseCL = KE x Vd -AUCFigure 20 shows elimination curves of VPA and [ 2H6]VPA in serumtotal, serum free and saliva which were measured with NCI techniques.10050^100^150^200^250^300^350HOURS10.000(t)4,1.000(C1/7EJ'o's^0.100o_0.010 50^100^150^200^250^300^350HOURSo^1.000oc01000.0500^10^20^30^40^50HOURSFig. 20: Elimination Curves of VPA (A ) and [2H6]VPA (I ) in serum total(top), serum free (middle) and saliva (bottom) which were measured withNCI techniques.101From the elimination curves shown in figure 20, [ 2H6]VPA shows verysimilar pharmacokinetic behavior to VPA. Table 9 presents thepharmacokinetic parameters of both VPA and [ 2H6]VPA measured in thisvolunteer under steady state conditions.These [ 2H6]VPA (Table 9) parameters are in agreement with theresults that Acheampong et al. obtained for a pulse dose of [ 2H6]VPA(1984). The pharmacokinetic equivalency of the labelled and unlabeledVPA might indicate that the two main metabolic pathways, glucuronidationand 0-oxidation, cannot be affected by primary deuterium isotope effectsin the metabolism of [ 2H6]VPA.To help demonstrate the equivalency of VPA and [ 2H6]VPA withrespect to metabolism via the fl-oxidation metabolic pathway, the timecourse (12 hours) of both labelled and unlabeled 0-oxidative metabolitesof VPA are illustrated in Figure 21.Table 10 lists the terminal elimination half-life and eliminationconstant values of the 0-oxidation and other metabolites of VPA and[ 2H6]VPA. Deuterium labelled 2-ene, 3-keto and 3-0H VPA have verysimilar elimination behaviors as their unlabeled analogues.102Table 9: Pharmacokinetic Parameters of VPA(I) and [2N6]VPA(II) measuredby NCI technique in seruT and saliva samples of one subject after 5 oraldoses of 700 mg of VPA:[ NOPA (50:50).t1/2 KE CL AUC VD(h) (h-1) (L/h/Kg) (mg.h/L) (L/Kg)Serum TotalI 19.8 0.035 0.0079 636 0.226II 20.4 0.034 0.0078 642 0.229Serum FreeI 15.1 0.046 0.134 37.22 2.92II 14.7 0.047 0.154 32.38 3.29SalivaI 13.6 0.051II 13.6 0.052103100. 00010.000 - A^At^s1.000-Li\u00E2\u0080\u00A20.10\u00C2\u00B0-0.050^O VPA[2HOVPA^ V 2-ene VPA^ v [2H6]2-ene VPAA_A 3- keto VPA\u00E2\u0080\u00A2 -\u00E2\u0080\u00A2 [2H6]3-keto VPA3-0H VPA\u00E2\u0080\u00A2 \u00E2\u0080\u00A2 [21i]3-0H VPA1040 2^6^8^10^12HOURSFig. 21:^Time courses (12 hours after last dose) of labelled andunlabeled fl-oxidation m4abolites of VPA in the subject administered 5doses of 700 mg of VPA:[H7]VPA (50:50).105Table 10:^Pharmacokinetic Parameters of some deuterium labelled andunlabeled metabolites of VPA measured in a healthy volunteer understeady state conditions, all data were based on NCI results.Metabolites of VPA Apparent Apparentt1/2^(h) KE^(h -1 )4-ene 15.0 0.046[ 2H6]4-ene 14.7 0.0473-ene 80.4 0.0086[ 24]3-ene 75.2 0.0092(E)-2-ene 31.2 0.0222(E)-[ 2H6]2-ene 29.7 0.02372,4-diene 50.1 0.0139[ 2H5]2,4-diene 41.0 0.0169(E,E)-2,3'-diene 27 0.0259[ 2H6](E,E)-2,3'-diene 26 0.02693-keto 28.5 0.024[ 2H6]3-keto 32.1 0.02164-keto 32.7 0.0212[ 2H6]4-keto 34.8 0.01993-0H 35.7 0.0194[ 24]3-0H 32.8 0.0211The half-life values of 3-keto and 2-ene VPA determined in thisstudy are very close to the reported values of Pollack et al.'s studies(1986).^Basically, elimination of the metabolites of VPA were slowcompared with the parent drug.^If one or more of these metabolitesexerted a significant anticonvulsant action, their presence mightexplain the general clinical observation of a slow onset of maximalanticonvulsant effect and a prolonged duration of action of VPA. Asdiscussed in the introduction, the unsaturated metabolites 2-ene VPA, 3-ene VPA, 4-ene VPA (Loscher, 1981; Loscher et al., 1985) and 2,3'-diene(Abbott et al., 1988) were found to have significant anticonvulsantactivity in rodent models.3.4 Isotope Effects of [ 2H6]VPA Metabolism.When used as a 'pulse' dose to investigate the pharmacokineticparameters of a drug, the stable isotope labelled analogue should notshow isotope effects with respect to metabolic reactions and should havethe same pharmacokinetic behavior as the unlabeled analogue. There aretwo main reasons for these requirements. First, the pharmacokinetics ofthe labelled analogue must represent those of the unlabeled drug if thepulse dose method is to prove successful. Secondly and most important,the labelled drug should not be expected to switch metabolic pathways asa result of introducing a stable isotope, otherwise, severe toxicity maybe caused. Because isotope effects are possible, these should beevaluated based on the labelling position of stable isotopes andmechanism of the drug metabolism, and confirmed with experiments beforeuse of the labelled drug in patients.106Since [2H6]VPA has been labelled with deuterium on the terminalcarbons, the two main metabolic pathways, a-oxidation andglucuronidation should not be affected by any deuterium isotope effect.However, for w-oxidation which occurs at terminal protons, isotopeeffects were expected for this metabolic pathway.Potential isotope effects of [ 2H6]VPA were investigated in thehealthy volunteer from a) serum data based on the AUC ratio of [2H0]VPAand its metabolites to [ 2H6]VPA and its deuterated metabolites, and b)urine data based on the recovery ratio of [2H0]VPA and its metabolitesto their stable isotope labelled counterparts during the 12 hoursfollowing the last dose. All urine samples were measured by both El andNCI. Tables 11 and 12 show data of the serum AUC ratios and urinerecovery ratios of VPA and its metabolites to their deuterium analogues.Several conclusions can be drawn from the results.107Table 11: Area under curve (AUC) ratios of VPA and VPA metabolites totheir deuterium labelled analogs over 12 hours after the final dose inserum samples of a healthy volunteer administered 5 doses of 700 mgVPA:rHOVPA (50:50). All values were based on NCI results.AUC^(mg.h/l) AUC RatioMetabolites 2H0 2uH6 2H0/24VPA 636.26 493.8 1.284-ene 1.675 1.294 1.293-ene 10.08 6.768 1.49*(Z)-2-ene 6.021 4.827 1.25(E)-2-ene 31.45 26.26 1.202,4-diene 1.538 1.03 1.49*(E,E)-2,3'-diene 7.365 6.663 1.104-keto 3.424 1.78 1.93*3-keto 46.84 33.11 1.413-0H 3.063 2.363 1.294-0H 3.402 2.663 1.285-0H 1.075 0.028 38.4*2-PGA 1.268 N.D. N.D.*: Potential isotope effects based on AUC ratio.108109Table 12:^Steady state urinary recovery molar ratio of ,VPA and itsmetabolites to their deuterium labelled analogs,^[ 2H 6]VPA andmetabolites in a healthy human volunteer administered 5 doses of 700 mgof VPA:[9-16]VPA (50:50), based on 12 hour urine collected following thefinal dose*.MetabolitesNCI + Base lRatio 2H0/ 2H6NCI + Enzyme 2^EI + Base3 EI + Enzyme4VPA 1.12 1.21 1.10 1.094-ene 1.08 1.25 N.D. N.D.3-ene 1.29 1.20 1.34 1.22(Z)-2-ene 0.97 1.28 1.27 1.23(E)-2-ene 0.97 1.13 1.30 1.27(E)-2,4-diene 1.36 1.42 1.59 1.63(E,Z)-2,3'-diene 0.87 1.03 N.D. N.D.(E,E)-2,3'-diene 1.00 1.04 1.26 1.234-keto 0.90 1.20 0.90 1.133-keto 1.14 1.45 3.23 3.123-0H 1.10 1.15 1.27 1.674-0H 1.00 1.22 2.33 2.985-0H 6.50 6.38 6.04 5.892-PSA 1.11 1.00 1.20 1.202-PGA 15.5 16.3 14.6 15.2N-Acetyl^Cysteine Conjugate of (E)-2,4-diene (NCI) 1.54* Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [ 2H6]VPA.1. Urine samples were hydrolyzed by alkali and analyzed by NCI.2. Urine samples were hydrolyzed by glucuronidase and analyzed by NCI.3. Urine samples were hydrolyzed by alkali and analyzed by EI.4. Urine samples were hydrolyzed by glucuronidase and analyzed by EI.From serum data, [21-105-01-1 VPA shows prominent isotope effects,while deuterated 3-ene, 2,4-diene and 4-keto also show slight isotopeeffects based on this AUC ratios.According to the urine data, a large isotope effect was observed inthe metabolic formation of [ 21-105-OH VPA and [ 21-13}2-PGA, which agreeswith the finding of Acheampong et al. (1984). This result was expectedbecause 5-0H VPA and 2-PGA are the products of metabolic w-oxidation bymixed function oxidative enzymes. As mentioned in the introduction, 5-OH VPA is formed via abstraction of a hydrogen from position 5 to form acarbon-centered free radical(Rettie et al., 1987). An isotope effect ispredicted if the extraction of hydrogen is the rate limiting step.Since 5-0H VPA and 2-PGA only account for a small portion of thetotal metabolites, the decrease in the formation of [ 2H05-01-I VPA and[21-3]2-PGA did not markedly affect the elimination kinetics of [2H6]VPA.No major isotope effects were observed in the other metabolicpathways, based on urine data, including the formation of 4-ene VPA,where one deuterium is lost to form the product. This result supports arecently reported mechanism for the cytochrome P-450 desaturationmetabolism (Rettie et al., 1988) of VPA in which a carbon centeredradical at C-4 serves as an intermediate, and this step is rate limitingin the formation of 4-ene VPA.Surprisingly, a small isotope effect was apparent for the formationof [ 2H6](E)-2,4-diene VPA, from both serum and urine data.^It is110believed that 2,4-diene VPA is formed via 2-ene and 4-ene VPA (Porubeket al., 1988). The formation of [ 2H6](E)-2,4-diene VPA should not showany isotope effect since the formation of [ 2H6]4-ene VPA does not. Itwas also found by my colleague Dr. Kassahun that the ratio of the N-acetylcysteine conjugate of (E)-2,4-diene VPA to its deuteriumcounterpart was as high as 1.54 (Table 10), when the same urine sampleswere analyzed. Based on this information and our findings of a smallisotope effect for 2,4-diene VPA formation from [ 2H5]VPA, we thereforeproposed that the formation of 2,4-diene VPA might occur partly from 3-ene VPA. Removal of a deuterium atom from the C-5 position in [ 2H03-ene VPA may be the first step in the formation of 2,4-diene VPA.Consequently, an isotope effect is expected for this pathway, and thusmight explain the observed result. An experiment was designed to testthis proposal, and the results of that experiment will be discussedlater.3.5 Isotope effects with respect to [ 13C4]VPA metabolism:A healthy human volunteer participated in this study. He was givena single oral dose consisting of 700 mg of VPA:0 3C4]VPA (50:50). Thestructure of [ 13C4]VPA (8) is illustrated below.CH3-CH2- 13CN3CH- 13COOHru^1CH3-2 13ru-^ 2111( 8 )Thirteen blood samples were collected at 0, 0.5, 1, 1.5, 2, 2.5, 3,5, 7, 9, 12, 24, 48 and 72 h after the single dose and allowed to clot.The blood samples were centrifuged to obtain serum samples. A few urinesamples were collected at convenient time blocks. All urine and serumsamples were analyzed using NCI techniques, and the potential isotopeeffects of [13C4JVPA were studied based on the concentration ratio ofVPA and its metabolites to [13C4]VPA and its metabolites in urine orserum samples. The results are presented in Table 13. No apparentisotope effects were observed, thus qualifying [ 13C4]VPA to be used inpharmacokinetic studies of VPA metabolites in pediatric patients. Theratios of [13C0/13C4]VPA for 4-0H VPA and 5-0H VPA wre different fromunity, but these differences were accounted for by a high back groundfor the unlabeled VPA metabolites.112Table 13: Metabolic equivalence of [ 13C4]VPAAnd VPA based on mean TICpeak area ratio of VPA metabolites to their [\"C4]-labelled analogs (13serum samples and a urine sample collected 3-9 hr after the dose from ahealtbx^human^volunteer^administered^a^single^dose^of^700^mg^ofVPA:[\"C4]VPA (50:50) were analyzed by NCI techniques).Metabolites \u00E2\u0080\u00A2Ratio 13^13^.(^Co/^C4)Serum Urine4-ene VPA 0.954 0.9743-ene VPA 0.982 1.154(Z)-2-ene VPA 0.780 0.884(E)-2-ene VPA 1.018 1.110VPA 1.006 0.9872,4-diene VPA 1.020 1.112(E,E)-2,3'-diene VPA 1.041 1.1044-keto VPA 1.041 1.0453-0H VPA 1.038 1.1224-011 VPA 1.413* 1.335*3-keto VPA 0.891 0.9562-PGA 0.934 0.8945-0H VPA 6.331* 1.675** Area ratio of 4-OH and 5-011 VPA are over 1 due to high background atthe same retention time as 4-OH and 5-011 VPA when monitoring ion m/z231.1133.6 Urinary recoveries of VPA and its metabolitesThe urinary recoveries of VPA, [21-16]VPA and their metabolites inthe study of multiple doses (one dose every 12 hr for 2 and half days)were measured in urine samples collected for 12 hours after the finaldose.This study found that VPA glucuronide and 3-keto VPA are thepredominant urinary metabolites, which is consistent with previousstudies (Abbott et al., 1986; Pollack et al., 1986; Dickinson et al.,1989) on VPA metabolites in humans. About 50% of the VPA dose isrecovered as conjugated metabolites, while 25% is recovered via the /3-oxidation pathway. The remaining oxidative metabolites in urine, theunsaturated and hydroxyl metabolites, together account for only a smallfraction of the recovered VPA-derived products in urine.Tables 14 to 17 present the metabolite concentrations measured byEl or NCI after hydrolysis with either glucuronidase or sodium hydroxidesolution. The conjugated fraction will be discussed in section 3.7.114115Table 14: Steady state urinary recoveries of VPA, [ 2H6]VPA andmetabolites (free plus * conjugated) in the urine collected 12 hrfollowing the final dose , hydrolyzed with NaOH solution, and analyzedby NCI GCMS.Metabolites Recoveries(% of dose) **2Ho 2H6VPA 59.9 53.34-ene 0.056 0.0523-ene 0.036 0.028(Z)-2-ene 0.037 0.038(E)-2-ene 0.89 0.92(E)-2,4-diene 0.99 0.73(E,Z)-2,3'-diene 1.66 1.90(E,E)-2,3'-diene 1.26 1.264-keto 1.64 1.833-keto 26.6 23.43-0H 2.03 1.844-0H 2.83 2.845-0H 1.24*** 0.192-PSA 0.30 0.272-PGA 3.10*** 0.20* Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [ 2H6]VPA.** Recoveries are calculated on a molar basis.***Qualify as an isotope effect.116Table 15: Steady state urinary recoveries of VPA, [2H6]VPA andmetabolites (free plus ionjugated) in urine sample collected 12 hoursfollowing the final dose , hydrolyzed with glucuronidase and analyzed byNCI GCMS.Metabolites Recoveries(% of dose)**2Ho^2 H6VPA 48.3 39.84-ene 0.045 0.0363-ene 0.024 0.020(Z)-2-ene 0.041 0.032(E)-2-ene 0.87 0.77(E)-2,4-diene 0.94 0.66(E,Z)-2,3'-diene 2.09 2.02(E,E)-2,3'-diene 1.11 1.074-keto 2.01 1.673-keto 16.4 11.33-0H 2.09 1.814-0H 4.40 3.605-0H 1.34*** 0.212-PSA 0.29 0.292-PGA 3.26*** 0.20* Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [2H ]VPA.** Recoveries are calculated on a molar basis.***Qualify as an isotope effect.117Table 16: Steady state urinary recoveries of VPA, [2H6]VPA andMetabolites (free plus ionjugated) in the urine collected 12 hoursfollowing the final dose , hydrolyzed with NaOH solution, and analyzedby El GCMS.Metabolites^ Recoveries(% of dose)**2Ho 2un6VPA 45.6 41.64-ene 0.065 N.D.3-ene 0.055 0.041(Z)-2-ene 0.047 0.037(E)-2-ene 1.13 0.86(E)-2,4-diene 0.54 0.34(E,E)-2,3'-diene 1.45 1.154-keto 2.25 2.503-keto 29.8 9.223-0H 2.70 2.124-0H 3.39 1.455-0H 5.80*** 0.962-PSA 0.24 0.202-PGA 2.92*** 0.20* Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [2H6]VPA.** Recoveries are calculated on a molar basis.***Qualify as an isotope effect.118Table 17:^Steady state urinary recoveries of VPA, [ 2H6]VPA andmetabolites (free plus conjugated) in the urine collected 12 hoursfollowing^the^final^dose*,by EI GCMS.hydrolyzed with glucuronidase,^and^analyzedMetabolites Recoveries(% of dose) **2Ho 24VPA 42.1 39.24-ene 0.055 N.A.3-ene 0.028 0.023(Z)-2-ene 0.042 0.034(E)-2-ene 1.00 0.79(E)-2,4-diene 0.49 0.30(E,E)-2,3'-diene 1.15 0.934-keto 1.31 1.163-keto 18.7 5.983-0H 2.11 1.264-0H 3.70 1.245-011 1.59*** 0.272-PSA 0.30 0.252-PGA 3.80*** 0.25* Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [ 2H6]VPA.** Recoveries are calculated on a molar basis.***Qualify as isotope effect.3.7 Conjugated Fraction of VPA and Its Metabolites in Urine SamplesVPA and most of its metabolites undergo phase II conjugation priorto excretion. The most important conjugate in urine is the glucuronicacid conjugate, which is susceptible to hydrolysis with fl-glucuronidase,and with alkali or strong acid (Dickinson et al., 1985a).Studies by Dickinson et al. (1979; 1982) of the disposition of VPAin the rat, monkey and dog revealed that, in bile and urine samples, aproportion of the total conjugated VPA, as determined by alkalinehydrolysis, was resistant to cleavage by fl-glucuronidase, suggestingthat nonglucuronide conjugates were present. Further studies byDickinson et al. (1984) indicated that acid- and base- catalyzedintramolecular acyl migration of the valproate moiety in valproateglucuronides away from the C-1 position could occur. In the subsequentprocess of ring-opening mutarotation and lactonization, six structuralisomers and lactones could be formed which were not substrates for fl-glucuronidase, but could be hydrolyzed in strong alkaline media. Thedisposition of these rearranged glucuronides in vivo differs from thatof the primary isomer (Dickinson et al., 1985a; 1985b; 1986), and it isof some interest that abnormally high concentrations of VPA conjugates,consisting largely of the rearranged glucuronide isomers, were detectedin the plasma of a patient diagnosed with VPA-associated hepatobiliaryand renal dysfunction (Dickinson et al., 1985b).In the present study, we used both enzyme fl-glucuronidase andalkali NaOH solution to hydrolyze VPA and its metabolite conjugates.119Whether any glucuronidase-resistant conjugates exist or not weredetermined based on results of these two hydrolysis methods. Theconjugated fractions were obtained from the concentration differences ofrecovered drug and metabolites in hydrolyzed (total) and unhydrolyzed(free) urines. All urine samples were analyzed with both the EI and NCImethods. Table 18 shows the conjugated fractions of VPA and itsmetabolites obtained with the different hydrolysis and assay methods.About 95% of VPA in urine was in the form of the glucuronideconjugate and all unsaturated metabolites were excreted mainly as theirglucuronide conjugates. However, other metabolites of VPA particularlypolar metabolites show relatively low or no conjugation. These resultsare in agreement with the findings of Kassahun et al. (1989) for theexcretion of VPA and metabolites in the urine of pediatric patients.120Table 18: Conjugated fraction (%) of VPA and its metabolites in urinesamples collected for 12 hours after final dose measured by differenthydrolysis and assay methods.Metabolites^ Conjugated Fraction (%)Enzyme + NCI *^Base + NCI *^Enzyme + EI * Base + EI *VPA^96^97 93 934-ene 96 97 89 913-ene^79^86 61 80(Z)-2-ene^99 99 92 92(E)-2-ene 99^99 89 90(E)-2,4-diene^98 98 84 86(E,Z)-2,3'-diene^96 95 N.D. N.D.(E,E)-2,3'-diene^96 97 81 854-keto^34 19 0 353-keto 0^0 0 03-0H^47 45 44 564-0H 22^0 65 625-0H^22 15 0 492-PSA 0^0 20 02-PGA^7 0 24 0* Base:^Hydrolyzed with NaOH solution,Enzyme: Hydrolyzed with glucuronidase,NCI:^Analyzed by NCI GCMS,EI:^Analyzed by EI GCMS.121No apparent differences were observed between the fl-glucuronidaseand alkaline catalyzed hydrolysis (Table 18), as measured by a paired t-test. Table 19 presents the t-test results. No significant differencewas indicated between glucuronidase and alkaline hydrolysis (p-values:0.4069, 0.1141), with samples analyzed with either NCI or El.^We cantherefore,^assume that there was little glucuronidase-resistantconjugate present in the urine samples of this subject after beingstored at -20 \u00C2\u00B0C for about 2 months. A greater number of subjects wouldbe required to give this finding statistical significance.Table 19:^P-values of paired t-test over different hydrolysis andanalysis methods.*Enzyme + NCI ^*Base + El *Enzyme + El^0.7776^0.1141* Base + NCI 0.4069 0.8115* Base:^Hydrolyzed with NaOH solution,Enzyme: Hydrolyzed with glucuronidase,NCI:^Analyzed by NCI GCMS,El:^Analyzed by El GCMS.1223.8 Comparison of analysis and hydrolysis methodsAs discussion above, all urine samples from the multiple dose studywere hydrolyzed with both NaOH solution and glucuronidase, and analyzedby both EI and NCI techniques. The data from different methodcombinations were compared to see if the same results were obtained.Table 19 presents the t-test results of comparison of four groups ofurine data measured by different method (hydrolysis and analysis)combination. No significant difference (p-value >> 0.05) among thosefour groups of data was indicated by this statistic results.Further comparison of these four groups of data were performed on aMIDAS based computer program containing methodological statistics.Table 20 lists the correlation coefficients r 2 between those four groupsof data.Alkaline and glucuronidase hydrolysis gave very good consistency,when samples were analyzed by the NCI technique. The results from theNCI and EI methods also gave good consistency except for 5-0H VPA, whenurine samples were hydrolyzed with enzyme. The poor agreement for 5-0HVPA could be due to EI analysis, since very good agreement was obtainedbetween the two hydrolysis methods when 5-0H VPA was analyzed by NCI.Comparing these four groups of data of total urine VPA and metabolites,we can say that the combined method of base hydrolysis and EI analysisis not as good as the other three.123124Table 20: Coefficients of determination (r2, n=11) betweenconcentrations of VPA urine metabolites measured by different hydrolysis(base or enzyme) and analysis (NCI or El) methods. Urine samples werecollected from a human,volunteer participated in the multiple dosesstudy (700 mg of VPA : rHOVPA (50:50) every 12 hr for 2.5 days).MetabolitesNCI*CorrelationEl*CoefficientsBase^Enzyme Unhydrolyzed*4-ene 0.98968 0.99668 0.98718 0.97977 0.67333**3-ene 0.97400 0.97742 0.96690 0.95566 0.79158**2-ene^(Z)- 0.96690 0.99339 0.97621 0.98726 0.35921**2-ene^(E)- 0.99391 0.99846 0.98287 0.99561 0.84806**VPA 0.97912 0.98785 0.98547 0.96284 0.81675**2,4-diene 0.99594 0.99777 0.98856 0.99397 0.54398**2,3'-diene 0.97460 0.99190 0.94098 0.94878 0.29505**4-keto 0.95869 0.89630 0.95808 0.94769 0.977063-0H 0.98686 0.89371 0.95017 0.96591 0.918514-0H 0.95681 0.59668** 0.91728 0.95648 0.871083-keto 0.96737 0.99358 0.99383 0.96563 0.992715-0H 0.99854 0.52438** 0.61801** 0.62679** 0.957982-PSA 0.95541 0.99230 0.95195 0.95161 0.985702-PGA 0.99005 0.99584 0.99193 0.99827 0.98440NCI: Correlation between alkaline and glucuronidase hydrolysisresults, when measured by NCI GCMS.El:^Correlation between alkaline and glucuronidase hydrolysis,when measured by El GCMS.Base: Correlation between NCI and El analysis methods whenhydrolyzed with NaOH solution.Enzyme: Correlation between NCI and El analysis methods whenhydrolyzed with glucuronidase.Unhydrolyzed: Correlation between NCI and El analysis results of**^non-conjugated components in urine samples.Poor correlationFor unhydrolyzed (free) urine samples, two analysis methods gavedifferent correlation values for different metabolites. VPA and itsunsaturated metabolites which exist mainly as conjugates in urine showpoor agreement between the two analysis methods. Other metaboliteswhich are not conjugated or have low levels of conjugates in urine showvery good consistency when analyzed by the two different methods. Sincestrong acidic solution can dissociate the conjugates, it could bepossible that some conjugates were hydrolyzed when urine samples wereadjusted to pH=2 during extraction. The partial hydrolysis results inthe variation of free fraction of VPA and its unsaturated metabolitesthat are excreted into urine mostly as their conjugated forms.3.9 A Pharmacokinetic study of VPA in sheep using [ 13C4]VPAThe pharmacokinetics of VPA in sheep were studied by giving asingle dose (I.V.) of 1000 mg of VPA:[ 13C4]VPA (50:50) to each of twosheep.^Blood samples were collected and allowed to clot beforecentrifuging to collect serum samples.^All serum samples were thenanalyzed by EI GCMS quantitatively, using the [ 2H7]VPA and itsmetabolites that were synthesized as internal standards. When stableisotope-labelled analogs are applied as internal standards, SIM mode isthe ideal method for quantitative analysis. Figure 21 illustrates theSIM chromatograms of VPA [ 13C4]VPA and [ 2H7]VPA. These three analogsshow very sharp peaks and can be readily differentiated based on theirion masses. As mentioned in the introduction, there should be no125natural isotope interference since the mass difference is equal orhigher than 3 mass units. The separation and quantitation of VPA, VPAmetabolites and their C-13 labelled analogs were completed in a singlechromatographic run of 29.5 minutes in length, C-13 labelled metaboliteshad very similar retention time as their unlabeled analogs, with about0.01 min difference. Table 21 presents the retention times and m/zvalues of the (M-57)1- diagnostic ions for VPA and its metabolites.126Table 21: The retention time and m/z values of the (M-57)+ diagnosticions of VPA and its metabolites iso14ed from serum samples of sheepdosed with single dose of 1 g of VPAT'COVPA (50:50).Metabolites* Retention time(min)Ion monitored(m/z)VPA 13.64 2014-ene VPA 13.88 199(E)-3-ene VPA 14.05 199(Z)-3-ene VPA 14.29 199(Z)-2-ene VPA 16.58 199(E)-2-ene VPA 14.64 199(Z)-2,4-ene VPA 17.05 197(E)-2,4-diene VPA 17.78 197(E,Z)-2,3'-diene VPA 17.80 197(E,E)-2,3'-diene VPA 18.71 1973-keto VPA 21.79 329b4-keto VPA 20.03 2153-0H VPA 19.66, 19\u00E2\u0080\u00A293a 2174-0H VPA 12.21, 12\u00E2\u0080\u00A241a 100c5-0H VPA 22.14 331b2-PSA 22.18 331b2-PGA 22.50 345babcTwo isomersDiderivatives7-Lactone*^[ 13C4]VPA and its metabolites were monitored by SIM withdiagnostic ions which were four mass units higher than those oftheir unlabeled analogs.12713.00 14.00 17.0011.00 12.00 16.0015.00Time ->Abundance150000-100000-50000-:Ion 205.00: 1001010.DAbundance150000100000500000Time ->Ion 208.00: 1001010.DAbundance15000010000050000Ion 201.00: 1001010.DTime ->^11.00^12.00^13.00^14.00^15.00^16.00^17.0011.00 12.00 13.00 14.00 15.00 16.00 17.00128Fig. 22:^SIM Oromatograms of [ 2H7]VPA (top, internal standard), VPA(middle), and [ IJ C4]VPA (bottom).Excellent standard curves were obtained for most of themetabolites. The coefficients of determination obtained for calibrationcurves of metabolites isolated from standard urine samples are presentedin Table 22.As Table 22 shows, the calibration curves have very good linearity.The coefficients of determination r2 for all metabolites, were greaterthan 0.994, except for 3-0H and 3-keto VPA. Calibration curves for 3-0Hand 3-keto VPA could be expected to improve had [2H7]3-0H VPA and[2H7]3-keto VPA been used as internal standards. These two internalstandards were not synthesized until after this analysis had beencompleted. Our results thus support the contention that optimalcalibration curves are obtained by using stable isotope-labelledanalogues as internal standards when samples are analyzed with GCMS.Unfortunately, [2H7]4-ene VPA could not be used in this assay because ofinterference from [13C4]VPA with a mass difference of only I mass units.Retention times were very close and the sample contained a highconcentration of [ 13C4]VPA. Thus to apply [2H7]4-ene VPA as an internalstandard, some modification in the GC conditions would be necessary toadequately separate VPA and the 4-ene VPA peaks. The NCI method shouldbe suitable for application of [2H7]4-ene VPA as an internal standardbecause the 4-ene precedes VPA in retention time.129130Table 22: Linearity of calibration curves for the quantitative assaysof VPA, VPA metabolites and their C-13 labelled analogues isolated fromurine,,samples of sheep dosed I.V. with a single dose of 1000 mg ofVPA:[\"C4]VPA (50:50).Metabolites *^r2^Internal StandardsVPA^ 1.000^[2H7]VPA4-ene VPA^0.999^[2H7]VPA3-ene VPA 0.999^[2H7]VPA(Z)-2-ene VPA^0.999^[2H7]VPA(E)-2-ene VPA 0.999^[2H7]VPA(E)-2,4-diene VPA^0.999^[2H7]VPA(E,Z)-2,3'-diene VPA^0.999^[2H7]VPA(E,E)-2,3'-diene VPA^0.996^[2H7]VPA3-keto VPA^0.975^[2H7]4-keto VPA4-keto VPA 0.999^[2H7]4-keto VPA3-0H VPA^ 0.988^[2H7]VPA4-0H VPA 0.996^[2H7]VPA4-0H VPA isomer^0.990^[2H7]VPA5-0H VPA^ 0.994^[2H7]5-0H VPA2-PGA 0.997^[2H7]VPA*^[ 13C4]VPA and its metabolites were analyzed using the calibrationcurves for the unlabeled analogs.Pharmacokinetic parameters for VPA, [13C4]VPA and the major serummetabolites, 2-ene VPA and [13C4]2-ene VPA were determined for the twosheep that were studied. The results are presented in table 23.Table 23: Pharmacoktnetic Parameters for VPA(I), [13C4]VPA(II), (E)-2-ene VPA (III) and [\"C4](E)-2-ene VPA (IV) meamred in two sheep dosedI.V. with a single dose of 1000 mg of VPAT'CWPA (50:50). Serumsamples were analyzed by El GCMS.t1/2 KE CL AUC VD(h) (h-1) (L/h/Kg) (mg.h/L) (L/Kg)Sheep 411I 5.09 0.136 0.122 55.9 0.897II 4.98 0.139 0.127 53.42 0.913III 5.63 0.123IV 5.63 0.123Sheep #2I 2.42 0.286 0.159 52.5 0.556II 2.41 0.287 0.160 52.0 0.557III 4.33 0.160IV 3.89 0.178131No apparent isotope effect of [ 13C4]VPA metabolism was observed inthese two sheep based on AUC ratio of VPA to [ 13C4]VPA, which is inagreement with the result we obtained from the study in a healthy humanvolunteer.Since few studies of VPA pharmacokinetics have been carried out insheep, very little information is available on the metabolic fate ofvalproic acid in this species. Nevertheless, this initial study of[ 13C4]VPA indicates that this compound should prove ideal for the studyof placental transfer and the determination of drug pharmacokinetics inboth mother and fetus.3.10 Metabolic studies of (Z)-and (E)-3-ene VPAAs discussed in the section 3.4., it was suggested that, 2,4-eneVPA may be partially formed from 3-ene VPA. To further investigate thishypothesis, we synthesized (Z)-3-ene and (E)-3-ene VPA for metabolismstudies in rats.The syntheses of (E)- and (Z)-3-ene VPA were discussed in section3.1.9. Both (E)-and (Z)-3-ene VPA were analyzed by GCMS and by NMR anddetermined to be of very high purity before they were used in metabolismstudies.(Z)- and (E)-3-ene VPA (150 mg/kg dose) were separatelyadministrated i.p. to two rats (adult male Wistar rats weighing 270 and308 g). A blood sample (about 200 ul) from each rat was taken 2 h after132the dose, allowed to clot, and centrifuged to obtain a serum sample.Urine samples were collected every 24 hours for two days. Samples werestored at -20 \u00C2\u00B0C until derivatized and analyzed by GCMS using Eltechniques.In the SIM mode used, m/z 197 and 199 were monitored to see thediene VPA metabolites and the parent drugs (E)- and (Z)-3-ene VPA. Fromthe assay results, two diene VPA metabolites were present in urinesamples of both sheep, one metabolite was confirmed to be (E,E)-2,3'-diene VPA. The other diene VPA could be either (E,Z)-2,3'-diene VPA or2,4-diene VPA since they had very close retention time under El analysisconditions. It was likely that (E,E)-2,3'-diene VPA was the main dienemetabolite of (E)-3-ene VPA, while (E,Z)-2,3'-diene or 2,4-diene VPAaccounts for most of the diene metabolites of (Z)-3-ene VPA. Table 24shows the peak area of all the ions monitored in urine samples. Since(E)-2,4-diene VPA and (E,Z)-2,3'-diene VPA were not differentiated wellunder that condition, it is not apparent that (E)-2,4-diene VPA is oneof metabolites of 3-ene VPA. Further analysis by using NCI mode has tobe done before making a final conclusion.The t-BDMS derivative of (Z)-3-ene VPA had a slightly longerretention time than that of (E)-3-ene VPA, when run on an OV-1701column.^It is usually the (E)-isomer of unsaturated fatty acids thathas the longer retention time with this stationary phase.^The reasonfor this reversal of elution order by the (E)- and (Z)- isomers of 3-eneVPA is not readily apparent.133134Table 24:^Retention time and peak area of monitored^ions m/z^199 and197 which represent parent drug 3-ene VPA and diene metabolitesrespectively isolated from urine of rats dosed with either (Z)- or (E)-3-ene VPA (150 mg/kg).JZ)-3-ene VPA jE)-3-ene VPAMetabolites^(tR) 0-24h^24-48h 0-24h^24-48h(Z)-3-ene VPA 178442944 21974892 N.D. N.D.(8.53 min)(E)-3-ene VPA N.D. N.D. 243174752 9520370(8.38 min)* (E,Z)-2,3'-diene VPA 66036848 3494949 4874712 118144(10.82 min)(E,E)-2,3'-diene VPA 21874212 1503089 17302344 548160(12.83 min)*or (E)-2,4-diene VPA4. Summary and Conclusions4.1 GCMS conditions for both El and NCI were optimized to obtainoptimal resolution and sensitivity for VPA metabolites and their stableisotope-labelled analogs. A single temperature program with a run timeof 47 min was established for NCI analysis of PFB derivatives of VPA,VPA metabolites and their [24] labelled analogs. Two temperatureprograms were investigated for t-BDMS derivatives of VPA and VPAmetabolites and their [24] labelled analogs, one with a run time of 35min was used for VPA unsaturated metabolites, the other with a run timeof 20 min was for more polar metabolites of VPA.4.2 In the 12h period immediately following the dose, t112 and AUCvalues for VPA and [2H6]VPA in the human volunteer were not different.AUC values and urinary recoveries of unlabeled and labelled metabolitesover the same time period were equivalent except for 5-0H VPA and 2-propylglutaric acid (2-PGA). Significant isotope effects were observedfor 5-0H VPA and 2-PGA. No apparent isotope effect was observed for 4-ene VPA, which supported the mechanism that a carbon centered radical atC-4 of VPA serves as an intermediate, and this step is rate limiting inthe formation of 4-ene VPA.A small isotope effect was observed for 2,4-diene VPA. It was thenproposed that the formation of 2,4-diene VPA might occur partly from 3-ene VPA. An experiment was designed to test this proposal.1354.3 (E)- and (Z)- 3-ene VPA were synthesized. Both were used inmetabolic studies in rats. The metabolites diene VPA and parent drug 3-ene VPA were monitored in urine samples of rats dosed with either (E)-or (Z)-3-ene VPA by SIM EI mode. Both (E)- and (Z)- isomers of 3-eneVPA were shown to have two diene VPA metabolites. One is confirmed tobe (E,E)-2,3'-diene VPA, the other could be either 2,4-diene VPA or(E,Z)-2,3'-diene VPA since they had the same retention time. The (E)-3-ene VPA had a higher level of metabolite (E,E)-2,3'-diene VPA, while(Z)-3-ene VPA was metabolized into more (E,Z)-2,3'-diene VPA (or 2,4-diene VPA).4.4 More than 90% of VPA and its unsaturated metabolites (2-ene,3-ene, 4-ene, 2,4-diene and 2,3'-diene-VPA) were in the form of theirglucuronic acid conjugates when excreted into urine. Metabolites 3-0H,4-0H and 5-0H VPA were excreted partly as glucuronides, while, 3-keto,4-keto VPA, 2-PSA and 2-PGA were excreted mostly as free metabolites.4.5 No difference was observed between the concentrations of totalVPA and its metabolites when urine samples were hydrolyzed withglucuronidase and alkali NaOH solution. Therefore, it seems that therewas no glucuronidase-resistant conjugate present in the urine samples ofthis healthy human volunteer after urine samples were kept at -20 \u00C2\u00B0C forabout two months.NCI and EI analyzing techniques show very good agreements for mostmetabolites in urine samples.^PFB derivatives of VPA metabolites136analyzed by NCI technique give higher sensitivity and better resolutionthan t-BDMS derivatives of VPA metabolites analyzed by El methods.4.6 No apparent isotope effect was observed in the metabolism of[13C4]VPA based on concentration ratios of VPA and its metabolites totheir [13C4] labelled analogs in serum and urine samples of a healthyhuman volunteer. Thus it would appear that [ 13C4]VPA is suitable forthe pharmacokinetic studies of VPA metabolites in pediatric patientswhen given as a \"pulse\" dose.^4.7^Eight deuterium labelled compounds that included [2H7]VPA,[2H7]4-ene , [2H7]4-keto, [2H7]4-0H, [2H7]5-0H, [2H7]3-keto, [2H7]3-0H,and [2H7]2-ene VPA were synthesized and used as internal standards in aGCMS assay of VPA, [ 13C4]VPA and their metabolites for thepharmacokinetic studies in sheep.4.8^Pharmacokinetics of VPA was studied in sheep dosed withVPA:[13C4]VPA (50:50).^No isotope effect was observed for the[13C4JVPA.^The elimination half-life of VPA in these two sheep wasestimated to be approximately 5.0 and 2.4 hr respectively.1375. ReferencesAbbott, FS., and Acheampong, AA., (1988) Quantitative structure-anticonvulsant activity relationships of valproic acid, relatedcarboxylic acids and tetrazoles, Neuropharmacology, 27, 287.Abbott, FS, Kassam, J., Acheampong, A., Ferguson, S., Panesar, S.,Burton, R., Farrell, K., and Orr, J., (1986) Capillary gaschromatography - mass spectrometry of VPA metabolites in serum and urineusing tert-Butyldimethylsilyl derivatives, J. Chromatogr., 375, 285.Abbott, FS., Kassahun, K., and Panesar, S., (1987) Negative ion chemicalionization GCMS analysis of valproic acid in saliva and serum and itsmetabolites in saliva, presented at the 35th ASMS conference on MassSpectrometry and Allied Topics, Denver, Colorado.Acheampong, AA., (1982) Pharmacokinetic and metabolism studies ofvalproic acid using gas chromatography mass spectrometry. M.Sc. Thesis,University of British Columbia, p29.Acheampong, AA., (1985) Quantitative structure-anticonvulsant activitystudies of valproic acid analogues. Ph.D. Thesis, University of BritishColumbia, p73.Acheampong, A., and Abbott F., (1985) Synthesis and stereochemicaldetermination of diunsaturated valproic acid analogs including its majordiunsaturated metabolites, J. Lipid Res., 26, 1002.Acheampong, A., Abbott F., and Burton R., (1983) Identification ofvalproic acid metabolites in human serum and urine using hexadeuteratedvalproic acid and gas chromatographic mass spectrometric analysis,Biomed. Mass Spectrom., 10, 586.Acheampong, AA., Abbott, FS., Orr, JM., Ferguson, SM, and Burton, RW.,(1984) Use of hexadeuterated valproic acid and gas chromatography massspectrometry to determine the pharmacokinetics of valproic acid, J.Pharm. Sci., 73, 489.Bailer, M., Hussen, Z., Raz, I., Abransky, 0., Herishanu, Y., andPachys, F., (1985), Pharmacokinetics of valproic acid in volunteersafter a single dose study, Biopharm. Drug Dispos., 6, 33.Balazs, R., Machiyama, Y., Hammond, BJ., Julian, T., and Richter, D.,(1970) The operation of the GABA bypath tricarboxylic acid cycle inbrain tissue in vitro, Biochem. J., 116, 445.Bjorge, SM., and Baillie, TA., (1985) Inhibition of medium-chain fattyacid fl-oxidation in vitro by valproic acid and its unsaturatedmetabolite, 2-n-propyl-4-pentenoic acid, Biochemical and BiophysicalResearch Communications, 132, 245.138Bjorge, SM., and Baillie, TA., (1991) Studies of the a-oxidation ofvalproic acid in rat liver mitochondrial preparation, Drug Metabolismand Disposition, 19, 823.Bohan, TP., Millington, DS., Roe, CR., Yergey, AL., and Liberato, DJ.,(1984) Valproylcarnitine: A novel metabolite of valproic acid , Ann.Neurol., 16, 394.Bohan, TP., Tennison, MB., Rettenmeier, A., and Baillie, TA., (1987)Valproic acid metabolism in a boy with liver failure, ThePharmacologist, 29, 179.Bowdle, TA., Patel, IH., Levy, RH. and Wilensky, AJ., (1980) Valproicacid dosage and plasma protein binding and clearance, Clin. Pharmacol.Ther., 28, 486.Brown, HC., and Mandal, AK., (1980) Borane:1,4-oxathiane - A newconvenient hydroborating agent, Synthesis, p153.Buchhalter, JR., and Dichter, MA., (1986) Effect of valproic acid incultured mammalian neurons, Neurology, 36, 259.Caraz, G., Gau, R., Chateau, R., and Bonnin, J., (1964) Communication apropos des premiers essais cliniques sur lactivit\u00C3\u00AA anti-epileptique delacide n-dipropylacetique, Ann. Med. Psychol., 122, 577.Chapman, A., Keane, PE., Meldrum, BS., Simiand, J., and Vernieres, JC.,(1982) Mechanism of anticonvulsant action of Valproate, Prog. Neurobiol(Oxford), 19, 315.Chadwick, DW., (1984) Concentration-effect relationship of valproicacid, Clin. Pharmacokinet., 10, 155.Claeys, M., Markey, SP., and Maenhaut, W., (1977) Variance analysis oferror in selected ion monitoring assays using various internalstandards, A practical study case, Biomed. Mass Spectrom., 4, 122.Cloyd, CJ., Kriel, RL, Fischer, JH, Sawchuk, RJ., and Eggerth, RM.,(1983) Pharmacokinetics of valproic acid in children: I. Multipleantiepileptic drug therapy, Neurology, 33, 185.Corredor, C., Brendel, K., and Bressler, R., (1967) The mechanism of thehypoglycemia action of 4-pentenoic acid, Proc. Natl. Acad. Sci., USA,58, 2299.Cregge, RJ., Herrmann, JL., Lee, CS., Richman, JE., and Schlessinger,RH., (1973), A convenient one flask procedure for ester alkylation.Tetrahedron Lett., 2425.Dickinson, RG., Eadie, MJ., and Hooper, WD., (1985a) Glucuronidase-resistant glucuronides of valproic acid: consequences to enterohepaticrecirculation of valproate in the rat, Biochem. Pharmacol., 34, 407.139Dickinson, RG. Harland, RC., Ilias, AM., Rodgers, RM., Kaufman, SN.,Lynn, RK., and Gerber, N., (1979) Disposition of valproic acid in rat:dose-dependent metabolism, distribution, enterohepatic recirculation andcholeretic effect, J. Pharmacol. Exp. Ther., 211, 583.Dickinson, RG., Harland, RC., Kaufman, SN., Lynn, RK., and Gerber, N.,(1982) An osmotic explanation for valproic acid induced choleresis inthe rat, dog and monkey, Arzneim. Forsch. Drug Res., 32, 241.Dickinson, RG., Hooper, WD., Dunstan, PR., and Eadie, MJ., (1989)Urinary excretion of valproate and some metabolites in chronicallytreated patients, Ther. Drug Monit., 11, 127.Dickinson, RG., Hooper, WD., and Eadie, MJ., (1984) pH dependentrearrangement of the biosynthetic ester glucuronide of valproic acid tofl-glucuronidase resistant forms, Drug Metab. Dispos., 12, 247.Dickinson, RG., Kluck, RM., Hooper, WD., Patterson, M., Chalk, JB., andEadie, MJ., (1985b) Rearrangement of valproate glucuronide in a patientwith drug -associated hepatotoxicity and renal dysfunction, Epilepsia,26, 589.Dickinson, RG., Kluck, RM., Wood, BT., Eadie, MJ., and Hooper, WD.,(1986) Impaired biliary elimination of fl-glucuronidase-resistant\"glucuronides\" of valproic acid after intravenous administration in therat. Evidence for oxidative metabolism of the resistant isomers, DrugMetab. Dispos., 14, 255.Diliberti, JH., Farndon, PA., Dennis, NR., and Curry CJ., (1984) Thefetal valproate syndrom, Am. J. Med. Genet., 19, 473.Dodson, WE., and Tasch, V., (1981), Pharmacology of valproic acid inchildren with severe epilepsy: Clearance and hepatotoxicity, Neurology,31, 1047.Dreifuss, FE., Santilli, N., Langer, DH, Sweeney, KP., Moline, KA. andMenander, KB., (1987) Valproic acid hepatic fatalities, a retrospectivereview, Neurology, 37, 379.Dren, AT., Giardina, WJ., and Hagen, NS., (1979) Valproic acid in:Pharmacological and biochemical properties of drug substances, edited byGoldberg, ME., APA, Washington.Fariello, R., and Smith, MC., (1989) Valproate mechanism of action, in\"Antiepileptic drugs\", 3rd Ed, edited by Levy, R., Mattson, R., Meldrum,B., Penry, JK and Dreifuss, FE., Raven press Ltd, New York, p567.Fariello, RG., and Tichu, MK., (1983) Minireview. The perspective ofGABA replenishment therapy in the epilepsies: A critical evaluation ofhopes and concerns, Life Sci., 33, 1629.Franceschetti, S., Hamon, B., and Heineman, U., (1986) The action ofvalproate on spontaneous epileptiform activity in the absence of140synaptic transmission and on evoked changes in [Cal and [K1 in thehippocampal slice, Brain Res., 386, 1.Frey, HH., and Loscher, N., (1976) Di-n-propylacetic acid profile ofanticonvulsant activity in mice, Arzneimettelforsch, 26, 299.Gibaldi, M., and Perrier, D., (1982), One-compartment model, in:Pharmacokinetics, Marcel Dekker Inc., New York, pl.Godin, Y., Heiner, L., Mark, J., and Mandel, P., (1969) Effects of di-n-propylacetate, an anticonvulsive compound, on GABA metabolism, J.Neurochem., 16, 869.Gram, L., and Bentsen, KD., (1984) Controlled and comparative trials ofVPA performed in Europe and Asia, Epilepsia, 25 (Suppl. 1), S32.Gram, L., and Bentsen, KD., (1985) Valproate: An updated review, ActaNeurol. Scand., 72, 129.Granneman, GR., Wang, S.-I., Machinist, JM., and Kesterson, JW., (1984)Aspects of the metabolism of valproic acid, Xenobiotica, 14, 375.Granneman, GR., Marriott TB., Wang, SI, Sennello LT., Hagen, NS., etal., (1984a) Aspects of the dose-dependent metabolism of valproic acid.In Levy et al. (Eds) Metabolism of antiepileptic drugs, Raven Press, NewYork, p97.Gugler, R., and von Unruh, GE., (1980) Clinical pharmacokinetics ofvalproic acid, Clin. Pharmacokin., 5, 67.Harding, GFA., Herrick, CE., and Jeavons, PMA., (1978) A controlledstudy of the effect of sodium valproate on photosensitivity epilepsy andits prognosis, Epilepsia, 19, 555.Harrison, NL., and Simmonds, MA., (1982) Sodium valproate enhancesresponses to GABA receptor activation only at high concentrations,Brain. Res., 250, 201.Harvey, PKB., Bradford, HF., and Dadisson, AN., (1975) The inhibitoryeffect of sodium n-dipropylacetate on the degradative enzymes of theGABA shunt, FEBS Lett., 52F, 251.Herrmann, JL.,^Kieczykowski, GR.,^and Schlessinger,^RH.,^(1973)Deconjugative alkylation of the enolate anion derived from ethylcrotonate, Tetrahedron Letters, 26, 2433.Hoffman, F., Von Unruh, GE., Jancik, BC.,^(1981) Valproic aciddisposition^in epileptic patients during combined antiepilepticmaintenance therapy, Eur. J.. Clin. Pharmacol., 19, 383.Jaeger-Roman. E., Deichl, A., Jakob, S., et al., (1986) Fetal growth,major malformations and minor anomalies in infants born to womenreceiving valproic acid, J. Peds., 108, 997.141Kapetanovic, IM., and Kupferberg, HJ., (1980) Stable isotope methodologyand gas chromatography mass spectrometry in a pharmacokinetic study ofphenobarbital, Biomed. Mass Spectrom., 7, 47.Kassahun, K., Burton, R., and Abbott, FS., (1989) Negative ion chemicalionization gas chromatography/mass spectrometry of valproic acidmetabolites. Biochemical and Environmental Mass Spectrometry, 18, 918.Kassahun, K., Farrell, K., Zheng, J., and Abbott, FS., (1990) Metabolicprofiling of valproic acid in patients using negative-ion chemicalionization gas chromatography-mass spectrometry, J. Chromatogr., 527,327.Kassahun, K., Farrell, K., and Abbott, F., (1991) Identification andcharacterization of the glutathione and N-acetylcysteine conjugates of(E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid,in rats and human, Drug Metab. and Dispos., 19, 525.Kerwin, RW., Olpe, HR., Schmutz, M., (1980) The effect of sodium-n-dipropyl acetate on /-aminobutyric acid-dependent inhibition in the ratcortex and substantia nigra in relation to its anticonvulsant activity.Br. J. Pharmcol. 71, 545.Kesterson, JW., Granneman GR., and Machinist, JM., (1984) Thehepatotoxicity of valproic acid and its metabolites in rats. I.toxicologic, biochemical and histopathologic studies, Hepatology, 4,1143.Kinsley, E., Gray, P., Tolman, KG., and Tweedale, R., (1983) Thetoxicity of metabolites of sodium valproate in cultured hepatocytes, J.Clin. Pharmacol., 23, 178.Kuhara, T., Inoue, Y., Matsumoto, M., Shinka, T., Matsumota, I.,Kitamura, K., Fuji, H., and Sakura, N., (1985) Altered metabolicprofiles of valproic acid in a patient with Reye's syndrome, Clin.Chim. Acta., 145, 135.Lee, RD., (1987) The synthesis of 2-((E)-1'propeny1)-(E)-2-pentenoicacid and its metabolites and pharmacokinetics in rats, M.Sc. thesis,University of British Columbia, p43.Lee, RD., Kassahun, K., Abbott, FS., (1989) Steroselective synthesis ofthe diunsaturated metabolites of valproic acid, J. Pharm. Sci., 78, 667.Levitt, MJ., (1973) Rapid methylation of micro amounts of nonvolatileacids, Analytical Chem., 45, 618.Levy, RH., and Lai, AA., (1982) Valproate absorption, distribution, andexcretion in: Antiepileptic drugs, 2nd edition, edited by D.M. Woodbury,J.K. Penry, and C.E. Pippenger, Raven Press, New York, p555.Levy, RH., and Shen, DD., (1989) Valproate absorption, distribution andexcretion, in \"Antiepileptic drugs\", 3rd Ed, edited by Levy, R.,142Mattson, R., Meldrum, B., Penry, JK and Dreifuss, FE., Raven press Ltd,New York, p583.Li, J., Norwood, DL., Mao, L. and Schulz, H., (1991) Mitochondrialmetabolism of Valproic acid, Biochemistry, 30, 388.Lockard, JS., and Levy, RH., (1976) Valproic acid: Reversibly actingdrug? Epilepsia, 17, 477.Loscher, W., (1981) Anticonvulsant activity of metabolites of valproicacid, Arch. Int. Pharmacodyn., 249, 158.Loscher, W., and Nau, H., (1985) Pharmacological evaluation of variousmetabolites and analogues of valproic acid. Anticonvulsant and toxicpotencies in mice, Neuropharmacology, 24, 427.MacDonald, RL., (1986) Mechanisms of anticonvulsant drug action , in:Recent advances in epilepsy, edited by Pedley, TA., and Meldrum, BS.,Churchill Livingstone, Edinburgh, pl.McClean, MJ., and MacDonald, RL., (1986) Sodium valproate, but notethosuximide, produces use and voltage-dependent limitation of highfrequency repetitive firing of action potentials of mouse centralneurons in cell culture, J. Pharmacol. Exp. Therap., 237, 1001.McMahon, RE., Sullivan, HR., Due, SL., and Marshall, FJ., (1973) Themetabolite pattern of a-propoxyphene in man, The use of heavy isotopesin drug studies, Life Sci., 12, 463.Meunier, H., Carraz, G., Meunier, Y., and Eymard, P., (1963) Proprietespharmacodynamiques de l'acide n-dipropylacetique, Therapie, 18, 435.Minns, RA., Brown, JK., Blackwood, DHR., et al (1982) Valproate levelsin children with epilepsy, Lancet, 1, 677.Morre, M., Keane, PE., Vernieres, JC., Simiand, J., Roncucci, R., (1984)Valproate: Recent findings and perspectives, Epilepsia, 25 (Supp1.1):S5Mortensen, PB., (1980) Inhibition of fatty acid oxidation by valproate,Lancet, II, 856.Mortensen, PB., Gregersen, N., Kolvraa, S., and Christensen, E., (1980)The occurrence of C6 -Clo-dicarboxylic acids in urine from patients andrats treated with dipropylacetate, Biochemical Medicine, 24, 153.Mutani,^R.,^and^Fariello,^RG.,^(1969)^L'azione^dell'acidon'dipropilacetico (DPA) sull \"caudate spindles\" corticali. Boll. Soc.Ital. Biol. Sper., 45, 1416.Nau, H., and Krauer, B., (1986) Serum protein binding of valproic acidin fetus-mother pairs throughout pregnancy: Correlation with oxytocinadministration and albumin and free fatty acid concentrations, J. Clin.Pharmacol., 26, 215.143Nau, H., and Loscher, W., (1982) Valproic acid: Brain and plasma levelsof the drug and its metabolites, anticonvulsant effect and GABAmetabolism in the mouse, J., Pharmacol. Exp., Ther., 220, 654.Nau, H., Wittfoht, W., Schafer, H., Jakobs, C., Rating, D., and Helge,H., (1981) Valproic acid and several metabolites: Quantitativedetermination in serum, urine, breast milk, and tissue by gaschromatography-mass spectrometry using selected ion monitoring, J.Chromatogr., 226, 69.Nau, H., and Zierer, R., (1982) Pharmacokinetics of valproic acid andmetabolites in mouse plasma and brain following constant rateapplication of the drug and its unsaturated metabolite with an osmoticdelivery system. Biopharm. Drug Dispos., 3, 317.Nowack, WJ., Johnson, RN., Englander, RN., and Hanna, GR., (1979)Effects of valproate and ethosuximide on thalamocortical excitability,Neurology, 29, 96.Pellegrini, A., Gloor, P., Sherwin, AL., (1978) Effect of valproatesodium on generalized penicillin epilepsy in the cat, Epilepsia, 19,351.Perry, TL., and Hansen, S., (1978) Biochemical effects in man and rat ofthree drugs which can increase brain GABA content, J., Neurochem., 30,679.Pfeffer, PE., Silbert, LS., and Chirinko, JM., (1972) a-Anions ofcarboxylic acids. II. The formation and alkylation of a-metalatedaliphatic acids. J. Org . Chem., 37, 451.Philbert, A., and Dam, M.,(1982) The epileptic mother and her child,Epilepsia, 23, 85.Phillips, NI., and Fowler, LS., (1982) The effects of sodium valproateon GABA metabolism and behavior in naive and ethanolamine-O-sulphatepretreated rats and mice, Biochem. Pharmacol., 31, 2257.Plasse, JC., Revol, M., Chabert, G., and Ducerf, F., (1979) Neonatalpharmacokinetics of valproic acid, in Progress in clinical pharmacy,edited by Schaaf, D., and van der Kleijn, E., Elsevier/North-HollandBiomedical Press, Amsterdam, New York, p247.Pohl, L., Nelson, S., Garland, W., and Trager, W., (1975) The rapididentification of a new metabolite of warfarin via a chemical ionizationmass spectrometry ion doublet technique, Biomed. Mass Spectrum, 2, 23.Porubek, DJ., Barnes, H., Theodore, LJ., and Baillie, TA., (1988)Enantioselective synthesis and preliminary metabolic studies of theoptical isomers of 2-n-propyl-4-pentenoic acid, a hepatotoxic metaboliteof valproic acid, Chem. res. Toxicol., 1, 343.144Prickett, KS., and Baillie, TA., (1984) Metabolism of valproic acid byhepatic microsomal cytochrome P-450, Biochemical and BiophysicalResearch Communications, 122, 1166.Ramsey, RE., (1984) Controlled and Comparative trials with valproate -United States, Epilepsia, 25 (Suppl. 1) 40.Rettenmeier, AW., Gordon, WP., Barnes, H., and Baillie, TA., (1987)Studies on the metabolic fate of valproic acid in the rat using stableisotope techniques, Xenobiotica, 17, 1147.Rettenmeier, AW., Gordon, WP., Prickett, KS., Levy, RH., and Baillie,TA., (1986a) Biotransformation and pharmacokinetics in the rhesusmonkey of 2-n-propy1-4-pentenoic acid, a toxic metabolite of valproicacid, Drug Metab. Dispos., 14, 454.Rettenmeier, AW., Gordon, WP., Prickett, KS., Levy, RH., Lockard, JS.,Thummel, KE., and Baillie, TA., (1986b) Metabolic fate of valproic acidin the rhesus monkey, formation of a toxic metabolite, 2-n-propy1-4-pentenoic acid, Drug Metab. Dispos., 14, 443.Rettenmeier, AW., Howald, WN., Levy, RH., Witek, DJ., Gordon, WP.,Porubek, DJ., and Baillie, TA., (1989) Quantitative metabolic profilingof valproic acid in humans using automated gas chromatographic/massspectrometric techniques, Biomed. and Environ. Mass Spectrometry, 18,192Rettenmeier, AW., Prickett, KS., Gordon, WP., Bjorge, SM., Chang, SL.,Levy, RH., and Baillie, TA. (1985) Studies on the biotransformation inthe perfused rat liver of 2-n-propy1-4-pentenoic acid, a metabolite ofthe antiepileptic drug valproic acid, evidence for the formation ofchemically reactive intermediates, Drug. Metab. Dispos., 13, 81.Rettie, AE., Rettenmeier, AW., Howald, WN., and Baillie, TA., (1987)Cytochrome P450-catalyzed formation of 4-ene VPA, a toxic metabolite ofvalproic acid, Science, 235, 890.Rettie AE., Boberg, M., Rettenmeier, AW., and Baillie, TA., (1988)Cytochrome P450-catalyzed desaturation of valproic acid in vitro, J. ofBiol. Chem., 263, 13733.Rowan, AJ., Binnie, CD., de Beer-Pawlikowski, NKB., et al., (1979a)Sodium valproate: Serial monitoring of EEG and serum levels, Neurology,29, 1450.Rowan, AJ., Binnie, CD., Wafield, CA., et al., (1979b) The delayedeffect of sodium valproate on the photoconvulsive response in man,Epilepsia, 20, 61.Schappel, GJ., Beran, RG., Doecke, CJ., (1980) Pharmacokinetics ofsodium valproate in epileptic patients: Prediction of maintenance dosageby single-dose study, Eur. J. Clin. Pharmacol., 17, 71.145Schechter, PJ., Trainer, Y., and Grove, J., (1978) Effect of n-dipropylacetate on amino acid concentrations in mouse brain:Correlations with anti-convulsant activity, J. Neurochem., 31, 325.Schreiber, BA., (1981) A proposed mechanism for the anticonvulsantaction of valproate, Med. Hypothesis, 7, 1377.Slater, GE., and Johnston, D., (1978) Sodium valproate increasespotassium conductance in Aplysia neurons, Epilepsia, 19, 379.Sullivan, HR., Marshall, FJ., McMahon, RE., Angard, E., Gunne, CM., andHolmstrand, JH., (1975) Mass fragmentographic determination of unlabeledand deuterium labelled methadone in human plasma, Possibilities formeasurement of steady state pharmacokinetics, Biomed. Mass Spectrom.,2, 197Tatsuhara, T., Muro, H., Matsuda, Y., and Imai, Y., (1987) Determinationof valproic acid and its metabolites by gas chromatography massspectrometry with selected ion monitoring, J. Chromatogr., 399, 183.Thurston, JH., Carroll, JE., Hauhart, RE., and Schiro, JA., (1985) Asingle therapeutic dose of valproate affects liver carbohydrate, fat,adenylate, amino acid, coenzyme A, and carnitine metabolism in infantmice: Possible clinical significance, Life Sciences, 36, 1643.Thurston, JH., Carroll, JE., Norris, BJ., Hauhart, RE., and Schiro, JA.,(1983) Acute in vivo and in vitro inhibition of palmitic acid andpyruvate oxidation by valproate and valproyl-coenzyme A in livers ofinfant mice. Annals of Neurology, 14, 384.Ticku, MK., and Davis, WC., (1981) Effect of valproic acid on H-diazepamand H-dihydropicrotoxin in binding sites at the benzodiazepine-GABAreceptor-ionophore complex, Brain Res., 223, 218.Tsuji, J., (1984) Synthetic Application of the palladium-catalyzedoxidation of olefins to ketones, Synthesis, 369.Von Unruh, GE., Jancik, BC., and Hoffmann, F., (1980) Determination ofVPA kinetics in patients during maintenance therapy using atetradeuterated form of the drug, Biomed. Mass Spectrom., 7, 164.Walter, DS., Boardman, SP., Henry, EJ., et al., (1980) The comparativeeffects of single or multiple doses of sodium valproate on mouseanticonvulsant activity, Plasma and brain valproate and brain GABA, inWada, JA., and Penry, JK., (eds): Advances in epileptology, 10thepilepsy international symposium, New York, Raven, p359.Worms, P., and Lloyd, KG., (1981) Functional alterations of GABAsynapses in relation to seizures, In Neurotransmitters, Seizures, andEpilepsy, edited by Morselli, PL., Lloyd, KG., Loscher, W., Meldnum,BS., and Reynolds, EH., Raven Press, New York, p37.Zafrani, ES., and Berthelot, P., (1982) Sodium Valproate in theinduction of unusual hepatotoxicity, Hepatol., 2., 648.146Zimmerman, HJ., and Ishak, KG., (1982) Valproate induced hepatic injury:analysis of 23 fatal cases, Hepatology, 2, 591.147"@en . "Thesis/Dissertation"@en . "1993-05"@en . "10.14288/1.0302328"@en . "eng"@en . "Pharmaceutical Sciences"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Metabolism and pharmacokinetic studies of valproic acid using stable isotope techniques"@en . "Text"@en . "http://hdl.handle.net/2429/2582"@en .