SFFECT ·o·F EXPERIMENTAL HYPERPHENYLALANINEMIA ON . - . MYELIN METABOLISM AND NEUROTRANSMITTER . SYNTHESIS AT LATER STAGES OF BRAIN DEVELOPMENf BY E. HOWARD TAYLOR SUBMITTED TO THE FACULTY OF THE SCHOOL OF GRADUATE STUDIES OF THE MEDICAL COLLEGE OF GEORGIA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF·. DOCTOR OF PHILOSOPHY AUGUST. 1982 EFFECT OF . EXPERIMENTAL HYPERPHENYLALANIN'EMIA ON .. MYELIN .'METABOLISM ANP NEURQT.RANSMITTER SYNTHESIS 'AT .·LATER ·STAGES .OF BRAIN DEVELOPMENT This dissertat:i,on · submi.tted by E.- Howarcl Taylor has been examined and.approved by an appointed·committee pf the faculty of the School of ' Gra<;luate Studie·s of the Medical Coilege of Georgia·. · The signatures which appear below veri:fY the ·fact that all requi.red . ·changes have been incorporated artd.that the dissertation has received full approval with reference to cohtent, form and ac·curacy of presentation. This dissertation is therefore accepted in partial fulfillment; of the requirements for the degree of Doctor of Philosophy. en.~ 110~ ·u Date Advisor ii 124696 ACKNOWLEDGID1ENTS I wishto express .my. sincer~· thanks to my advisor, Dr. Frits Hommes,· - . .. - . whose encouragement throughput the y¢ars made this disseJ:tation' possible.· . ··~~ ! appreciate the guidance and. advice p..tovided by members of my .Advisory Committee. Many individ'l.lals have provided expe:J;tise in cert(;lin areas to enable me to complete this work •.. ·I owe a special thanks to Dr. Margaret Kirby for devoting· time· and assistance with the electron microsco\pe. I wish to thank the entire Gracewo<?d laboratory, .especially Dr. August Roesel for ma:ny useful sc·ientific discussions, ·Paul Blankenship for assistance with the amino acid ana lysis, and also Dr.. Marga·ret Coryell for many use- ful ideas with gas chromatography. John Craig's assistance and many discussions with the GC/MS were invaluable. Dr. Dan Doran's expertise in statistical analysis and the SPSS package saved countless hours and to him I owe a special thanks. Gary Eller's friendship and encouragement for the past three years as a lab partner shall not be forgotten. I wish to dedicate this dissertation to my pare_nts, Eugene H. Taylor and Eteanor w. Taylor, whose love.aild encouragement hetped me make this goal a reality. iii T~LE OF CONTENTS Page INTRODUCTION 1 A. Statement 'of the Problem • • . .' . .. •· . 1 J- . -.ft B~ Review-of Related Literature .. .. .. •. ·_ . 3 1. Metaboli-c Defect in PKU • • • • . .. 0 • • . • • • 3 2. Animal .-Model$ • .• • ... • • • • • • • • • • • • • • • • • • 7 3. Effect on Myelin • • • • • • • • • • • • • • • • • • • • 9 4. E~fect on Cerebral Protein Synthesis • • • • • • • • • • 13 5. Effect on Serotonin and the Catecholamines •••••• , 16 6. Phenylalanine Metabolites • • • • • • • • • • • • . • • • • 24 MATERIALS &'ill METHODS 32 A. Animals .and Diets • • • • • • • • • • • • • • • • • • • • • • 32 B. Exi?,eriments Involving Radio labeled Compounds • • • • • • • • • 3.2 C. ~J.\mino Acid Analysis '• • • • • • • • • • • • • _ • • • • • • • • 33 D. Brain Proteins and Myelin • • • • • • • • oe • • • • • • 34 E. Electron Microscopy • • • • • • • • • • • • • • • • . • • 3.5 F. Determination of Phenylalanine Metabolites • • • • • • " • 36 G. Determinations of Catecholamines and Serotonin • • • • • • • • 40 RESULTS 44 ·A. Amino Acid Analysis . • • • • • • • • • • • • • • • • • _• • • 44 . B. PhenylalanineMetabolites ••••••••••••••••• 47 ------C-•. __ Catecholamine and- Serotonin Synthesis •. • • - • •. • • •. • • • 53 D. Myelin Metabolism • • • • • • • • • • • • .• • • • • • • • • 64 DISCUSSION 81 .A•. Myelin.Metabolism •.•••••••• . .. .81 B •. Effect of Phenylalanine Metabolites . .. • • • 85 c. Neurotransmitter 'Synthesis • • • • • • • • • • • • • • . .. 86 SUMMARY- 93 REFERENCES 95 iv - . - .. LIST OF ABBREVIAtiONS . AADC aromatic amino· acid decarboxylase AGBA ct-aminoguaiiidinobutyric acid ala alanine aMP a~methyl_phenylalanine AL~OVA analysis of variance includi11g Duncan range test arg arginine ATP adenosine 5'-triphosphate B6 pyridoxal phosphate · BAS Bioanalytical Systems BSTFA bis-( trimethylsilyl) trif louroac·etamide COMT catechol-o-methyltransferase CRM cross reacting material CSF . cerebrospinal fluid dopa 3,4 dihydroxyphenylalanine dopamine 3,4-dihydroxyphenylethylamine dpm disintegrations·· per minute . ·. DHBA dihydroxybenzylamine DHPR dihydropteridine reductase ED'rA ethy1enediaminetetraacetic acid · EEG electroencephalogram EV electron· volts GABA . Y-aminobutyric acid GC/MS Gas chromatograph/mass spectrometer gly glycine his-tidine 5HIAA 5-hydroxyindoleacetic acid v vi . HPLC·. ·high performance liquid chroma togr~ph · 5HT 5-hydro_xytryptamine. (serotonin) HVA homovanillic acid . HyPhe hyperphenylal~ninemi~ inducing diet of _normal ~chow supple- ·merited with 5% phenylalanine and • 4% a-methylphenylalanine ileu isoleucine IQ intelligence quotient · INCOS Data system for Finnigan 4023 GC/MS I.S. internal standard K Michaelis constant rn· leu . leucine log logarithm base 10 log (% max) log of percent maximum incorporation lys . lysine.· MHPG 3•methoxy-4-hydroxyphenylglycol na nanoamps NADH reduced nicotinamide adenine dinucleotide NADPij .reduced nicotinamide adenine dinucleotide phosphate NCS trade name of tissue solubilizer (Amersham) . ' MA mande lie acid · MAO monoamine oxidase .met methionine o-OHPAA o-hydr.axyphenylacetic acid PAA phenylacetic acid PCA perchloric acid PCPA p-chlo.rophenyla lanine _phe phenylalanine ·vii . PKU .phenylketonuria PLA 'phenyllactic acid PPA phenylpyruvic acid z. psi pounds/inch Rf detector ·response fattor·- SD standard deviation ser serine SE slope standard error of the slope SPSS statistical package for the social sciences tl .half.-life ~ TCA trichloroacetic acid TGA cycle tricarboxylic acid cycle thr threonine TMS trimethyls.ilyl tRNA transfer ribonucleic acid tRNAmet f formylmethionine transfer ribonucleic acid tryp tryptophan tyr tyros'ine v volt val valine v maximal velocity in Michaelis Menton Kinetics m VMA vanillylmande.lic acid (4-hydroxy-3-methoxymandelic acid) 'WMC weight-matched control dihydropterin XH2 LIST OF FIGURES Figure _ _Page 1 Pheny:Lalanine Hydroxylase_ Reaction· 5 2 PhenylalanineMetaboJ,ites viC!~~~e Transaminase_Pathway 26 . .· .. -'; ~-; 3a · Ghroma,togram from·· a Var-ian 3700 .@as Chromatograph of TMS Derivatives· -of Organic Acids from the Urine· of a Rat on a Hyperphenylalaninemia Inducing Diet 50 3b Chromatogram from a Varian 3700 Gas_Chromatograph C)f TMS Derivatives ··a-£ ···or-ganic--Acid-s from the Urine of a Rat on Norma+ Chow Supplemented with .4% a.-methylphenylalanine 51 4a .Mass Chromatogram from a Finnigan 4023 Gas Chromatograph/ Mass Spectrdtneter. of Plasma Extract of ·Organic Acids from a Rat-Maintained on a Hyperphenylalaninemia Inducing Diet 54 4b Mass Chromatogram from a Fin-nigan· 4023. Gas Chromatogt"aph/ Mass Spectrometer of Brain Extract of Organic Acids from a Rat Maintained on a Hyperphenylalaninemia Inducing Diet 55 Sa Chromatogram from an.HPLG.of Catecholamines from Acid ··soluble Extract of Brain from a 35 Day Control Rat· 57 5b Chromatogram from an HPLC_of Catecholamines from Acid Soluble E~tract of Brairi from a 35 Day Old Rat Treated with 5% phe + · .4% a.Ml? Diet for 10 Days Previously 6.8 6a Chroma-togram from an HPLC of serotonin and 5H!AA from Acid Soluble Extract fr8m Brainstem of .35 Day Old Control Rat 61 6b Chromatogram from an HPLC of serotonin and SHIM from Acid Soluble Extract-- -from Brainstem of 35 Da:y Old Rat Treated with 5% ph~ + .4% a.MP Diet for 10 Days Previously 62 7 · _Brain WE!_ight of Rats Maintained_ on Diets 66 8 Myelin· ·Protein Content (mg/ g) ·of Brain 6 7 · 9 Turnover of Myelin P-roteins 69 10 Turnover of TCA Precipita_ble Whole Brain Proteins 70 11 Short T·erm Incorporation of Lysine into Myelin -Proteins · 71 12 Short Term Incorpora-tion of Lysine into Brain Proteins 72 13 Myelinated AXons from Lower Midbrain at the Pon"t;ine Junction- of a 45 Day Old Rat -:on a Norinal-- Diet 76 viii. ix Figure ·Page 13a High Magnification of My~lin Sheath froni Control Rat 76 14 -Myelinated Axoris from LowerM:idbra~n at the Pontine Junction of 45. Day Old Rat, o.p a Diet Supplemented of· 5%·phenylalanine and 0.4% aHP-for the Previous 20 Days 77 14a High .Magnification of Myelin Sheath from Hyperphertyl­ - alaninemic Rat 77 15 Longitudinal.~ection of Myelinated AXon from Hyerphenyl­ alaninemic Rat 79 LIST OF TABLES · Table Page .I Plasma Amino Ac~d Levels 46 II Amino Acid Levels in Bra:L~:w · 48 III Phenylalanine Metabolites. 52 IV .Brain Catecholamine Levels 59 ·v Serotonin and SHIAA Levels from Brainstein 6) VI . Body Weights of Rats ~65 VII Half-Lives of Various Fractions from Brain 75 VIII Effect of Hyperphenylalaninemia on Myelin Morpholo.gy 80 X ·: t', · INTRODUCTtoN· A. STATEMENT OF THE PROBLEM Pheriylketonuria (PKU) is an autbsomal recessive inborn ·ertoi of metab~lis~ due to a defici~ncy·of phenylalanirie 4-rnono-oxygenas~ (EC 1.14.16 .1) ,· resulting in an· inability to convert phenylalai'line t.o tyrosine. This deficiency of enzyme activity causes plasma phenylalanine to exceed r.s rnM (25 mg%) whereas a normal individual h~s.levels· of approximately .05 mM ( • 8 rng %) ( Hs i a 19 7 0 ) ., As with,many other .inborn errors of metabolism, the developing brain is affected, resulting in sevete mental retardati6n if the disease is untreated. However, PKQ is unique in that the metabolic defect occurs in liver ~nd kidney and the pathologidal effe~ts of elevated phenylalanine are primarily manifest in brain