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 Gracewoamino 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 (Gaull et al. 1975} ~ Untreated PKU patients show abnbrmal EEG patterns, increased muscle tone, hyperactive

ten~on reflexes, tremors and hyperkinesis (Knox 1972). Disturbed behavior, sudden outbursts, and violent activity are common.

These neurologic~l abnormalities, in~luding the mental

. . retardation can be prevented with a pheriylalanine restricted diet, provided this diet is instituted .at a sufficiently young age (Bibkel et al. 1954). If this dietary regime.is not begun soon after birth.during the periodof rapid development or the

·"growth spurt 11 of the brain,' th~·· neurolog·ical damage is

1 2 irreversible.

The brain is most vulnerable to potentially toxic substances when it passes through its most rapid phase of growth. This period of "brai;·vulnerability" can be divided into two phases: first, there is a period of neuronal multiplication occurring at 12-18 gestational weeks in the human, and the second occurring near birth. be·nc.r it ic arborization, establishment of synaptic connections, and glial multiplication occurs during this second period of growth, followed by myelination (Dobbiri~ 1974). It is this second period of growth in human brain development which is most critical in PKU, as it occurs about the time of birth.

Therefore, the timing of dietary therapy is most critical in order to avoid permanent impairment of brain function-due to an

insult during this "growth spurt".

The question of when to begin dietary treatment of PKU is a very simple one as it is known that the earlier the low phenylalanine diet is begun, the higher the IQ later in life­

(Bickel et al. 1954) • By means of a mass screening program, a

PKU individual is identified and then placed ori a low · phenylalanine diet, preferably irt the first thr~e weeks of life.

The question of when to terminate dietary treatment is a much more difficult one which is not easily answered. Current opihion is to relax or terminate the dietary treatment at 6-8 years of age , \.,hen the bra in i s thOught to be s u f f i c i en t 1 y mature to tolerate higher levels of phenylalanine. There is . ·. . : . treatme:nt. The·re. is no agreement on. the consequences of the . . ·. "...... i·ncreased intake of ph~ny"lalan{ne at this age on behavior and i_ntellectual de·velopment. s~h.lk irtvesti9.ators find no difference

in intellectual developm.~nt ·after diet termination ·(.Solomons et ·

·. al. 1966, Kang et al •. ·· 19 70, Holzmann et al ~ 1975,-. Koff et ·al ~

1~79) 1 while others demonstrate a drop.in IQ (Hudson and.

- . . Hawcroft 1973, Cab~lska et al. 1977, Smith et al. 1978, Berry

et al. 1979) •

. . ' ' The research described *h this disser.tation was undertaken to study the effects of increased phenylalanine levels:in brain_

using a suitable anim~l modei system, at a time in brain

development equivaLent to that of a PKU· individual b~ing removed from a low phenylalanine diet at approximately 6 years of_ age-. Changes due to the following parameters of excess phenyLalanine were investigated: 1. myelin metabolism and morphology 2. free amino acid pool of brain 3. neurotransmitter production- 4. levels of phenylaLanine metabolites

B. REVIEvv OF RELATED LITERATURE

Met~bolic Defect in PKU

Phenylketonuria is an autosomal recessive inborn error of

metabolis~ whieh occurs in _approxim~tely 1 in 11,000 births .4 .

.(Blckel 1980t •. The condition was· first described by. Falling as.

"imbecillitas phenylpyruVica" (Falling 1934) and was also c~ll~d phenylpyruvica oligophrenia. The uririe from PKU ~atients had a must~ smell and the urine tu~~~d green upon·the addition of

_Fecl , due to the presenc~ of phenylpy~uvate. The name of 3 . \ phenylketoriuria was given due to the presenc~ of phenylketones in the urine of such patients (Pentrose and Qua~tei 1937). The metabolic block.was discovered to be a defect in the enzyme which converts phenylalanine to tyrosine (Jervis 1953) • At this time little was known about the enzyme system .which converts phe~ylalanine to tyrosine and Jervis assumed that only one enzyme was involv~d.

More information of the enzymatic block in PKU had to wait until detailed knowledge of the mechanism of the conversion of . phenyalanine to tyrosine was known {Kaufman 1957,1963). The current knowledge of the phenylalanine hydroxylating system is described by Kaufman (1977). Figure 1 illustrates the complexity of the phenylalaninine hydroxylase reaction •.

Although DNA sequence analysis of the phenylalanine hydr6xylase gene iD PKU indivi~uals has- not b~en per£orm~d,·a classical PKU patieht.may possess a structurally altered bydroxylase with very low catalytic activity, 0.27% of normal

{Friedman et al. 197~), rather than a complete deletion of the s~ruetural gene or a low amount of.a normal hydroxylase {Kaufman.

1977). ·cross reacting material (CRM) was not observed in

response to antibody to rat liver hydroxylase in a biopsy from a Figure .1: Phenylalanine hydroxylase reaction. H . H NAD(P)+ NAD(P)H H .

H. N .. ·· .· NYNH;l NVNH, ~ + H+ H N N NH . H 7 8 I . """"'I Ht ~1 • / . y- CH,-GH-GH{.f;NH CH3 -cH-CH x;NH tdohydoorledd;ne oeduclase) CH,--CH~CH! X NH I I N I I I HN I 1'1 HN ~ OH OH . O . OH OH H O OH OH II - ' / 0 7 ,8-0ihydrobiopterin tt / 5,6, 7,8-Tetrahydrobiopterin

Ouinonoid dihydrobiopterin

(dit)ydrotolale NADPH + H+ reducta~e) NADP+ ~ <: •H~O

.· NH2 . NH 2 C)-CH,~6H-cooH HO~YCH,-'6H--COOH

Phenylalanine Tyrosine

FIGURE 1 ' .., ·''• '. ' '•f .~ ..··: 'l ' ' :

' ' ' ' . . hot-n~ve-r, Barthoiome ·and Ert·e_l· (1978). used ·a very highly_ purif.ie-d .. · . - - . '. -_ . . . rabbi_t anti~monk-~y hydroxiase. ·to visua1iz~- .a ring of precipitat~ . - . ' .. . ' : ·: -' in Ouchterlony plates~-- corifi]~mfng th~ presence of phenylalard.ne .. · . . -.. :.~·:·;{?~. hydroxylase in an Inactive f~rm •.._A very. sensitive in vivo.

me~surement by ~re£z et al. (1979) ·showed a small residual'

'hydroxylase: activity by means of' admi-nistration of del,iterated

- . ' '. . . phenylalanine and measuring. in-corporation int:o. tyrosine, thus·

showing an ~-ctivi ty. of 1 ~ 5% and 0. 3% in two· PKU patients. · A

mut~ted enzjme can·ha~e vary~ng· degr~es -of enzyme activity

depending on the- site of the mutation in the· prot·ein. This

condition leads to a-variant form-of PKU with approximately 5%

of the norma-l- hydroxylas-e activ1ty · (Barthblome- et al.- 1975)

which i_s. well below the range. for hetetozygotes of 10~50%

activity {Kaufman 1977). Variant forms also exist if there- i$ _a

defect in the dihydropteridine reductase. {DHPR) · or dihydropterin _

biosynthesis (XH ) . A DHPR deficient or XH patient shows 2 2

symp-toms of hyperph~nylalaninemia similar to those of cla-ssical

PKU· (Smith et al. 1975, Grobe et al.. 1978); ·however,. DHPR

d~ficiency is very severe since this reductase is also a ·necessary. component of . .the tyros{ne {Shiman et·al. 1971) ·and

trypt6phan hydroxyl·a.ting systems (F~iedma_n- et ·al. ·1972a) -~ A·

' . - . pa-tientwith.hyperphenylalaninemia du~ to OHPR. deficiency, developed neurological symptoms despite a low phenylalanine,diet

and this patient had· le~s than· 1% of normal DHPR acti~fty (Kaufman et al. 197.5). . ·~ .. ~

7 .··

The .foc~s Of ~he researbh· iri this disseitatiOn is devoted

...... to the neurologi~al problems. and treatmen:t associated .with·

classical phenyi~~tonuria.

Anima'! Models

Since experimentation in humans to stu~y the pathological alteration. in PKU is not possibl·e, it is necessary· to'. us·e· an· .animal model. No true genetic animal model for PKU exists and

therefor~ an altern~tive mod~i mtist be employed. It is pb~si6le . . to supplement normal laboratory chow with_very large quantities

of phenylalanine or to inject phen~lalanine::howev~r, this· presents a p.roblem_ because high_ levels of tyrosine are also

produced which: is not analogous to th~·human conditi6n. · A

partial solution to this problem is to utili~e an inhibitor of phenylalanine hydroxylase in· addition to. excess phenylalanine •

. ; . . . ,. l Parachlorophenylalanine. (PCPA) inhibits phenylalanine hydroxyla-s.e '(Lipton et al.l967) , and the addition of 0.12% PCPA . . . : . . ' and 3% phenylalanine to norm~l laboratory rat chow provides an effective. means of inducing hyperphenylalaninernia (Berry et_al. ·

. . . · 1975) • Unfo.rtunately PCPA. has. the metabolic sipe · effeqts such

as inhibition .of tyrosine hydroxylase (Koe an~ -Weisman 1966.)',

ttypt6phan hydroxylase (Jequier et al~ 1967) , and pyruvate kiria$e (Schwark et al·. ·1970) •. Other side effects have been ·

· noted such as cataracts, high mortality, skin lesiori~, ioss· of

body hair and re~udtion in brain biogertic ~min~s- (Lane et ~1. . I - ,, ...

•,j L

·.\. ,' ·a

.1980, Delvalle et ··al ~ . 1978) •. . . :. - . . .. An alternative.· model. utilizing: Cl;_methy}.phenylalanine .·

. . ..· .. · . ; : : . (aMP),. to inhibit phenylal~nine· hydroxy: ~se: has .been developed .. '' .. ·.· .. '<> 'i.. : ... ·.. ,. '' :' . (Greengard ·et al. 1976) and ··later modi_fied (Lane and· Neuhoff

1980) • This inhibitory model seems to be ftee of many of the side effects of th'= PCPA model, and thus re$embles mo:re qloselY. the human condition. Both·· the PCPA ario aMP models suffer from

. . .. •. one s~ribus problemJ neither is effective in redu~ing.the phenylalanine hydroxylase to near zero. ·. PCPA leads to

approximately 70% inhibi tion·"'.fn both 1 iver and kidney. The aMP

irihibits the liver phenylalanine hydroxylase however, the

kidney enzyme is unaffected (Delvalle e~ al. 1978)~ · In the ' ·human, total acti~ity of kidney phenylalanine.hydrbxylase

(measured i~ ·vitro) , is less than· 5%· that of the liver

value.s (Ayling ·.et al. 1975). In the rat, the kidney levels of

biopterin are 26% of that of liver (Rembold 1964). It is

there·fore likely that the kidney phenylalanine hydroxylating ability is insignifican·t when compared· ·to the hydroxylating

levels .of live·r. · An elevated tyrosine level is p.re.sent in boi;:h · the PCPA and aMP models; howev·e;r; r'ess. is present than in ·models ...... · ...... ' . . . . · in which: exc~s_s phenylalanine was the s.ole m~ans ()f p'roducirtg · .·. hyperphenylalaninemia. Therefore, a tat animal.model in which

·normal .. laboratory chow is supplemented with a.M~ ana· e_xcess

phenylalanin~ has been utiliied in this di~~ertati6n •

. The age of· r~ts used is. also.- very critical.. In order ·to

appro~imate tBe human conditio~ of a ~ year bld Pku patient, on~ ·- ._ .. ' l!~ •.

9

must c~refully ~at6h the ap~ropriate stages in braiti maturation~

- . . . In iats, derebral ~~uronal mul~iplication occrirs before. birth

while gli?l.proliferatiort, dendritic:arboriza~ion and·

myelinattqn _ocd4rs postnatai'l:'y (Dobbing 1974) ~ The peak in the

- . rate of ~yelin~tion in rats occurs: at appioximately.20 days

(Norton and Poduslo 1973a) ~ thus t~ts of 25 days are beyond the most rapid phase of myelination:-, although sti11 continuing to

accumulate ·myelin. ·These· r,:~.if:s·:. of 25 a_;sys of age would

approximate the growing human brain at 5-8 years of age (B~rger et al. 1980, Hommes et al. i982a), when dietary therapy is relaxed. Myelination continues into the second decade o-f life in humans (Dodge et al. 1975), and similarly the rat has been

shown to accumulate ~yelin up· to 425 days (Norton and Poduslo

1973) • Rats of 25 days of age simulate the human stage of brain development when the peak rate of myelination has passed, yet myelin still continues to accumulate.

Effect on myelin

-An altered myelin morphology was demonstta·ted by Alvord et al. (1950). _They observed a -marked lack of-. myelin in th.ree of

five PKU patients and proposed that a ~efect or an arrest in

myelin~tion could be p~rtly responsible for the mental

deficiency. Further interpretation attributed the ~ltered

- ~orphology fo be of a degenetative nature (Bend~ 1952) and the

demyelinating lesions "typic-al of Schilder's disease" in a 25 ·year old pdult (J~rvis 1954). Scholz. (1957). considered .an

. . ' . . abnormal glial function as a. cause .for dysmyelin?tion, that· is, an abnormally formed myel in as opposed to_··a c1emyel.ination in . . .- . . . ,• . . . ~~hich there is. increased br~a}:{do'"l!. of a. normally_ synthesized

myelin ~heath~ Spqngy and ~emyelinating foci with the absence .

-.of breakdown products prompted Poser ~nd Van· Bogaert (1959) to

_consider PKU_as a dysrnyelinating disease characterized· by a disturbance jn anabolism. Extensive patches of diffuse demyelination accompanied by_ intense gliosis and sudanophillic breakdown_products were

observed in demyelinating lesions (Creme 1962~). Spohgy and

demyelinating foci bave been described for ot~er cases {Bechar

et al. 1965, MalamUd 1966). These findings of an altered myelin morphology were also shown to occur in a PCPA

. - experimental model system in· optic tracts. (Avins et al. 1975) in which neonatal rats were injected with phenylalanine and PCPA

daily between 5 and 20 days of life and myelin sheaths exa~ined up to two years later. Phenylalanine injections bet\veen 1 and 7

days produc~d neuropathologic lesions limited to the cerebe~lum · when the animals were sacrificed at 50 days (Adelman et q.l.

1973). Howe~er~ in the case of PKU, it is the ·upper cortical function which is most .dev·astatingly ·impa-ired with relatively minor. abno·rmal i ties· in cerebellar function. Thus, hyperphenylalaninemia sustained during the neonatal period during the period of rapid myelin development can lead to an abnormal myelin sheath. Studies· on myelin-ultrastructure of 11 bYP~rphenylalaninemia instituted b~yond the ~0 day peak in myelination have· not been done and ·.this is· one objective .of .th~s dissertation.

The total lipid content -~6£ \>Thi te rna tte-r from bra ins of PKU patients was shown to be deficient in cholesterol and in.the cerebrosides (Creme et al. 1962b). However., the composition of lipids isolat~d from p~rified myelin of PKU patients did not vary from that of normal ·brains even.though a 40% de-crease in the amount of myelin was observed (Shah et al. 1972b). 'The ratio bf cbolestetol to galad~6lipid. w~s significantly higher in the white matter from brains of PKU patients versus normal brains. Therefore,· Shah et al. (1972b), attributed ·the normal compo~ition of myelin lipids and the absence of cholesterol esters in white matter to an arrest in myelination rather than a ~emyelinating condition.

Hyperphenylalaninemia induced early in the neonatal period. caused myelin to be abnormal with respect to sulfatides (Grundt and Hole 1974, Sprinkle and Rennert 1976, Shah and Johnson 1978) and galactolipids (Shah et al. 1972a). Inhibition of brain sulfatide .formation has been attributed in one case to diminished availability of adenosine 3-p~osphat~ 5-phosphosulfate, a substrate necessary in the formation-of sulfatide from cerebroside (Chase atid O'B~ien l970). No difference was seen in a PCPA rat model in cholesterol and cerebroside~ at-the 15th. postn~tal day, just prior to the burst in myelination (Grundt and Hole 1974) :·however, a difference was seen at the 23rd " l . : ~ \'

12

postnatal.. day_ for ·rats . tre.ated from. day 7 with a high

phenylalanine d_iet ·(. Grundt and Hole 1974 ). · A decrease in-the

formation of-~yelin (Prensky et al. 1971) was also observed when stibjecting rats to hype~~~enylalaninemia fr6rn 13 to 31 day$

. postpartum (i. e.· the time of inyelinatio·n) ~ Therefore, a

hyperphenylalaninemi~ present during_ the suckli~g period dan

severely inhibit a normal myelin metabolism. 14 The in vivo rate of incorporation of c- glucose into

brain lipids in a PCPA ~nimal model is reduced (Shah et al. . . 1970) • Incorporation oi mev~i~nate. into sterols is also decreased (Shah et al. 1969). A defect in the elongation of

fatty acids has also been suggested (Johnson and Shah 1973) •

Shah.and coworkers proposed a defect in myelination due to a

reduction in cholesterOl synthesis in hyperphenylalaninemia

(Shah et al. 1972a). These ideas of a faulty myelin anabolism

suggest a ·deficit in the synthesis of myelin rathe~ than ~n

increased turnover of myelin.

Myelin is composed of approximately 70% lipid and 30%

protein. Another explanation for altered myelin morphology

could be abnormal myelin protei,n metabolism. An increase in

whol~ myelin turno~er (Berger et al. 1980) and in myelin

protein turnover {Hommes et al. 1982) has been demonstrated in

a PCP~ rat model system. A half-life of 37 days~ uncorrected ·

(Sabri et al. 1974) . fast component of control myelin, was

observed \vhile the high phenylalanine treated group showed a

half-life of only 2 days (Eommes et al.· 1982a). · A reduced .. · :, ..·: .. :I '

I ·,:

- ~ ' - • • • ' < • short te-rm· incorp()rafton of 3H.:..lysin'"e · .r'nto myelin prOtein has been· sho~ri (Berger .et a;.· 1980, Hommes ~t. a1.:L9$2a) ~1ith i·no· ' . . . . change in: the .·shor-t. term ·inqorporation· into total brain proteins . . . .(Berger ·et ai.·· 1980) •.. Agraii}~i:et al. (1970) demonstrated that·. ·hype.rphenylalaninemic rats had a severe reduction of incorporatio·ri -of. 35 s-me~hioni~e·and 14 c-:-leucirie-into:~yelin and non-myeiin cerebral protein fractions while l.ncorporat-ion of ac,etate into

cerebral lipids_was unaffected. Figlewicz and.Druse.~(l98d)

observed a deficiency of myelin prot~in in adult iats made

hyperphenylalaninerriic from 3~·is· days of age, and ·these

irivestigators proposed a deficit in myelin basic protein which

could not be ~tt~ibuted to a decreas~d synth~sts. ·Thus,

interfer~nce· in myelin protein metabolism can a~so. occur just as

interferece in myelin lipid metabolism.

Effect on cerebral protein synthesis

Numerous theories exist to account for abnormal ce~ebral

: protein metabolism (for revie\'1 see Kaufman 1977, Vorhees _et Cit~.

1980). Phenylalanine shares a common mechanism of uptake across

. . . ·. . . ' ·the "b.lood .... brain · barrier'i with :the lar_ge neutral amiho acids:

.· . . . : '·. . ' ' tyrosine, leucine, isoleucine, methionine, tryptophan, valirte,

and threonine_ (Blassberg and· Laj tha 1965) • : ~xcess phenylalanine

can ·lead to inhibiton of the other large neutral amino acids (Blassberg. and Laj tha 1966) · and saturation by phenylalanine of ·

' . ' ' . . . ' this transport mechanism can lead to a decr-eased uptake. or the '\

14

'I , , ,· , . r .. branched _:chain amino· acids~ methionine and the aroinatics . . . (Oldendor,f 197>3) '•. Alteration ~n the f.ree ·amino.: acid poo_l in . . . .· ' . ' . . . . . ' ... . expe~imeritalhyperphenalaninemi~·.has. al.so been "demonstrated with ...... :i;·\if~.- . . . . ' . . . ·...... · . . ' . a reductiOt1 by 38--64% of the ·!oran¢hed cha.in. amino acids (McKean

·e~ al~ 1968) • Thi~ ef£ect was jfrdged to b~ more severe on . . . . · i~mature anitnals as th~ _transpo:rt system become.s saturated more. · rapidly· w_i th excess phenylalanine from brain slices of. yo·unge·r ·

rats than· from older animals' (Vahvelainen and Oja 1975) ,; .

Antonas and Cou.lson (1975) . observed a so·% reduction :t:ollowing . int~aperitoneal injection o~~~~C-leu~ine in both uptake and incorporation into cerebral proteins which was parallel to a

reduction in the acid soluble pool of brain .. How~vet, no such

changes w~re observed follo~ing intracerebral injection o£ 14 . "14 . . h d d f h c- glyc1ne or c-1ys1ne. T ~y condlu~e rom t ese

e~periments that the effects of hyperphertylalaninemia on

cerebral protein synthesis ~eie primarily due to a decreased availability of the large neutral amino acids and that·other

effects ort p~otein ~ynthesis were small.

I The comparisons betwe~n animal models'andhuman PKU

' . . ' ' . patierits have yielded similai ~indings. Lowered levels of

' . . . . tryptophan and tyrosine.have been-observed in cerebral cortex' . . (McKean 1972). In vivo uptake.of 75se-Selenomethionine. into brains of·PKU patients was depressed (Oidendorf et al •.

1971) and similar resUlts were see~? in· animal models for PKU

. ' . . . (Agrawal et al.. 1970, B~rger· et al. 19 78). Inhibi ton of 11 c--methionine upt,ake was t'ever.sed in PKU children followin·g ·: ,: •"ll'•' .... - .. ·~"' · ··rs _·._

low ·phenytaia-nine dl~tary · ·ther·apy ·(coma_~ et_ al. ·.1981) • Treatment._ of· 'pr{u pa.t.tents with die·ts supplemen.te·d with

c,.. • • ••· ' ·, • • tryptoph_an, tyrosine, meth·~_onine and .the branched chafn ·.amino. . .-. . . . . ' ·. ~fh;J·~· ·. ' . . . ·.· .. . ·acids·. hits been proposed (Banos- et·· al .. 197 4, Pratt 1980). and

...... : practiced_ ~.,i th success -(Berry ·et ·al. 1977) •

. . · . ·. . . . .· . . . ' . . ~he effects of hyperphenylalarii?emia have be·en observe.d. at

. . •' .. the level· of translation in protein synthesis w'i th increased . . ( . . ·...... :- : . . . disaggregation of brain polysomes after ·exposing_.fetal rats

(Aoki and Siegel J970) and young . rats (Siegel· et al.. 1971) to

excess phenylalanine. . These .i;iJffe~·ts :.':'lere mo1:=e profout{d Oh free

rather than bound. polysomes with ~erebra.l cortex being. most

affected· (Taub and Johrtson 1975) ~- The iricrease ·in the

percentage of monosomes w·as.~ pa.rtially reversed in a PCl?A rat

. . . model sy~tem following a single inj~ction of .the seven large

. . . . n·eutral amino acids while the other common amino acids had no

effect, indicating the limiting-nature of the availability of

the large. neutra·l a-mino acids (Hug'hes ·and J-ohnson 1·977') •

Disaggregation of polysomes .was. also show.n with an aMP model

system with r.educed rates of polypeptide chain elongation (Binek

et al. 1981). Increased lysosomal· ribonucleases in hyperphenyl-

. . 1-,. alaninemia' hav~ also ·been observed (Roberts, and M_ore·los 1976) -~

. . . - .. Alt~rations in th~ in!tiation ~~ocess .of protein synth~sis

...... may contribute to' art ·altered pr·otein··s·ynthes·i·zing mechanism

(Hughes and· j.ohnson 1978~) possibly by a de-creased aminoacy_lat1on f·

·of tRNA f Met . since ·no effec;::t of.hyperphenylalaninemia on

alanyl~,: lysyl-, or leucyl-- · tRNAs· was demons·trated. More '.,- !_ •

. ' . - -_ . ~ . ' ·,, ...... _.:· .: ··, . ··16' <. .. . ., ...· ' ' '·. ' .·

. re~ently, chan~es in.· r ibbsorilal protein phosphorylation in· ..• cerebral ribosomes;, and polysom~s ~ w~re seeri a,nd these ch~nges partly ·reversed' after. f3~pplement·at1on. with the seven· neutral . · amino aci.ds (Rober:ts: and r~or.~ilios ·19aO) •. •·· ·It. is: po~sible. that the . ,· •, • ·• • , . 'II ;·· , -

disaggregation of polysomes and ~iterations iri ~longatibn ~nd

. ' • I .. ' ' • , initiation of protein synthesi~ are·effebts ~ather'than a cause for an altered cerebral protein syn-thesis (Macin-nes and . Schlesinger 1971) •

Effect .on serbt6nin and the:&atecholam1nes ,,:,; .· .

The int-erference_of excess phenylalanine ·with tryptophan

and tyrbsine uptake acr6ss the blood-brain·barrier.extends -~

. ' beyond. cerebral protein synthesis as tryptophan ~nd tyrosine are

ptecursors for rte~rotransmitters. Serotonin or 5-hydroxytryptamine. (SHT) is synthesized from tryptophan: ) '

( 1) ( 2) .

. tryptophan -~~.- 5-h-ydroxytryptophan ---... serotonin ·

The enzYme-in teaction.(11 is tryptophan hydroxylase (~C

' . . . . . ·1.14 .16 .4) ;: which re9.uires the sa.~e cofadtors· 'as the ·pb_enylalanine ·

· hydroxylating. syst:ern, i~ e. tetrahydrqbiopterin whi_ch~ is pr-ovided

by the actibn of dihydopteridihe reductase. (EC 1.6.99~~). . on . .. . - . - . . . . '• q~i~oid dihidr6biopt~rin in the ptesen6e of,~ADH·Dr NAD~H· (for

review see Kaufm~n 1~77, Scri~~r and Clew 1980) ~ Re·action. (2) . is

catalyzed by Aromatic Amino Acl.d Decarboxylase, AADC. CEC· 17

.4.1.1.28)· •. Serotonin is degrad~d to·s-hydr6xyindoleaceti6 adid

( 5HIAA) . Th i. s react-ion is catalyzed by monoamine oxidase,

MA.O, ( EC 1 • 4 • 3 • 4) .

·:·' In PKU·patients, a r~d~~ed amount of SHIAA excreti~n was .. observed (Armstrong arid Robinson 1954) along with lowered· biood levels of serotonin (Pare et al. i957). ·Decreased levels of serotonin in PKU patients were also seen in brain stem·, occipital cortex, and caudate nucleus (McKean 1972).

These effects of decreased serotonin and 5HIAA levels in brains of PKU patients have been reproduced in brains of

\qeanl ing rats (age· 17 days) which. were fed a,. high phenylalanine diet (Green et al. 1962, Yuwiler and Louttit 1961, McKean et al.l967). A decrease in serotonin was seen in. rats treated from birth in a PCPA model system; however, this effect '"as shown to· be due to the presence· of PCPA alone, i •. e. ih the absence of excess phenylalanine {Wapnir et al. 1970, Hole 1972a, Berry. et al. 1975) because of the inhibition of tryptophan hydroxylase by PCPA (Koe and ~.Veisman 1966) • A decrease in serotonin was also observed in weanling rats but ndt in neonatal rats following treatment from birth in an aMP m9del system (Lane et al. 1980) •· Treating adult tats (100 days old) for 10 d~ys with·

5% phenylalanine did not alter brain serotonin levels appreciably from those of controls (Yuwiler and Geller .1969).

Phenylalanine can decrease the try~to~han cdntent (McK~an 1972)

in brain by its competition across the blood brain barrier, and this.depletion of substrate is offered as a possible explanation_ 18. for the decrease·in brain serotonin (Fernstrom and wurtman 1971).

A decreased uptake of. 5-hytl:roxytryptophan (NcKean et. al.

1962) and serotohin .. (Loo. 1974) into brains of hyperphe~yl~ alaninemic rats has been obi~fved; Since· br~in cont~ins all enzymes nece~sary for the ~ynth~sis of serotonin. frbm tryp~ophan, ·it see~s unlikely that transport cari fully acdount for the decreased levels of serotonin (Kaufman 1977) •.

Ihhibition of AADC, by excess p~enylalanine has been described

(Huang and Hsia 1963, Leo 1974); however,· no i.nhibiton of AADC was observed aftet tre~ting ~~ariling rats with ~% phenylalanine

(Yuwiler et al. 1965). Kaufman (1977) does not bel{eve that

such a slight degree of inhibition at the AADC step could

account for such reduced levels of serotonin since the rate

limiting step in serotdnin synthesis is the hydroxylating step

(Jequier 1967) • Phenylalanine inhibits tryptophan hydroxylase

(Levenberg et alv 1968·) as·well as being a compet~ng substrate

(T6ng and Kaufman 1975) •

The degradation of serotonin does not seem to be increased

as monoamine oxidaae activity was unaffected by a 5%

phenylalanine diet (Yu~iler· and Geller 1969) , although the

·possibility of an alt~red mech~nis~ for reuptake has not beeh

investigated •. Studies using pargyline, an inhibitor of

monoamine oxidase, also.revealed no diff~rence in the rate of

degradation (L6o 1974). Therefore,· the primary effect of

phenylalanine on serotonin levels.must be on the availability of

tryptophan and the inhibition at the.rate limiting step of 19 hydroxylation.

Evidence eqtiating serotonin deple~ion with mental

/' ' . deficiency was offered by _Walley and Van der Hoeveh (1964a) . \vho

·showed a reduced ability of ·_m"ice to 1e.arn a. maze when they were·

~reated from birth ~ith phenylalanine plus tyrosine~ Similar

r.esults were obtained by feeding· reserpine, which increases the

release of biogenic amines from storage vesicles, and by feeding

chlorpromazin~, which blocks th~ amine receptors. This ·learning

deficit co~ld be reversed upon treatment with the serotonin

congeners, melatonin or 5-hy~roxytryptophan (Walley ~nd Van der

Hoeven 1964b) and these investigators concluded that -serotonin

depletion was the cause of the learing impairment. In these

studies, learning deficiencies were produced. after treating·

neonatal mice, and the deficiencies could not be reproduced in

weanling rats. Yuwiler and Louttit (1961) were unable to

show a correlation between poor maze preformance.and decreased

levels of serotonin after feeding weanling rats a high phenyl-

alanine diet.

Following injection of PCPA during the first 7 weeks of

life in a rat. animal model,. b~ain.serotonin~levels decreased to

20% o·f control .values (Hole 1972b). However,· 1::here -were no

learning deficits or motivational changes associated with _this

PCPA grorip, although decreased arousal levels were obser~ed.

There were behavioral deficits as well as decreased arousal

levels in rats fed a 5% ph~nylalanine diet-from -~8 days of age

(Hole 1972a). A diet of 0.12% PCPA produced. similar s~rotonin '·20

.· _ .. · :· .. level.s ·as· a cti€H: of 3%. phenylalat:ine. + · 0,.12% PCP.A. in neonatal ' . . . . ·.r:at·s ·(Berry l975) , ·a~though ·behci~/ior·al :studies by .this grqup

. . ' . ' ,· $·hawed- differ(9rices in water ·maze learning·_be·tween · t;.he ·PCPA and_

. ' : ...... the PC.PA +. phenylal?nine... tr~·~ited group (Butcher et al. +970) ~- · . F.rorn these· studies i.t is. difficult' to establish _a cause. ·and .

effect relationship between'decreased level$·Of serotonin and -- m~ntal·r~tarC,Iation~ Studies on· 50 d?iY old and 2 year.old :~;at~

. . ' . made_ hyperphenylalaninemic· by 7% phenylalanine in th~ chew from··

r • ' • ' ' • • ' day 2 2 showed decre·ases in both serotonin arid·· SHIAA with ' . . ' ( . . impaited l~arning ability~ ~~tet th~ levels of serotonin and

SHIAA retur~ed to normal upon return to a norm~l diet, the \ . . '

learning deficit could not be altered SKo~aka and Tsukada 1979).

Perry et al. ( 1970) compared the urinary exdre·tio_n of

serotonin and SHIAA of two bro-the·rs., both· with untreated -

classical PKU •. B6th had ohly 1d~201 of the l~vels.o£ serotonin

and SHIAA as- normal· .adults; however, one bro:ther was. retarded

.and the· other was not. Therefore,I . . Perry. concluded that

decreas~d levels· of s~rotonin co~ld not ·explain.th~ mental

defect in the retarded brother. The catechol·amines are ·syn,thesize·a· fr6m tyrosine:

(1) (2) (3) . (4)

.. tyrosine ---dopa -~ .... dopamine --· norepinep~r ine ---.eptnep}1r ine

. ' . . . . . The enzyme_in reaction (1) is ctyrosine 3....;hydroxylase (EC

1 .• 14 .16. 2) ·.which requires. the'. sante -cofactors as·. phenylalanine

. . . ' hydro*yl~~~ and tryptoph~n hydro~ylase i. e. tetrahyd~obiop~erin, 22 dehydrogenase (EC 1.2.1.3) to vanylmandelic _acid (VNA) or through a re~uction by an aldehyde reductase (~C 1.1.1.1) to the alcohol,· 3-methoxy-4-hydroxylphenylglycol (M!!"PG)-. Dopamine is degraded similarly to homov~nJ.lic acid (HVA) via a pathway analogous to the formation of VN:A from norepinephrine.

Weil-Malherbe (1955) .was the first: to suggest . .that PKU patients have an abnormal catecholami-ne metabolism whi-ch m"ight . relate to the-mental deficiency. Decreases i:n excretion·o:f

. . dopamine, norepinephrine, and epinephrine by PKU patients led

Nadler and Hsia ( 1961) to pc:H~tulate in vivo. inhibition of dopa decarboxylase by phenylalanine or-its metabolites since tyrosine levels were unaltered ·by excessive phe·nylalani.ne. Dopa levels, bowever, were not measured in this study. The levels of_ serotonin, dopa.rtiine, and norepineph.. r i ne :in necr-opsied bra ins of un·treated PKU pat_ients were only one third of normal while a

40-50% d~crease was found in -their amino ~cid precutsors tryptophan and tyrosine. The reduced levels .of biogenic a-mines we·re rever,sed, as measured indirectly using the probenecid technique, following restriction of phenylalani"ne intake (McKea-n

1972) •

oeuterated tyrosin:e was given oraily to two PKU patients, one- hytrerphenylala-ninemia patient and one normal control. The levels of HVA, ·vMA,_and MHPG in the urine· were low at high phenylalanine concentrations and. inversely TIJ:er.e high when plasma phenylalanine was low (Cur.tius et al. 1972) • An --·-··-.in vivo inhibition of dopa formation was· postulated _to be due to 23

inhibition of tytosine 3-hydroxylase by phenylalanine. It has · been proposed (Wurtman et al. 1974) that the tyrosine concentration in -brain can control .the activity of tyrosine hydroxylase. by limitatio.n o:(;i.!3ubstrat.e which could be important

in a PKu·p~tient. The catecholamines are seemingly unaffected in rat animal

models as treatment with St phenylalanine .in weanling_ r~ts did not alter the brain levels· of dopamine: or norepiriephrine at a

. dose that ~aused depletio~ of serotonin (Yuwiler et al~ 19~5)~ Norepinephtinewa.s not signt£icaritly .altered with a diet nine times- the. basal diet of phenylalanine intake:· ho~never_, . dopamine· was increased (.Green et al. 19·6 2) • The effects of. phenylalanine on the levels of biogenic amines a·t 10 and· 30 days in an aMP

mod·el system was ~imited to decreased levels c).f ·serotonin at1d

SHIAA while those of popamine. and norepinephrine were. un-aff-ected

(Lane et al. 198n). Udenfriend ·et al~ (1965) obse~ved aM~ to be capable of inhibiting tyrosine hyd:roxylase in vii:rQ and subsequent studies showed aM.P-to-be-an-inhibitor in vivo -.~

following ac~te injection (~orchi~na-et al. 1970) •. There i's no evidence that the decrease in brain aatecholamines plays a role in th·e mental deficiency of PKU. However, depletion.of dopamine.ano norepinephrine by administration of 6-hydroxydopamine intraventicularly and- ihtracisternally has been reported to cause increased·

irritability~ reduced motor activity, desire for isolation, acute hypothermia, l:ethargy, reduced self-stimulation and 24 behavior resembling catatonia (for review. see Gaull 1975).

Further b~havioral tests are-necessary to relate catecholamine depletion to mehtal disorders although the synthesis of

6,...hydroxydopamine in vivo h~s been implicated as a biochemical.

. e~planation for the onset o£ schizophrenia (Stein and .Wise_l971).

Phenylalanine Metabolites

The metabolism of phenylalanirte is divided between three

pathways: 1) 'protein synthesis 2) hydroxyl~tion and 3}

transamination. In the case of PKU, a defective phenylalanine hydroxylase can force phenylalanine towards the other two

The K V pathways. m and · max are shown in the following table

(from Table 3, Kaufman 1977) ~ Transaminase

Phe prot~in mitocho.ndr ion cytoplasmic hydroxylase synthesis phe tyr phe tyr

Km (mM) .OS .02 12 12 80 1.5

vmax .04 .01 .13. .oa .04 .03 (uinole/min/g)

It is evident from· this table that .the protein synthetic pathway is quickly saturated at high phenylalanine concentration:s

and an increas·ed net protein synthesis can n·ot rid the· body of excess pbenylalafiine.· Therefore, an increase in the phenyl- alanine transaminated metabolites occurs in an attempt to 25

. . di.spose of the exces·s ·phenylalanine. At normal serum l·evels of

50 uM, phenylalanine is metabolized almost exclusively via the· hydroxylation pathway and only after a phenylalanine load can

the· transaminated m;etabo1it~s account for a significant fracti.oh. _of· the· phenylalanine metabolism (Raufman _1977) • ·The formation · of these metaboli t·es in the humain is shown in Figure 2. ·

The metabolites '~hich are excr-eted in increased- amounts in

urine of PKU patients are phenylpyru~ic acid {PPA)I phenyllactic acid (l?LA) 1 phenylacetic acid (PA;A)-, mandelic acid (MA) and ·o~oa phenylacetic acid (o-OH-PAA) , (Blau 19701 Vavich a!'ld Howell 1971,

Wadman et al. 1971~ Hill et al. 1972). These increas~~ levels -

exceed normal values by a factor of 20 to 20n fold and thus give

PKU urin~ a unique pattern (Va~ich and Rowell 1971) • An increased

. . - renal cle~rance f6r PPA has been demonstrated and it is thought

that.the rapid elimination of this compound by the kidney is

related to the fact that PPA is not reutilized as an energy

source and is not necessary for metabol i$ril ( Z.elnicek and·

. ,· Podhradska 1969) • These aromatic acids have .also bee-n

demo.nstrated in uri.nes of. hyperphe'l'1ylalaninemia var ian·ts . (Koepp

and Hoffma-n 1975) a.s well as following a· phenylalanine loading

test in hete~ozygote pare~ts of affected patients (Berrv et al.

1957, Ko'epp a·nd Hoffman :t,~75) •

PPA 1 PLA, and o-OH-PAA have also bee:n 'found in the. blood of

PKU patients (Jervis 1952 1 Jervis and Prejza 1966, Coburn et al~

1971, Clark and Land 1973) • Values· for CSF were reported by ·Clark and Land (1973). In another study of untreated PKU t.

Figure 2: Phenylalanine metabolites via the tr~nsatninase pa:thway. Phenylacetic acid is conjugated with glutamine tn humans and conjugated with glycine in rat~ (from: Vavich and Howell, 1971). PHENYL~_~ANINE METABOLITES

~- ~H2. · ~ .NH2 . ~CH 2 -CH -C02 H .~ HO-Q;

PHENYLALANINE PHENYLALANINE TYROSINE I HYDROXYLASE 7 @-cH2-tCOzH------• @-CH2-C02H

PHENYLPYRUVIC ·ACID PHENYLACETIC ACID jGLUTAMINE . . ·... ·. . .• . ·. .• l· · OH .. · 0. 0 NH2 · n u I · H CH· -C-NH-C-CH _-CH -CH-CO h @~H -C0 H . ~. CH. -tH--CO . 2 . . 2 2 2 2 2 ~ 2 2 @- OH 0--HYOROXY-­ PHENYLLACTIC ACID PHENY.LACETYLG·LUTAMINE PHENYLACETIC ACID

FIGURE 2

mN 27

patients, PPA was· not detected in CSF (Partington and Vickery 1974 The concentration of metabolites of phenylalanine in the

brains of PKU patients ha.s not been det·e·rmi-ned as rev:ie·wed by. Gau11· ( 1975) • . In or·a.er ·to 'aetermine the levels of these metabolites in brain. tissue, animal models of h'yperphenyl-.-

alanirtemia have been employed~ Leo and Mack (1972) demonstrated

increased labeling of an acidic fr~ction ef brain wi~h 14 c-phenyl~lan~ne and later confirmed the presence of PAA :( 85.9 ninole/g) , .M·A ( 3 .12 nmole/g) , PLA ( 7. 8 0 ·nmole/gf, and PPA (13 .• 75 nmole/g) in bra.in (Lao. et al. 1976). In their model, 5 day old ~ats w~re.treated with PCPA and phenylal~nine until d~y- 15; animals were then given a subcutaneous injection of

phenylalanine (400-450 mg/kg) and were sabrificed 6 hours later~

Phe rose to 2.12 um61e/q in brain. Edwards and Blari (1972)

pr·etreated 23 day old rats for 23 hours with PCPA, and 1/2 hour with pargyline, an inhibitor of MAO. A. single injection of lg/kg of phenylalanine caused plasma phenylalanine to ri.se to.

4.7 mM. _PLA (2.6 nmole/g) and PAA (1.9 ninole/g) ~>~ere present in

·brain while o--oH-PAA and MA were not detected~ Phenylethylamine has also been demonstrated at leveis of 19.7 nmole/g (Edwards··

·and Blau 1973) and has been impli~ated in vitamin B6 depletion (Loo and Ritman 1967) • using intr.ape·ri toneal i.njection of phenylalanine (1g/kg)

every 2 hours for 14 hours, .Gqldstein (1961) was able to show the presence of PLA (.33 umole/g) in brains of 18 dq.y old and

younger rats. However, PLA was not detectable in brain~ of 25, 28

40, and 75 day old rats. From these studie~ it is possible to

conclude that the influertce of the blood br~in baxrier may

affect levels of th~se rnetabolites·-in braih, ·sin~e the

. ) -~;, c:oncentration of phenylalanine in plasma r·pse to levels of

7. 5 m..l'v!,. fr.om 40 day old. -rats; yet no PLA ~vas detected.

The_ question arise~ as to the source .of the neu~otoxic ,

effects of phenylalanine or its metaboli te.s.. In an a~t te-mp.t to

answer this question, numerous in vitro models have be-e:n . . . . --. utilized: -(for review see :La·ne and Neuhoff 1980). To interp,ret

leve·ls of phe·nylalanine· and 'its rn:e·tabol i tes- in t-he in vi trq

setting, ·l-evels in vivo should be considered:. The following

is a table of ranges of blood and CSF valu.es from 6 untreated

PKU patients (taken from Clark and Land, 1973):

Serum (mM) CSF(mM-) Phe 1.56-2.79 • 81--1 .• 89

o-OH-PAA trace-.033. .099 PLA .024--.205 .018-.223 ·p·pA .024-.195 .006-.671

Studies designed to evaluate the "metabolite theory" have shown in vitro inhibiton of a large group or enzymes. A 3 . · decreased in-corporation of H- glucose into _proteins, nuclei-c

acids, . and 1 ipids has been ob.servec1 in the p·re.sen-c·e of 2. 5-20 mM

PPA in rat brain sl.ices, in fetuses and 5-2'0 mM in adult an.imals

(Weber et al. 197'0}. In a cell free supernatant systemJ . 29

concentrations of 16. and 11 mM of PPA, caused 50%·inhib{tion of

glycolysis, presumably by inhibiting hexokinase (EC 2.7.1.1) and pyruvate kinase (EC 2.7.1.40), (Glazer arid Weber 1971). The TCA

cy.cle enzymes, 4:-ketoglutarat~ dehydrogenase (EC 1. 2. 4. '2) and

malate dehydrogenase iEC 1.1.1.37), are inhibited by PP~ although the concentrations used were not reported (Patel ana

Arinze 1975) . Xnhibition of the glycolytic pathway and

impairment of the TCA cycle would certainly le~d to interference ih ATP and energy production. Miller et al. (1973) showed that

·the glycolytic metabolite pattern in quic·k froz~n brain samples· from hyperphenylalaninemic rats was consistent with irihibition of hexokinase and pyruvate kinase, but no.t necessarily impaired

glycolysis, since ATP product ion t.a~as unchanged.

PPA (.77-7.5mM) can also a~fect fatty acid synthesis by in vitro inhibition of. citrate synthetase· (EC 4.1.3. 7), acetyl CoA carboxyla-se (EC 6.4.1.2) and fatty acid sy!lthetase in rat

b~ain slices (Land and Clark 1973) • The in vit~o incorporation of· 14c-glucose into cerebral lipids from rat brain hornogenates·.­ was shown to be inhibited byPLA, PPA, and PAA at concentrations

of 5 to 35 rnM~·phenylalanine was only effective a~ 35 mM and caused a 10% decrease in incorporation {Shah et a1. 1970). A decreased bicarbonate fixation was observed at 5 mM PPA .(Patel et al. 1973) thus inhibiting pyruvate carbo'xylase leading to a

reduction of acetyl GoA.. There ~qas no effect when SmM phenylalanin~

was present and this finding of Patel and cowork~rs prompted

them to state that PPA ~-1as more toxic to the brain than 30

phenylalanine. The inhibition of sulfate incorporation into galactollpid in spinal cord culture in the presence of 0.5 mM PPA. resulted in·

7_0% inhioition,. \orhile phenylalan-ine· (1. Irhl\1) resulted. in 51% · ·inhibiton; phenylacetate and phenyllactate had no-effect (Sprinkle

and Rennert 197·6). Silberberg (1967) showed phenylacetate to be slightly cytotoxic to rat cerebellar cells in cultures at

concentrations of 2.2 mM and 4.4 mM, and lethal at 6~6 mM;

however, no effect ~-1as seen at· 1.1 :rru~ and lower levels. PPA was

not toxic ·at 2 mH.; howeve'r, a severe decrease in mye·lin concentration was observed at 3.2 mM. Phenylalanine had no

effect at 9 mM; however, the greatest .effect on the· ce-re-bellar culture was from the indole acids. An animal model has been developed using direct injection

. ' of PAA for studying its.effect.on neuronal development (.Wen et

al. 1980). High levels of phenylacetate c.aused a retarded maturation of synapses in the cerebrum of an adult rat when treated for the first 21 days of life, while no effact was

observed with PPA (Lao et al. 1980). Wen and cowork~rs

observed an alter~tion in the cerebellar vermis and .fewer

myelinated axons aft~r treatment with phenylacetate. Deficiencies in myelin and myelin synthesis were seen in both a phenylacetate model and a PCPA plus phenylalanine· model_ when

compared to controls (Lao et al. 1978.). This observation of Loa and coworkers suggested that phenylacetate may be the primary neurotoxin associated with PKU. 31

The- e-ffects of PAA on the morphology and cerebral

metabolism were_shown to have a behavioral corr~late. PAA,

phenylethylamine, or PCPA and phenylalanine treatment resulted

in behavioral deficits such~~~ poor perforrnahte by rats in a

-~"later maze a-nd a shuttle _box-, while animals treated \vi th PPA,

PCPA and MA displayed normal behavior (Fulton et al. 198D).

Treatment of rats 2-21 days of age, with ~ach of the metabolites and then behavioral tes-ting at 9 weeks of age did not yield s.itnilar res-ults (Kaplan et al •. 1981). The PCPA plus phenylalanine t·reated· rats required significantly more trials than co_ntrols, while those a.nimal:S injected w.i th each metabolite

did not behave diff~rently from controls.

"The neurotoxin" in PKU has y.et ·t~ be demonstrated.

Conflicting obs-ervations of_ lo~-'1 levels of these metabolites in serum or CSF of PKU patients and the inhibition of a plethora of enzymes make a difficult case for_ "the metabolite theory". Lo.o and coworkers have made the most promising progress in equating behavioral performance testing with levels of phenylacetate. MATE:RIALS. AND !1-~ETHODS

Animals and Diets:

Female. Harlan. Wistar rats, (l"TI) BR, were obtained from

Harlan Sprague _Dawley, Indianapolis, Indiana.· At the age of 25 days, the animals received one of the follo,#ing dl.ets: a) normal laboratory chow; b) normal laboratory chow supplemented with 0. 4% Cl-methylphenylalanine ( ar....Y:P) , Research Organics,. Cleveland, Ohio;, a,nd c) normal laboratory chow supplemented- w·ith

0. 4% aMP and 5%. phenylalanine-.· · All diets were pr-epared by

Ralston Purina Co.,. Richmond, Indiana. In experiments involving myelin ancl the whole brain proteins, animals i-n group (a) were weight-match.ed -with those of group '(c).-. · A:nimals of groups (b) and (~) received unlimited access to food. Some animals -used in the electron microscopy_ experiments rec.eived -normal laboratory· chow supplemente_d with 3% p~eny]_alanine plus 0. 4% aMP (group. d) and normal laboratory chow suppl·emented with 7. 5% ·phenylalanine plus 0.4% aMP (group e). Anim~ls of groups .(a), (c), (d), and

(e) were given unlimited access to food in the ~lectron microscopy e~perimertts~

Experiments involving radiolabeled .compounds

Two types of experiments were conduc-ted: · 1) long term

turnover study, ~nd 2) shott term incorporation •. In both cases

rats were injected with ( 4, 5--3H) .~L-lysine . {The. Radiochemical

32 33

Centre, Amersham; specific activity 40 Ci/mrnole) intravenously via the tail vein"{ at:. a'. dose of 1 uCi per gram body weight. In the lohg term study, animals were placed on each of the three­ 3 diets (a), (b), or (c}- and vh~-re injected with· H-lysine at 25. days· of age (day 0 on diet). Animals· ~1ere ·sacrificed at various· times and a decay curve was plotted as log of % maximum

in~orporation {Sabri ·et al. 1974) versus the days on diet. The short term incorporation experiment co·nsisted of injecting 3 animals with H-1ysine while at various days on their respective diets and as dpm/mg protein plotted. ve_rstfs· days on diet.

Amino Acid Analysis: •

Solid sulfosalicylic acid \'las added to plasma samples (30 mg/ml) and the mixture centrifuged at 3000 x g for 10 minutes at

4°c. The supernatant(.4 ml) was mixed with 1.5 ml of lithium citrate buffer {.21 M, pH:::2.8) and 0.1 m1 of an internal

standard, 4-aminoguanidinobutyri.c acid, (AGBA, 500 nmole/ml in

10% isop.ropanol) • The instrument used was a JEOL Amino Acid

Analy~er (JEOL, u.s.A., Cranford, N. J.) -with an Autolab- Computing Integrator (Spectra-Physics, Santa Clara, Calif.) •. Chromatography was done with a four buffer gradient of lithium citrate from .21 r1 to 1.4 M and a temperatu're gradient of 32°C-58°C. The glas-s column was .a em x 48 em packed with

JEOL LCR~2 resin. Detection was made by means of the ninhydrin 34

reaction at 95°C a-nd spectrophotometric quantitatiori at 570 nm.

Brain samples 'llere prepared for -.~mino acid analysis by

placing ~hol~ br~ins, whicih were tapidly dissected ~fter

decapitation~ into liquid nitrogen and homogeriizin~ them in 5 ml

of 0.1 N perchloric acid- (PCA)· ~nd ceritrifuged at 21;ooo x g for

15 minutes in ~ Beckman J2-21 centrifuge with a J~2n rotor. A

mixture of 1.9 ml supernatan~ and 0.1 ml of the AGBA solution . was chromatog·raphed using a JEOL Amino Acid analyzer· under the same condit-ions as described for t·he pla-sma samples. Tryptophan

~-.,as d.etermined in the supernatants from -the brains by the

revised method of Denkla and Dew~y (Bloxam and Warren 1974)_~- Tryptophan reacts in the presence of ferric chloride and

· fbrmaldehyde to give a p~oduct which fluoresces and can be measured o.n a fluorescenc-e spectrophotometer, excitation at 3-60 ·nm and emmission· at 452 nm. Unknowns were calculated from a standard curve of 25, 50, 75, and 100 ul-of a standard

co.ntaining 40 nmole/ml, each added to 4 ml of the TCA assay mixture.·

Brain Proteins and Myelin:

Brains were weighed and homogenized in 25 ml ~old 0.32 M ,sucrose in a Dounce homogenize·r. An aliquot of this .homog:enate .

. . was deproteinized ~·Ti th an equal VOlume of 12% tr-ichloroaCetic acid (TCA) and centrifuged for 10 minutes at 3000 x g in a clinical centrifuge. The pellet was washed twice with 6% TCA.- 35

The lipids were e~tradted by th~ee washings ~ith acetone and th~

0 pellet dr-ied 16 hours at .100 C. Ne~t, the pel1et y1as weighed

and dissolved with 0.4 ml 90% NCS tissue solubiliz~r {The

' - . . . Radiochemi_ca1 Center,. Amer.sham) • at 50°C for· 3 hours in. a

scintillation vial. To thi~ soluble mixture was added·s ml

Scintiverse {Fisher Scientific) and 100 ul of conc~ntrated

glacial :acetic -acid to prevent chemiluminescence. Valu-es are · expressed as dpm/mg protein.

The remainder of the homogenate was lised for ,-the ·isolation

of myelin on a discontinuo·us- sucros·e.· gradient of • 32 M and .85 M

sucrose accor.d ing to. Norton and Poduslo { 197'3b) • The volume of

myelin was reco-rded and protein determinations were done by the

method of Lowry et_ al. · -(1951).. The quanti,tative yield of myelin protein per gram of wet brain we.ight· was next calculated.

An aliquot of the myelin was counted and .results :expr_essed as

dpm/mg. The pu-rified myelin was subsequently fractio.nated

(r·1atthieu et al. 1973) by means of a discontinuous sucrose

g_radient of 7 ml of .70 M sucrose, 18 ml o·f .62 M sucrose, and

10 ml of .32M sucrose in which the myelin was dissolved. ·The

myelin separated into a pellet and two bands representing heavy,

medium·, and light fractions, respectively.

Electron Microscopy:

Tt<~o rats ,.,ere ·maintained on each of· the four respective

diets for 20 days. At 45 days of age, ~he animals were 36

~n~sthetized with nembutal (70mg/kg) and perfused with cold 4%

glutaraldehyde in -0.157 M cacodylate buffer ·(pH 7. 4) through the left ven·tr icle for five minutes. .·Brains "'re·re removed ana

sections of- white matte·r wer·e. dissected from lower midbrain at

~he pontine jUnction and· immersion- fixed overnight·in.4%

glutaraldehyde~ The tissue was postfixed in osmium and stained en bloc with 0.5% uranvl acetate for 16 hours, and then embedded ----- ·...... in Araldite (Electron Microscopy Sciences, Fort washington~ Pa.}. Thin sections were stained with lead citrate and viewed . . and photographed on a Phillips 400 elec.tron microscope. For quantitative analysis of disrupted myelin,.random

fields .(x26,008) ·containing at least 10 myelinated axon~ were pho'tographed.. Abnormal myelin sheaths were judged according. to one or more of the following criteria: 1)_ incomplete or disrupted myelin sheath surrounding an axon, 2) splaying of

lamellae, 3) widening of lamellae. by focal dilations, 4) · abnormal whorls of myelin. Ap,proximately 300 axons· 'llere examlned.for each diet group and results were then expressed as. percent of. total. Controls versus expetimentals we.re compared using analysis of covariance·.

Determination of Phenylalanine Metabolites:

Animals -were maintained ort each of diets (a:), (b), and '(c)

for 9 days~ then placed. i~ a metabolic cage {Nalgene, Rochester, N.Y. ) and u r ilie was collec·ted fo_r 2 4 hours.· An aliquot of 37

tirine was takeri for creatirtine determinatiohs usihg the Jaffe reaction, (Bonsne'S and Taus sky 19_45) ·- , The remainder of the urine sample was adjusted to pH=l2 with- 5N NaOH and 200 rng of hydroxylamine was- added., . 'rhe'· urine ~1as. heated at 60°C for 30 ·

minutes to prepare the oxime derivative of-pheny.lpyruvate. . ,, .

. ' . (Lancaster et al. 1973). The urine was acidified to pH=l with

6N HCl. An aliquot of urine cont~ining _3-4 mg creatinine was saturated with NaCl and added to 250 ug of the· internal

standard, phehylbuty.ric acid. This mixture w~s -extract.ed four

times with t1~6 volumes of· ethYlacetat·e. The combined extracts · were dried with s-odium -sulfate and ·evaporated to dryne-ss_ on a

rotary evaporator. It- was transfer~ed-with ethylacetate to a Reacti--Vial (Pierre Scientific, Rockford, Illinois) and dried

· .. 0 under'a ~trea~ of-nitrogen on~ biock heater at 6n c.

Trimethylsilyl (Tr~S) derivative.s were prepared by adding. 100 ul of bis-{ttimethylsilyl) trifluoroacet·amide (BSTFA,. Pierce Chemical Company) and heating for 50 minutes at 70°C. The s'amples were stored over o·rierite in a sealed container and_ kept frozen for no longer than tw.o days until chroma-tographed.

A Varian 370Q gas chromatograph (Varian Instruments 7 Houston, Texas) was used for the analysis of urines. Instrument conditions were as follows: injector temperature.230°C: a teflon lined stainless steel column (8 ft. x 0.125 in.- i.d.)

packed with 5% SE-52 on a chrornosorb WAWDMCS ( 10.0/120') support­ (.Anspec Compal')y of Ann Arbor, Michigan) ; temperature ·programming

fr.om 75°C for 10 minutes increas~n9 to 200°C at 2°C per minute; -·~ ', 38-

th· e carr1er.. gas was n1. . t regen a t 30 . m_1 ;·h, r: fl_arne 10n1za. . t 10n.. i. detector at 300°C; the strip chart recorder at 15 in./hr; the attenuator adjusted be.tween 8 and 32 x 10~ 11 • TMS derivatives

of the standards ~ere prepar~d in duplicate with the following. . . ',, mean retention. times {mirttites): 1. phenylacetate ( 24 .1.) · 2.· phenylbutyrate (37.0)

3. o~OH phenylacetate (39.2)

4. phenyllactate (4~.6):

5. phenylpyruvate. oxime (44.0)

Chromatograms were. integrated ''~i th a MOP.;;.l electronic integrator (Zeiss Instruments) ano a detector re.sponse 2 . factor (Rf) for ·each compound calculated: (Rf) -=mm area 2 . standard/rom area of internal standard. Amount. (ug) ·of each metabolite was calculated according to the following formula:

2 ug=(pea~ area unknown/Rf) x _(lug/1T5mm area of internal standard)

Mass units (ug) w-ere conve·rted to u,moles and normal·ized pe~ mrnole creatinine. Brain samples were ftozen into liquid nitrogen and

homogen-ized with 4.5.ml of 0.1 ·M NaCl to which has been added 80

mg of hydroxylamine and extraction of metabo-lites 'lias done according to Lob et al. (19761 except that malonic acid·was used as an irtternal standard. The metabolites were extracted

four time~ wit& two ~olumes of ethylacetate and twic~ with two 39

volu~es of ether, rather than with carbon absorption~ The: combined organic phas·es were treated as were the urine samples.

The instrument used w-as a Finnigan 4023 _gas chromatoqraph/ mass spectrometer .with an INCOS data system. The column was a 6 ft long, 2 mm diameter glass column packed with 3% SP-2250,

100-200 meshf on a Supelco port.solid phase. The injector . 0 temperature-was: set at ·250 c· and flow rate of helium carrier ' ' 0 0 gas toT as 25 ml/min. Tempera-ture programming \'las 70 C - 200 C 0 .· ' increasing at 4 C per minute. The mass spectrometer was set as follows: ion source set at maximum sensitivity at uriit

resolution, source optimized for electron impact at 70 eV. The 7 ion source was at a pressure of 1.8 x 10- torr and the electron mu~tiplier at 1400 ·eV. Analysis of peaks was done by means of spectral analysis of the derivatized biological samples

and compared to spectra in a library of TMS derivatives prepared

from the standards. Those compounds were: 1) phenylacetic

acid; 2) malonic acid (i.s.); 3) phenylbutyric acid; 4) o-OH phenylacetic acid; 5) phenyllactic acid; 6) p-OH phenyl~cetic

acid ~ 7) phenylpyruvate oxime. Mass chromatograms of specific

ions were analyzed as a means of improving sensitivity for

detection of compounds in plasma and brain.

Plasma samples (.3-.4ml) were treated as urine sample£

except 80 mg hydroxylamine was· used and proteins removed by

centrifugation after precipitation with 6 N HCl at pH=l~

Malonic acid was used as an internal standard. Chromatography

was done as described for the brain samples. Results were 40 calculated by the same formula as urine values·~xcept areas were calculated manually by the INCOS data sys.tem and the area of 175 · 2 . mm was· replaced by the internal standard ar.ea. of malonic acid which was deri~ed from manual integration·with .the INCOS dat~ system.

Determinations of·catecholamines and Ser6tonin

Wate~ used in these determinations ~as HPLC grade (tisher ;., Scientific) • Catecholamine ~tandards and alumiria were obtained from Bioanalytical Systems (West Lafayette, Indiana). All other chemicals were purchased fro·m Fisher Scientific· (At':.1.anta.," Ga.).

A stock stand~rd solution c·ontaining 0. 5 mg/ml in .1 N perchloric a·cid (PCA) ·of no~epinephrine, dopa, epineJ?hrine ·and dopamin~ was prepared and then diluted to give five concentrations of 12.5 ng/ml t·o 500 ng/ml. The· internal stand~rd, dihydroxybenzylamine (DHBA), was prepared at a concentration of 5 mg/L and 10 ul ·of this suspension was add~d to 0.5 ml of each dilute standard, 25 mg of alumina and 0.3 ml of 2.5 M'Tris. ·This suspension was vortexed 2 minutes and the pH adjusted to 8.6-8.7. After centrifugatio~ in a microfiltration. centrifuge ·(BAS,. ~,rest Lafayett, ·Indiana) , the pellet was washed twice with 2 rnl H2o. The catechoJ.amines were eluted with 0 •. 5 rnl 0.1 N PCA by vortexin·g 4. 5 minutes and centr ifugatio.n. Supernatants w.ere then transferred to ·a microfilter tube (BAS) ·and centrifuged in the BAS · .. f

41

rnic'rocentrifuge. T\'lenty ul of· supernatant ~t~as inj·ected ·into the.·

HPLC·apparatus'!

Rat~ of ~5 days of age wer~·maintained on each.of the 3 diets (a), (q), -and (c) for ·r:o days and:.sacrific-ed by ·means o.f decapitation. Each whole brain was di.~sect.ed, and piaced into liquid nitrogen. The brain -was later weighed and homogenized in

5 ml bf 0.1 N PCA and centrifuged at ~7~qoo x g fo~ 15.minutes in a Beckman J2-21 centrifuge with a J-20 rotor. Art aliqtiot 6f a·. 5 ml was take·n from the supernatant and proce·ssed jus·t as the standard solutions in the· ab6ve procedure.

The mobile phase consisted qf 0.15 M· monocholoracetic acid.,

1 mM·Na EDTA and25 ml/L sodium octylsulfateat pH=3.0. This 2 solution was f-iltered through·a 0.2 micron.ce'llulose nitrate membrane (Fisher Scientific) a·nd degassed under a vacuum with. a magnetic stirring bar.. Chromatog-raphy was done on a Beckman r-1odel 332 gra.dient liquid chromatograph with a flow· rate of -2

1_ ml/min and a pressure of 4000 psi with. a· Beckma·n ODS ul trasphere reversed phase column. The detector was an_ LC-4A.amperometric detector (BAS) with a potential of + a·.7o V. Detections were done at o.s,· 1., or 2 nanoatnps (naY, with an offset of approximately 3D. The chromatographic tracings were done on a s:trip chart recorder at· a speed of 5 mm/min. . Unknown peak hei~hts were read off 6f the standard curve (na x.peak height vs. ng) and converted into units o.f nmol~ per gram wet weight of brain. 42

The determinatibn o£ serotortin and 5HIAA-was also done by the method of HPLC. Standards of serotonin and 5HIAA were prepared in similar concentrations~ as thos·e for. the datecholamine determination._ ·Th~ standards were diluted t6 give si~ concentration~ of 500 ng/ml through 5 ng/ml. The internal· standard, N.-methylserotonin, was prepared at a concentration of

10 ug/ml~ To 500 ul of each of the dilute standards were-added

10 ul of ·internal standard and 20 ul of this solution was injected into the HPLC. Rats were sacrificed as the rats ,_used ·

in the catecholamine determirl~tions, except th~t only the

brainstem was dissected. ,Each brainstem wa~ homogenized in 2 ml

of 0~1 N PCA in a polytron tissue homogenizer (Brinkman­

Instruments, Westbury N.Y) • Samples 'toTer~ centrifuged as described in the catecholamine ass.ay, and 500 ul of the supernat_ant was added tq 10 ul of internal standard. · T'IITenty ul was used to inject into the·HPLC apparatus. Chromatographic conditions·were the same as described· in the catacholamine assay·

with the_ ·exception of the mobile phase wh~ch consisted of 0.1 M chloroacet:ic acid, 1 mM EDTA,. 9% 'methanol, and pH=3 •. a. Standard

curves ~..rere plotted and unknowns determined as described in the- catecholamine assay.

Statistical Analysis

Analysis of variance, ANOVA, which included'the Duncan r~nge test, and Students T-test were performed using the SPSS £tatistical 43 packageG Regression analysis which included standard eirors of t: slope were also perform~d using ·the SPSS statistical package. RESULTS

Amino acid analysis

In order to approximate· the plasma· phenylalanine· values o.f a PKU patient, an appropriate animal model must prove effective to mimic the PKU syndrome. To avoid some of the side effects of using p-chlorophenylalanine as an inhibito-r of phenylala·ni.ne hydroxylase, a more sui table model using a·-methylphenylala,nine (aMP) was investig.ated. A diet supplemented with 3% phenylalanine + 0.4% aMP was attempted; however, plasma phenylalanine reached a level of 9Q3 + 58 uM (n=3) for 35 days old rats which-were maintained on this diet for 10 days, while nqrmal ra~s had.a plasma phenylalanine level of 50 uM. This plasma phenylalanine value of 900 uM was considerably less than the 1500 uM observed in a classical PKU patient (Hsia 1970). Next, a diet supplemented with 7.5% phenylalanine + 0.4% aMP diet was employed and plasma phenylalanine was quite high at 5129 + 537 umolar (n=3) after 10 days on th.is diet. 1 The extremely high plasma phenylalanine was very toxic and animals survived on-ly about,three weeks on this diet. After 2 week~ on a diet suplemented· with 7.5% phenylalanine + .4% aMP, rats we~e not able to maintain their starting body weight of approximately 60 g at 25 days of age.

44 .':·

45

On a diet supplemented with 5% phenylalanine + 0.4% aMP

(HyPhe), the plasma phenylalanine was 40 times normal, 2.2mM.

versus ~05 mM for control animals (Table I) • · This elevation in

·plasma phenylalanine is comparable to a value of 1.8 IID.\1 obtained

by Berry et.al. (1975) in a 3% phenylalanine + 0.12% PCPA model. The effectiveness of aMP as an ---in vivo inhibitor of phenylalanirte hydroxylase was apparent in a group of rats fed a

diet Sl1PPlemented with only 0.4% aMP (aMP group), '¥hich had an

elevated plasma phenylalanine of .15 rnM. Although the at-1P is not effective alone as a model for PKU, this value of .15 mM was

3 times the control plasma phenyl~lanine value. When aMP is

used in dornbination with excess phenylalanirie, a suitable ~lasma phenylalanine value is attained to approximate that of the PKU patient (Table I). One drawback to this model, which is also evident in the model of Berry et al. (1975), is the elevated plasma tyrosine in· the HyPhe group (Table I). This increase is likely due to the inability of aMP to completely block phenylalanine hydroxylase. This drawback is evidentfrom the phe/tyr ratio of

50:1 in PKU patients (Perry et al. 1970), while 5:1 was

observed in this HyPhe ~odel. The plasma amino acid profile in Table I is very ·similar to the pattern of amino acid values' · observed for PKU patients (Perry et al. 1970). No changes in glycine and serine and decreases in leucine, isoleudine, valine,

threonine, alanine, histidine,- and arginine were evident (Table I) o

Perry et al. (1970) observed a slight drop in plasma lysine and '···,' 46

TABLE" I

PLASMA AMINO ACID LEVELS IN

EXPERIMENTAL HYPERPHENYLALANINEMIAa

.;~ .; ' -~ . . . ·.... · b Controls (n=3) . .4% aMP(n~4) 5% phe + .4% a.MP(n=4)

Thr 284 ±. 13c 316 ± 16 239 :t 37

Ser 264 ± 27 277 :t 74 287 :t 14

Gly 370 :t 99 346 :t 45 398 :t 23

Ala 480 :t 87 46"9 :t 40 371 ± 40*

Val 232 :t 22 238 ± 49 130 :t 25**

Met 43 ± 2 43 :t 6 41 :t 7

I leu 112 :t 16 91 :t 12 50 ± 8**

Leu 187 ± 17 181 :t 29 83 :t 12** + Tyr 90 ± 10 108 ± 14 423 :t 41

Phe 52 :t 5· 147 :t 23** 2217 ± 889+

Lys 498 :t 59 463 :t 39 498 :t 54

His 56 :t 5 59 :t 12 41 ± 5*

Arg 214 ± 6 240 ± 38 85 ± 18+

a) Animals of "35 days on respective.diets for the previous· .10 days (for explanation of diets see materials and methods).

b) Numbet of animals

c) Values expressed as 11M: mean ± SD

* p < .05 with respect to controls. (ANOVA) **P ·. < -.01 with respect to -controls (AN OVA) + p < .001 with respect .to controls (AN OVA) 47 methionine in untreated PKU patients, neither of which was observed in this study. Decreases in lysine and methionine 'ilere not seen in a PCPA + phenylalanine model (Berry et al. 1975).

The HyPhe treatment caused the intracerebral le~els of phenylalanine to rise approximately 30 fold (T.able II). The elevation of pla~ma tyrosine also resulted in increased values of intracerebral tyrosine, which were three times the control value. Table II also shows intracerebral levels of the branched chain amino acids and methionine, which were substantially decreased to approximately half the control values. Tryptophan levels in brain were not different among the three diet groups.

Interestingly, the brain levels of glycine, a putative inhibitory neurotransmitter, were double that of controls without ~n. increase in plasma glycine (Table I) , or intracer~bral serine, the precursor in glycine synthe·sis (Table II.)~ -'Y-aminobutyric acid, GABA, also a putative inhibitory neurotransmitter, was not significantly different from the control valu·es. The levels of lysine in the free a~ino acid pool of brain did not vary among the three diet groups (Table II). Arginine was decreas·ed by 30%

in brain, but the decrease was not as severe as that in plasma •.

~h~nylalanine met~bolites

As the hydroxylase pathway becomes saturat~d, it is of interest to determine the levels of phenylalanine metabolites which originate via the transaminase. pathway in urine, plasma, 48

TAB~E II

AMINO ACID LEVELS IN BRAIN IN

EXPERIMENTAL HYPERPHENYLALANINEMIAa

. . b Controls (n.=4) • 4% ~P(n=5) . . 5% phe + .4%

Asp 3.55 :t .51c 3.71 ± .72 3.08 ± .39

Th:,; ~63 :t .18 .52 :t .• 04 .49 :t .13

Ser .78 ± .29 .64 :t .03 .83 ± .23 ** ++ Gly · 1 •. 11 ± .28 .73 :t .08 2.15 ± .40 ' + Ala .80 :t .16 .77 ± .07 1.05 :t .19

Val .03 ± .01 .05 ±· .01* .02 :t .02

Met .03 :t .01 .041 :t .010* .018 :t .oo6+ * .. ** + !leu .020 :t .006 .036 :t .01 .006 :t .003 ,+ • Leu .050 :t .014 .070 :t .024 .041 ± .006+ **,++ Tyr .065 ± .029 .092 ± .026 .192 ± .04 * Phe .042 ± .005 .083 :t .026 1.26 ± .sz**,++

GABA 1.88 ± .35 1.92 :t .31 2.21 ± .36

Lys .29 ± .04 .24 ± .06 .22 ± .03

His .057 :t .003 .053 ± .01 .067 :t .012 ,+ Arg .108 :t .014 .105 :t .023 .070 :t .013*

Tryp .020 ± .001 .019 ± •:002 .018 ± .002

a) Animals of 35 days on respective d!ets· fo,r the previous 10 days (for explanation of diets see materials and methods). b) Number of animals

c) Values expressed as }Jmole/g: m~an ± SD.

* p < .05 with respect to con·trols (AN OVA) + p < .05 with respect to aMP group (ANOVA) ** p < .01 with respect to cont·rols (ANOVA) ++ p < .01 with. respect to etMP group (ANOVA) 49 and brain. After Maintaining animals on a HyPhe.diet for 10 days, excretion of phenylacetate, o-OH phenylacetate, phenyllactate, and phenylpyruvate were easily detectable in the HyPhe group by gas chromatogr;aphy (Figure 3A) ..and quantified . . (Table III) . The values of o-OH phenylacetate·, phenyl.acetate, and phenylpyruvate fro~ urine (Table III) approximated the phenylalanine metabolite levels in a PCPA + Phenylalanine .rat model and also similar to levels of the phenylalanine metabolites of 15 PKU patients (Lane et al. 1980) ~ although in this same study, the values 6btained for phenylpyruvate a.nd. phenyllactate in a aMP + phenylalanine mod~l were toughly 3 times the values in Table III. Plasma valties of PLA and PPA were in the range obtained for PKU patients· (Land and Clark

1973) wi.th the exception of 0. 2 ur-1 for· one determination of PPA •.

The value of 20.5 uM was in agreement with the valu,es in ·the experimental model of Lane et al. (1980); however, the values 2.4 uM and 0.2 uM observed in this study (Table III) were slightly below the mean of PPA observed by Lane et al. (1980) following a single injection of aMP + phenylalanine. Only a very small peal< for phenylacetate (5.4 .±. 5.3 umole/mmole creatinine) could be· detected in urine from that of the at-1P group, but~ a considerable phenylpyruvate peak (209 + 29~5 umole/mmole creatinine) was also evident (Figure 3B). The presence of the phenylalanine rnetabolit~s could not be-detected. in urine from control rats with the excepti6n of phenylpyruvate

(180.6 + 47.1 umole/mmole cteatinine). The volume of urine :: ._:... ~

Figure 3A: Chromatogram from a Var;i.an 3700 gas·chromatograph,of TMS de..;. rivatives· of organic aci~s.from. urine of a rat on a hyper­ phenylalaninemia inducing diet.

A 35 day old rat was fed n()rmal laboratory chow supplemented with 5% phe +- .4% a.MP for the previous 10 days. Attenuati.on was set at. J2 (see materials and·methods for chromatographic conditions)~

Identification of peaks:

1) phenylacetic· acid (PAA) 2) phenylbutyric acid (I.S.) 3) o-OH ph~nylacetic acid (oOHPAA) · 4) phenyllactic acid (PLA) 5) Oxime of phenylpyruvic acid (PPA) 50

r.t)

~ Q "~if'

N

fn -CD ,::z: ("~':) -=c Q g ~ ··"" 0· CD t.-' e H i= ~·

,..Q Figure 313: Chromatogram from a Vari&rl 3700 gas chromatograph of TMS de­ rivatives of organic a·cids from urine of a rat fed normal chow supplemented with .4% a.-methylp-henylalanine. A JS day old rat was fed:·this diet for the previous 10 days. Attenuation was set at 8 (se~ materials ·and methods for chro­ matographic conditions).

Identification of peaks:·

1) phenylacetic acid (PAA) 2) phenylbtityric acid (I .S.) 5) Oxime of phenylpyruvic acid (PPA) 51

\

c;; CD fii -c= M Q ('I) g ~ Q) .:::::> (.!) e H ·i= r..

.,..Q 52

TABLE III PHENYLALANINE METABOLITES IN

. EXPERIMENTAL HYPERPHENYLALANINEMIAa

.b Metabolite Urine(n=4) Plasma(n=3) Brain(n=4)

. d Phenylacetate 86.7 ± 48.7c nd nd o-OH Phenyiacetate 33.3 ± 17.6 nd nd e Phenyllactate 110.9 ± 61.4 93,33,80 nd

Phenylpyruvate 392.9 ± 105.5 . 20.5,2.4,0.2 nd a) 35 days old animals maintained on diet supplemented with 5% phe + .4% a.MP for the previous 10 days. b) Number of: animals c) Urine values expressed as ~mole/mmole Creatinine: mean ± SD. d) nd = not detected e) Plasma values expressed as ].1M. 53 used in the aMP group, and control animals T..Vas. normalized to the creatinine p·resent in approximately 2 ml of urine from

the HyPhe ani~als.

A gas chromatograph/mass spectrometer (GC/MS) w~s utilized to determine the values of phenylalanine metabolites in plasma and brain. Phenyllactate and phenylpyruvate were detectable irt plasma (Table III) but, not in brain. The limits of ·sensitivity -13 . are 1 x 10 g/ul or approximately 10 pg per brain. The characteristic· ions 193 and 220 of.phenyllactate at the

retention time of interest (23:32), demonstrate the ·presence of phenyllactate in plasma (Figure 4A): however, it is just below detection in a sampl~ from br~in {Figure 4B) • Using this very

.sensitive method of mass chroma tog rams., phenylpyruvate was alSO' . - not detected in brain, while phenylacetate and o-OH phenyl­ acetate could not be detected in plasma or brain (Table III) •

Catecholamine and Serotonin Synthesis

Since experimental hypetphenylalaninerni~ ~auses increased

levels of tyrosine ~n brain and phenylalanine is known to competitively inhibit tyrosine hydroxylase, it is of interest to investigate the effects of experimental hyperphenylalanemia on

catecholamine synthesis and compar~ these findings with thbse iri patients. '!'he separationof norepinephrine, dopa, epinephrine, and dopamine of brain of control animals at 35 days of age is shown in Figure 5A. The epinephrine peak.was noticeably absent· .I

Figure 4A: Ma~s chromatogram from a Finnigan 4023 gas chromatograph/Ina.s.s . spectrometer of a plasma extract ·of organic a~ids from a rat maintained on a hyperphertylalaninemia inducing diet. Plasma extract of organic acids from a 35 day old rat·fed nonna.l laboratory chow supplemented with 5% phe + .4% aMP for the previous 10 days. This figure -_shows the presence of the TMS deriva_tive of ph.enyllactate at· a retefltion time of 23:32 minutes, with scan 11709. The top chromatogram is a mass chromatogram of peaks which contain the ion of mass 193, pre­ sent in the TMS derivative of phenyllactate. The middle chromatogram is of peaks which contain ions of mass 220, also present in the THS derivative of phenyllactate. The bottom chromatogram is a reconstructed ion chromat.ogram (RIC) containing ions of mass 1.;..450. RIC + I'IASS CHROI'IATOGAAMS DATA: tfllPLASMA ltl SCANS 680 TO 818 81/28/82 21:30:00 CALI: ~4KJ11 .2 SAI'lPLE: .., lfLASM RANGE: G L 1000 LA8EL: H e, 4.9 QUAN: A 9, 1.9 BASE: U 20, 3 709 100.8 {\ 2676 193 193.85: I \ . * 8.591 J '----737 . 687 788 _/ - 799 13.3 357

220 228.00 I t 8.591 I ' 737 · I J "-:~~-]55 764 _llL___ ..?~2 7~8 I I I -- I I -~- "'"F =~-- ..___ -t-- =·--- . 710 . . 787 . 42243 . . 775

(\ ·. 764 / . \ 73a /~~ ~IC ~ ·~

688 788 729 748 768 780 see SCAN 22i40 23:29 24:00. ·24:40 25:20 26:a0 26:48 Til£

l11 FIGURE 4A ~ Figure 4B: Mass chromatogram from a Finnigan 4023 gas chromatograph/mass spectrometer of brain extract of organic acids from a rat maintained on a. hyperphenylalaninetn.ia inducing diet.

Brain extract of organic acids from a 35 day old rat fed normal laboratory chow supplemented with 5% phe + .4% aMP .for · the previous 10 days. The mass chromatograms for ions of· mass 193 and 22.0 and the RIC are describe4 . in Figure 4A.- Scan 11705 at retention time 23:32 minutes on the chromatogram of mass 220 ·is present in levels ( < 12 ·.ions) which is just below detection for an aQcurate.identif.ication of phenyllac­ tate. RIC + tiHSS CHROMTOGRAAS DATA: HPlBRAIN 1708 SCANS 680 TO 810 01/20/82 23:3:3:00 CALla CAL4iUl1 f2 SAtiPLE: ..,IBAAIH RANGE: G 706L 1000 LABEL:. N e.' 4.0 QUAN:. A 0, 1.0 BASE: U 20. :3 J~b

193 193.05! :t 0.501

786 792.~896

12

229 220.00 t 9.591

708 92672 \\ J '\. ;uc· ...... ·------...... ___-.... n9 . 7S7 893 . 767

680 7El9 720 740. 769 780 890 sc~ · 22:49 23:29 24:09 24:49 25:29 26:09 26:49 TitlE

FIGURE 4B lJl U1 56 in brains from 35 day old animals t,vhich hac been treated with a nbrmal diet supplemented with 5% phenylalanine + •. 4% aMP from day 25 (Figure SB) . The question as to the effects of aMP on catecholamine synthesis must be evaluated before conclusions can be drawn about the effects of experimental hyperphenylalaninemia. The aMP proved to be a very potent_ inhibitor of tyrosine hydroxylase in vivo, since animals treated with a diet supplemerited with .4% aMP showed substantially decreased levels of dopa in brain (Table IV). The reason for ·the discrepancy of .. decreased dopa and increased dopamine in the HyPhe group is not known. Dopamine :a-hydroxylase is also inqibited by aMP as norepinephrine values decreased to half the control values, and this decrease was not due to depressed le~1els of dopa, as· dopamine values were slightly lower in controls (Tab~e IV) ~ Thus, aMP appears to inhibit the hydroxylases in catecholamine metabolism in addition to inhibition of phenylalanirte hydroxylase. Surprisingly, aMP also caused-a decrease in epinephtine to about 25% of control values. Since the norepinephrine w-as a little more then 50% of controls in the aMP group, inhibition of phenylethanolamine N-methyltransferase (EC 2.1.1.28) by aMP may occur b.eyond.the decreased availability of norepinephrine for epinephrine _synthesis. Even though the.tyrosine level in brain of the.Hyphe group was 3 times the value in the aMP group (Table II) , there was no difference in dopa concentrations in brain, suggesting that the substantially increased levels of phenylalanine had little- .Figure SA: Chromatogram from a Beckman Model 332 HPLC of catecholamines from acid soluble extract of brain from a 35 days control rat.

Identification of peaks:

1) Norepinephrine 2) Dopa 3) Epinephrine 4) Dihydro-xyben.~ylamine (internal standard) · 5) 5-Hydro-xyindoleacetic acid ·6) Dopamine 57 .

CD 4 ·~··. ·-·' Q, c. U) a:CD 6· ..-0 u ....CD c.CD

5 3. 2

30 20 10 . 0 Tim·e (minutes)

FIGURE SA Figure 5B: Chromatogram from a Beckman Model 332 HPLC of catecholamines from acid soluble extract of brain from a- 35 day. old rat fed a diet of normal laboratory chow supplement.ed with 5% phe + .4% ctMP for 10 d·ays previously.

Identification of peaks:

1) Norepinephrine 2) Dopa 4) Dihydroxybenzylamine (internal standard) 5) 5-Hydroxyfndoleacetic acid 6) Dopamine 58

Cl). U)· c: a.0 en CD a: 6

"

5· . 2

30 20 10 0 Time (minutes)

FIGURE SB 59

TABLE IV

BRAIN CATECHOLAMINE LEVELS IN

EXPERIMENTAL HYPERPHENYLALANI~EMIAa·

Diet Group. Dopa Dopamine Norepinephrine Epinephrine

b Control(n=4) .239 ~ 0.24c 3.39 ~ .51 2.00 ~ .23+ .087 ~ .054

.4% ·aMP (n=S) .086 ~ 0.59* 2.94 ~ .35 1.08 ± .14+ .021 ± .004*

5% phe + .4% aMP (n=.Sr .0.67 ± .005* 3.78 ± .68 1.51 ± .29+ not detected

a) Animals of 35 days on respective diets for the previous 10 days (for explanat;i.on of diets see materials and methods).

b) Numher of animals

· c) Values expres.sed as nmo le/g: Illean ± SD. • * p < .05 with respect to controls (ANOVA)

+ p < .05 with respect to· all groups (ANOVA) 60 effect on further depressing the dopa levels beyond those . observed in the aMP group. The levels of norepinephrine wer~ significantly different among all diet groups, and the increased norepinephrine ih the HyPhe group versus the aMP group may be related to increases in dopamine bbserved in the HyPhe_group

(Table IV) i Hyperphenylalaninemia depresses the epinephrine levels in brain more than that observed in the aMP group (Ta~le

IV), ·as epinephrine could not be detected in th~ HyPhe group (Figure SB) • In PKU patients, decreased plasma values of serotonin (5HT) and 5-hydroxyindoleacaetic acid (SHIAA) do occur. Therefore, one can ask how experimental hyperphenylalaninemia and aMP affect serotonin metabolism. The separation of serotonin and 5HIAA pres-ent in an extract of normal rat brainstem is shown in Figure 6A and it is apparent that a substantial reduction of serotonin and SHIAA occurs in the hyperphenylalaninemic condition (Figure 6B). The aMP group did not vary from the control group in concentrations of serotonin or SHIAA (Table V) • Although no difference i.n intracerebral values of tryptophan in the HyPhe group versus controls (Table II) ,.,as observed., a substantial reduction (greater than 50%) of both serotonin and

SHIAA was demonstrated in the P.yPhe group which \'Jas independent of the presence of aMP. This inhibition was indeed due to the presence of excess phenylalanine (Table V) . Figure 6A: Chromatogram from a Beckman Model .332 HPLC of serotonin or 5-Hydroxytryptamine .(SHT) and·S-Hydroxyindoleacetic acid (5HIAA) from ac.id soluble extract from b:rainstem of 35 day old control rat.

IS = internal standard (N-methylserotonin) 61

·CD en c I.S. 0 Q.. .5HT en. CD: a: SHIAA

14 12 10 8 6 4 . 2 0 Tlme ·(m.inute.s.)

FIGURE 6A Figure .6B: Chromatogram from a ·B~ckman Model 332 HPLC of serotonin or 5-Hydroxytryptamine (5HT) and 5-Hydroxyindoleacetic acid (5HIAA) from acid soluble extract from brainstem of 35 day old rat fed a c;liet of normal laboratory chow ·supplem~nted with 5% + .4% aMP for 10 days previously.

IS = internal standard (N~methylserotonin) .62

I.S. G)· en .c Q· ·C.. Cl) a:.CD· .....0- -U· CD ....G) c SH·T

14 12 10 8 6 4 2 0 Time (minutes)

FIGURE 6B 63

TABLE V SEROTONIN AND 5HIAA LEVELS FROM BRAINSTEMS IN EXPERIMENTAL HYPERPHENYLALANINEM!Aa

. . b Controls(n=5) 2.30 ± .27 2.73 ± .48

.4% aMP(n=5) 2.26 ± .47 2 .• 14 ± • 73

5% phe + .4% aMP(n=4) 1.07 :t .29* .87 ± .23*

a) Animals of 35 days on respective diets for the previ-ous \. 10· day$ (for explanation of diets see materials and methods).

b) Number of animals

c) Values expressed as nmole/g: mean ± SD.

* p < .001 with respect to controls (ANOVA). 64

Myelin metabolis~

Animals maintained on the HyPhe diet avoided eating the ·

chow. Weight gain was very sl6w, when compa~ed to the normal

control group or ~he aMP group {Table VI) . A comparison of the body weight data (Table VI) , shows that the .aMP group wa.s identical to the control group, while a PCPA treated group

showed small deficits in body t~eight gain (Hommes et al. 1982b). The brain weights of the aMP group did not vary from the ad 1.!.£:_ fed controls and no difference w-as observed in

the brain w·eights of a PCPA group versus controls (Hommes et al.

1982b). A weight-matched control (WMC) group 'Has necessary to

insure tha·t changes observed in .. the HyPhe group were not . . consequences of nutritional deprivation. The body weights· of

the W!'..YC group averaged appro:timat.ely 6% difference from the body weights in the HyPhe group ove.r the 70 days of the study (Table VI] although some discrepancy was observed on day 0 due to different shipments of rats. Brain weight was decreased in both the WMC group and also in the HyPhe group when compared to an ad lib. diet

supple~ented \'lith • 4% a~4P. However, the decreases in brain weight of the HyPhe group were greater than that due to nutritional deprivation in the WMC group ·(figure 7). The brain

weights of the aMP group did not vary from normal controls (ie. an ad lib. control .diet). Even though the WMC group had a lower brain weight than·the aMP group, the concentration (mg/g) 65

TABLE VI BODY WElGHTS OF RATS IN EXPERlMENTAL HYPERPHENYLALANINEMIAa

Days on Diet Controlb HyPhe e

0 4·8. 7 :t 6.1 54.2 :t 13.1 65.6 :t 4.8 68.7 :t 10.9

3 79.5 :t 2.6 67.7 ± 4.2 63.7 :t 9.9

5 76.9 ± 7.9 74.3 ± 5.0 76.1 ± 4.5 63.5 ± 9.9

7 101.1 ± 2.5 100.0 ± 9.8 66.5 ± 3.2 63.4 ± 9.8

10 98.2 ± 7.7 120.0 ± 15.2 68.7 :t 4.3 64.5 ± :Lo.o

19 151.3 ± 10.3 160.9 ± 12.5 69.4 :t 3.1 69.9 ± 15.8

30 190.8 ± 14.8 184.7 ± 19.5 79.7 ± 3.4 82.0 ± 18.6

50. 229.6 ± 19.0 236.5 :t 9.9 . 95.0 ± .2.9 89.2 ± 18.6

70 263.3 ± 26.2 251.7 ± 6.5 102;.3 ± 5.7 101.0 ± 39.7 a) for explanation of dietary models see materials and methods b) Values are g :t SD for 40 rats at day 0 decreasing to 4 for the 70 days value. c) aMP group not .statistically different from controls except on day 0 (p < • 05, ANOVA) d) Weight matched control (WMC) by restricting intake of control chow to match body weight gain of group (c) overall an Clverage deviation of 6.3% was observed when compared. to the HyPhe group •. WMC group not statistically different from HyPhe group exc~pt at day 5 (p < .OS, ANOVA) •. e) HyPhe/WMC group statist:i.cally different from controls (p < .001,. ANOVA) for all days. except day 5. Figure 7: Brainweight of rats maintained on diets. For explanation of diets see materials and methods. Day 0 corresponds to rats of 25 days of age Points are the mean and standard deviationof 4 animals. All groups were significantly different at p < .• 001 (ANOVA). 2.0 ..

II e4°/oQMP 1.9 • VVMC A 5°/oPHE+ .4°/oQMP .- _g 1.8 ....., ..c CJ) ~ 1.7 c: (lS '- 1.6 0)

1.5

1.4~--~~~~--~~---.--~.---~----~---- 30· 40 50 Days on Oiet

(J\ FIGURE 7 (J\ Figure 8: Myelin protein .content expressed as mg/g wet weight of brain. Day 0 corresponds to rats of 25 days of age • .For explanation of dietary treatment see materials and methods. Point·s are ·the· mean and standard d~viation. of 4 animals. The HyPhe group was significantly different at p < .001 (ANOVA). ( 67

Qm 0 ~· co 0 0~ ~ ~ ~. 0 a.. + 1'- ~ w 0 (J :::t:· ~··~ 0~ 0 ~ ~-~ c.c

iii::- ., ~- r---= 0 ...... 1.0 Q) 0 c 0 o· co ~ ~- rn ~ 8· ctS H J::.! 0 Q M

0 1.()

D/DW U!9lOJ.d- UH8Afi\J 68 of myelin protein did not vary between the two ~roups (Figure 8). The HyPhe group showed a distinct deficit of myelin protein which was independent of nutritional deprivation or the presence of .4% aMP in the diet (Figure 8). This loss of myelin protein could occur due to a decreased synthesis of myelin protein, an increased degradatioh of myelin protein, or a combination· of both mechanisms.·

Two types of e~periments were undertaken to investigate the pot:ential source for the loss of myelin protein. The-first expetiment involved a long term turnover study in which 3H-lysine ·was inject·ed at 25 days o~ age and animals then placed on the respective diets, HyPhe, aMP or ·VJMc. Age 25 days

was designated day·o on the diet. The loss of lab~l was followed up to 70 days following injection of the label of both myelin proteins (Figure 9) and whole brain proteins (Pigure 10) •

The rate of protein synthesis could also be estimated by means of a short term labeling experiment. After injection of 3H-lysine (luCi/g), rats were sacrificed 1 hour later and the incorporatiort of label into both myelin proteins (Figure ·Ll) and whtile brain proteins {Figure 12) was d~termined. 3 H~lysine was chosen as a precursor label to study both the long term turnover and rate of protein sy:nthesis since the free lysine pool was not significantly different in any of the brains between the three diet groups (Table II) . Also, plasma lysine was not altered between the three diet groups (Table I) , elimi-

nating the availability of label as a potential source of error~ Figure 9:. !urnover of myelin protein ·following itijection of 1 11Gi/g of H-lysine into the tail vein of rats 25 days of age (day.O on diet).

For ·explana·tion. of dietary treatment see materials and methods.

LOG (% maximum) = Logarithm of percentage of maximum incor­ poration of label. Each point is the-mean of 4 animals. In the WMC group, the standard deviation averaged 12% of the mean and the correlation coefficient was 0.92. In the aMP group, the standard deviation averaged 14% of the mean and the correlation coefficient was 0.93. In the group 5% phe + .4% aMP, the standard deviation averagf:!d 21% o~ the mean and the correlation coefficient was 0.98.

Diet Group· Slope SE slope intercept 3 -4 WMC -6.54 X 10... 7•• 49 X 1.98 2 10_3 aMP -9~40 X 10-· 1.70 X 10_2 1.98 lO-l. HyPhe* -1.75 X 3.18 X 10 2.86

*p · < • 05 with respect to WMC group 2.0

...-WMC -...-.4°/ooMP E 1.9 +. 5°/o PHE: + .. 4°/o od\IIP ::J ' E ·x 1.8 ctS ~

"'ifi..._.. 1 . 7 Ol 0 _J 1.6

1.5·' I I I I I I I ""=0'!"1 10· 20 30 40 50 '60 70 80 Days on Diet

m FIGURE 9 w Figure 10: Turnover of TCA precipit~ble who.J..e brain pro.teins following injection of 1 1-!Ci/g of H-lysine in:to the tail vein of rats of 25 days of age (day 0 on diet).

For explana-tion of dietary treatment see mater;i.als and . methods •.

LOG (% maximum) = Logarithm of percentage of maximum incorpo­ ration of label. Each point is the mean of 4 animals. The average standard deviation for the WMC group was 9% of the mean. The correlation coefficient for the WMC group was 0.99 and· the t~ is li days. The averag.e standard deviation for· the 5% ·phe + • 4% Ctt1P group was 14% of the me~n. The correlation coefficient was 0.98 and the t~ is 25 days.

Diet Group Slope SE slope intercept 2 4 WMC -1.56 X 10- 5.79 X lo- 2.04 2 4 HyPhe* -1.27 X 10- 7.53 X lo-:- 2.06

*P < .05 with respect to WMC group 70

Q;.~ :E; 0 ?fl. ~ •· 0 +

Rats ·of ·various ages (dayO on diet corresp311ds to rats of 25 days of age) were injected with 1 /J..LCi/ g H-lysine via the tail vein. Animals were sacrificed .one hour later. Myelin was isolated as described in materials·· and methods.. Results are expressed as dpm/mg. Points are the mean· and standard· deviation of 4 animals. The two groups were not statis.tically different (ANOVA). 1000

800 • CfJntrols • 5o/o·PHE+ .. 4°1oOcMP 0) E aoo --E c.. u 400 l I 200 A

4 8 12 16 20 Days on Diet

-....) FIGURE 11 I-' Figure 12: Short term incorporation of 3H-lysine into·TCA precipitable brain proteins. ·

Rats of various ages -(day 0 on diet corrresyonds .to rats of 25 days of age) were inje.cted with 1 1JCi/g H-lysine via the tail vein. Animals were sacrificed one·hour later. Brai-n proteins were precipitated as described in materials and methods. Results are expressed as dpm/mg of protein. Points are plotted as mean and standard. deviation of 4 animals. No statistical difference was observed between the two groups (ANOVA). 72

·~ .. ·t

·a 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.0 ~ ("') C'\J· ,... owrwdp 73

. t . ..!:! 3 f! 1 . ' b . . Th e s1or1 t t erm 1ncorpbra 10n or 1- ys1ne ~nto ra1n proteins (Figure 12) and into myelin proteins (Figure 11) of

HyPhe animals did not vary from control animal~ during the 20 days of this study, suggest!rig no ch~nges in the rate of synthesis of myelin proteins or·of tqtal brain proteins when. animals were subjected to a diet supplemented with 5% phenylalanine + .4% aMP. The long term turnover of label in the HyPhe group was strikingly different from that of the WMC or aMP group (Figure 9). Maximum incorporation irito rnyeliri proteins occurred at 5 days on the diet (age 30 days) as shown in Figure 9. A half-life (t ) of 36 days was observed for the uncorrected 112 fast compon:ent of whole myelin in the WMC group while the half life of whole myelin was reduced to 3 days in the HyPhe group (Figure 9). A very rapid turnover of part of the myelin proteins is evident w~ich is independent of nutritional deprivation (l;-'1MC), or the presence of .4% aMP added to the diet.

Although a slig·htly increased turnover is evident in the ar-1P group (Figure 9), it is not comparable to the H.yPhe group. The turnover of myelin proteins appears to be rather specific as the turnb\"e·r of label in TCA precipitable ,.,hole brain proteins of the HyPhe group did not vary from WMC animals (Figure 10) . From Figure 9 and Figure 10 it can be concluded that the dramatically increased rate of turnover in hyperphenylalaninemia is specific for the myelin proteins, which was not observed in the turnover of whole brain proteins. 74

The n~xt question ~as the specificity of the increased turnover, that is, was it.due to an increased turnover of newly

· synthesized myelin or to an increased turnovet of mature my~lin.

Matthieu et al. (1973) hav~ proposed that myelin can be subfractionated into a heavy (immature myelin), medium, and light (-most mature myelin) fractions. It was found that the increased turnover was generalized for all three subfractions of·

myelin and the decre~sed t . of 3 days, which was evident 112 with whole myelin, was also sho\o~n in all subfractions (Table VII). Thus, a generalized demyelination appears to occur .which is not dependent on the maturity of the myelin proteins. An increased "turnover of myelin protein could lead to alteration in the myelin sheath. Gross abnormalities in the myelin sheath were indeed observed after rats were maintained on various hyperphenylalaninemia inducing diets from 25 days of age and sacrificed at age 45. Control animals showed densely packed myelin sheaths with typical concentric lamellae (Figure 13) with easily distinguishable major dense line and iritraperiod line· (Figure 13A) . The myelin sheaths of HyPhe animals are grossly disfigured with axons undergoing· demyelination (Figure ·14). The axons 6haracteristically show disruption of the myelin and splayirig of the lamellae. This disruption of the myelin architecture does not enable one to differentiate the major dense line and intraperiod line in the affected area (Figure 14A) . There was no evidence of infiltration of phagocytic cells causing this des·truction of myelin. The extent of demyelination 75

TABLE VII

HALF LIVES (IN DAYS) OF VARIOUS FRACTIONS FROM BRAIN

AFTER 3H-LYSINE INJECTION INTO RATS OF 25·DAYS OF AGEa

b. Fraction WMC .4% a.MP 5% phe + .4% ctMP

c whole myelin 36 26 3 light myelinC· 67 4 mediummyelinc 43 3 heavy myeline 33 3 brain proteins· 17 25 a) Animals injected with (llJCi/g) 3H-lysine and placed on respective diets at 25 days. For explanation of diets see materials and methods. b) Whole myelin was taken from sucrose gradient by method of (Norton and Poduslo, 1973) and subfractionated into light medium,. and heavy myelin (Matt.ieu, 1973). Brain proteins isolated from brain homogenate prior to isolation of myelin. c} Uncorrected fast components (Sabri et al., 1974). Figure 13: Myelinated axons of a 45 day old rat on a normal diet. Sec­ tion of white·matter was taken from lower midbrain at the pontine junction. (x 28,000)

Figure 13A (inset): Myelin sheath of control rat showing easily distin­ guishable major dense l:f_neand intraperiod line. (xl65,000). 76

FIGURE 13 Figure 14: Myelinated axons of . 45 . day. old.· rat fed normal laboratory chow supplemented with·S%.pbe·+ .4%·aMP diet for the previous 20 days. ·Section ·of: wh:li~ .matter. from lower midhrain .at the pontine ju1:1ction~ (x:28.t000) ·.

Figure 14A (inset): $playing. of· lamellae observed with HyPhe diet.

FIGURE 14 78

in the HyPhe condition is illustrated in a longitutional section of an.axon (Figure 15). Increasing the phenylalanine content

from 3% to 5% and even to 7.5% resulted in extensive damage to myelin sheaths (Table VIII) • The presence of abnormal myelin sheaths was similar in all HyPhe gro~ps and abnormal myelin composed approximately 63% of the total (Table VIII) • Figure 15: Longitut:ional (:Jection o~ ·a m.yelinated axon fr.om a rat fed a normal diet supplemented with 3% phenylala~ine + .4% aMP for the. preceedin:g 20 .days. _· Section of white matter taken from lower midbrain at the pontine junction. (x6 ,500). ::. 79 80

·TABLE VIII EFFECT OF HYPERPHENYLALANINEMIA ON

MYELIN MORPHOLOGYa

Myelin Morphology .(%) c Diet Group.b Normal Abnormal

Control 97.6 2.4

3% phe + .4% aMP 37.5 62.5

5% phe + .4% aMP 33.8 66.2

7.5% phe + .4% aMP 40.0 60.0 a) Animals were ·placed on each diet at 25 days of age and sacrificed at 45 days. Approximately 300 axons were photographed in each diet . group •. b) For explanation of dietary treatment see mat~rials and methods. c) Each myelina:ted axon was classified as abnormal or normal (se~-materials and methods for details) and results expressed as percent of total. Number of abnormal ·axons in control and experimental g;toups were compared using analysis of covariance. The HyPhe groups are signifi- . ·- cantly· different from control (p < .005). DISCUSSION

C·

Myelin Metabolism:

This study was undertaken to investigate the

neuropathologieal alterations which occur in PKU, particularly

during later stages of brain development. Myelination is the .

most impor.tan.t proce.ss in postnatal maturation and during this

period, the growing infant passes through a "vulnerable period",

(Dobbing 1974) in which irreversible brain damage can occur if

the infant is subjected to a potential neurotoxin. There is

considerable evidence that PKU or hyp~rphenylalaninernia can

contribute just such a neurotoxic envir6n~ent to myelin

metabolism. In humans~ myelin accumulates most rapidly until 4

years of age (Dobbing 1974); however, myelin still continues to

accumulate into the teens (Dodge et al. 1975). It is of

interest to understand the implications of this vulnerable

period with respect to PKU ·and myelination.

An altered myelin morphology and marked lack of myelin has

been observed in untreated PKU patients (Alvord et al. 1950)

with the presence of demyelinating lesions accompanied by

sudanophillic breakdown products. Similar abnormal findings

have been observed by Avins et al. (1975) fo·r a hyperphenyl­

alaninemic condition instituted in a rat animal model during the

peak in the velocity of myelination at 5-20 days. One can

conclude that hyperphenylalaninemia instituted during this

81 82 neonatal period of rapid myelin development can lead to abnormal myelin sheath formation. The present study shows that at a relatively mature age, the myelin sheaths are still very susceptible to the influence of hyperphenylalaninemia. · When rats are subjected to the 5% phenylalanine + .4% aMP diet from ages 25-45 days, a massive deterioratio.n of the myelin sheaths was observed (Figure 14 and 15) , suggesting a profound effect of hyperphenylalaninemia on myelin beyond the most rapid phase in myelination which occurs at 20 days in the rat (Norton 1973b) a These effects were observed in·· six animals on a HyPhe diet while the two control animals showed no abnormalities in the doncentric wrappings of the myelin lamellae around the axon (Table VIII) • The question arises as to the source of this abnormal myelin morphology, ie. hypomyelination, dysrnyelination or demyelination. The amount of myelin protein was decre.ased in the HyPhe group throughout th" 70 days of the study (Figure 8). This loss of protein could be caused by a decreased synthesis of myelin protein, an increased degradation or a combination of both mechanisms. Although no decrease in the rate of synthesis of myelin proteins was observed in this phenylalanine + aMP model, a slight decrease was observed in the HyPhe group in a phenylalanine + PCPA model (Hommes et al. l982a) and thes·e effects may be attributed to the effects of the various enzyme inhibitors used in the two studies. ~rundt and Hole (1974) did observe a decrease in protein synthesis in brain proteins and in myelin proteins after PCPA injections into neonatal rats. Even 83

thoug~ decreased levels of the branched 6hain amino acids were present in brain of the HyPhe rats, no differences were noted in the rate of synthesis into the whole brain proteins or myelin proteins. From this evidence, it is unlikely that this massive deterioration of the myelin sheath can be attributed solely to decreased myelin synthesis or to hypomyelination.

It is more likely that the disruption of the normal myelin morphology is caused by a destabilization of the myelin membrane as a result of the abnormal environment created by chronic hyperphenylalaninemia. The loss bf myeliri protein can be explained-by an increased turnover of myelin (Figute 9) with dramatically decreased half-lives (Table ·vii) for the uncorrected

fast component of myelin (Sabri et al. 1974). The data for the

turnover of whole myelin ·for WMC rats are in agreement with

those reported by Sabri et al. (1974) but differ from the

reports of Singh and Jungalw.ala ( 1979) • Differences in the age of animals, route of injection and isotope used could explain

the differences. In the study by Sabri et al. (1974) the same

isotope, route of injection, and r.ats of about the same ages were used. Similar observations_of increased myelin protein

turnover in a phenylalanine + PCPA model have been observed

(Berger et al. 1980, Hommes et al. 1982a). Therefore, the

increased myelin turnover in the presence of hyperphenyl­

alaninemia can not be attributed to the type of inhibitor used,

since it was observed in both a phenylalanine + PCPA and also in

a phenylalanine + aMP model system. PCPA alone (Hommes et al. 84

.l982a) or a~·1P alone had no eff"ect ·on myel in protein turnover .. This increased turnover appears to be specific for the myelin membrane as turnover of label in the whole brain proteins was not different in the HyPhe group versus the WMC group. A source of this increased turnover may be the instability of tha myelin membrane in the presence of excess phenylalanine. Modification of tubulin by replacement of the carboxyl terminal tyrosine by phenylalanine has been observed (Rodriguez and Boisy

1979) and a similar modification by phenylalanine on the myelin proteins may occur resulting in unstable myelin leading to dysmyelination.

The HyPhe treatment can also h~ve a profound effect on myelin specific lipids such as the sulfatides (Chase and O'Brian

1970, Sprinkle and Rennert 1976, Shah and Johnson 1978, John.so.n

and Shah 198D) and galactolipids (Creme ~t al. 1962b, Prensky

et al. 1974) as well as proteolipids (Prensky et al. 1971). Other abnormalities, such as a defect in the elongation of fatty

acids (Johnson and Shah 1973) , reduced concentrations of unsaturated fatty acids in phosphatidyl choline and phosphatidyl

ethanolamine (Shah and Johnson 1978) or a reduction in

cholesterol synthesis (Shah et al. 1972a) which may also contribute to the instability of the myelin membrane .,,.,as suggested. The interaction of lipid and protein may well contribute to the stability of the myelin membrane. It has been hypothesized that sulfatides and other acidic lipids can protect myelfn basic proteins from enzymatic hydrolysis (London et al. 85

1973a,· 1973b) and the abnormal.ities in sulfatide synthesis may offer a plausible explanation .for the decreased stability. This· abnormal lipid metabolism ~ay lead to unstable myelin or i~creased protein turnover may lead to abnormal incorporation and assembly of lipid into myelin. Either mechanism could explain an increased protein turnover.

Effects of Phenylalanine Metabolites:

Deficiencies in myelin and myelin synthesis were seen in neonatal rats in both a phenylacetate model and in a PCPA + phenylalanine ~odel (too et al. 1978). This observation prompted the suggestion. that phenylacetate may be the neurotoxin

~ssociated with an abnormal myelin metabolism~ To investigate this theory, mass chromatograms were run to determine minute levels of phenylalanine metabolites in brain. Phenylacetate, o-OH phenyl-acetate, phenylpyruvate or phenyllactate could not be detected in brain and therefore are ptob~bly not associated with the loss of myelin and increased myelin turnover of the HyPhe group at this later stage of brain development. These conflicts with the studies of Loa et al. (1978) may be related to the state of maturation of the blood brain barrier, as phenyllactate could be detected in brains of 18 day old and younger rats but not in brains of 25, 40, and 75 day old rats (Goldstein 1961). This evidence would indicate that the development of the blood brain barrier does affect the levels of the phenylalanine 86

metabolites in brain~ 1 4 . - C-labeled phenylpyruvate injected into the carotid artery of an adult animal was not taken up into brain (Conn and Steele

1982). Similar results were obtained in this study: PLA and.PPA

~ere detected -in plasma but not in brain, thus eliminating the metabolites of phenylalanine as contributors to the increased myelin protein turnover. Thi~ evidence d0€5 not preclude the metabolit~s of phenylalanine from being involved in the mental retardatiori associated with PKU at earlier stages of brain development.

Neurotransmitter synth~sis:

The influence of aMP and the aMP + phenylalanine treatment on neurotransmitter synthesis was also investigated. Reductions of norepinephrine. and epinephrine '.vere observed in plasma

(Nadler and Hsia 1961) and the reductions of dopamine and

norepinephrine were accompanied by decreased amounts of tyrosine

in autopsied human brain of PKU patients (:r-1cKean 1972). One major difference in this experimental model and the human PKU patient is the tyrosine value. The lack of substrate could account for decreased catecholamine synthesis and this effect would not be observed in an animal model due to the presence of elevated tyrosine, after feeding a diet of excess phenylalanine. The differences noted in the catecholamine determinations from whole brains do not preclude regional variations in certain 87

areas of brain, since McK~an (1972) observed severely decreased

I. levels of dopamine in the dopamine-rich area of the caudate

nucleus with no change evident in brainstem.

Udenfriend et a1. (1965) demonstrated inhibition of

tyrosine hydroxylase by a-methylphenylalanine in vitro and

Torchiana et al. (1970) demonstrated inhibiton in vivo.

The .4% aMP group also showed substantially reduced levels of

dopa in brain suggesting inhibition of tyrosine hydroxylase as

well as phenylalanine hydroxylase. These reductions in dopa

were observed even in the presence of increased tyrosine in the

phenylalanine + aMP group, possibly due to in increased

competition by the excess phenylalanine. Phenylalanine is

present in the HyPhe group at 30 times the value of the control

group: phenylalanine is 40 times less potent than ~MP as an

inhibitor of tyrosine hydroxylase (Udenfriend et al. 1965).

The presence of a less potent inhibitor (phenylalanine) at 30

times the control value could act to depress the dopa values

even in the presence of elevated tyrosine, so that no difference

was noted in the aMP group versus the phenylalanine + al\1P group.

Although dopa was decreased in the aMP group~ dopamine did net

vary amoung the· 3 diet groups, confirming results of L.ane et al.

(1980) in a phenylalanine + aMP model system.

A 50% decrease in norepinephrine was observed in the aMP

group which would indicate aMP also has an inhibitory effect on

dopamine B-hydroxylase. It is interesting that the ~ddition of

excess phenylalanine increased the norepinephrine concentration 88

between the aMP group and the control group and this effect may ·account for the fact that no differences were ·observ·ed by Lane

et al. (1980) with controls and phenylalartine + aMP tr~atment. aMP also had a profound influence on epi.n·ephrine production,

possibly by interference with phenylethanolamine N-methyl­ transferase by phenylalanine and aMP since the presence of excess phenylalanine depressed the levels of epinephrine

beyond those observed with aMP alone. This evidertc~ of

decreased synthesis of th~ catecholamines in the presence of 5%

phenylalanine + .4% aMP should be extrapolated to the PKU syndrome with great care, be,cause the decreased l-evels of dopa,

norepinephrine, and epinephrine weie also observed in the .4% aMP treated group thus attributing these deficits to the

presence of the enzym~ inhibitor and not to an increased phenylalanine. In PKU patients, a reduced amount of 5HIAA excretion (Armstrong and Robinson 1954) , and decreased blood levels of serotonin (Pare et al. 1957) were observed. Reduced amounts of serotonin in brains tern, occipital cortex, and caudate nucl-eus have been observed (McKean 1972) " These effects of decreased serotonin and SHIAA are also· seen in brains of w.eanling rats fed

a hig~pheny1alanine diet (Green et al. 1962, Yuwiler and Louttit 1961, MdKean 1967)·. The decrease in serbtonin observed in rats treated from. birth in a PCPA model was due to ·the presence of PCPA alone, i.e. in the absence of excess phenylalanine (Wapnir et al. _1970, Hole 1972a, Berry et al. 89

1975) because of the inhibition of tryptophan hydroxylase by PCPA (Koe and Weisman 1966) • This side effect was eliminated by · using aMP, since no difference irt serotonin or 5HIAA was observed in the aL~P group ver·sus controls. The mechanism ·of r~duction of serotonin in the phenylalanine +·aMP group is probably due to the inhibition of tryptophan hydrdxylase by phenylalanine rather than a lack of substrate, as tryptophan in brain did not vary betweeri th~ 3 diet groups. This situation is riot analogous to the human condition in which tryptophan values from the brain of a PKU patient were decreased to 1/2 the levels in controls with corresponding decreases in serotonin (McKean 1972)

The Km values of 3 2 uM for tryptophan and 287 u~1 for phenylalanine for tryptophan hydroxylase indicate that phenylalanine can act as a competing sub~trat~ fbr tryptophan hydro~ylase (Tong and Kaufman 1975) • The phebylalanine concentration of 1. 26 umole/g or approximately 1260 ur~ was sufficiently above th~ Km of 287 uM to result in inhibition of tryptophan hydro~ylase, by phenylalanine. This hypothesis confirms results of Yuwiler et al. (1965) in which tryptophan hydroxylase activity was shown to decrease 75%· in artimals fed a 5% phenylalanine diet. ·The possibility of increased degradation of serotonin has been ruled out since monoamine oxidase (t-1AO) activity was unaffected (Yuwiler and Geller 1969) • A similar decrease in serotonin was observed in weanling rats treated from birth in a phenylalanine + aMP model {Lane 1980) , and the control group of aMP in this study verifies. the results being 90 due to excess phenylalanine and not due to the presence of the inhibitor.; The decreases in serotonin observed in this study are relevant to the PKU patient, since aMP had no effect on serotonin synthesis and the results of decreased serotonin can be attributed to phenylalanine alone. Also, the absence of the phenyl~lanine metabolites in brain makes it clear that th~ serotonin depletion is due to the presence of phenylalanine itself competing with the tryptophan hydroxylase. Although no decrease in tryptophan was observed in this study, the possibility that tryptop~an supplementation can increase brain levels of serotonin is a plausible solution (Fernstrom and Wurtman 1971) and behavioral testing of such supplementation ~.v·i th untr.eated PKU pat·ients is being performed (Roesel et al. 1982). There is evidence for GABA and glycine functioning in brain as putative neurotransmitters (Roberts and Hammerschlag· 1976). GABA was not significantly different among the three diet groups. The implication involving GABA in PKU is from in vitro inhibition of glutamat~ decarboxylase by means of phenylacetate

(Tashian 1961) • The .absence of pheriylacetate in brain is supportive of this hypothesis. Glutamate decarboxylase is not inhibited by phenylalanine itself as .GABA.values did not differ among the 3 diet groups even in the presence of a hyperphenylalaninemia.

Glycine in brain of the HyPhe ~as double the values in - 91 control brain and this change was the greatest of any amino acid other than phenylalanine or tyrosine. This increase in intracerebral glycine has been described previously in neonatal rats (Dienel 1981) and similar increases are observed here for rats of 35 days of age~ The increased intracerebral. glycine occurs without increased plasma glyciri~ and also serine, the metabolic precursor to glycine, was not elevated in- either plasma or brain. An -iricrease in phospho~erine phosphat~se actiVity was observed in hyperphenylalaninemia (Delvalle et al.

1978): however, this increase could not account for ·the total increase in intracerebral glycine (Dienel 1981) • The mechanism of gly-cine increase remains to be elucidated; ho~"leve.r, it does function as a neurotransmitter and it is associated with mental retardati6ri in non~ketotic hyperglycinemia (Nyhan 1972) •

The evidence presented in this dissertation suggests a profound influence on myelin metabolism, serotonin synthesis and intracerebral levels o-f the branched chain amino acids and also abnormal intracerebral glycine metabolism in experimental hyperphenylalaninemia during later stages of brain development.

These effects are apparently hot due to the intracerebral-values of metabolites of phe·nylalanine, or the presence of the phenylalanine hydroxylase inhibitor cz~methylphenylalanine, but can be attributed to phenylalanin~ itself.

This stage of brain development in the rat model is similar to that of a PKU child (5-8- year old) , :at ~J'lhich time the low phenylalanine dietary therapy is relax~d or terminated. To ! 92

extrapolate these results to the human cbndition would strongly

suggest continuation of dietary therapy, although an age at

which excess phenylalan-ine no longer interferes with myelin·

metabolism has y~t to be established. To Bickel (1980) it seems

logical to relax or terminate the diet around the age of 12 to

15; however, continuation for so long· into the ·school a~e may

create problems. A compromise at the ag~ of 10 is suggested

while on a relaxed but still protein restricted diet (Bickel

19 80) .

Tryptophan suppl~mentation_to increase serotonin synthesis

. (Roesel et al. 1982) and also supplementation with the branched

chain amino acids is being applied successfully (Berry et al.

1977). These new approaches togethe~ with dietary phenylalanine • therapy may prov~de·an e£fective means of tieatme~t to overcome

many of the neurochemical abnormalities observed in this study.

The nature of the mental retardat{on remains unresolved;·

however, the evidence presented here may contribute to the

elucidation of some underlying ne·urochemical abnormalities

associated with PKU. SUMMARY Hyelination is the most important process in postnatal ma_!:uration of th~ nervous system and during this period the growing infant passes through a "vulnerable period" in which irreversible brain damage cah occur if the neonate is subjected to a potential neurotoxin. This study was undertaken to investigate the rnechani srns by 'ftlhich chronic hyperphenylalaninemia interferes with myelin metabolism, beyond the neonatal period of ·rapid myelination, at a time when myelin continues to accumulate. Rats of 25 days of age were placed on a hyperphenylalaninemia

(HyPhe) iridu~ing diet of 5% phenyla~anine plus .4% a-methylphenylalanine (aMP) at 25 days of age to approximate

plasma· phenylalanine levels of a human PKU patient ( 1.5 mM). Elec.tron microscopic observations of myelin sheaths from

animals after 20 days on the HyPhe diet were characterized by an

incomplete or disrupted myelin and spla~ing of the lamelLae, whereas axons from control animals exhibited normal concentric wrappings of myelin. The HyPhe group exhibited a lower brain

weight and approximately a 15% decrease in the amount of myelin protein throughout the 70 days of the study. The rate of 3 incorporation of H-lys ine into both TCA precipitable "''hole brain proteins or myelin proteins did not vary from the HyPhe

group and a ~'Ieight matched control group noJr.t!C) • Therefore,. this loss of myelin cannot be attributed to a hypomyelination. The

turnover of whole brain proteins also was unaffected by the HyPhe treatment; ho't1ever, the turnover of myelin proteins in the HyPhe group \'laS dramatically different (t =3 aays) from that 112

93 94 of·the WMC group (t =36 d~ys) or a group treated with only 112 aMP (t =26 days). The increased turnover could not be 112 attributed to the presence of the phenylalariine metabolites:. phenyl aceta-te, o-OH-phenylacetate·, phenylpyruvate or p~enyllactate since these compounds could not be detected in brain using mass fragmentography. Therefore, phenylalanine must cause decreased stability of the myelin membrane, resulting in

increased ~urnov~r. The effects of· hyperphenylalaninemia on catecholamine and serotonin synthesis were also investigated. A group treated with aMP alone, as well as the HyPhe group, showed decreases in dopa, norepinephrine, and epinephrine and these decreases could be attributed to the presence of the inhibitor, aMP, rather than the HyPhe condition. No differences in dopamine were· observed.

Serotonih and 5-hydroxyindoleacetic acid (5HIAA) were d~creased 50% in the HyPhe condition and were unaffected in the presence of aMP alone. The concentrations of tryptophan in the acid soluble pool of brain did not vary from the HyPhe and control groups, thus the decrease· in serotonin synthesis is due to inhibition of tryptophan hydroxylase by phenylalanine, rather than decreased availability o£ substrate. Increased intracerebral glycine as well as decreased amounts of the branched chain amino acids and methionine were also observed. These abnormalities in myelin metabolism, serotonin

synthesis and increas~d intracerebral glycine at later stages

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