The Role of Acetone in the Anticonvulsant Actions of the Ketogenic Diet in Rats

Kirk Jon Nylen

This thesis is submitted in conformity with the requirements for the degree of

Master of Science in Pharmacology

Graduate Department of Pharmacology

University of Toronto

© Copyright by Kirk Nylen (2004)

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ABSTRACT

The Role of Acetone in the Anticonvulsant Actions of the Ketogenic Diet in Rats

Kirk Nylen, M.Sc. (2004)

Department of Pharmacology, University of Toronto

The ketogenic diet (KD) is used to treat drug-resistant seizures. The goal of the

present studies was to understand the KD’s mechanism of action. Experiments 1 and 2

compared the ability of 2 KD’s to elevate seizure threshold in adult rats and rat pups. It

was concluded that the KD does not have anticonvulsant actions in rats. We then

hypothesized that acetone is the anticonvulsant mechanism of the KD, and that rats on the

KD do not develop therapeutic levels of acetone. Experiment 3 determined that seizures

themselves decrease in vivo acetone levels, therefore, measuring acetone post-seizure

should be performed cautiously. Experiment 4 demonstrated that acetone is not elevated

in KD-fed rats, but it is elevated in KD-fed rats when acetone’s metabolism was

inhibited. Finally, it was determined that acetone suppressed focal cortical and

generalized convulsive seizures in the kindling model, but not amygdala focal seizures.

ii

ACKNOWLEDGEMENTS

It has been an honour working in the Burnham laboratory for these past two years.

Patty, Deborah, Claudia, Sesath, Brian, Elan, Mani and Sofia: Our conversations have left

me richer. A special thanks to Jerome — in the face of a lab full of pillagers, he always

kept things bountiful. Thank you to my “ketogroup” colleagues (Peter, Sergei, and

Jasper). You were integral in helping me complete this important work. A special thanks

to Sergei for his brilliant and critical insight to this extremely interesting, challenging

field.

A special “thank you” to my advisor, Dr. Woodland, for asking the important

questions and giving great advice.

Perhaps most of all, I would like to thank my wife for her continual support of me

and my “mad science”. “Thank you” to my parents, siblings, and friends for allowing me

to determine whether that interocetor could withstand a sudden charge of 20 000 volts.

I would like to thank my Supervisor and mentor, Dr. Burnham. I will be forever

mystified by the amount of knowledge you command and the ease and finesse with which

you convey it. Thank you for being an incredible support, both academically and

personally.

Finally, “thank you” to rats that have made the lives of epilepsy patients much

richer and more hopeful. They are truly amazing creatures.

My research was funded, in part, by the Margaret and Howard Gamble

Scholarship, OSOTF.

ili

TABLE OF CONTENTS

ABSTRACT... ...ccccccscscscscccccvsnsececessescccccnccsvacrovcescsseooveseoncverecousessenopovssscoovusesrscccesseccccssccesesazes il

ACKNOWLEDGEMENTS ....ccccssocscscssscsscancsssessccnecsccccssnsecsrcsssovsccescescersccscosecccoccevscasceese ili

TABLE OF CONTENTS ....ccccccsesencccccsccescsocccsccccccccccccesaccccsscccatecsceccesensccccccecscccccescesocecane iv

LIST OF TABLES ........cccccescssecsccccssccsensccccccsssrcenssacvoveessvonestevocosescosccessseuceussrecccessovccscovones ix

LIST OF FIGURA|G ......cccccoscssccssssccesscccccsceissccevevcuacnvaseccassatenscussceccssesoceesescsavensescccccasecoessss x

ABBREVIATIONS... sees sovcceaccssorccesccsvcnsescusvasesooesanescsvessocpecsssccccesssceasssece xii

LIST OF CHEMICALS AND DRUGS .........cccessccccrccscccccsccsccccrscscccscccccensece Xiv

CHAPTER 1: GENERAL INTRODUCT ION......ccuccosscsssccnscaccvaccsssssoscsccscccccccsescacccsncees 1

INTRODUCTION TO EPILEPSY. ..ssccsccsssssssscscssscesssesceveecscssscesvscessccescnccesassansnssecusesersacosacsssneessecs I

WHAT IS EPILEPSY? 1

DISTINGUISHING BETWEEN “EPILEPSY” AND “SEIZURE” 1

THE ECONOMIC COST OF EPILEPSY 1

THE HUMAN COST OF EPILEPSY 2

INCIDENCE AND PREVALENCE OF EPILEPSY 2

WHO GETS EPILEPSY? 2

CAUSES OF EPILEPSY visccccsccssssssssvsscosscecscssosccsssscscnsssccccccssseccsccesscccescencutccoctacsnecesescescaceseasaccncse 2

SYMPTOMATIC EPILEPSY 2

IDIOPATHIC EPILEPSY 3

SEIZURE TYPES uscccscccccssscsccvscscccsececseccccseccosesnccssssvacceccuvaccessscneceqasaccenaceseessavensoscuenesssvoaveesecsacnens 3

PARTIAL SEIZURES 3

GENERALIZED SEIZURES 4

ANIMAL SEIZURE PREPARATIONS wsssossssessscccvsssccneccesecscessroccsscccssnccasszccessensrscsosvsceassesnctessaceeees 5

GENERALIZED SEIZURE PREPARATIONS 5

PARTIAL SEIZURE PREPARATIONS 7

PHARMACOLOGICAL TREATMENT OF EPILEPSY ...sccccscvccsrscccsrsencssecsnsecssnscsnscesaccsosnessecenss 8

BRIEF HISTORY 8

ANTIEPILEPTIC DRUGS: MECHANISMS OF ACTION 9

NON-PHARMACOLOGICAL TREATMENTS FOR EPILEPSY ........sssscsrsocssssonsccsesersessssessseces IO

NEUROSURGERY 11

iv

DEEP BRAIN STIMULATION 11

VAGAL NERVE STIMULATION 12

THE KETOGENIC DIET 12

THE KETOGENITC DIET ....cccccssccvsesscscesnsscceecsactsnssansesessocosressseetsnsecssenensssssestsscssnscenacoevecessaasecoees 13

EVOLUTION OF THE KD 13

CLINICAL PROFILE OF THE KD 14

TYPES OF KD 16

SIDE-EFFECTS OF THE KD Suen tg 16

SUMMARY OF SIDE EFFECTS AND THE KD 18

KD: POTENTIAL MECHANISMS OF ACTION. sccssssccsssssstssssccesenssscessoscsssesessessscesssescessseccens I9

THE BRAIN LIPIDS HYPOTHESIS 19

THE ACIDOSIS HYPOTHESIS 19

THE ENERGY SUBSTRATE HYPOTHESIS 20

THE GABA SHUNT HYPOTHESIS 21

THE KETONEMIA HYPOTHESIS 22

THE ACETONE HYPOTHESIS 23

RESEARCH GOALS & HYPOTHESES. .....s.sccsscssssscsssssssssessssersscsssssssoeessesscsssnoessnesenssescnsnansoess 24

CHAPTER 2: GENERAL METHODS oc ccesecceeesresscccoseesneccecnscesnccessnescnsersecconsee 27

SUBIECT S venseccccesvsnreccessnssrcecsssceccnscneccnsnaccesessonseseusavencnsnecaensnedasessscesesteteessssecsessecensneaesasesessenseceress 27

DIETS. ..ccssenversscccnsrstecnscasecsscccssascesssscsonsssscscesuconsavoneacesssossaesessaneesssusesesscuesssssaeceseusenssonseseaseocarosnoeses 27

THE 4:1 KETOGENIC DIET 27

THE BALANCED CONTROL DIET 28

THE 6.3:1 KETOGENIC DIET 28

THE STANDARD RAT CHOW CONTROL DIET 29

ADMINISTRATION OF DIETS wiscccscssssssscssscsccssesonsnsesscnscseccossseronsesscecaneussessssccsnseasonsceuseeerseceenes 30

DETERMINATION OF GLUCOSE AND B-GHB wiscsssscsssscccensesnsessnncesssseccssnsnencscenoscaesentaatessauess 30

DETERMINATION OF ACETONE .0...ssscccssssseosssscssccesesscncevsensescnsncsssssnccovscerscsseasecseussssevessarseeses 3l

SEIZURE TESTS ...sseccsssecsenssssscsrncscnnsensnassessssessascescacssaceoesbeceoscesnsassanessuoeessssessasevscesosnseeseasevocseasees 32

FITS TOL OGY ..ecesccccsssvsssnrornsscersssccssnsencconncccssssescsccconsccncserassocseeescnssossessesssosansssenesovcsusesseserasesessoesces 35

DATA ANALYSIS we.serssessssrsrcscssesssesensccescacsnsscssnocevsacesssossaccssssoctseossvennacanavesoussossesoatscveuseseseeseseeaseoss 36

CHAPTER 3: EXPERIMENT 1 ........cccccssesesccconessesconcssoscscoesccccesccccescsenssesesssccessenescnses 37

RATIONALE. ..cccccsessscccnssccsnascceranccenanctasssseecsceesonssesssssncessoasssssssanseusensucssunaesvsnnessusseussessvaneasesssssaeses 37

METHODS. ....ccessossssecrrssssnrecnsnssssnasscnssccecsssscsssessoascesssssansssevensassesensesansscssosscesssecenseasooseanescoosssasenee 38

SUBJECTS AND GROUPING 38

DIETS 39

PTZI SEIZURE TEST 39

GLUCOSE AND BETA-HYDROXYBUTYRATE MEASUREMENTS 39

STATISTICS 39

RESULTS ...cccccossccscssvsrssssncssessacsceacsscensssscncessessacsocenssssesoossesssccnsesscccscassoseueravouscsecsneusessoosasensensaussees 40

WEIGHTS 40

BETA-HYDROXYBUTYRATE LEVELS 40

GLUCOSE LEVELS 41

PTZ THRESHOLD DOSE 41

CHAPTER 4: EXPERIMENT 2 .......ccccccccsssccssscccnscreccsesscccsnctoncosnscsscessscessscseacsecnscersees 49

RATIONALE vicscsescecsscsssscsssncessccessscanasesscessesonsnsvecesesseseseosessooneessoneesssnesosoeussutessscecennesesserasneseauenses ss 49

MEE THODSS vrccccesevececerecsesscesnnersncsrancsnstescesoacesssseeersessonacesnscccssseuessscurensecsoasevesesseusensonsseseseesnseseneseess 49

SUBJECTS AND GROUPING 49

DIETS 50

PTZI SEIZURE TEST 50

GLUCOSE AND BETA-HYDROXYBUTYRATE MEASUREMENTS 50

STATISTICS 51

RESULTS Vo .cccscecesssscccovsesecsevecssssnnccccssosrsnsaccssousesessosacccceseecusassussuscussssoesasscessncseseessasecscussonsensnessaeseaes 51

WEIGHTS 51

BETA-HYDROXYBUTYRATE LEVELS 51

GLUCOSE LEVELS 52

PTZ THRESHOLD DOSE 52

DISCUSSION ssssscecssssecserenscsssrsccsssncsssscccssonnctancsssccsaceacassensecscussanaesenocsssaascssaccceseesarscosseueraceuseeeess 60

CHAPTER 5: EXPERIMENT 3 .......ccsssccccscscessssecssesscscsccsnsccssessanssenecsssenecesceeessacceesseaes 64

RATIONALE ....cccccssessvsennsesssnsnsccerscencssstcevsacorssssaseuacessosssceuecensooausseeeevnoparessssnageesnssneceeaserarscesssseeeaee 64

vi

METHODS, ..snccsersersesssvnecsecscersevcnconscscsconscesenseesensessasvansessscanecsdosesaceseseacoeesenseasensseesecssoutocssseasorsessees 65

SUBJECTS AND GROUPING 65

ACETONE INJECTIONS 66

MES TESTING 66

SCPTZ SEIZURE TEST 66

MEASURING ACETONE IN BLOOD 66

STATISTICS 67

RESULTS... seccessesscsssccsssenocssenssenseseonsassononensscsseassasesencesssseessssesenssesencueesasareessssesseseneansessepensaseesases 67

MES GROUP 67

SCPTZ GROUP 67

DISCUSSION. ..ccssssscesssssssscesssccsnsessceconesnenenessessacsnssseenssssssussssencsossessessseeasesasasosonscesnenssenesessneeessaes 71

CHAPTER 6: EXPERIMENT 4 ......ccssssssssccessccssssvessssserecseesresssesessesnsesesensnsessresscaseonses 72

METHODS. ..1.scsssssssssoorssscsncssecsecsscsnsssssonsosescsesscasssenesssssscscossacesscnneasensnscacsncosencensaceneatsatsecseseesesscues 73

SUBJECTS AND GROUPING 73

DIETS 73

DIALLYL SULFIDE ADMINISTRATION 73

BLOOD SAMPLING 73

GLUCOSE AND B-OHB MEASUREMENTS 74

ACETONE MEASUREMENTS 74

STATISTICS 74

RESULTS. cecsneccsssssscccsencssccssessncecscsdcceseseccdoccnacaseessgceecessensssessneseeansesersueeessscenaneaecsenasensesssnanesensensess 76

WEIGHTS 76

B-OHB LEVELS 76

GLUCOSE LEVELS 77

ACETONE LEVELS 77

DISCUSSION... .s-ssscsesssccssssssssestecssveasscsssscessessssasessssscasonsonssvesssesssedsessnssansessesooscssesbvatartsessseeensones 83

CHAPTER 7: EXPERIMENT 5 .........:scccssscsesoscsssscsecssossscersnscacesescsscsesrssessossesscsonecsssors 86

RATIONALE... .sssessscscssercesvccsesonsonccsccsseccionsecacossesecscesssnoocccsosccacoussseseesesscousoesessovceaceacoasssessossooseenaeee 86

METHODS. ...1ssscssssesssecccescvssvecccscoovensssnsbeccogeessassecssncuescsasecscedscsercessossensececeseeeseeessorssascosccoucnseseesnes 87

SUBJECTS AND SURGERY 87

Vil

ACETONE DOSES AND INJECTIONS 88

SCORING OF ATAXIA 88

KINDLING 87

RESULTS ..sceccccccccrscsecssensesransccncenccconscscsecessansccacnececcsdaasosasseessrseenessensseaseaeeeesassacseonsenesessssesocesonsnsoes 89

AMYGDALA GENERALIZED AND FOCAL SEIZURES 89

CORTICAL GENERALIZED AND FOCAL SEIZURES 89

TOXICITY AND THERAPEUTIC INDEX 90

HISTOLOGY 89

DISCUSSUONcccsssossssssvsssssssssesssovsssccssssnssessasesessnessssssnnsssenssssvsssssseunasssennaseesssnssunessessensnnese 93

CHAPTER 8: GENERAL DISCUSSION ...... sossssarscessenssscoonscescssscscarssonensecessnssssscacseoees OD

EXPERIMENT |. A COMPARISON OF TWO KDsS IN ADULT RATS 94

EXPERIMENT 2: A COMPARISON OF TWO KDS IN RAT PUPS 96

EXPERIMENT 3. THE EFFECTS OF SEIZURES ON ACETONE LEVELS IN VIVO 99

EXPERIMENT 4. LEVELS OF B-OHB, GLUCOSE AND ACETONE IN RATS ON THE KD/DIALLYL

SULFIDE 101

EXPERIMENT 5. ACETONE DOSE-RESPONSE IN THE KINDLING PREPARATION 102

OVERVIEW Qua sscsssssssscsssscssscsrecscsssersssnecacenssasocsneceassaccessseasonesasssssesseasssencsaceaseasoeseesseasneusseoessnsenaces 105

FUTURE EXPERIMENTS .uosesccsessossssssscecssssccnssnssscsseuscacsacsscessncnceassacusvacssenssaceacsnsecenacensenssass 107

1. SHOULD WE BE USING “THRESHOLD DOSE” CALCULATIONS OR “SEIZURE LATENCIES”? 107

2. DO SEIZURES AFFECT IN VIVO ACETONE LEVELS? 107

3. DOES CHRONIC ADMINISTRATION OF DAS FURTHER ELEVATE BLOOD-ACETONE LEVELS? 108

4. DAS SUPPLEMENTATION IN PATIENTS ON THE KD 108

5. DOES ACETONE CORRELATE WITH SEIZURE PROTECTION IN CHILDREN ON THE KD? 109

6. WHAT IS ACETONE’S ANTICONVULSANT MECHANISM OF ACTION? 109

REFERENCE SG. ........cssssccsscsccoesscsssssonesesssosnscsssssencssesssessoesoeesveccseessonsseeseees soceseneeserecsnece 109

Vili

LIST OF TABLES

Table 1. Composition of 6.3:1 KD and stCTRL/adlibCTRL diet ........0...0...0.0.c. eee

Table 2. Composition of 4:1 KD and balCTRL diet ........0... 0... cece cece e cece teeta e es

Table 3. The Therapeutic Profile of Acetone

ix

LIST OF FIGURES

Figure 1. Historical Efficacy of the Ketogenic Diet .......00.0.... occ cee e eee ee ee ees 14

Figure 2. Clinical Outcomes in Patients of Varying Ages Fed the Ketogenic Diet ...... 15

Figure 3. Breakdown of a Typical American Diet vs. a Ketogenic Diet ................... 16

Figure 4. Weight Gain in Adult Rats 0.000.000... cc cence eee e ene ee eee neee ents en eetnnes 43

Figure 5. B-OHB Levels in Adult Rats ...........0.00... EMR eee ec ete ee ene eee eee ene eees 44

Figure 6. Glucose Levels in Adult Rats 0.0.0... cceceee cece eee eceee eect ee enetaeeneenensas 45

Figure 7. PTZ Threshold Dose Calculations in Adult Rats ...............000... veceeeeenwees 46

Figure 8. Weight Gain in Rat Pups ........... cc cccce cece e nce e nent eden ee eee tence eee eneea ease 54

Figure 9. B-OHB Levels in Rat Pups ........... 0... cce cece cence eee ee ence eee eeneeaeeeenaeaaes 55

Figure 10. Glucose Levels in Rat Pups ............. cece cece eee eee e ee eee ne eee e eee ee etn eneeaes 56

Figure 11. PTZ Threshold Dose Calculations in Rat Pups ..........0.0 cece ee eeteee ee 57

Figure 12. Latency to Seizure in Adult Rats 2.0.0... 0.00.0 cece cece ec ee eens eceeeeceee trees: 58

Figure 13. Latency to Seizure in Rat Pups ............ 0. ccc ce se eee eee ne cence e eee ee teeta en enaes 59

Figure 14. Blood Acetone Levels in Adult Rats Pre- and Post- MES Seizure ............ 69

Figure 15. Blood Acetone Levels in Adult Rats Pre- and Post- scPTZ Seizure ........... 70

Figure 16. HPLC chromatograms both with and without acetone ......................05045 75

Figure 17. Acetone Biotransformation Pathways ................0. ccc cece ee ee eee ee eee e eee 78

Figure 18. Weights in Adult Rats Fed a 4:1 KD Ad libitum ...........0..0. ccc 79

Figure 19. Time-course of B-OHB in Adult Rats Fed a 4:1 KD Ad libitum ............... 80

Figure 20. Time-course of Glucose in Adults Rats Fed a 4:1 KD Ad libitum ............. 81

Figure 21. Time-course of Acetone in Adult Rats Fed a 4:1 KD Ad libitum .............. 82

Figure 22. Acetone Dose-Response for Amygdala Seizures ...............0.:0cecee ee eeeees

Figure 23. Acetone Dose-Response for Cortical Seizures

XI

ACAC

AD

adlibCTRL

ADT

AED

ANOVA

ATP

AUC

baiCTRL

8-OHB

CYP2E1

DAS

DBS

DNPH

EDso

EEG

GABA

GABAa

GLUT-1

HPLC

IP

IV

' ABBREVIATIONS

Acetoacetate

Afterdischarge

Ad libitum Control Diet

Afterdischarge Threshold

Antiepileptic.Drug

Analysis of Variance

Adenosine Triphosphate

Area Under the Curve

Balanced Control Diet

Beta-Hydroxybutyrate

Cytochrome P450 2E1

Diallyl Sulfide

Deep Brain Stimulation

2,4-dinitrophenylhydrazine

50% Effective Dose

Electroencephalograph or Electroencephalogram

Gamma Amino Butyric Acid

Gamma Amino Butyric Acid Type A Receptor

Glucose Type-1 Transporter

High Performance Liquid Chromatography

Intraperitoneal

Intravenous

Xli

KD

MCT KD

MES

NMR

MRI

PTZ

PTZi

S.E.M.

stCTRL

scPTZ

TDs0

Tl

UV

VNS

Ketogenic Diet

Medium Chain Triglyceride Ketogenic Diet

Maximal Electroshock Test

Nuclear Magnetic Resonance

Magnetic Resonance Imaging

“Number of Subjects per Group

Postnatal Day

Pentylenetetrazole (a.k.a. Metrazol)

Pentylenetetrazole Infusion Test

Standard Error of the Mean

Standard Control Diet

Subcutaneous Pentylenetetrazole Test

50% Therapeutic Dose

Therapeutic Index

Ultra Violet

Vagal Nerve Stimulation

Xili

LIST OF CHEMICALS AND DRUGS

Chemicals

Chemical

Acetone

Acetonitrile

Diaily! Sulfide

2,4 — dinitrophenylhydrazine

Hydrochloric acid 6.000 N

Pentylenetetrazole

Drugs

Drug

Buprenorphine Hydrochloride (Buprenex® Injectable)

Ketamine Hydrochloride

(Ketalean®)

Lidocaine (with Epinephrine)

(Xylocaine®)

Xylazine

(Rompun®)

Sodium Pentobarbital

(Somnotol®)

CAS

67-64-1

75-05-8

592-88-1

119-26-6

7647-01-0

54-95-5

DIN

Company

SIGMA

Omni-Solv

SIGMA

SIGMA

VWR

SIGMA

Company

12496-0757-1 (NCD) Reckitt & Colman Ltd.

00612316

036641

02169592

00141690

Bimeda-MTC

AstraZeneca

Bayer Inc.

MTC Phamaceuticals

XIV

CHAPTER 1: GENERAL INTRODUCTION

INTRODUCTION TO EPILEPSY

What is Epilepsy?

Epilepsy is a neurological disorder characterized by a chronic low seizure

threshold that results in two or more spontaneous seizures (Burnham, 2002). A seizure is

a state of self-sustained, synchronous neuronal hyper-excitation (Burnham, 2002). During

a seizure, there is a disruption of normal brain function (Fisher et al., 2000). The

particular brain function that is disrupted depends on where the seizure originates.

Seizures originate in different areas of the brain, have many different etiologies,

and are often classified according to their idiosyncratic electrographic properties

(Pulliainen et al., 2000). For example, absence seizures are electrographically

characterized by a bilateral, synchronous, three per second spike and wave discharge

(Manning et al., 2003). Further, seizures can be classified according to the behaviour they

elicit, if any. For example, absence seizures are often typified by a blank stare, often

mistaken for daydreaming (Manning et al., 2003).

Distinguishing Between “Epilepsy” and “Seizure”

Having epilepsy means one has seizures. Having a seizure, however, does not

mean one has epilepsy. A seizure can occur in anyone who receives enough excitation or

loses enough inhibition in the brain (Engel, 1989).

The Economic Cost of Epilepsy

In 2002, the direct and indirect costs associated with epilepsy were $12.5

billion/year in the United States alone (Wiebe, 2003). With people living longer and with

the increased incidence of late-onset epilepsy, this cost is estimated to rise (Wiebe, 2003).

The Human Cost of Epilepsy

If uncontrolled, epilepsy has a significant, negative impact on patients’ quality of

life (Baker et al., 1997). Patients with uncontrolled epilepsy are not allowed to drive,

preventing them from pursuing many vocations. They also experience social rejection

and a wide spectrum of epileptic co-morbidities (Burnham, 2002). Epilepsy has been

described as a disorder that “robs patients of what they value most;independence,

relationships, respect, control over their own life, and the pursuit of vocational and

educational aspirations” (Wiebe, 2003).

Incidence and Prevalence of Epilepsy

The adjusted annual incidence of epilepsy is between 30 and 60 per 100,000 and

the prevalence is approximately 6 per 1000, making epilepsy one of the most common

neurological disorders (Wiebe et al., 1999; Fisher et al., 2000).

Who Gets Epilepsy?

Men and women are equally likely to develop epilepsy (Wiebe et al., 1999). Onset

of epilepsy is greatest in children and the elderly, however, one can develop this disorder

at any stage in life (Burnham, 2002).

CAUSES OF EPILEPSY

Symptomatic Epilepsy

Approximately 30-40% of patients with epilepsy have “symptomatic” seizures.

These are seizures associated with a specific structural abnormality in the brain. Such

abnormalities can include tumours, brain injury, infections, scars, and blood vessel

malformations (Browne & Holmes, 2001). Symptomatic epilepsies are the hardest to treat

and are often drug-resistant (Kwan & Brodie, 2000).

Idiopathic Epilepsy

Sixty to seventy percent of patients with epilepsy have “idiopathic” seizures.

These are seizures that occur in an apparently normal brain (Burnham, 2002). Idiopathic

seizures are thought to be caused by a subtle biochemical.gr.jonic imbalance, probably

inherited (Burnham, 2002). These seizures tend to respond favourably to antiepileptic

drugs (Kwan & Brodie, 2000; Perucca, 2001).

SEIZURE TYPES

Seizures are classified as “partial” (focal, local) or “generalized” (global)

(Burnham, 2002).

Partial Seizures

Partial seizures, initially, involve only a portion of the brain. The three forms of

partial seizures are simple partial, complex-partial and partial seizures that secondarily

generalize (Browne & Holmes, 2001).

Simple partial seizures are usually non-motor seizures that involve certain

sensations (e.g. flashing lights). Those who experience a simple partial seizure remain

conscious and alert throughout the seizure (Burnham, 2002). These seizures typically last

less than two minutes. Simple partial seizures may secondarily generalize (spread) to

other brain structures (Burnham, 2002).

Complex-partial seizures begin as a focal or “partial” seizure and spread to

become partially generalized, which causes impairment of consciousness. The patient is

La

not unconscious, but is unaware of the environment around him (Burnham, 2002).

Impairment of consciousness makes the seizure “complex” (Leung et al., 2000).

Complex-partial seizures are often preceded by an “aura”, that warns the patient the

seizure is going to occur. The aura is actually the simple partial seizure that triggers the

complex-partial attack. During the seizure, “automatisms” may occur. These non-reflex

movements can involve oral automatisms €e:g—.chewing, lip smacking, and swallowing)

or “ambulatory automatisms” (e.g. rubbing or picking hand movements, running or

walking) (Leung et al., 2000). Patients have no memory for the period of the seizure

(Burnham, 2002). Complex-partial seizures typically last between 30 seconds and 2

minutes but can leave the patient mentally hazy or confused for hours (Leung et al.,

2000).

Generalized Seizures

Generalized seizures involve the entire brain. There are various types of

generalized seizures, including absence seizures, myoclonic seizures and tonic-clonic

seizures (Browne & Holmes, 2001).

Absence seizures are characterized, behaviourally, by a sudden loss of

consciousness accompanied by brief staring spells (Manning et al., 2003).

Electrographically, absence seizures are characterized by a three per second spike and

wave discharge (Manning et al., 2003). Absence seizures tend to be very short (i.e. 3-10

seconds) and they can occur many times in a day (Manning et al., 2003).

Myoclonic seizures involve a sudden jerking movement of the body (Wheless &

Sankar, 2003). These seizures tend to only last a second or two but can recur frequently.

Tonic-clonic seizures are what many people think of when they hear the word

“epilepsy”. They are seizures involving unconsciousness and generalized convulsions.

The words “tonus” and “clonus” apply to the muscle actions involved in the convulsions.

Clonus refers to a rapid succession of muscle contraction and relaxation, leading to

jerking-like movements. Tonus refers to a constant state of contraction. This usually

causes the limbs to stiffen and'to:flex or extend. Tonic-clonic seizures tend to last

between one and two minutes, although they may last longer (Leung et al., 2000).

ANIMAL SEIZURE PREPARATIONS

Experimentally, normal animals can be made to seize when given a powerful

epileptogenic stimulus. This stimulus is usually electrical or pharmacological. Animal

seizure preparations are designed to model the seizures seen clinically. Researchers use

animal preparations to study the physiological, pharmacological, biochemical, and

electrographic features of seizures (Fisher, 1989).

Similar to the human seizures, animal seizures are classified as either

“generalized” or “partial”.

Generalized Seizure Preparations

Generalized seizure preparations model the generalized seizures seen in humans.

The two most common forms of generalized seizures in humans are tonic-clonic seizures

and absence seizures (Burnham, 2002).

There are several animal preparations used to model generalized seizures. These

include photosensitive seizures (e.g. Papio papio baboons), audiogenic seizures (e.g.

Frings mice), Drosophila shakers, maximal electroshock seizures, pentylenetetrazole,

picrotoxin, bicuculline, penicillin seizures, and febrile heat-induced seizures (Fisher,

1989). The most commonly used tonic-clonic animal seizure preparation is the maximal

electroshock seizure test (MES). The most commonly used absence seizure model is the

subcutaneous pentylenetetrazole test (scPTZ).

The Maximal Electroshock Test (MES). It has been known since the late 1800s

that electrical shocks can trigger seizures in animals. MES involves the administration of

electrical current to the brain via corneal electrodes or ear clips. In rats, the stimulus is

usually a 150mA, 60Hz sine wave current, which is given for 0.2 seconds (Fisher, 1989).

MES seizures are characterized by tonic extension of the fore and hind limbs.

They are generally scored as either “present” or “absent”. A maximal seizure is “absent”

if the hind limbs fail to extend beyond a 90 degree angle during the tonic component of

the seizure. Conversely, a maximal seizure is said to be “present” if the hind limbs extend

beyond 90 degrees during the tonic period of the convulsion (Likhodii et al., 2003).

Not all naive rats, however, demonstrate hind limb extension. These rats are

termed “non-extenders”’. In drug studies, animals are said to be “protected” if hind limb

extension is absent in MES-stimulated animals. Non-extenders must be excluded from

experiments to reduce the potential for false-positive results. For this reason, all rats

should be pre-screened before the experiment began. MES seizures model tonic-clonic

attacks in humans (Fisher, 1989).

The Subcutaneous Pentylenetetrazole Test (sePTZ). Pentylenetetrazole, also

known as “Metrazol®”, is a convulsant drug that works by non-specifically antagonizing

the y-amino-butyric acid A-type receptor (GABAa).

PTZ can be given at a high dose that yields maximal seizures that resemble MES

seizures. It may also be given at a low dose that elicits minimal seizures. This is known

as the threshold or “‘scPTZ test”. Only threshold scPTZ tests are used in this thesis.

scPTZ involves the injection of PTZ into the subcutaneous tissue, often between

the shoulder blades. scPTZ seizures take between 15 and 30 minutes to evolve. These

long latencies make it easier to detect differences between groups.

The scPTZ test models absence attacks in humans (Fisher, 1989).

The Pentylenetetrazole Infusion Test (PTZi). The PTZi infusion test involves

the infusion of PTZ into the subject’s tail vein. Compared to the scPTZ test, the PTZi test

is much quicker at causing seizures. This allows for much more rapid testing, however, it

is often harder to detect differences between groups.

Similar to the scPTZ preparation, PTZi is thought to model absence seizures

(Fisher, 1989).

Partial Seizure Preparations

Partial seizures are most commonly modeled using the kindling preparation of

complex-partial seizures.

Kindling. Kindling involves the application of a mild electrical stimulus to a

discrete brain region via a chronically indwelling bipolar electrode (Goddard, 1967;

Racine, 1972a and 1972b; see surgery methods below). The electrical stimulation

intensity is much lower than the MES stimulus, and is sufficient only to evoke a focal

seizure, or “afterdischarge” (AD). Generally, an AD is defined as an electrographic

seizure outlasting the stimulation by at least three seconds. When ADs are repeatedly

evoked in the kindling model, they begin to yield increasingly intense seizures.

Eventually, these intensified seizures spread widely in the brain, culminating in a

generalized motor seizure.

Amyedala kindling has been pharmacologically validated as an animal

preparation modeling complex-partial seizures (Albright and Burnham, 1980).

Motor seizures in kindled animals are rated according to Racine’s scale (Racine,

1972). This scale is discussed in the General Methods section.

PHARMACOLOGICAL TREATMENT OF EPILEPSY

Brief History

There are both drug and non-drug therapies for patients with epilepsy. The first

line of therapy for most cases of epilepsy is, however, antiepileptic drug (AED) therapy

(Fisher et al., 2000).

Until the mid 1990s, the most commonly prescribed AEDs were the “traditional”

AEDs. These include phenobarbital (Luminal®), primidone (Mysoline®), phenytoin

(Dilantin®), carbamazepine (Tegretol®), ethosuximide (Zarontin®), clonazepam

(Rivotril®) and valproate (Depakene®) (LaRoche & Helmers, 2004). Although these

“first-line” anticonvulsant drugs are familiar and efficacious, they are only effective in

60-70% of patients. They may also be associated with severe side-effects (Browne &

Holmes, 2001; LaRoche & Helmers, 2004).

In the past decade, a number of new drugs have been introduced to the market.

These include felbamate (Felbatol®), fosphenytoin sodium (Cerebyx®), oxcarbazepine

(Trileptal®), gabapentin (Neurontin®), lamotrigine (Lamictal®), zonisamide

(Zonegran®), levetiracetam (Keppra®), tiagabine (Gabitril®) and topiramate

(Topamax®). Although these newer drugs have fewer side effects, they do not appear to

be more effective anticonvulsants (Cole, 2004). As such, the problem of drug-resistant

epilepsy still remains.

Antiepileptic Drugs: Mechanisms of Action

The new AEDs may not be more effective than the old AEDs because they tend to

have the same mechanisms of action. ~-- -----— rr Be

AEDs generally work to decrease excitation or increase inhibition in the brain.

This is usually accomplished by one of three mechanisms: enhancing GABAergic

activity, decreasing the activity of voltage-dependent sodium channels, or decreasing the

activity of T-type voltage dependent calcium channels (Burnham, 1998).

GABA, Potentiators. GABA, potentiators bind the pentameric GABA, receptor

ionophore. They help keep the channel to open and allow the influx of negatively charged

chloride ions. Simultaneously, positively charged potassium ions are effluxed (Cutrer,

2001). This maintains the neuronal membrane in a polarized (inhibited) state (Cutrer,

2001). Although GABAzg potentiators are effective at stopping seizures, they may also

decrease memory and alertness. A number of the anticonvulsants enhance the activity of

GABA, often indirectly. Examples of AEDs that potentiate GABAergic activity are:

phenobarbital, primidone, topiramate, diazepam and tiagabine (Burnham, 1998; LaRoche

& Helmers, 2004).

Sodium Channel Blockers. “Blockade” of voltage-dependent sodium channels

affects the production of action potentials in neurons (Catterall, 1987). “Sodium channel

blockers” is a misnomer as the drugs do not actually block the channel. Rather they keep

the sodium channel in its inactive state for a longer period. This prevents rapid re-

polarization of neurons, therefore limiting the rapid firing required to sustain a seizure

(Catterall, 1987). Examples of voltage-dependent sodium channel blockers are phenytoin,

carbamazepine, felbamate, lamotrigine, oxcarbazepine, topiramate and zonisamide

(Burnham, 1998; LaRoche & Helmers, 2004).

Calcium Channel Blockers. Calcium channels, when open, allow the passage of

calcium into the cell. One calcium channel, the T-type calcium. channel, is thought to

cause rhythmic firing associated with the 3/second discharges of absence epilepsy (Kulak

et al., 2004).

T-type calcium channels are highly concentrated in the thalamus. Therefore, drugs

that act on these receptors are usually used to treat thalamo-cortical absence seizures.

Examples of calcium channel blockers are ethosuximide, trimethadione, felbamate,

lamotrigine, topiramate and zonisamide (Burnham, 1998; LaRoche & Helmers, 2004).

As noted above, some drugs have multiple mechanisms of action. Other drugs,

such as gabapentin, valproate and levetiracetam, have an unknown mechanism of action

(LaRoche & Helmers, 2004).

NON-PHARMACOLOGICAL TREATMENTS FOR EPILEPSY

If drug therapy creates harmful side effects or is ineffective at stopping seizures,

then a non-drug alternative therapy may be tried. Four commonly used non-drug

therapies are neurosurgery, deep brain stimulation, vagal nerve stimulation and the

ketogenic diet.

10

Neurosurgery

Neurosurgery for seizures can range from removing a small piece of cortical

tissue to removing an entire hemisphere of the brain. The goal of neurosurgery, in the

treatment of epilepsy, is to reduce or eliminate the tissue producing seizures, while

removing the least amount of tissue possible. This serves to improve the patient’s quality

of life without significantly compromising: cognitive function (Spencer, 1996; Reid et al.,

2004). Neurosurgery is currently viewed as the only treatment that can potentially stop

seizures altogether.

Deep Brain Stimulation

The newest non-drug treatment for epilepsy is deep brain stimulation (DBS).

Electrodes are implanted using magnetic resonance imaging (MRI), extracellular field

recordings, and electroencephalographic activity to ensure the accurate placement of the

electrode The placement is usually in a subcortical centre. Electrodes are attached to a

subcutaneously implanted, battery-powered, programmable stimulator (Theodore &

Fisher, 2004).

In humans, DBS is most commonly applied to the cerebellum, caudate, thalamus,

(centromedian nucleus, anterior thalamic nucleus) or subthalamic nucleus. DBS can also

be applied directly to an epileptic focus (Theodore & Fisher, 2004). A recent study by

Chabardes et al. (2002) demonstrated a 67-80% decrease in seizure frequency in five

patients receiving DBS to their subthalamic nuclei. DBS inhibits seizure activity through

various, complex and poorly understood inhibitory pathways (Theodore & Fisher, 2004).

11

The animal literature supports the clinical findings, showing that the stimulation

of various sub-cortical nuclei can abort or even prophylactically treat seizures (Velasco et

al., 2001; Nanobashvili et al., 2003; Hamani et al., 2004).

Vagal Nerve Stimulation

Vagal nerve stimulation (VNS) is also used to treat drug-resistant seizures

(Cramer et al., 2001). A simulator is implanted subcutaneously into the left chest muscle

with wires sending intermittent impulses to vagus nerve (FineSmith et al., 1999).

VNS has been reported to attenuate drug-resistant seizures by 15-30% (Ben-

Menachem et al., 1994; The Vagus Nerve Stimulation Study Group, 1995; FineSmith et

al., 1999). This is thought to be significant, given that candidates for VNS have failed

drug therapy.

There also exists evidence, however, that questions VNS’s effectiveness (Cramer

et al., 2001). A recent study by Wennberg (2004) found a >25% reduction in seizures in

patients whose vagal stimulator was no longer working. Wennberg (2004) argues that any

benefits of VNS are probably a placebo effect. VNS is often chosen as the last-line of

treatment for drug-resistant seizures (Cramer et al., 2001).

The Ketogenic Diet

The ketogenic diet (KD) is a carefully formulated high fat, low carbohydrate and

adequate protein diet used to treat drug-resistant seizures. The KD is the primary focus of

this thesis. It will be discussed in detail below.

12

THE KETOGENIC DIET

The fact that fasting can stop seizures has been known for more than 2000 years.

In the Bible (King James Version), Matthew 17, 14-21, the idea that seizures can be

treated through “prayer and fasting” was first introduced.

In the early 1900s, two French physicians, La Marie and Guelpa, postulated that

seizures were causced.from “excessiveness” (i.e. excessive eating) (Vining, 1999). It was

thought that the intestines needed cleansing, and thus fasting was successfully employed

as the treatment for seizures. In the 20" Century, Bernarr Macfadden, a faith healer, and

Dr. Hugh Conklin, an osteopathic doctor, used prayer and fasting to successfully treat

seizures (Vining, 1999).

In 1921, Geyelin adopted a 3-week fasting protocol that was very successful in

treating seizures in his patients. Some of his patients remained seizure free, even after

their normal diets were resumed (Vining, 1999).

Evolution of the KD

In 1921, Wilder developed a diet that mimicked the physiological effects of

fasting. Wilder intended this diet to have the “ketogenic” foods (i.e. fats) and “non-

ketogenic” foods (e.g. carbohydrates and proteins) at a 2:1 ratio, or ideally, a 3:1 ratio

(Vining, 1999; Nordli, 2002). This diet was termed “the ketogenic diet” due to its ability

to elevate systemic levels of the ketone bodies. The ketone bodies, acetoacetate, beta-

hydroxybutyrate, and acetone are products of fat metabolism.

The KD served as a major treatment for seizure disorders until the introduction of

the modern AEDs. Interest in the KD has recently revived due to its ability to suppress

seizures that resist the AEDs (Lefevre & Aronson, 2000).

Clinical Profile of the KD

Clinically, the KD is used to treat patients that have failed to respond to AEDs

(Nordli & De Vivo, 1997). Early studies of the KD suggested that approximately 55% of

patients achieved complete seizure control, while 26% achieved marked decreases in

seizure frequency and severity (Livingston, 1977). More recent studies have suggested

that.10-15% of patients on the KD become seizure free, and up to 60% experience a

greater than 50% reduction in seizure frequency (see Figure 1; for reviews, see: Vining,

1999; Thiele, 2003).

The inhibition of seizures in patients that have failed drug therapy suggests that

the KD may have a novel mechanism of action. This is to say, that if the KD controls

seizures in patients that fail to respond to GABA enhancements or sodium or calcium

blockers, it may have a mechanism of action different from GABA enhancement or

sodium or calcium blockade.

Figure 1. Historical Efficacy of the Ketogenic Diet

Historical Efficacy of the Ketogenic Diet

Y, of Pts.

60 66 .. 86

>90%

50 w>5oy ** 40 - m<50%,

34

30

20 4

10 ~

0 4.

Heimhoiz

(827}

c Go am ES

a Be Go a,

Withins

(1937

Livingston

(498

4)

Sitka:

:

(19SHACT)

insiman

(4992)

Efficacy of the KD and varying degrees of seizure control obtained in

patients (Pts) treated with the KD. From: Vining, 1999.

14

The KD is most commonly used in children (Vining, 1999). Although the

strongest anticonvulsant effects are seen in children between the ages of 2 and 10 the KD

has also been shown to have anticonvulsant actions in adults (Livingston, 1977; Swink et

al., 1997; Vining, 1999; Sirven et al., 1999). Sirven et al. (1999) have recently reported

that 3/11 adult patients had a >90% reduction in seizures, that 3/11 had a >50% reduction

in seizures and that 1/11 had a <50% reduction in seizures. 4/10 adults in the Sirven

study, however, discontinued the KD due to the KD’s unpalatable nature. Unpalatibility

is postulated to be one of the main reasons why adults tend not to do well on the KD

(Vining, 1999). Data on the age-related efficacy of the KD can be found in Figure 2.

Figure 2. Clinical Outcomes in Patients of Varying Ages Fed the Ketogenic Diet

JHMI KETOGENIC DIET:150 , Outcomes by Age:>50% Improved

of subjects 0

60

“2Yr. |

g 2-5. Yr.

w 5-8 Yr.

y i 8-12 Yr.

3 MO. & MO. 12 MO.

Seizure control after 3, 6, and 12 months on the KD in patients of varying

ages. This graph is shown divided by age groups and is derived from the

1998 prospective Johns Hopkins Medical Institutions Study. From: Vining,

1999.

15

Types of KD

Several forms of the KD are used clinically. KDs vary according to their ratio of

fat to combined carbohydrate and protein (e.g. 3:1, 3.5:1, 4:1 etc.). They also vary

according to the type of fat used (e.g. long chain fatty acid, medium chain triglyceride

KD, polyunsaturated fatty acid KD, etc.). One common variant, the “MCT” KD, uses

medium chain triglycerides as the source of fat (Carroll & Koenigsberget; 1998).

In most clinics, patients are started on a 4:1 KD, with the fat being provided by

long chain fatty acids (e.g. meat and dairy fats). This is termed the “classic” KD (see

Figure 3; Thiele, 2003).

Figure 3. Breakdown of a Typical American Diet vs. Ketogenic Diet

American Diet Ketogenic Diet

Carbs Protein

Distribution of the nutrients in a typical American diet and a typical 4:1

KD. From: Thiele, 2003. (Carbs = Carbohydrates)

Side-Effects of the KD

A number of side-effects of the KD have been documented. These side-effects

tend to be infrequent, and do not appear to occur in a dose-related fashion (Swink et al.,

16

1997). This is in contrast to the AEDs that cause many, potentially serious, dose-related

side-effects (Swink et ai., 1997).

Short-term risks often involve vomiting, hypoglycemia, dehydration, and refusal

to adhere to the diet’s protocol. Due to these risks, patients are hospitalized while the KD

is initiated (Vining, 1999).

Long-term risks of the KD involve hyperlipidemia;kidney stones, bone

demineralization, constipation, stunting of growth and ketoacidosis (Vining, 1999).

Hyperlipidemia. Some studies have suggested that the KD elevates plasma levels

of total cholesterol and triglycerides (Dekaban, 1966; Chesney et al., 1999). Other

studies, however, have failed to show this elevation (Schwartz et al., 1989; Katyal et al.,

2000). Kwiterovich et al. (2003) have recently reported that the KD causes a significant

increase in atherogenic apoB-containing lipoproteins, and a decrease in anti-atherogenic

high-density lipoproteins.

Kidney stones. Patients on the KD often have hypercalciuria, acidic urine, and

low urinary citrate excretion. Liquid intake is often restricted in patients on the KD. This

is believed to facilitate the anticonvulsant effects of the KD (Vining, 1999). When liquids

are restricted, however, approximately ~5% of patients will develop kidney stones

(Betsey et al., 1990; Herzberg et al., 1990; Furth et al., 2000; Nordli, 2002; Kossoff et al.,

2002). These stones are usually small and easily “passed”, and they do not contraindicate

the use of the KD for treatment of intractable seizures (Kossoff et al., 2002).

Bone Demineralization. Bone demineralization is a risk in patients maintained in

ketoacidosis for long periods of time (Nordli, 2002). Chronic administration of the KD

17

has been shown to decrease bone density. This, however, may be reversed through the

use of vitamin D supplementation (Hahn et al., 1979).

Constipation. Constipation is the most common side-effect observed in patients

on the KD. This is thought to occur from fluid-restriction, however, rather than from the

KD itself (Swink et al., 1997).

Stunting of Growth. Studies havé-repezted that children fed the KD have stunted

growth (Freeman et al., 1990). Recently, however, it has been shown that if children on

the KD are given vitamin and mineral supplements they experience normal growth in

height, although they do not gain weight at the same rate as children on a normal diet:

(Liu et al., 2003).

Ketoacidosis. Patients on the KD-experience elevated ketone bodies. Since

ketone bodies are acidic, this may cause ketoacidosis, which is usually tolerable in a

healthy patient. When patients develop an illness (e.g. influenza or a bad cold), however,

they may become too ketoacidotic (Swink et al., 1997). This may be prevented by

increasing oral fluid intake, or in extreme situations, by intravenous administration of

fluids (Swink et al., 1997; Vining, 1999).

Summary of Side Effects and Discontinuation of the KD

Thompson et al (1998) determined that <6% of patients discontinue the KD due to

side-effects. The most common reason for discontinuing the KD is lack of efficacy (i.e.

discontinuation is high in patients that do not achieve seizure control on the KD). Eighty

percent of patients experiencing a >90% reduction in seizures stay on the KD for at least

a year. Only 20% of patients experiencing <50% reduction in seizures stay on the diet for

at least a year (Vining, 1999).

18

Most patients are taken off the KD after 2 or 3 years, however, due to fears about

its long-term effects on health (Vining, 1999). The KD, though efficacious, does not offer

the long-term control achieved by the AEDs.

KD: POTENTIAL MECHANISMS OF ACTION

The mechanism of action-efthe KD is currently unknown. A number of different

theories have been proposed:

The Brain Lipids Hypothesis

Clinically, the KD has been shown to increase blood cholesterol and triglyceride

levels (Dekaban, 1966; Chesney et al., 1999; Kwiterovich et al., 2003). It is hypothesized

that this increase in lipid levels contributes to the anticonvulsant actions of the KD.

Rabinovitz et al. (2004), for instance, have suggested that lipids alter the structure and

function of neuronal membranes, causing changes to membrane fluidity, ion channel

functioning and receptor-ligand affinities. They suggest these changes have

anticonvulsant effects.

A problem with the brain lipids hypothesis is that not all KDs elevate lipid levels.

For example, the medium chain triglyceride KD (MCT KD) does not elevate blood

triglyceride levels, but it still has good anticonvulsant activity (Carroll & Koenigsberger,

1998).

The Acidosis Hypothesis

Acidosis was first hypothesized as the anticonvulsant mechanism of action for the

KD in 1931 by Bridge and Iob. When initiated on the KD, the patient’s metabolism

switches from the use of glucose to the use of ketoacids as an energy substrate. These

19

ketoacids were hypothesized to lower blood pH in patients on the KD, which was thought

to have anticonvulsant effects.

More recently, it has been further hypothesized that a low pH would inhibit pH-

sensitive NMDA-type glutamate receptors, decreasing excitation in the brain (Traynelis

& Cull-Candy; as cited in Schwartzkroin, 1999).

The acidosis-hypothesis has largely been abandoned, however, as clinical studies

have failed to show long-term KD-induced changes in pH (Huttenlocher, 1976). Animal

studies have confirmed this finding by demonstrating that there is no change at all in

brain pH in animals fed a KD (Al-Mudallal et al., 1996).

The Energy Substrate Hypothesis

Before the energy substrate hypothesis can be discussed, it is necessary to briefly

discuss the way the brain gets energy. The adult mammalian brain obtains its energy from

breaking carbon bonds in the macronutrients: protein, carbohydrate, and lipid (Dioguardi,

2004). Energy from these sources is created through either anaerobic metabolism or

aerobic metabolism (Greene et al., 2003).

Anaerobic metabolism — metabolism in the absence of oxygen — occurs when

glucose molecules are oxidized into two pyruvate molecules outside of mitochondria

(Dioguardi, 2004). This process is known as “glycolysis”. Glycolysis yields a small (~8

mol of adenosine triphosphate, ATP), but immediately available, source of energy for the

cell (Dioguardi, 2004).

Aerobic metabolism requires oxygen and occurs in mitochondria via the citric

acid cycle. Most of the brain’s energy is normally derived from the aerobic oxidation of

glucose, which provides higher levels (~30 mol) of ATP (Greene et al., 2003; Nehlig,

20

2004; Dioguardi, 2004). When carbohydrates are not abundantly available through the

diet — as on the KD — the brain begins to use ketone bodies for energy. Ketone bodies can

be converted to pyruvate, which can then be used in the citric acid cycle to make ATP.

The conversion of ketone bodies to pyruvate, however, does not release ATP as does

glycolysis.

<*. Normally, glucose serves as the preferred energy substrate for the brain. In

patients fed a KD, however, ketones can supply the brain with up to 60% of its energy

needs (Veech, 2004). Greene et al. (2003) hypothesized that glucose yields both “slow”

energy (via citric acid cycle) and “fast” energy (via glycolysis). Ketones, however, yield

only “slow” energy (via citric acid cycle). The energy substrate hypothesis suggests,

therefore, that although ketones provide sufficient energy for regular brain activity, they

do not provide enough “fast” energy to sustain seizure activity.

The GABA Shunt Hypothesis

The GABA shunt hypothesis suggests that the KD leads to higher levels of

GABA in the brain. Nordli (2002) argues that the KD causes increased levels of a-

ketoglutarate in the brain. Excess a-ketoglutarate can be used to produce GABA. Nordli

(2002) points out that elevated GABA levels would then elevate seizure threshold in the

brain.

One of the strongest lines of reasoning opposing the “GABA shunt hypothesis” is

that the KD is often successful in patients that have already failed GABAergic AEDs.

Therefore, if GABA agonists do not control the patient’s seizures and the KD does, it

would be a non-sequitur to assume that the KD works by elevating GABA levels.

21

Further, animal studies have shown that GABA levels are not increased in the

brains of animals fed the KD (Appleton & DeVivo, 1974; Al-Mudallal et al., 1996).

The Ketonemia Hypothesis

The “ketones” B-OHB, ACAC, and acetone are elevated in patients on the KD

(Kossoff, 2004). Although B-OHB and ACAC are considered ketones because of their

interconversion with acetone, acetone is the only “true” ketone. B-OHB and.-ACAC are

organic acids with an extra alcohol group and an extra ketone group, respectively

(Likhodii & Burnham, 2004).

The ketonemia hypothesis postulates that ketone bodies themselves are

anticonvulsant, and that the KD is effective because it elevates ketone bodies in the blood

and brain. No specific mechanism of action, however, has been suggested, i.e. no

“receptor” is known that ketones might bind to confer their anticonvulsant activity (for

review, see: Prasad et al., 1996).

Some clinical studies and animal studies have reported significant correlations

between levels of B-OHB or ACAC and seizure protection (Huttenlocher, 1976; Bough et

al., 1999a; Bough et al., 1999b; Whendon et al., 1999). Other studies, however, have

reported a lack of correlation between B-OHB or ACAC and seizure protection (Likhodii

et al., 2000; Bough et al., 2000; Eagles et al., 2003). At present, the relationship between

ketosis and seizure control remains unciear.

Opponents to the ketonemia hypothesis have argued that seizure control is seen

very quickly in fasted patients, whereas B-OHB and ACAC levels can take some days to

rise (Dekaban, 1966; Schwartz et al., 1989; Sirven et al., 1999). Further, Rho et al. (2002)

have failed to show that B-OHB had anticonvulsant effects in vitro.

22

Historically, however, researchers have neglected the possible role of acetone in

the anticonvulsant mechanism of the KD. The acetone hypothesis will be discussed

below.

The Acetone Hypothesis

Acetone is a ketone elevated in patients on the KD (Likhodii et al., 2002). The

idea that acetone has anticonvulsant properties was first-proposed by Hemholtz and Keith

in 1930 (as cited by Likhodii et al., 2002). The idea was ignored for some years.

Recently, however, Seymour et al. (1999) have reported that acetone is elevated in the

brains of children on the KD. Also, Likhodii et al. 2003) have found that acetone has a

wide spectrum of anticonvulsant effects in animals. Likhodii et al. (2003) have now

proposed “the acetone hypothesis”, which states that an elevation of acetone is the

anticonvulsant mechanism of the KD.

There are two lines of evidence for the acetone hypothesis. Firstly, acetone is

elevated in fasted patients and patients fed the KD (Seymour et al., 1999; Likhodii et al.,

2002). It is known that both fasting and the KD have anticonvulsant properties (Vining,

1999),

Secondly, acetone has been shown to have a broad spectrum of anticonvulsant

action, similar to that of the KD. Intraperitoneal injections of acetone have been shown to

be anticonvulsant in the amygdala kindling preparation, which models complex-partial

seizures, the MES preparation, which models tonic-clonic seizures, the AY9944

preparation, which models atypical absence seizures and the scPTZ preparation, which

models typical absence seizures (Likhodii et al., 2003).

N wo

RESEARCH GOALS & HYPOTHESES

The initial aim of the present research was to develop a more clinically accurate

animal model of the KD. The KDs used in most animal studies model poorly the KDs

used clinically.

The 6.3:1 KD has been used in many previous studies (Bough et al., 1999a;

Bough et al., 1999b; Bough et al., 2000; Zhao et al., 2004). Likhodii (2001) has

demonstrated, however, that the 6.3:1 KD is not balanced for vitamins, minerals and

protein with the standard rat chow control diet used (see: “standard rat chow control

diet”, below). As such, the anticonvulsant effects of the 6.3:1 KD are not necessarily

attributable to ketosis alone. Further, the 6.3:1 KD has a higher fat to carbohydrate and

protein ratio than any KD used clinically. This makes it a poor model of the KD used

clinicaily.

The 4:1 KD, developed by Likhodii (see Likhodii, 2001), was balanced with its

control diet in terms of vitamins, minerals and protein. This KD accurately reflects the

fat, carbohydrate and protein intake of patients on the KD.

It was believed that a clinically accurate animal preparation of the KD would be

important in determining the anticonvulsant mechanism(s) of the KD. Once the

mechanism of the KD is determined, a less rigorous treatment — e.g. a revised diet or a

drug — could be devised to replace the KD.

Experiment 1. Experiment | was designed to compare the ability of two KDs to

elevate seizure threshold. These KDs were: 1) a clinically accurate 4:1 KD developed by

Likhodii (1999); and 2) a 6.3:1 KD used by Bough and Eagles (1999). We hypothesized

that the 4:1 KD would have anticonvulsant actions similar to those seen clinically and

24

that the 6.3:1 ketogenic ratio was unnecessary. Aduit rats served as subjects in this

experiment, as in studies by Bough and colleagues (1999-2003).

Experiment 2. When both KDs failed to elevate seizure threshold in adult rats,

we repeated Experiment 1, only in rat pups. It was thought that young rats would produce

higher levels of ketone bodies. We hypothesized that both the 4:1 KD and the 6.3:1 KD

would have anticonvulsant actions in rat pups.

Experiment 3. Experiments | and 2 both failed to show clear cut anticonvulsant

effects of the KD. We know the KD has anticonvulsant effects in humans, but in our

hands, it lacks anticonvulsant effects in rats. This seems to suggest a species difference in

response to the KD.

Likhodii & Burnham (2004) have reported that the KD elevates acetone levels

much more in humans than in rats. It seems possible, therefore, that the KD lacks

anticonvulsant activity in rats because rats on the KD do not develop high levels of

acetone.

To explore this possibility, future studies would require us to measure acetone in

rats on the 4:1 KD. This could be done either pre-seizure testing, or post-seizure testing.

It is not ideal to take blood samples before seizure testing, as blood sampling itself is

likely to affect the seizure threshold. Therefore, most blood sampling has been performed

after seizure testing. It is, however, unknown whether seizures themselves influence

acetone levels in vivo.

Experiment 3 was designed, therefore, to determine whether seizures alter levels

of acetone in vivo. Both the MES and scPTZ seizure preparations were employed. We

hypothesized that acetone levels might decrease as a result of a seizure.

25

Experiment 4. The purpose of Experiment 4 was to replicate the studies of

Likhodii (manuscript in preparation) that suggest acetone is not significantly elevated in

rats fed a KD. The balanced 4:1 KD was used. Experiment 4 was also designed to

determine whether blood-acetone levels could be significantly elevated in rats by

inhibiting the metabolism of acetone. We hypothesized that rats would not develop high

levels of acetone on the 4:1 KD. Further, we hypothesized that inhibiting acetone’s

metabolism would elevate blood-acetone levels in rats fed the KD.

Experiment 5. It is known that the KD has a broad spectrum of anticonvulsant

activity. If acetone is responsible for the KD’s anticonvulsant effects, then acetone should

share the KD’s broad spectrum of anticonvulsant action.

Experiment 5 was designed to test whether acetone shares the KD’s broad-

spectrum of anticonvulsant activity. To do this, we tested the anticonvulsant activity of

five different doses of acetone in both the amygdala (limbic) kindling and cortical

kindling preparations of partial seizures. We hypothesized that acetone would have a

similar, broad-spectrum of anticonvulsant action as the KD. Specifically, acetone

should suppress both cortical and amygdala kindled seizures.

26

CHAPTER 2: GENERAL METHODS

SUBJECTS

Male Wistar albino rats (upon arrival, adults weighed ~250g and rat pups weighed

~50g), obtained from the Charles River breeding farm, served as subjects (Charles River

Laboratories, Saint-Constant, Quebec). Upon arrival, all rats were allowed one week to

acciimatize to the vivarium before being handled. During the following week, all rats

were handled on a daily basis until experimental procedures began. Water was provided

to all subjects ad libitum. In most groups, however, food was present for only 2.5 hours

per day (see below).

The vivarium had a 12-12 light/dark cycle, with lights on at 7am. All

experimental protocols were approved by the Animal Care Committee of the University

of Toronto’s Faculty of Medicine. Experiments were carried out in accordance to the —

guidelines of the Canadian Council on Animal Care.

DIETS

The following KDs were used in Experiments 1 and 2. Only the 4:1 KD was used in

Experiment 4.

The 4:1 Ketogenic Diet

The composition of the 4:1 KD is presented in Table 1. The 4:1 KD was

composed of 4 parts fat to one part of combined carbohydrate and protein. It was

developed by Likhodii (see Likhodii, 2001) to accurately reflect the fat, carbohydrate and

protein intake of patients on the KD. The 4:1 KD was obtained from Dyets Inc.,

Bethlehem, PA (product # 101092).

27

The Balanced Control Diet

The balanced control diet composition is also presented in Table 1. The control

diet was carefully balanced with the 4:1 KD in terms of vitamins, minerals, and protein.

This balanced control diet will be referred to as “balCTRL”. balCTRL was also obtained

from Dyets Inc., Bethlehem, PA (product # 101091).

Table 1. Composition of 4:1 KD and balCTRL diet

Macronutrient Micronutrient Diet

BalCTRL 4:1 KD

Protein Casein 87.225 142.09

L-Cystine 3 4.887

Carbohydrate Corn Starch 465.7 -

Sucrose 137.5 -

Dextrin 165 30

Fiber Cellulose 41.431 66.087

Vitamins AIN-93G 0.33 0.538

Choline bitartrate 2.5 4.073

Minerals AIN-93G 27.3 44.472

Fat Soybean oil 70 114.03

Lard - 187.8

Butter - 406

Antioxidant tert-butylhydroquinine 0.014 0.023

This table shows the composition of the 4:1 KD and its respective control diet, the

balCTRL diet. The ratio of caloric densities between the balCTRL and 4:1 KD is 1:1.629. The AIN-93G vitamin mix contains no carbohydrates. Soybean oil came from the vitamin mix where it serves as a carrier for fat-soluble vitamins. The type of fat (i.e.

butter or lard) can be varied. In this case, butter served as the major fat source.

The 6.3:1 Ketogenic Diet

The 6.3:1 KD composition can be found in Table 2. This diet was obtained from

BioServe Inc., Frenchtown, NJ (product # F3666). The 6.3:1 KD has been used in many

previous studies (Bough et al., 1999a; Bough et al., 1999b; Bough et al., 2000; Zhao et

al., 2004). Likhodii (2001) has demonstrated, however, that the 6.3:1 KD is not balanced

28

for vitamins, minerals and protein with the standard rat chow control diet used (see:

“standard rat chow control diet”, below). As such, the anticonvulsant effects of the 6.3:1

KD are not necessarily attributable to ketosis alone. Further, the 6.3:1 KD has a higher fat

to carbohydrate and protein ratio than any KD used clinically. This makes it a poor model

of the KD used clinically.

The Standard Rat Chow Control Diet

The composition of the standard rat chow control diet (stCTRL) can also be found

in Table 2. The stCTRL diet (Purina, #5001, Purina Mills, St. Louis, MO) has served as a

control diet for the 6.3:1 KD in a number of studies (Bough et al., 1999a; Bough et al.,

1999b; Bough et al., 2000; Zhao et al., 2004).

The stCTRL diet also served as the ad libitum control diet (adlibCTRL). The only

difference between the stCTRL and adlibCTRL diets was the feeding regime (i.e. calorie

restricted, limited access versus ad libitum access, respectively).

Table 2. Composition of 6.3:1 KD and stCTRL diet

Macronutrient Micronutrient Diet

stCTRL 6.3:1 KD

Protein - 234 95

Carbohydrate Dextrose 490 7.6

Fiber - 53 50

Mineral Mix AIN-76 69 38

Vitamin Mix AIN-76 54 20.9

Fat Lard 15 475

Butter 0 199.5

Corn Oil 85 114

This table shows the composition of the 6.3:1 KD and its respective control diet, the stCTRL diet. This diet formulation also serves as the adlibCTRL diet. The ash content is

derived from formula AIN-76 mineral mix #F8505. The vitamin mix is derived from

formula AIN-76 vitamin mix #F8000. stCTRL is Laboratory Rodent Chow, Purina #

5001.

29

ADMINISTRATION OF DIETS

In keeping with previous studies, all animals, save those fed the adlibCTRL diet,

were fed using a calorie restricted, limited access paradigm (Bough & Eagles, 1999;

Bough et al., 1999; Bough et al., 2000; Thavendiranathan et al., 2003). Limited access

subjects receive food for only 2.5 hours each day (9:00am-11:30am) and are fasted for

the remainder of the day.

Calorie restricted subjects are limited to 90% of their daily caloric intake. The

average daily caloric intake for a laboratory rat is 0.3 keal/g body weight/day (Rogers,

1979). Therefore, rats were restricted to 0.27 kcal/g body weight/day. Briefly, the caloric

density of a KD is nearly twice that of a control diet (~8 kcal/g vs. ~4 kcal/g,

respectively). Therefore, a 300g rat would receive roughly 10g of KD diet per day (0.27

kcal/g body weight/day x 300 g body weight / ~8 kcal/gram body weight) while the

control diet fed animals received roughly 20g of food each day (0.27 kcal/g body

weight/day x 300 g body weight / ~4 kcal/gram body weight).

DETERMINATION OF GLUCOSE AND f-OHB

Blood glucose and B-OHB levels were always taken after a seizure had been

elicited. A surgical plane of anesthesia was induced using an i.p. injection of sodium

pentobarbital (100mg/kg; Somnotol®, MTC Pharmaceuticals, Cambridge, Ontario). A

cardiac puncture was then used to withdraw approximately ImL of blood. A small

quantity of this blood was used to determine glucose levels. Glucose levels were

determined using a MediSense® Precision Xtra'™ glucometer (Abbott Laboratories,

Bedford, MA). This instrument reliably and accurately detects glucose between 2.0 mM

and 24.0 mM (linear range).

Whole blood was then centrifuged (International Equipment Company,

Micromax®) for 15 minutes at 3000g. Plasma was extracted and used to determine B-

OHB levels. B-OHB was determined using the Ketosite Stat-Site® (GDS Diagnostics,

Elkhart, IN). This diagnostic instrument reliably and accurately detects B-OHB between

0.01 and 2.0 mM (linear range). Within this window, reagent methods correlate to the

Ketosite State-Site® at r= 0.985.

B-OHB was used as a measure of ketosis in Experiments 1 and 2, because it is the

measure traditionally used in KD animal experiments. Acetone was measured in

Experiments 3 and 4.

DETERMINATION OF ACETONE

Blood samples were taken and plasma was obtained as described above. All

plasma samples were then derivitized using 2,4-dinitrophenylhydrazine (DNPH; Sigma)

according to the method of Likhodii et al. (2003). Deproteinization was performed by

adding 1001 acetonitrile to SOul of plasma followed by centrifugation (Micromax®,

International Equipment Company) at 10°g for 10 minutes. 50pl of DNPH reagent was

added to 50ul of clear supernatant and vortexed for 15 seconds. The recipe for DNPH

was as follows. Two and one-half milligrams of DNPH were dissolved in a 2.5 ml

mixture of hydrochloric acid and distilled water (4:6 vol/vol) by warming in water at

60°C for 20 minutes. DNPH derivitizes acetone to hydrazine in a 1:1 ratio. HPLC was

then performed to determine the concentration of hydrazine.

Derivitized samples were analyzed using an Agilent 1100 Series HPLC system

equipped with an autosampler (Agilent, Palo Alto, CA). Samples were injected into a Cj

column Symmetry (5m particle size, 25 cm x 4.2 mm I.D.; Waters, Milford, MA). An

ultra violet (UV) absorbance detector was set to 365nm. The mobile phase was 63:37

(vol/vol) acetonitrile to water. The flow rate was set at 1.0 ml/min.

SEIZURE TESTS

MES Seizures. The MES test was used in Experiment 3. In preparation for the

MES stimulation, a local anesthetic (0.9% lidocaine + epinephrine, AstraZeneca,

Mississauga, Ontario) was placed on the eyes. The current was then given using an

electroshock stimulator (model #8888, Biomedical Engineering Sunnybrook Health

Services Centre, Toronto, Ontario). The stimulus was given via corneal electrodes.

The MES stimulation consisted of a 60Hz sine-wave current of 105mA with a

train duration of 0.2 seconds. Maximal seizures were scored as either “present” or

“absent”. The absence of a seizure was defined as the absence of hind limb extension

beyond 90 degrees (Likhodii et al., 2003).

When rats are used for MES, it is necessary to pre-screen all subjects using MES

stimulation before experimentation can begin. Not all naive rats produce hind limb

extensions beyond 90 degrees when given the MES stimulation. Therefore, it is important

to eliminate “non-extenders”. Otherwise, one will not know whether a lack of hind limb

extension means the drug has protective effects, or whether it simply means that the

animal was a “non-extender”’.

32

Pre-screening for “non-extenders” was performed in all animals | week prior to

the actual seizure tests.

Subcutaneous Pentylenetetrazole Test (secPTZ). The scPTZ test was used in

Experiment 3. The threshold scPTZ dose is defined as the minimal dose of

subcutaneously injected PTZ required to cause clonic seizures in all rats, no earlier than

15 minutes post-injection and no later than 30 minutes post-injection (Fisher, 1989).

For scPTZ testing, subjects were restrained in a cloth to maximize their comfort.

The neck area was exposed and the subjects were injected subcutaneously with a dose of

50mg/kg PTZ. This dose, although lower than the standard scPTZ dose, was determined

in pilot studies to be just above threshold. PTZ was injected into the loose fold of skin

between the subject’s shoulder blades. A large injection volume (7ml/kg of body weight)

was used to minimize irritation.

Immediately following scPTZ injection, subjects were placed in square open-field

boxes (size 1x1m) and videotaped.

Pentylenetetrazole Infusion Test (PTZi). The PTZi test was used in

Experiments | and 2. On the day of testing each subject was weighed and heated for 5

minutes using a heat lamp to induce vasodilation. This facilitated accurate needle

placement into the tail vein. All subjects were restrained in a towel to minimize

discomfort during the procedure. PTZ solution (10mg/ml) in physiological saline was

infused via a syringe pump (model 351, Sage Instruments, Freedom, CA) at a continuous

rate (1ml/min) into the tail vein of each rat until a seizure was evoked. A seizure was

defined as fore limb clonus. Latency to seizure onset was recorded, and used to calculate

G2

wo

the amount of PTZ infused. This amount was divided by the subject’s body weight to

yield a “threshold dose” (mg/kg).

Kindling. The amygdala kindling and cortical kindling tests were used in

Experiment 5. In preparation for kindling, all subjects were surgically implanted with a

chronic indwelling electrode. All subjects were weighed prior to surgery. Subjects then

received a 10 mg/kg intraperitoneal (i.p.) injection of ketamine hydrochloride

(Ketalean®, Bimeda-MTC, Cambridge, Ontario)/xylazine (Rompun®, Bayer Inc.,

Toronto, Ontario) anaesthetic, supplemented as needed with an ip. injection of 20 mg/kg

of sodium pentobarbital (Somnotol®, MTC Pharmaceuticals, Cambridge, Ontario).

Lubricating eye ointment (Tears Naturale® P.M., Alcon Inc., Canada) was applied to the

eyes during surgery to prevent the eyes from drying.

Upon obtaining a surgical level of anaesthesia, subjects were implanted

stereotaxically with a biplor stimulating/recording electrode. A 10-mm nichrome-plated

stimulating/recording electrode (Electrode SS 2C TW, Plastics One Inc., Roanoke, VA. #

MS303/1) was implanted into the left basal lateral amygdala or the left motor cortex

using standard stereotaxic techniques (Skinner, 1971). Four stainless steel jeweller’s

screws were set into the skull to serve as anchors. The electrode was then affixed with

dental acrylic. All animals were given |ml/kg body weight of buprenorphine

hydrochloride (Buprenex® Injectable, Reckitt Benckiser Healthcare, Hull, UK) for post-

surgical analgesia. Animals were then maintained in an incubator at ~25 degrees Celsius

with ad libitum food and water for 12 hours before being returned to the vivarium. All

animals were given at least a one week recovery period before any experimental

procedures were performed.

During kindling, subjects were connected to a recording/stimulating lead and

placed in a testing chamber (18”x18”x19”’). Electrical stimulation was administered using

a Grass $8800 stimulator (Grass Instruments, Quincy, MASS.). Stimulation consisted of

a one-second train of constant-current balanced biphasic square-wave pulses (1-msec

duration, 60/sec) delivered at a current chosen by experimenter (below). The duration of

AD was measured using a Grass Model 6 EEG, (Grass Instruments, Quincy, MASS.) and

behavioural seizure stages were classified according to Racine’s scale (Racine, 1972a).

Briefly, stage 1 involves mouth clonus. Stage 2 entails mouth and head clonus.

Stage 3 involves forelimb clonus and stage 4 involves forelimb clonus and rearing. Stage

5 entails loss of postural control, i.e. falling (Racine, 1972a).

HISTOLOGY

Histology was performed on all kindled subjects (Experiment 5) to determine the

accuracy of the electrode placements. Subjects were deeply anesthetized (pentobarbital

100mg/kg) and perfused intracardially with physiological saline, followed by 10%

formalin. Brains were removed and stored in 10% formalin.

One week later, the brains were frozen and sectioned at 40m using a Leika

Cryostat (Leica, Jung CM3000, Germany). Sections were subsequently stained with

cresyl violet. Sections were later examined under a microscope. Only subjects with

correctly located electrodes were used in subsequent data analyses.

DATA ANALYSIS

One-way analyses of variance were used to compare group means. Where

statistically significant differences were found, Tukey’s post-hoc tests were used for the

comparison of pairs of means. Sigma Stat v.2.0 (Jandel Corporation, San Rafael, CA)

was used for all statistical analyses. Alpha was 0.05 for all comparisons.

CHAPTER 3: EXPERIMENT 1

A COMPARISON OF THE ANTICONVULSANT EFFECTS OF A 4:1 KD AND A 6.3:1 KD IN ADULT RATS

RATIONALE

Despite its anticonvulsant efficacy, the KD plays a limited role in the treatment of

epilepsy due to its rigorous nature and concerns about its long-term effects on health

(Sirven et al., 1999; Kwiterovich et al., 2003). Much of the recent research on the KD

aims to elucidate the KD’s anticonvulsant mechanism(s) of action. Once the KD’s

mechanism(s) of action is (are) understood, an equally efficacious, more practical

treatment can be devised. Much of the research examining the KD’s mechanism of action

has involved animals, particularly rats (for summary, see Stafstrom & Bough, 2003).

Recent work in rats has involved several formulations of the KD. The most

common is the 6.3:1 (fat to combined carbohydrate and protein) KD introduced by Bough

and Eagles (1999). Likhodii (2001) has pointed out that the 6.3:1 KD, besides containing

more fat than clinical versions of the KD, is not balanced for vitamins, minerals and

protein with its control diet (standard rat chow). As such, effects seen in experiments with

the 6.3:1 KD are not necessarily attributable to ketosis. Rather, they may be caused by

differences in vitamins, minerals or protein.

Our laboratory has attempted to formulate a more clinically relevant diet for

animal studies (Likhodii et al., 2000). Early experiments with a 3.6:1 KD, however,

revealed that the amount of food consumed by the KD and control diet fed rats was

different. Further, increasing the KD ratio above 3.5:1 led to a decrease in protein and

micronutrients in the KD (Likhodii et al., 2000; Likhodii, 2001). To ensure an accurate

comparison between KD and control groups, a 4:1 KD and a corresponding balanced,

non-ketogenic control diet were finally developed (Likhodii, 2001). This KD was

modeled after the 4:1 KD used clinically.

If the balanced 4:1 KD has anticonvulsant effects against PTZ seizures, it may

serve as a more accurate model of the KD used clinically. A lack of anticonvulsant

effects with the 4:1 KD would suggest that a higher fat: carbohydrate and protein ratio

(i.e. 6.3:1) is required for anticonvulsant effects in rats. Alternatively, it might suggest

that the low protein and micronutrient content of the 6.3:1 KD are responsible for its

anticonvulsant effects.

Ad libitum control groups were employed in both KD experiments to represent

normal laboratory rats. This group controls for the effects of limited access and calorie

restriction (see Experiments | and 2).

Experiment 1, therefore, was designed to compare the anticonvulsant activity of a

clinically accurate 4:1 KD to that of a 6.3:1 KD used by Bough ad Eagles (1999). We

hypothesized that the 4:1 KD should have anticonvulsant actions similar to those seen

clinically and that the 6.3:1 ketogenic ratio is unnecessary. Subjects were testing using

the PTZ infusion test.

METHODS

Subjects and Grouping

Ninety adult male Wister rats (~250g) were randomly divided into one of 5 diet

groups: (1) 4:1 KD [N=20]; (2) balCTRL [N=20]; (3) 6.3:1 KD [N=20]; (4) stCTRL

[N=20]; (5) adlibCTRL [N=10]. As discussed in the General Methods, all diets were

administered using a limited access, calorie restriction paradigm, except for the

adlibCTRL diet, which was given ad libitum.

Diets

The compositions of the two KDs and two control diets used in the present

experiment are presented in Table 1 and Table 2, and have been discussed in the General

Methods section of this thesis. Adult rats were maintained on the diets for 21 days as per

Bough et al. (2000) and Thavendiranathan et al. (2003).

PTZi Seizure Test

After 21 days on the diets, the PTZi was performed as discussed in General

Methods. The latency to seizure onset was measured, and the total amount of PTZ,

infused was calculated, and then divided by each animal’s body weight to obtain the

milligram per kilogram (mg/kg) dose of PTZ. This has been characterized as a “threshold

dose” (Pollack & Shen, 1985).

Glucose and Beta-Hydroxybutyrate Measurements

Subjects were sacrificed following the PTZi test, and blood samples were

obtained. Glucose levels were determined from whole blood and B-OHB levels were

determined from plasma, as discussed in the General Methods section.

Statistics

A one-way ANOVA was performed on both “pre-diet” weights and “testing day”

weights (see Figure 4, p.43). Where statistically significant differences were found, a

Tukey’s post-hoc test was used for the comparison of pairs of means. The ad libitum

control group was omitted from “pre-diet” analysis, as weight data only exist for the

“testing day” weights.

B-OHB levels, glucose levels, and seizure latencies were compared using one-way

ANOVAs. Where statistically significant differences were found, a Tukey’s post-hoc test

was used for the comparison of means.

RESULTS

Weights

Weights of adult rats are presented in Figure 4 (p.42). All groups had statistically

similar weights before the experimental diets were initiated (p>0.05 in all cases). On the

day of seizure testing, however, a one-way ANOVA revealed significant differences

between the group means (p<0.001). Tukey’s post-hoc analyses revealed that the 4:1 KD

group was significantly lighter than the balCTRL group (p<0.05). Similarly, on the day

of seizure testing, the weight of the 6.3:1 KD group was significantly lower than the

stCTRL group (p<0.05). No “pre-diet” weights were available for the adlibCTRL group.

On day of seizure testing, however, the adlibCTRL group was significantly heavier than

all other diet groups (p<0.05, all cases).

Beta-hydroxybutyrate Levels

8-OHB levels are presented in Figure 5 (p.43). As indicated, the levels of B-OHB

(average mM concentration + S.E.M.) were: 4:1 KD (0.58 + 0.18); balCTRL (0.3 + 0.10);

6.3:1 KD (0.53 + 0.15); stCTRL (0.5 + 0.16) and adlibCTRL (0.22 + 0.08). A one-way

ANOVA determined that there were significant differences among the group means

40

(p<0.001). Tukey’s post-hoc analyses revealed that subjects in the 4:1 KD group and the

6.3:1 KD group had statistically higher 8-OHB levels than the balCTRL group or the

adlibCTRL group (p<0.05, all cases). Curiously, the stCTRL group also had a

statistically higher 8-OHB level than the balCTRL or the adlibCTRL groups. The 4:1 KD

group, 6.3:1 KD group and stCTRL group did not differ significantly from one another

(p>0.05, all cases). Also, the balCTRL group and the adlibCTRL group were not

statistically different (p=0.79).

Glucose Levels

Glucose levels are presented in Figure 6 (p.44). As indicated, the average mM

concentrations (+ S.E.M) for each group were: 4:1 KD (7.78 + 1.52); balCTRL (7.48 +

2.65); 6.3:1 KD (7.8 + 1.71); stCTRL (5.51 + 1.70) and adlibCTRL (10.65 + 3.50). An

ANOVA revealed significant differences among the group means (p<0.001). Tukey’s

post-hoc analyses revealed that the balCTRL group and the stCTRL group had

significantly lower glucose levels than the 4:1 KD group, the 6.3:1 KD group or the

adlibCTRL group (p<0.05, all cases). The 4:1 KD group and the 6.3:1 KD group had

lower glucose levels than the adlibCTRL group. This did not, however, reach statistical

significance (p=0.74, p=0.71, respectively).

PTZ Threshold Dose

PTZ threshold doses for adult rats are presented in Figure 7 (p.45). The average

doses (mg/kg + S.E.M.) required to elicit clonic-tonic seizures were: 4:1 KD (22.78 +

3.66); balCTRL diet (26.08 + 3.05); 6.3:1 KD (23.45 + 3.98); stCTRL diet (21.35 4 2.52)

and adlibCTRL diet (21.21 + 4.11). An ANOVA revealed that there were significant

differences in the mean doses of PTZ required to elicit a seizure in the different groups

(p<0.001). Tukey’s post-hoc tests revealed that rats fed the balCTRL diet required a

significantly higher dose of PTZ than all other groups (p<0.05). This was contrary to

expectations. Rats fed the 6.3:1 KD required a somewhat higher dose of PTZ to elicit a

seizure than did the stCTRL group. This was not statistically significant, however

(p=0.32). Still, these results were in the expected direction. No other differences were

found among the groups (p>0.05, all cases).

Weight (g

rams

)

Figure 4. Weights of Adult Rats

440

420

400

380

360

340

320

300

280

260

y | —w-- stCTRL

—@— balCTRL —O- 4:1 KD 4 —¥— 6.3:1 KD # .

—#— adlibCTRL

i T T T t T T T 1 T T Y

pre stat d2 d5 d7 dQ d1i di3 d15 d18 d20_ test

All groups tended to lose weight upon initiation of experimental diets and regain it as the diets proceed. Animals fed the 6.3:1 KD regained the least amount of weight. Rats

fed the 4:1 KD regained all their weight but did not thrive like the control diet fed groups. Those fed limited access, calorie restricted control diets regained their lost

weight and continued to thrive. Rats fed the adlibCTRL control diet grew normally and were significantly heavier than all other groups (data only available for testing

day). * signifies a significant difference from the related control group. *# sionifies a significant difference from all other groups.

Beta

-hyd

roxy

buty

rate

Le

vels

(m

M)

Figure 5. B-OHB Levels in Adult Rats

0.7

4:1 KD balCTRL 6.3:1 KD stCTRL adlibCTRL

Rats fed a KD had higher levels of §-OHB than rats fed a control diet, with the exception of

the stCTRL group. Although B-OHB levels were elevated in some cases, all levels were in the low, “non-therapeutic” range. * signifies a significant difference from the related control group and the adlibCTRL group.

*# signifies a significant difference from the adlibCTRL group.

44

Figure 6. Glucose Levels in Adult Rats

Gluc

ose

Leve

ls (m

M)

14

—

NO

—

©

oo

4:1 KD balCTRL 6.3:1 KD stCTRL adlibCTRL

Blood glucose levels remained in the normal range for all adult rats, except those fed the adlibCTRL diet. Subjects fed the adlibCTRL diet had significantly higher glucose

levels than subjects fed the balCTRL diet or the stCTRL diet.

* signifies a significant difference from the adlibCTRL group.

45

Dose

of Pentylenetetrazole

(mg/

kg)

Figure 7. Dose to Seizure in Adult Rats

30

25

20

15

10

i

4:1 KD balCTRL 6.3:1 KD stCTRL adlibCTRL

The balCTRL diet appeared to significantly elevate seizure threshold compared to the 4:1 KD. Subjects fed the 6.3:1 KD had moderately, but not significantly, elevated

seizure thresholds compared to stCTRL fed subjects. Animals fed the adlibCTRL diet

appeared to have lower PTZ seizure thresholds. * signifies a significant difference from related control group.

46

DISCUSSION

Experiment 1 was designed to measure the ability of an unbalanced 6.3:1 KD and

a balanced 4:1 KD to elevate PTZ infusion thresholds, relative to controls, in adult rats.

Neither the 6.3:1 KD nor the 4:1 KD was able to significantly elevate threshold doses in

adult rats.

Although the 6.3:1 KD did elevate the threshold dose to a certain extent, it was a

very modest increase that did not reach statistical significance. These data are in

disagreement with previous studies, which have shown significant effects of the 6.3:1 KD

in adult rats (Bough & Eagles, 1999; Bough et al., 1999).

The 4:1 KD did not elevate threshold dose at all. In fact, compared to the

balCTRL diet group, the 4:1 KD appeared to have significantly proconvulsant effects.

Significantly proconvulsant effects of a KD have been found previously using an MCT

KD (Thavendiranathan et al., 2000), but never using a balanced 4:1 KD. A 4:1 KD has

been reported to elevate thresholds in the PTZi test. The 4:1 KD in that study, however,

was not balanced with its control diet in terms of micronutrients and macronutrients,

which casts some doubts on the results (Bough et al. 2000). To our knowledge, no other

study has examined the anticonvulsant effects of a balanced 4:1 KD.

Why did both KDs fail to elevate seizure thresholds? It is possible that both KDs

failed to elevate seizure thresholds as B-OHB levels were “sub-therapeutic”. Although the

6.3:1 KD and the 4:1 KD both significantly elevated B-OHB levels, as compared to

controls, the elevations were very modest with the highest reaching <O.6mM. It has been

shown that B-OHB levels between 2-4mM are required to confer anticonvulsant activity

47

in humans (Huttenlocher, 1976). Therefore, it is possible that B-OHB levels were simply

too low to confer anticonvulsant activity in rats.

How could we observe higher B-OHB levels in rats? It was decided that using rat

pups might be the answer. It has been reported that young humans develop much higher

levels of ketosis than adult humans (Cremer, 1982). Similarly, young rats develop much

higher levels of ketosis than adult rats (Nehlig, 2004). It was decided, therefore, to repeat

the comparison of the 6.3:1 and 4:1 KDs in rat pups.

48

CHAPTER 4: EXPERIMENT 2

A COMPARISON OF THE ANTICONVULSANT EFFECTS OF A 4:1 KD AND A 6.3:1 KD IN RAT PUPS

RATIONALE

Clinically, the KD is generally administered to children. Accordingly, animal

models mainly used rat pups until it was reported that the KD also has anticonvulsant

properties in adult rats (Bough and Eagles, 1999; Likhodii et al., 2000). Subsequently,

much of the research has been done in adult rats, as they are generally easier to work

with. Given that we were unable to demonstrate anticonvulsant effects in adult rats, we

decided to compare the 6.3:1 KD and 4:1 KD in rat pups. Rat pups develop much higher

levels of ketosis than adult rats and have been reported to display stronger anticonvulsant

effects on the KD (Bough et al., 1999).

With the aim of developing a clinically relevant animal preparation of the KD,

Experiment 2, therefore, compared the anticonvulsant effects of the 4:1 KD and the 6.3:1

KD in rat pups. We hypothesized that both the 4:1 KD and the 6.3:1 KD would have

anticonvulsant actions in rat pups.

METHODS

Subjects and Grouping

Seventy-five male Wistar rat pups (~50g) (Charles River Laboratories, Saint-

Constant, Quebec) were used in Experiment 2. Rat pups, arriving on post-natal day 9,

were housed with their dams until post-natal day 16. On post-natal day 16, they were

individually housed and weaned onto Purina rat chow. After 5 days of being fed rat chow

49

(post-natal day 21), pups were switched to an experimental diet. Pups were randomly

divided into one of 5 diet groups: (1) 4:1 KD [N=15]; (2) balCTRL [N=15]; (3) 6.3:1 KD

[N=15]; (4) stCTRL [N=15]; (5) adlibCTRL [N=15].

Diets

The compositions of the two KDs and two control diets used in the present

experiment are presented in Table 1 and Table 2, and have been discussed in the General

Methods section. As in Experiment 1, all pups, except those fed the ad libitum control

diet, were fed using a calorie restricted, limited access paradigm (Bough & Eagles, 1999;

Bough et al., 1999; Bough et al., 2000; Thavendiranathan et al., 2003).

PT Zi Seizure Test

Rat pups were seizure tested after 10 days on their diets. This allowed the test to

occur while they were still pre-pubescent (before ~post-natal day 34). Previous studies

have reported anticonvulsant effects of the KD in subjects fed the KD for 10 days

(Thavendiranathan et al., 2000; Uhlemann & Neims, 1972).

The PTZi test was performed as described in the General Methods section. The

total amount of PTZ infused was calculated, and then divided by each animal’s body

weight to obtain the milligram per kilogram (mg/kg) dose of PTZ. This has been

characterized as a “threshold dose” (Pollack & Shen, 1985).

Glucose and Beta-Hydroxybutyrate Measurements

Following the seizure test, subjects were anaesthetized and blood samples were

taken. Glucose levels were determined from whole blood and B-OHB levels were

determined from plasma, as discussed in the General Methods section.

50

Statistics

A one-way ANOVA was performed on both “pre-diet” weights and “testing day”

weights (see Figure 8, p.66). Where statistically significant differences were found, a

Tukey’s post-hoc test was used for the comparison of means.

8-OHB levels, glucose levels, and seizure latencies were compared using one-way

ANOVAs. Where statistically significant differences were found, a Tukey’s post-hoc test

was used for the comparison of means.

RESULTS

Weights

Rat pup weights are presented in Figure 8 (p.53). There were no statistically

significant weight differences between group means before experimental diets were

initiated (p>0.05). On the day of seizure testing, however, an ANOVA demonstrated

significant differences between the group means (p<0.001). Tukey’s post-hoc analyses

revealed that pups fed the 4:1 KD were significantly lighter than those fed the balCTRL

diet (p<0.05). Similarly, pups fed the 6.3:1 KD were significantly lighter than those fed

the stCTRL diet (p<0.05). Rat pups fed the adlibCTRL diet were significantly heavier

than all of the other diet groups on the day of seizure testing (p<0.05).

Beta-hydroxybutyrate Levels

8-OHB levels (average mM concentration + S.E.M.) for each group are presented

in Figure 9 (p.54). They were: 4:1 KD (4.69 + 1.14); balCTRL (0.06 + 0.089); 6.3:1 KD

(3.32 + 2.3); stCTRL (0.08 + 0.08) and adlibCTRL (0.03 + 0.08). An ANOVA indicated

51

that there were significant differences among the group means (p<0.001). Tukey’s post-

hoc tests revealed that both the 6.3:1 KD and the 4:1 KD groups were significantly higher

that the three control groups (p<0.05). The 6.3:1 KD and the 4:1 KD did not, however,

differ significantly from one another (p=0.192). The three control groups were also not

statistically different from each other (p>0.05, all cases).

Glucose Levels

Glucose levels (average mM concentration + S.E.M.) for each group are presented

in Figure 10 (p.55). They were: 4:1 KD (5.31 + 0.89); balCTRL (8.05 + 0.71); 6.3:1 KD

(5.45 « 0.94); stCTRL (8.76 + 1.47) and adlibCTRL (8.27 + 1.38). An ANOVA

demonstrated that there were significant differences among the group means (p<0.001).

Tukey’s post-hoc analyses revealed that both the 6.3:1 KD group and the 4:1 KD group

had significantly lower glucose levels than the three control groups (p<0.05). The two

KD groups did not, however, differ significantly from each other (p=0.99). The three

control groups were also not statistically different from each other (p>0.05, all cases).

PTZ Threshold Dose

PTZ threshold doses in rat pups are presented in Figure 11 (p.56). As indicated, the

average doses of PTZ required to elicit a seizure (expressed as mg/kg + S.E.M.) were: 4:1

KD (33.9 = 4.04); balCTRL (38.32 + 6.67); 6.3:1 KD (39.57 + 6.83); stCTRL (30.57 +

5.73) and adlibCTRL (25.2+ 2.90). An ANOVA indicated that there were significant

differences among the groups (p<0.001). Tukey’s post-hoc tests revealed that the 6.3:1

KD group had significantly higher thresholds than the stCTRL group and the adlibCTRL

group (p<0.05, all cases). Curiously, the balCTRL and the 4:1 KD also had higher

thresholds than the stCTRLs or the adlibCTRLs (p<0.05, all cases). The 4:1 KD group,

52

balCTRL group and 6.3:1 KD group, however, did not differ statistically from each other

(p>0.05, all cases). Also, the stCTRL group and adlibCTRL group were not statistically

different (p>0.05).

Weight (g

rams

)

Figure 8. Weights of Rat Pups

180

160

140 ~+

120 +

100 -

80 -

60 + 40

—@— 4:1KD —O— balCTRL —w— 6.3:1 KD oes xf —y— stCTRL | —m— adlibCTRL

T I I ] I T t T q T T T

pre start d2 d3 d4 d5 d6 d7 d8 d9 4d10 test

All groups lost a small amount of weight upon initiation of diets. Pups fed the 6.3:1 KD regained the least amount of weight. Those fed the stCTRL regained the most

amount of weight. Rats fed the 4:1 KD and balCTRL diet regained similar amounts of weight, although they had statistically different weights on day of seizure testing. Pups

fed the adlibCTRL diet grew normally and were significantly heavier than all other

groups.

* signifies significant difference from related control group.

*# signifies significant difference from all other groups.

Figure 9. B-OHB Levels in Rat Pups

Beta

-hyd

roxy

buty

rate

Le

vels

(m

M)

==

4:1KD balCTRL 6.3:1 KD stCTRL adlibCTRL

Pups fed either KD had significantly higher levels of B-OHB than control diet fed animals. Rat pups developed much higher levels of B-OHB in contrast to adult rats. These levels are within what is thought to be the “therapeutic range”’.

* signifies significant difference from all control groups.

Gn nA

Gluc

ose

Leve

ls (m

M)

Figure 10. Glucose Levels in Rat Pups

10

4:1KD balCTRL 6.3:1 KD stCTRL adlibCTRL

Blood glucose levels remained in the low-normal range for pups fed a KD. Control

diet fed subjects had significantly higher blood glucose levels that fell within the

normal range. Asterisks signify statistical significance (a=0.05).

* sionifies significant difference from related KD-fed group and adlibCTRL group.

56

Dose

of Pe

ntyl

enet

etra

zole

(m

g/kg

)

Figure 11. Dose to Seizure in Rat Pups

50

4:1 KD balCTRL 6.3:1 KD stCTRL adlibCTRL

The balCTRL diet raised seizure threshold compared to the 4:1 KD group, although this is not statistically significant. The 6.3:1 KD group significantly elevated seizure threshold compared to the stCTRL group. Animals fed the adlibCTRL diet appeared to

have the lowest PTZ seizure thresholds.

* signifies significant difference from related KD-fed group.

*# signifies significant difference from all other groups, except the stCTRL group.

Latency

(sec)

Figure 12. Latency to Seizure in Adult Rats

60

40

30

20

10

4:1KD balCTRL 6.3:1 KD stCTRL adlibCTRL

When absolute latencies were used, the 6.3:1 KD no longer appeared to elevate seizure threshold. The directionality of the 4:1 KD and balCTRL group remained the same. The adlibCTRL group had a relatively higher seizure threshold than when threshold

dose calculations were performed.

* signifies significant difference from adlibCTRL group.

58

Late

ncy

(sec

)

Figure 13. Latency to Seizure in Rat Pups

30

4:1 KD balCTRL 6.3:1 KD stCTRL adlibCTRL

When absolute latencies were used, the significant anticonvulsant effects of 6.3:1 KD disappeared. The 4:1 KD and 6.3:1 KD appeared to have significantly proconvulsant effects. The adlibCTRL group had a relatively higher seizure threshold than when threshold dose calculations were performed.

* signifies significant difference from all control groups.

59

DISCUSSION

Experiment 2 was designed to measure the ability of an unbalanced 6.3:1 KD and

a balanced 4:1 KD to elevate PTZ infusion thresholds, as compared to controls, in rat

pups. The same diets were employed as in Experiment 1. Given the impact of this

experiment on future studies in this thesis, it will be discussed in detail at this point.

In Experiment 2, the 6.3:1 KD significantly elevated threshold doses in pups, as

compared to controls. In agreement with these data, anticonvulsant effects of the 6.3:1

KD have been reported elsewhere (Bough and Eagles, 1999; Bough et al., 2000a; Bough

et al., 2000b; Bough & Eagles, 2001; Harney et al., 2002; Thavendiranathan et al., 2003;

Eagles et al., 2003). These findings confirm our expectation that the 6.3:1 KD would be

more effective in rat pups (Experiment 2) than in adult rats (Experiment 1).

The 4:1 KD, however, did not elevate threshold doses in rat pups. As in

Experiment 1, the 4:1 KD appeared to have proconvulsant effects when compared to the

balCTRL diet. This difference, however, did not reach statistical significance.

Why did the 6.3:1 KD elevate seizure thresholds when the 4:1 KD did not? A

number of factors must be considered. One is the possibility that the 6.3:1 KD produced

higher levels of B-OHB. As indicated in Figure 9 (p.54), however, this was not the case.

Both the 6.3:1 KD and the 4:1 KD significantly elevated B-OHB levels as compared to

controls. B-OHB levels were elevated to much higher levels in rat pups than in adult rats

(Experiment 1) and were, in fact, within the “therapeutic range”. This range, 2-4mM,

represents the levels of B-OHB that correlate with seizure protection clinically

(Huttenlocher, 1976).

60

Interestingly, however, the 4:1 KD elevated B-OHB levels even higher than the

6.3:1 KD. Given that the 4:1 KD did not elevate threshold doses, it seems clear that there

is no direct relationship between B-OHB levels and elevation of seizure threshold. This

will be further discussed in the General Discussion section.

A second factor to be considered is glucose levels. A recent report suggests that

high blood glucose levels can have proconvulsiit effects (Schwechter et al., 2003), as

may very low blood glucose levels (Anuradha et al., 2003). Abnormal blood glucose

levels in the 4:1 KD group might cancel out the anticonvulsant effects of the KD. As

indicated in Figure 10, however, this was also not the case. In the present experiment

there was an initial decrease in glucose levels in animals fed the KDs. Blood-glucose

levels, however, stabilized and remained in the “low-normal” range throughout the

experiment. This is consistent with previous clinical data (Huttenlocher, 1976) and with

previous data from animal models (Appleton & DeVivo, 1974; Todorova, 2000; Likhodi

et al., 2000). Blood-glucose levels in pups were lower than blood-glucose levels in adults.

It cannot, however, explain the different effects of the 4:1 KD and 6.3:1 KD in pups,

since glucose levels were almost identical in the two groups.

Another possibility is that the unbalanced nature of the 6.3:1 KD might yield

anticonvulsant effects. Likhodii (2001) has suggested that protein, mineral and vitamin

deficiencies of the 6.3:1 KD employed by Bough et al. could affect measurements of PTZ

threshold doses. The 6.3:1 KD is deficient in vitamins, minerals and protein as compared

to the stCTRL diet, balCTRL and adlibCTRL. The 4:1 KD was balanced with its

balCTRL diet (see Likhodii, 2001). This possibility is further strengthened by a recent

report suggesting that a version of the 6.3:1 KD leads to impaired growth and brain

61

development (Zhao et al., 2004). It is possible that if a 6.3:1 KD could be balanced to its

contro! diet, its anticonvulsant effects would disappear. This possibility deserves further

investigation.

A final possibility to consider is that “threshold dose calculations” create the

appearance of “anticonvulsant effects” that do not actually exist (Likbodii & Burnham,

2003). Large weight differences exist between the groups at the time of seizure testing

(for adult rat weights, see Figure 4; for rat pup weights, see Figure 8). In the present

study, the KD fed groups were always lighter than the control diet groups. This difference

was more pronounced in Experiment 2 (rat pups) than in Experiment | (adult rats). Also,

the difference is larger for the 6.3:1 KD and stCTRL diet groups than it is for the 4:1 KD

and balCTRL diet groups. When calculating “threshold doses”, large weight differences

between the KD-fed and control diet-fed groups may skew the results. For example, the

average weight of pups fed the 6.3:1 KD was only ~60% that of the average weight of

pups fed the stCTRL diet (63g vs.102g, respectively). Future studies should consider the

impact of large weight differences between groups when using an intravenous infusion

seizure test. If weights were matched, apparent anticonvulsant differences might

disappear.

When “seizure latencies” (seconds to seizure) are used, a different picture

emerges. It is quite possible that latencies, rather than threshold doses, should be used.

Seizure latencies in the adult rats were less than 60 seconds, and in rat pups were less

than 30 seconds. At this time point, PTZ may be largely confined to the vascular system

and “vessel rich” groups (i.e. brain, liver, and kidney). Therefore, correcting for body

weights by calculating “threshold doses” may be irrelevant (see Likhodii & Burnham,

2003). We are unaware, however, of any PTZ pharmacokinetic data in an intravenous

preparation. If, instead of calculating PTZ “threshold doses”, one considers “‘absolute

latencies” (i.e.: number of seconds of PTZ infusion before seizure onset), then the

anticonvulsant effects of the 6.3:1 KD disappear altogether (see Figure 12, p.57 and

Figure 13, p.58). In fact, the 6.3:1 KD appears to be proconvulsant as compared to the

control diet fed animals: ‘This is in agreement with the suggestions of Thavendiranathan

et al. (2000), that “partial starvation” — i.e. calorie restriction and limited access feeding —

may be proconvulsant.

CHAPTER 5: EXPERIMENT 3

THE EFFECT OF SEIZURES ON ACETONE LEVELS IN VIVO

RATIONALE

During the period in which Experiments | and 2 were being conducted, the

suggestion was made that the KD is not anticonvulsant in rats, and that it is not

anticonvulsant because rats do not develop high levels of blood-acetone on the KD

(Likhodii, manuscript in preparation).

Past animal studies have tried to determine the relationship between B-OHB levels

and anticonvulsant activity on the KD. As in Experiments 1 and 2, no clear relationship

has been established (Bough & Eagles, 1999; Sirven et al., 1999; Schwartz et al., 1989;

Dekaban, 1966). Animal studies have also tried to determine the relationship between

ACAC and the anticonvulsant activity of the KD. Rho et al. (2002) demonstrated that

ACAC has anticonvulsant effects in vitro, however, no study has ever demonstrated

ACAC to have anticonvulsant properties in vivo. Few animal studies have tried to relate

acetone to the anticonvulsant effects of the KD. This is odd, since acetone has been

shown to have clear anticonvulsant effects in vivo (Likhodii et al., 2003).

In 1930, an article by Hemholtz and Keith first introduced the idea that acetone

might have anticonvulsant properties (as cited in Likhodii et al., 2003). This idea was

given little attention.

Recently, however, many studies have examined the anticonvulsant actions of

acetone (Likhodii & Burnham, 2002a; Likhodii & Burnham, 2002b; Likhodii et al.,

2003). Likhodii et al. (2003) found the KD to have strong anticonvulsant effects in

several seizure preparations. Acetone is elevated in patients on the KD. It has not,

64

however, been hypothesized to play a role in the KD’s mechanism of action until recently

(Likhodii et al., 2003).

A possible explanation for the failure of the 4:1 KD in rats is that it elevates levels

of B-OHB, but not acetone. We therefore proposed to measure acetone in rats on the 4:1

KD.

A challenge in determining the anticonvulsant effects of an endogenous ~*~

metabolite, like acetone, is that seizures themselves might affect acetone levels.

Experiment 3 was designed to test this possibility.

The purpose of Experiment 3 was to determine whether seizures alter in vivo

acetone levels. The present study used both the MES seizure preparation and the scPTZ

seizure preparation. Exogenous acetone was injected to ensure good levels for the assay.

We hypothesized that acetone levels would likely decrease as a result of a seizure. The

rationale for this hypothesis was that acetone may be used as a fuel source. We

hypothesized that acetone, therefore, would drop during an energy consuming event, such

as a seizure.

METHODS

Subjects and Grouping

Thirty-one male adult Wistar rats (~300g; Charles River Laboratories, Saint-

Constant, Quebec) were used in Experiment 3. On the day of testing, subjects were

randomly divided into the MES group (N=16) or the PTZ group (N=15). All subjects

received an initial injection of acetone (below). Half of the MES subjects (N=8) then

received MES stimulation while the remaining subjects (N=8) received sham-MES

65

stimulation. Similarly, eight of the PTZ subjects received a subcutaneous injection of

PTZ while the remaining seven subjects received a subcutaneous injection of saline.

Acetone Injections

All subjects were injected with an acetone solution. Acetone (580.08 mg/kg) was

mixed with 1Oml/kg of physiological saline. A large injection-volume was used to

minimize irritation from acetone. The acetone solution was injected i.p. Seizure testing

was performed 30 minutes after acetone injections.

MES Testing

Subjects were pre-screened and MES tested as described in the General Methods

section. The sham-MES animals were treated identically to the MES stimulated animals

except that the corneal electrodes were disconnected from the stimulator. As such, no

current was passed to the subject when the stimulator was activated.

scPTZ Seizure Test

Subjects were scPTZ tested as described in the General Methods section.

Measuring Acetone in Blood

Blood was sampled both pre- and post-seizure. Samples were obtained by

pricking the tail-vein. Approximately 0.5ml of blood was obtained from each subject two

minutes prior to the injection of acetone. Approximately 0.5ml of blood was obtained

from each subject 32 minutes after acetone injection, or approximately 2 minutes post-

seizure. Immediately after the second blood sampling, all subjects were euthanized using

a high dose of Somnotol® (100mg/kg). Blood-acetone levels were determined using

HPLC methods discussed in General Methods.

66

Statistics

One-way repeated measures ANOVAs were run on one factor, area under the

curve (AUC) values. Any significant differences between means were further analyzed

using Tukey’s post-hoc tests.

RESULTS

MES Group

Although all MES stimulated animals had tonic-clonic seizures, no hind limb

extension was observed in any of the animals. This was probably due to protection from

the acetone injections.

AUC values for acetone both pre- and post-MES seizure and pre- and post-sham-

MES seizure can be found in Figure 14 (p.68). The ANOVA demonstrated significant

differences between group means (p<0.05). Tukey’s post-hoc analyses revealed that pre-

MES AUC values were significantly higher than post-MES values (p<0.05). There were

no significant differences between pre-sham-MES and post-sham-MES AUC values.

Further, there were no significant differences between pre-MES AUC values and pre-

sham-MES values.

scPTZ Group

scPTZ-injected animals had very mild seizures, as acetone provided seizure

protection. Subjects showed only “wet dog shakes” and staring spells.

AUC values for acetone both pre- and post-PTZ seizure and pre- and post-sham-

PTZ seizure can be found in Figure 15 (p.67). An ANOVA revealed significant

differences between the group means (p<0.05). Tukey’s post-hoc tests revealed that pre-

67

PTZ AUC values were significantly higher than post-PTZ values (p<0.05). No significant

differences existed between pre-saline and post-saline AUC values. Further, there were

no significant differences between pre-MES AUC values and pre-sham-MES values.

68

Area

Un

der

the

Curv

e (A

UC)

Figure 14. Blood Acetone Levels in Adult Rats Pre- and Post- MES Seizure

600

500

400

300

200

100

MES-pre MES-post sham-pre sham-post

The MES group and the sham-MES group did significantly differ in terms of acetone

AUC values before seizure testing. MES tested subjects had significantly lower acetone AUC values post-seizure than pre-seizure (p<0.05). Sham-stimulated subjects

did not, however, have significantly lower acetone AUC values after the sham-MES

seizure than before the sham-MES seizure (p>0.05).

* signifies significant difference from related condition.

69

Area

Un

der

the

Curv

e (A

UC)

Figure 15. Blood Acetone Levels in Adult Rats Pre- and Post- PTZ Sub-Q Test

600

500

400

300

200

100

PTZ-pre PTZ-post saline-pre saline-post

The PTZ group and the saline injected group did significantly differ in terms of acetone AUC values before injections. PTZ tested subjects had significantly lower

acetone AUC values post-seizure than pre-seizure (p<0.05). Saline injected subjects did not, however, have significantly lower acetone AUC values after the saline

injection than before the saline injection (p>0.05).

* sionifies significant difference from related condition.

70

DISCUSSION

The present study was designed to determine the effect of seizures on blood-

acetone levels. We determined that MES seizures and scPTZ seizures both significantly

lowered acetone AUC values, as compared to sham-stimulated controls and saline-

injected controls.

Acetone levels dropped significantly in seizure tested subjects. Acetone levels in

MES stimulated subjects dropped by ~18%, while acetone levels in PTZ-injected subjects

dropped by ~16%. Seizures use more energy than any other brain activity (Cornford et

al., 2002). It seems possible that acetone was being used as an energy substrate, thus

explaining why acetone levels were significantly lower after a seizure.

A non-significant drop in blood-acetone levels was observed in both control

groups as well. Acetone levels in the sham-MES stimulated group dropped by ~3%

between pre- and post- measurements. Acetone AUC levels in the saline-injected group

dropped by ~9% between pre- and post- measurements. Acetone is excreted by the lungs.

It is probable that these small, non-significant changes were due to elimination of acetone

through respiration (EHC, 1998).

In summary, it appears that seizures significantly decrease acetone concentrations.

This suggests that blood-acetone levels measured after seizure testing will not accurately

reflect blood-acetone levels before seizure testing. This will be discussed further in the

General Discussion.

71

CHAPTER 6: EXPERIMENT 4

LEVELS OF fB-OHB, GLUCOSE AND ACETONE IN RATS ON THE KD / DIALLYL SULFIDE (pilot study)

RATIONALE

A number of previous experiments performed in our laboratory have suggested

that the KD significantly elevates blood acetone in human patients but not in rats —

(Likhodii et al., manuscript in preparation). In particular, Likhodii et al. used nuclear

magnetic resonance (NMR) and HPLC to demonstrate that the KD elevates blood-

acetone in rats to concentrations of less than 0.1-0.6mM (unpublished). This is not

sufficient to suppress seizures (Likhodii et al., 2003).

Experiment 4 was designed to confirm this effect and to determine whether

acetone levels can be elevated above 0.1-0.6mM, and into the “therapeutic range” by

inhibiting the metabolism of acetone.

Acetone is biotransformed into acetol via CYP2E1 (see Figure 16, p.76). Diallyl

sulfide (DAS) is a specific CYP2E]1 inhibitor in rats (Reicks, 1996). DAS has been

shown to elevate acetone levels in laboratory rats fed a standard control diet (Chen et al.,

1994). In Experiment 4, we administered DAS to adult rats fed a balanced 4:1 KD.

Rats were put on a 4:1 KD ad libitum and blood samples were taken every 2 days

and analyzed for B-OHB, glucose and acetone. We hypothesized, as per Likhodii

(manuscript in preparation), that rats do not develop “clinically relevant” levels of

acetone on the KD. Also, after 11 days of being fed the KD, subjects were given DAS.

We further hypothesized that acetone levels would be elevated by DAS. No seizure

72

testing was done since Experiment 3 had indicated that seizures affect blood acetone

levels.

Experiment 4 is a very smail scale study, involving only three subjects. It was

originally intended as a pilot study (a larger study is planned). It has been included in this

thesis because significant effects were found even with the small sample size.

METHODS

Subjects and Grouping

Three adult male Wistar rats were used in the present study (~250g). Subjects

were first fasted for 18 hours. Subsequently, they were placed on a balanced 4:1 KD (see

Table 1) for eleven days. Blood samples were taken at regular intervals (below). On the

evening of the tenth day, all subjects were gavaged with dially! sulfide.

Diets

The composition of the 4:1 KD used in the present experiment can be found in

Table 1.

Diallyl Sulfide Administration

A solution of diallyl sulfide and corn oil was prepared. 200mg/kg of DAS (Sigma)

was added to 4ml/kg of body weight of corn oil. This solution was drawn into a 5ml

syringe and administered intragastrically using a gavage. DAS administration was given

via gavage as per previous studies (Chen, 1994).

Blood Sampling

Blood sampling involved restraining animals by wrapping them in a cloth. A 25-

gauge needle was broken away from its hub. It was then inserted into the tail vein and

blood was collected in plastic Eppendorf vials. All blood samples were maintained on ice

until all three subjects had been sampled. This took approximately twenty minutes.

Approximately 0.5ml of blood was taken from the tail vein of each animal at

seven time points: 1.) The “pre” sample was taken in otherwise experimentally naive rats.

These levels were used to represent baseline B-OHB, glucose and acetone levels; 2.) The

“1 8hr fast” sample was taken after subjects had been fasted for 18 hours; 3-6.) The “d2”,

“5”, “d7” and “d9” samples were taken after subjects had been fed the KD for 2, 5, 7,

and 9 days, respectively; 7.) The “dil (DAS)” sample was obtained after animals had

been fed the 4:1 KD for 11 days. Subjects were gavaged DAS 12 hours prior to blood

sampling on d11. Previous studies have shown that maximal effects of DAS are obtained

after 12 hours (Chen et al., 1994). Weights were taken before blood was sampled on each

testing day.

Glucose and B-OHB Measurements

Glucose and B-OHB levels were determined according to the methods discussed

in General Methods.

Acetone Measurements

Acetone levels were determined according to the methods discussed in General

Methods. Figure 16 shows chromatogram both with and without acetone.

Statistics

A repeated measures one-way ANOVA was used to compare weights, B-OHB

levels, glucose levels, and acetone levels across the 7 sampling days (i.e. “pre”, “18hr

fast”, d2, d5, d7, d9, dl1(DAS)). Any significant differences were further analyzed using

Tukey’s post-hoc analyses.

74

Figure 16. Chromatograms of samples with and without acetone

No added

acetone

4.54 mM added

acetone

The top chromatogram is from a standard that lacked added acetone. The lower chromatogram is from a standard that was spiked with 4.54 mM of acetone. The acetone peak occurs approximately 6.8 minutes after sample injection.

75

RESULTS

Weights

Individual weights for subjects can be found in Figure 17 (p.77). A repeated

measures ANOVA revealed that there were significant differences between group means

across the testing days. Tukey’s post-hoc tests showed that subject’s weights on d5, d9,

and d11(DAS) were significantly higher than they svere-on “pre”, “18hr fast” and d2

(p<0.05, all cases). All subjects thrived on the 4:1 KD when fed ad libitum. Subjects, on

average, gained 5 grams of body weight per day.

p-OHB Levels

B-OHB levels can be found in Figure 18 (p.78). B-OHB levels increased during

fasting, but fell to near baseline levels when the KD was initiated. On d5 there was a

large spike in B-OHB levels. B-OHB then declined again and leveled out until DAS was

administered. On dil (DAS), B-OHB levels increased once again.

A repeated measures ANOVA revealed significant differences in the subject’s B-

OHB levels across the 7 testing days. Tukey’s post-hoc tests demonstrated that B-OHB

levels on d5 were statistically higher than B-OHB levels on “pre”, d2, d7, and d9 (p<0.05,

all cases). d5, “18hr fast”, and dl 1(DAS) B-OHB levels did not differ significantly.

Tukey’s post-hoc tests also revealed that d11(DAS) 6-OHB levels were significantly

higher than “pre” and d2 B-OHB levels (p<0.05, both cases). d11(DAS) B-OHB levels,

however, did not differ significantly from “18hr fast”, dS, d7, or d9.

76

Glucose Levels

Glucose levels for subjects in Experiment 4 can be found in Figure 19 (p.79).

Glucose levels dropped after the 18-hour fast. They then increased when animals were

started on the KD. Glucose levels appeared to be unaffected by DAS.

A repeated measures ANOVA was unable to detect significant differences

between group means across the 7 testing deys (p=0.067). Larger group sizes would

probably have resulted in statistically significant differences.

Acetone Levels

Acetone levels in Experiment 4 can be found in Figure 20 (p.80). Eighteen hours

of fasting caused acetone levels to more than double from the baseline level. Initiation of

the KD, however, caused acetone levels to drop to baseline levels. Acetone levels did not

increase until DAS was administered. DAS was able to elevate acetone to levels seen in

fasted animals.

A repeated measures ANOVA detected significant differences between group

means across the 7 testing days. Tukey’s post-hoc tests revealed that acetone levels on

“i 8hr fast” were significantly higher than on “pre”, d2, d7 or d9 (p<0.05). Levels of

acetone on “18hr fast”, however, did not differ significantly from d11(DAS) and d5

acetone levels. Tukey’s post-hoc tests also revealed that acetone levels on d11(DAS)

were significantly higher than “pre”, d2, d7 or d9 acetone levels. Acetone levels on

d11(DAS), however, did not significantly differ from acetone levels on “18hr fast” or d5.

77

Figure 17. Acetone Biotransformation Pathways

ACETONE

cytochrome P450 2E1

NADPH Ean acetone monooxygenase

NADP o

ACETOL

extrahepatic

acetone monooxygenase

cytochrome P450 2E1

METHYLGLYOXAL 1,2-PROPANEDIOL

2-oxoaldehyde-

dehydrogenase (NADP)

GLUCOSE

a — LACTATE PYRUVATE

ACETATE

PATH A PATH B

Approximately 70% of acetone is biotransformed. Acetone biotransformation plays a role in gluconeogenesis. Adapted from: EHC, 1998

78

Figure 18. Weights in Adult Rats Fed a 4:1 KD Ad Libitum

380 *

—@— Subject #1

360 + --©-- Subject #2 * “pop —ag— Subject #3 BES

340 +

. & oo ® 320 - ef 2 = © 300 - o S

280 +

260 +

240 T T T t T T T

pre 18hr fast d2 d5 d7 d9_~=«d11 (DAS)

This figure shows the time-course of weight gain in three adult rats fed a balanced 4:1 KD

ad libitum. All subjects gained an average of 5 grams of body weight per day, and appeared to thrive on the diet. Subjects were significantly heavier on d9 and d11(DAS)

than on previous days.

* signifies a statistical significance (a=0.05).

79

Figure 19. Time-course of Beta-hydroxybutyrate in Adult Rats Fed a 4:1 KD Ad Libitum

2.0 * *

_ 1.8 -

£ 16- ml) oO 3 1.4 -

mul ® 5 1.2 4

oo a 1.0 + >

S 5 0.8 5 >

& & 0.6 + oO

an]

0.4 +

0.2 i q T i 1 | q

pre 18hr fast d2 d5 d7 d9 = d11 (DAS)

This figure shows the B-OHB time-course of three adult rats fed a balanced 4:1 KD ad libitum. “Pre” depicts the baseline B-OHB levels. “18hr fast” represents B-OHB levels

after 18 hours of fasting. D2, d5, d7, and d9 show B-OHB levels after 2, 5, 7, and 9 days

on the KD. D11 (DAS) shows B-OHB levels after 11 days on the KD and twelve hours

after animals were intragastrically administered diallyl sulfide, a CYP2E1 inhibitor. p-

OHB levels were significantly higher on d5 and d11(DAS) than any other day.

* sionifies a statistical significance (a=0.05).

80

Figure 20. Time-course of Glucose in Adult Rats Fed a 4:1 KD Ad Libitum

7.0

6.5 4

= 60. £ 6.0

LL

£ @ 5.5 4 J @ ” 8 D 5.0 + Na

oO —@— Subject #1

4.5 - -.Q. Subject #2 —ay— Subject #3

4.0 '

q t i i

pre 18hr fast d2 d5 d7 d9 dit (DAS)

This figure shows the glucose time-course of three adult rats fed a 4:1 KD ad libitum. No

statistically significant differences in blood-glucose levels were found (i.e. “pre” blood-

glucose levels were not statistically different from “18hr fast” or d11(DAS) levels).

Figure 21. Time-course of Acetone in Adult Rats Fed a 4:1 KD Ad Libitum

Acet

one

Concentration

(mM)

1.0 k

6 —@— Subject #1 ae. : ‘O-- Subject #2 * ft —w— Subject #3

0.6 -

0.4 -

0.2 -

0.0 +

q T t t ' t T

pre 18hr fast d2 d5 d7 d9 d11 (DAS)

This graph shows the acetone time-course of three adult rats fed a 4:1 KD ad libitum. Blood-acetone levels were significantly higher on “18hr fast” and d11(DAS) then any

other day.

* signifies a statistical significance (a=0.05).

82

DISCUSSION

It has been suggested that rats on the KD do not develop high levels of acetone,

whereas humans do (Likhodii et al., manuscript in preparation; Likhodii & Burnham,

2004). Further, it has been shown that humans do develop “therapeutic levels” of acetone

on the KD (Likhodii & Burnham, 2004). Experiment 4 was designed to confirm these

data and to further determine whether acetonetexcls could be increased using DAS.

All subjects appeared to thrive on the balanced 4:1 KD. This is in contrast to the

adult rats in Experiment 1, who gained very little weight on the limited access, calorie

restricted feeding paradigm. Subjects in the present experiment gained, on average, 5

grams per day. This daily weight gain was similar to that of the adlibCTRL subjects from

Experiment 2.

B-OHB levels were elevated by fasting. This was expected and has been reported

to occur clinically (Huttenlocher, 1976). Interestingly, B-OHB levels increased

dramatically in subjects after five days on the KD, but then dropped dramatically.

Previous, unpublished research from our laboratory has also shown that B-OHB levels

peak after one-week and subsequently drop (Likhodii, personal communication). It is not

yet clear why this happens.

Fasting caused a large (but non-significant) reduction in blood glucose. Glucose

then rebounded upon initiation of the KD. Glucose levels appeared to stabilize in the

“low normal” range for the remainder of the experiment (Cremer, 1982). Glucose may be

derived in part from the KD — which is not carbohydrate free — and in part from

gluconeogenesis from ketone bodies — e.g. acetone (see Figure 16, p.76; EHC, 1998).

Baseline acetone levels were very low. This was expected, and has been reported

elsewhere (Likhodii and Burnham, 2004). Fasting caused a greater than 2-fold increase in

acetone levels in all cases. Blood-acetone levels then fell to baseline levels upon

administration of the KD. This is in agreement with previous data (Likhodii et al.,

manuscript in preparation). Further, this confirms our hypothesis that the KD does not

significantly elevate acetone in-rais:-Jt may explain the absence of anticonvulsant effects

in Experiments | and 2.

Chen et al. (1994) have reported that acute administration of DAS elevated

acetone levels two-fold above baseline levels. Their study, however, was performed in

rats fed a standard rat chow diet. We hypothesized that acute administration of DAS in

rats fed a KD would yield even higher levels of acetone. The present study demonstrated

that acetone levels were 5- to 8-fold higher, as compared to baseline levels, 12 hours after

DAS administration in rats fed the KD for 11 days. It appears that KD fed rats will

develop higher levels of acetone after DAS administration than rats fed a standard rat

chow diet. Potential clinical applications for DAS will be discussed in the General

Discussion.

Interestingly, B-OHB levels were elevated by DAS. Chen et al. (1994) reported

that DAS had no effect on B-OHB levels. B-OHB is not known to be metabolised by

CYP2E1. It appears, however, that B-OHB levels, in the present experiment, increased in

response to DAS administration. It is also possible, however, that 8-OHB was simply

spontaneously peaking again on d11(DAS) as it did on d5. Future studies will incorporate

a group of vehicle controls to help solve this question. Glucose levels did not appear to be

affected by DAS.

84

Chen et al. (1994) reported that chronic administration of DAS did not yield

higher acetone levels than acute administration of DAS. This, however, was in animals

fed a standard rat chow diet. Future research should examine the effects of chronic DAS

administration in rats fed a KD. It is possible that chronic DAS treatment could further

elevate acetone levels. If acetone levels can be pushed above the 2-4mM range, then it is

possible that the KL) would have anticonvulsant actions in rats. This would further

implicate acetone in the anticonvulsant actions of the KD.

85

CHAPTER 7: EXPERIMENT 5

ACETONE DOSE-RESPONSE IN THE KINDLING PREPARATION

RATIONALE

It is known that the KD has a broad spectrum of anticonvulsant activity. It

suppresses many types of seizures (Vining, 1999). The KD is effective against tonic-

clonic seizures, absence seizures, atypical absence seizures, and complex-partial seizures.

It is also used in many types of intractable epilepsy, for example Lennox-Gastaut

syndrome (Kinsman et al., 1992; Swink et al., 1997).

Recently our laboratory has proposed the acetone hypothesis (see Likhodii &

Burnham, 2002a). This hypothesis states that acetone is responsible for the KD’s

anticonvulsant mechanism of action. If this is true, then acetone should share the KD’s

broad spectrum of anticonvulsant activity. A recent study by Likhodii et al. (2003)

indicates that may be true. Likhodii et al. (2003) found that acetone suppressed MES

seizures, scPTZ seizures, amygdala kindled seizures, and AY-9944 generated seizures.

Perhaps the most significant finding of Likhodii et al. (2003) was the finding that

acetone suppressed the amygdala focal seizure in kindled animals. Amygdala kindling

has been pharmacologically validated as a rat preparation that models complex partial

seizures of temporal lobe origin (Albright & Burnham, 1980). This suggests that acetone

— used as a drug — might be effective against complex-partial seizures. The control of

complex-partial seizures is a major problem in epilepsy treatment (Burnham, 2002).

In the Experiment 5, we tested acetone against both focal and generalized seizures

elicited from the amygdala or the cortex. Cortical kindling has been pharmacologically

86

validated as a preparation of simple partial seizures of cortical origin (Albright &

Burnham, 1980).

The purpose of Experiment 5 was to confirm and extend the findings of Likhodii

et al., (2003), using the standard Albright and Burnham paradigm. EDsos (“effective

dose” at which 50% of subjects have no seizure), TDsys (“toxic dose” at which 50% of

subjects experience toxicity) and Tls (therapeutic index, EDs9 / TDso) were calculated...

We hypothesized that acetone would have anticonvulsant effects in both

amygdala and cortical kindling preparations.

METHODS

Subjects and Surgery

Twenty adult male Wistar rats were used for Experiment 5. Rats were randomly

and evenly divided into either the amygdala kindling group or the cortical kindling group.

Animals were then surgically implanted with chronic indwelling bipolar

recording/stimulating electrodes (see Surgery in General Methods). Half of the animals

(N=10) had electrodes aimed at the left motor cortex while half of the subjects (N=10)

had electrodes aimed at the left basal lateral amygdaloid nucleus.

Kindling

Kindling was begun 14 days after surgery. Subjects were kindled daily, 5 days per

week. All amygdala kindled subjects were stimulated at 400A. All cortically kindled

animals were stimulated at 800A. Stimulation parameters are outlined in the General

Methods section. All animals were stimulated until 10 stage 5 seizures had been reached

87

(see General Methods for seizure stages). Seizures become stable and predictable after 10

stage 5 seizures. Stability and predictability of seizures are critical for drug testing.

Acetone Doses and Injections

Solutions of acetone in physiological saline were prepared to give doses of 0

mg/kg, 290.4 mg/kg, 580.8 mg/kg, 1161.6 mg/kg, 1742.4 mg/kg and 2323.2 mg/kg. All

solutions were injected i.p. The total injection volume was 10mI/kg of-bady weight. This

large injection volume was used to minimize irritation from acetone.

All subjects were tested using the Omg/kg (control) dose on the first and last day

of testing to help control for any effect of day.

Scoring of Ataxia

Twenty-eight minutes following acetone injections, subjects were rated for ataxia

using the Léscher ataxia scale (Léscher et al., 1987). Briefly, subjects were placed in an

open field (size, 1x1m) and observed for 1 minute. A scale of 0-5 was used: (0) no ataxia;

(1) a slight ataxia in the hind limbs; (2) a more pronounced ataxia with a slight decrease

in abdominal muscle tone; (3) further ataxia with a pronounced decrease in abdominal

muscle tone; (4) marked ataxia with a loss of balance during forward locomotion, loss of

abdominal tone; and (5) very marked ataxia with frequent losses of balance during

forward locomotion, a loss of abdominal tone. Ataxia was scored as “present” when there

was an ataxia rating of 1 or higher.

Drug Testing

Subjects were stimulated 30 minutes after acetone injections. Subjects were

stimulated at the group-dependent stimulation intensities mentioned above. Seizures were

scored as being either “present” or “absent”. The generalized seizure was said to be

88

“present” if the subject had a stage 3 or higher seizure (see Racine’s seizure

classifications above). The focal seizure was said to be present if the subject exhibited

three or more seconds of spiking in the EEG record.

Histology

Histology for electrode placements was performed as discussed in the General

Methods.

RESULTS

Histology

All 10 amygdala electrodes and all 10 cortical electrodes were accurately placed

within their respective structures.

Amygdala Generalized and Focal Seizures

The dose response curve for acetone in the amygdala kindling preparation can be

found in Figure 21 (p.89). For generalized seizures, the EDsy was approximately 871.2

mg/kg and 100% suppression occurred at 2323.2 mg/kg. For amygdala focal seizures,

there was no suppression at any dose.

Cortical Generalized and Focal Seizures

The dose response curve for acetone in the cortical kindling preparation can be

found in Figure 22 (p.90). For generalized seizures, the EDs9 was approximately 232.3

mg/kg and 100% suppression occurred at 580.8 mg/kg. For cortical focal seizures, the

EDs was approximately 580.8 mg/kg and 100% suppression occurred at 1742.4 mg/kg.

89

Toxicity and Therapeutic Index

Table 3 gives the EDso, TDso, and TI values for acetone for focal and generalized

seizures in amygdala and cortically kindled animals. The TDs) was similar in cortical and

amygdala kindled subjects (1742.4 mg/kg). Any differences between TIs were caused by

differences in EDsos.

As indicated, acetone has the highest-TI for cortical generalized seizures, with

lower TI’s for cortical focal and amygdala-generalized seizures. A TI could not be

calculated for amygdala focal seizures.

Table 3. The Therapeutic Profile of Acetone

EDs TDs TI

Cortex (generalized) 232.3 mg/kg 1742.4 mg/kg 7.5

Cortex (focal) 580.8 mg/kg 1742.4 mg/kg 3

Amygdala (generalized) 871.2 mg/kg 1742.4 mg/kg 2

Amygdala (focal) >2323.2 mg/kg 1742.4 mg/kg *

This table gives the EDso (“effective dose”, dose at which 50% of subjects do not have

seizures), TDs (“toxic dose”, dose at which 50% of subjects experience toxicity) and TI (therapeutic index, calculated as the TDs9 / EDs9) values for acetone in the kindling

preparation.

90

Perc

ent

Seizure

Protection

Figure 22. Acetone Dose-Response for Amygdala Seizures

120

—@— Generalized Seizure

400 - --©- Focal Seizure

©

Oo 1

60 +

40 +

20 +

t t t t t T

0 290.4 580.6 1161.6 1742.4 2323.2

Dose of Acetone (mg/kg)

Vehicle alone did not elevate seizure threshold. Acetone was able to suppress

generalized seizures kindled from the amygdala. A toxic dose (2323.2 mg/kg), however, was required to suppress 100% of generalized seizures. No dose of acetone

could suppress focal seizures kindled from the amygdala.

91

Perc

ent

Seizure

Prot

ecti

on

Figure 23. Acetone Dose-Response for Cortical Seizures

120

100 -

80 -

60 -

40 4

—@— Generalized Seizure

-O-- Focal Seizure

t v t t v T

0 290.4 580.6 1161.6 1742.4 2323.2

Dose of Acetone (mg/kg)

Vehicle alone did not elevate seizure threshold. Acetone was able to suppress 100% of generalized seizures kindled from the cortex at a non-toxic dose (580.6 mg/kg). A 1742.4 mg/kg dose of acetone suppressed 100% of cortical focal seizures. This dose,

however, had toxic effects in some animals.

92

DISCUSSION

Experiment 5 was performed to confirm and extend the important finding of

Likhodii et al. (2003) concerning acetone’s ability to suppress amygdala focal seizures.

It was found that acetone is able to suppress cortical generalized seizures, cortical

focal seizures, and amygdala generalized seizures at non-toxic doses. Acetone did not,

however, suppress amygdala focal seizures. This does not agree with the results of

Likhodii et al. (2003). This will be discussed further in the General Discussion.

Our data do agree with Likhodii et al. (2003) in that acetone was able to suppress

amygdala generalized seizures. Full suppression, however, was only seen using a toxic

dose 2323.2 mg/kg. Likhodii et al. (2003) found suppression of amygdala generalized

seizures at non-toxic doses.

Experiment 5 also demonstrated that acetone has a different EDso in generalized

seizures triggered from the amygdala and the cortex. This is in contrast with Albright &

Burnham (1980), who reported that the traditional AEDs suppress both amygdala- and

control generalized seizures at similar doses. This is also discussed in greater detail below

(see General Discussion).

The results from Experiment 5 only partially support the acetone hypothesis. The

present study demonstrated that acetone is able to suppress generalized and focal seizures

triggered from the cortex. This is consistent with the acetone hypothesis as the KD is

effective at suppressing seizures of cortical origin (Vining, 1999). Our data, however,

suggest that acetone is ineffective at suppressing seizures of amygdala origin at non-toxic

concentrations. This is inconsistent with the clinical literature that suggests that the KD is

effective at suppressing complex-partial seizures of temporal lobe origin (Vining, 1999).

CHAPTER 8: GENERAL DISCUSSION

Each experiment conducted will be discussed in detail, followed by a general

overview.

Experiment 1. A comparison of two KDs in adult rats

Experiment | was designed to measure the ability of two KDs to elevate PTZ

infusion thresholds, relative to controls, in adult rats. These diets were the newiy*

developed 4:1 KD and balCTRL diet, the previously used 6.3:1 KD and stCTRU diet, and

an adlibCTRL diet. We found that the 6.3:1 KD showed a “trend” towards elevation of

threshold doses in adult rats. The 4:1 KD, however, did not elevate threshold doses in

adults.

The finding of a failure of the 6.3:1 KD to elevate threshold doses in adult rats

differs from the previous findings of Bough & Eagles (1999) and Bough et al. (1999).

The 6.3:1 KD, however, did at least show a trend towards elevation of threshold doses.

The formulation of a balanced 4:1 KD used in the present study was based on a

3.6:1 KD used previously (Likhodii et al., 2000) and was designed to accurately model

the 4:1 KD used clinically. In the present study, the 4:1 KD failed to elevate threshold

doses at all in adult rats. Therefore, the question that needs to be addressed is: Why did

the KD — and especially the 4:1 KD — fail in adults?

One possible explanation is that a 4:1 ratio of the KD is not high enough to

produce an elevation of PTZ seizure threshold in rats. It may be that rats require a higher

ratio. Bough et al. (2000), however, have shown that their version of a 4:1 KD was

effective in elevating PTZ seizure threshold, albeit in a different strain of rat (Sprague

94

Dawley). The 4:1 KD used by Bough et al. (2000), however, was not truly balanced in

protein, minerals and vitamins.

A second factor to be considered is levels of ketosis. In the present study, both

KDs were able to elevate blood B-OHB levels in adult rats. Interestingly, the 4:1 KD

produced slightly higher B-OHB levels than the 6.3:1 KD. B-OHB levels, however, did

not rise into the 2-4 mM range, perceived to be the “therapetitic‘raiige” in humans with

either diet (Huttenlocher, 1976). Non-therapeutic levels of B-OHB in adult rats might

explain the failure of both KDs to produce significant anticonvulsant effects.

A third factor to be considered is glucose levels. A recent report suggests that

high blood glucose levels can have proconvulsant effects (Schwechter et al., 2003), as

may very low blood glucose levels (Anuradha et al., 2003). Abnormal blood glucose

levels might have cancelled out the anticonvulsant effects of the KD. In Experiment 1,

there was an initial decrease in glucose levels in animals fed the KDs. Blood-glucose

levels, however, stabilized and remained in the low-normal range throughout the

experiment. This is consistent with previous clinical data (Huttenlocher, 1976) and with

data from animal models (Appleton & DeVivo, 1974; Todorova, 2000; Likhodii et al.,

2000). Altogether, blood glucose levels were neither very high nor very low, and offer no

explanation for the failures of the KD.

It was not clear, therefore, why the KDs failed to be anticonvulsant in Experiment

1. It was decided to repeat the experiment in pups, where larger anticonvulsant effects of

the KD are reported (Bough and Eagles, 1999; Likhodii et al., 2000).

95

Experiment 2: A comparison of two KDs in rat pups

Experiment 2 was designed to measure the ability of two KDs to elevate PTZ

infusion thresholds, relative to controls, in rat pups. We found that the 6.3:1 KD

significantly elevated thresholds doses in rat pups whereas the 4:1 KD did not.

Much of the following discussion is similar to the discussion of Experiment 2. It

is, however, important to revisit certain points.” ~°*"

In rat pups, the 6.3:1 KD significantly elevated threshold doses. This is similar to

the findings of others (Bough and Eagles, 1999; Bough et al., 2000a; Bough et al., 2000b;

Bough & Eagles, 2001; Harney et al., 2002; Thavendiranathan et al., 2003; Eagles et al.,

2003).

In the Experiment 2, however, the 4:1 KD failed to elevate threshold doses in rat

pups. Therefore, the question remains — why did the 6.3:1 KD elevate seizure thresholds

in rat pups whereas the 4:1 KD did not?

One possible explanation is that a 4:1 ratio of the KD is not high enough to

produce elevation of PTZ seizure threshold in rats. Once again, it is umportant to note that

others have reported anticonvulsant actions with a 4:1 KD (Bough et al., 2000), albeit an

unbalanced 4:1 KD.

A second factor to consider is levels of B-OHB. Both KDs were able to elevate

blood B-OHB levels in rat pups. The 4:1 KD, which did not elevate seizure thresholds,

elevated B-OHB levels even higher than the 6.3:1 KD, which did elevate seizure

thresholds. This suggests that B-OHB levels are not clearly related to seizure protection.

This is consistent with the findings of Bough et al. (1999a) and Likhodii et al. (2000).

96

Studies exist that have reported a strong correlation between B-OHB levels and

seizure protection in rats (Bough et al., 1999b). Other studies, however, have shown no

correlation between B-OHB levels and seizure protection (Bough et al., 1999a; Todorova

et al., 2000; Likhodii et al., 2000; Harney et al., 2002). The present study supports the

idea that there is little or no relationship between B-OHB levels and elevation of seizure

threshold. a whe ae

A third factor to be considered is glucose levels. There was an initial decrease in

glucose levels in animals fed the KDs, but not in the control groups. Blood-glucose levels

in the KD groups, however, stabilized and remained in the low-normal range throughout

the experiment. This is consistent with previous clinical data (Huttenlocher, 1976) and

with data from the animal models (Appleton & DeVivo, 1974; Todorova, 2000; Likhodii

et al., 2000). Levels in pups were somewhat lower than levels in adults (Experiment 1).

They were, however, very similar in the 4:1 KD group and the 6.3:1 KD group.

Differences in glucose levels, therefore, cannot explain the failures of the KD to have

anticonvulsant effects.

A fourth explanation is that it is the vitamin, mineral and protein deficiency of the

6.3:1 KD that produces the anticonvulsant effect. stCTRL diet could have caused the

observed elevation of seizure threshold. The fact that elevation of threshold doses was

seen in young developing rats — where protein and micronutrients are critical for healthy

development — and not in adult rats supports this possibility. This idea is further

strengthened by a recent report suggesting that a version of the 6.3:1 KD leads to

impaired growth and brain development (Zhao et al., 2004). This possibility deserves

further investigation.

97

A final possibility to consider, however, is that “threshold dose calculations”

create “anticonvulsant effects” that do not exist (Likhodiit & Burnham, 2003). Large

weight differences exist among the groups by the time of seizure testing (for adult rat

weights, see Figure 4; for rat pup weights, see Figure 8). The KD groups are always

lighter than the control diet groups. This difference was more pronounced in Experiment

2 (rat pups) than in Experiment 1 (adult rats). Also, the difference is larger between the

6.3:1 KD and stCTRL diet groups than it is between the 4:1 KD and balCTRL diet

groups. When calculating “threshold doses”, a large weight difference between the KD-

fed and control diet-fed groups may skew the results. For example, the average weight of

pups fed the 6.3:1 KD was only ~60% that of the average weight of pups fed the stCTRL

diet (63g vs.102g, respectively). Future studies should consider the impact of large

weight differences between groups when using an intravenous infusion seizure test.

Further to this point, seizure latencies in the adults were less than 60 seconds, and

in pups were less than 30 seconds. At this time point, PTZ may be largely confined to the

vascular system and “vessel rich” groups (i.e. brain, liver, and kidney). Therefore,

correcting for body weights by calculating “threshold doses” may be irrelevant (see

Likhodii & Burnham, 2003) (we are unaware of any present PTZ pharmacokinetic data in

an intravenous preparation. Such an experiment is proposed below under Future

Experiments). If, instead of calculating PTZ “threshold doses”, one considers “absolute

latencies” (i.e.: number of seconds of PTZ infusion before seizure onset) then the

anticonvulsant effects of the 6.3:1 KD disappear altogether (see Figure 12, p.57 and

Figure 13, p.58). In fact, the 6.3:1 KD appears to be proconvulsant as compared to the

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control diet fed animals. This is in agreement with the suggestions of Thavendiranathan

et al. (2000), that “partial starvation” may be proconvulsant.

It appears possible, therefore, that the KD is simply not anticonvulsant in rats,

although it is in humans. Before accepting this possibility, however, it must be noted that

studies have reported anticonvulsant effects of the KD in rats in experiments that did not

involve the calculation of threshold doses. There are the studies of Hori et al. (1997) and

Thavendiranathan et al. (2003).

In each of these studies, however, the threshold elevations were small (~20%). In

Experiment 2, it must be noted, four of the groups were calorie restricted, and the KD

groups were lighter than their controls. It is possible that this “partial starvation” in the

KD-fed groups may have produced proconvulsant effects that cancelled out the

anticonvulsant effects of the KD. An experiment to test this possibility appears below

(see Future Experiments).

Why should the KD be anticonvulsant in humans but not anticonvulsant — or only

mildly anticonvulsant — in rats? Recently, Likhodii et al. (2003) have shown that acetone,

a ketone elevated by the KD, has strong anticonvulsant properties. They have proposed

that this elevation in acetone produces the anticonvulsant effects of the KD. Preliminary

data from our laboratory suggest that humans fed the KD develop high levels of acetone

while rats fed the KD do not (Likhodii & Burnham, 2004).

Experiment 3. The Effects of Seizures on Acetone Levels In Vivo

Experiment 3 was performed to determine the effects of seizures on acetone

levels. Results from Experiment 3 demonstrated that both MES and PTZ seizures

significantly lowered acetone AUC values, as compared to sham-stimulated or saline-

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injected controls. This suggested that acetone levels would not be accurate in animals that

had been seizure tested.

It is not clear why acetone AUC values are lower post-seizure. It is possible,

however, that acetone is being used as an energy substrate. Given that the breaking of

carbon bonds fuels cells (Dioguardi, 2004), there is no reason to assume that acetone, a

three-carbon molecule, couldn’t serve as an energy substrate.

A second possibility is that the increased respiration, caused by seizures, increases

the excretion of acetone via the lungs. It is known that approximately 30% of acetone is

excreted through the breath (Haggard, 1944; as cited in EHC, 1998).

Finally, it is also possible that seizures cause an increase in acetone metabolism.

Approximately 70% of acetone is metabolised and incorporated into lipids, amino acids,

and glucose (Haggard, 1944; as cited in EHC, 1998). If seizures, for example, increased

cytochrome P450 2EI activity (CYP2E1), then acetone AUC values could be diminished.

Future studies could use radio-labeled acetone to determine its fate post-seizure versus

post-sham-seizure.

One question that was raised during review of the video tapes was whether the

PTZ subjects actually had seizures. Certainly, none of the PTZ-injected subjects had full-

scale seizures. The 10mM dose of acetone was sufficient to protect subjects, in part, from

PTZ. All of the PTZ-injected animals, however, did experience “wet dog shakes” or

myoclonic jerks. These occur in rats during sub-maximal seizures. Although these

animals did not have a tonic-clonic seizure, they were likely experiencing seizure

activity.

100

Similarly, none of the MES stimulated animals displayed full hind limb extension

after MES stimulation. This suggests that they were also protected by the anticonvulsant

actions of acetone. Nonetheless, all of the MES-stimulated animals did have a clonic

seizure. The simplest way to clarify this situation would be to repeat the study with a low

(non-anticonvulsant) dose of acetone. This is suggested below (see Future Experiments).

A final question is “if seizures lower acetone levels by ~1520%5- why not just

take blood samples post-seizure and add 20% to the level found in the assay?” Such a

procedure would be possible, but only if it were found that the drop is 15-20% at all

acetone concentrations.

In summary, it is important to know whether seizures themselves affect acetone

levels. It is often assumed that post-seizure test acetone levels are similar to pre-seizure

test acetone levels. Experimenters often take blood post-seizure, as the animal is

unconscious and often subsequently euthanized, allowing them to draw more blood. Also,

taking blood before seizure testing could have effects on seizure threshold, altering the

animal’s response to the seizure test. The present study, however, makes it clear that

acetone levels should be taken before seizure testing to avoid artificially low acetone

AUC levels, or employ a separate non-seizure tested group in which blood acetone levels

can be measured.

Experiment 4. Levels of B-OHB, glucose and acetone in rats on the KD/diallyl

sulfide (pilot study).

Experiment 4 was designed as a small study to confirm and expand upon the

results of Likhodii et al. (manuscript in preparation) that the KD is unable to produce

high blood-acetone levels in rats. Furthermore, Experiment 4 served to determine

101

whether blood-acetone levels could be pushed into the “therapeutic range” by inhibiting

acetone’s metabolism via CYP2E1.

Data from the present study confirmed that blood-acetone levels are only mildly

elevated in rats fed a 4:1 KD.

Further, results from the present study demonstrated that acetone levels can be

manipulated. Blood-acetone levels, however, were not:puszed into the therapeutic range

in Experiment 4. As discussed in the Discussion of Experiment 4, DAS might further

elevate blood-acetone levels if given chronically. Future experiments will be designed to

examine this (below).

It should be noted that DAS could have potential clinical applications. It is

possible that patients who fail to respond to the KD are patients who rapidly metabolize

acetone, and who, therefore, have low blood-acetone levels. DAS could be used to

increase acetone levels into the “therapeutic range” in those with non-therapeutic levels.

It is also possible that DAS would allow patients to consume a “less ketogenic”’

diet — i.e. lower fat — diet (e.g. an Atkin’s-like diet). Future experiments will be designed

to examine this possibility.

It should be noted that DAS is a garlic oil extract. It could probably be considered

a food additive, and could be introduced into clinical practice without having to go

through the stages of drug development.

Experiment 5. Acetone Dose-Response in the Kindling Preparation

The purpose of Experiment 5 was to confirm and expand upon the important

finding of Likhodii et al. (2003) concerning the ability of acetone to suppress the kindled

102

amygdala focus at non-toxic doses. This would suggest that acetone could be effective

against complex-partial seizures clinically.

The study was done using a modification of the paradigm of Albright and

Burnham (1980) in which anticonvulsants are tested for their ability to suppress: 1)

amygdala focal seizures; 2) cortical focal seizures; and 3) generalized convulsions

triggered from both foci. PRO,

We found that acetone could suppress cortical focal seizures and generalized

convulsions — but that contrary to our expectations — it could not suppress amygdala focal

seizures even at toxic doses.

The reasons why we failed to replicate Likhodii et al. (2003) are not entirely clear.

Experiment 5 used the same methodology as Likhodii et al. (2003). Further study of this

question will be required.

The results of Experiment 5 were in agreement with the findings of Albright and

Burnham (1980) that focal seizures of cortical origin are suppressed at lower doses of

anticonvulsant drugs than focal seizures of amygdala origin. In Experiment 5, acetone

suppressed cortical focal seizures at non-toxic doses. It is well known that limbic seizures

(e.g. amygdala) are more resistant to AED therapy than cortical seizures.

In contrast with Albright and Burnham (1980), however, the present experiment

demonstrated that cortical focal seizures and generalized seizures may have very different

EDsos. Albright and Burnham (1980) reported that traditional AEDs tend to suppress

cortical focal seizures and generalized seizures at similar doses. They also found similar

EDsos for generalized seizures regardless of kindled site. Experiment 5, however, found

acetone to be more effective at suppressing cortical generalized seizures than it was at

suppressing cortical focal seizures. It was also more effective at suppressing cortical

generalized seizures than amygdala generalized seizures. This further supports the idea

that acetone works by a different mechanism than the traditional AEDs.

In the present experiments, we found that acetone has quite a good TI against

cortical generalized seizures.

Also, the ataxia fésisised in the present experiment is more sensitive to motor

impairment than other, more commonly used tests, such as the rotorod test (Wlaz &

Léscher, 1998). As such, these estimates of TI may be conservative. TI in Likhodii et al.

(2003) ranged from 1.2 to 6.0. Further, a lower dose of acetone would likely be required

to suppress “naturally occurring” seizures in an epileptic brain than would be required to

suppress experimentally generated seizures.

It is known that the KD is effective at suppressing complex-partial seizures

(Kinsman et al., 1992). What is unclear is whether the KD is able to truly suppress focal

limbic seizures in humans. We know the KD suppresses complex-partial seizures, which

are partly generalized. Without the use of depth electrodes, however, it is not possible to

determine whether the KD actually suppresses the focal limbic seizures that trigger them.

Therefore, in terms of the acetone hypothesis, it is unclear whether acetone should be

expected to suppress the kindled amygdala focus. A future clinical study might be

designed to study whether acetone suppresses the temporal lobe “aura” as well as the

complex-partial seizure that follows. The kindled amygdala focus is actually a better

model of the temporal lobe aura than of actual complex-partial seizures (Burnham,

personal communication).

104

OVERVIEW

The initial goal of the present research was to establish an animal preparation that

accurately modeled the effects of the KD seen clinically. We then hoped to use this

preparation to study the KD’s mechanism of action. Once the KD’s anticonvulsant

mechanism of action was elucidated, we hoped that the diet might be reformulated into a

more palatableyhealthy form. Alternatively, the KD could be replaced by a drug of equal

or greater efficacy.

A number of studies have previously been performed on animals fed the KD.

Many of these have reported that the KD has anticonvulsant effects in rats. Likhodii

(2001), however, has pointed out that many of the studies had used a 6.3:1 KD (the

“classic” KD) that was not balanced with its control diet in terms of vitamins, minerals,

and protein. This confounds the anticonvulsant effects seen with the KD, as one cannot

eliminate the possibility that the deficiencies in vitamins, minerals, or protein are

responsible for the observed anticonvulsant effects. Further, the “classic” KD — in animal

studies — has a fat to carbohydrate and protein ratio of 6.3:1, which is higher than any

ratio used clinically.

We employed a carefully formulated, balanced 4:1 KD (Likhodii, 2001) that

accurately models the 4:1 KD used clinically. The 4:1 KD was balanced with its control

diet in terms of micro- and macro-nutrients. We then compared the effects of this 4:1 KD

and the commonly used 6.3:1 KD to their respective controls.

We failed to demonstrate anticonvulsant actions of the 6.3:1 KD in adult rats, but

did demonstrate apparent anticonvulsant actions in rat pups. We did not observe

anticonvulsant effects, however, with the 4:1 KD in either adult rats or rat pups. During

105

<berartifactual in nature. This possibility was strengthened by preliminary data suggesting »

the course of these experiments we began to question the procedure of dividing PTZ dose

by body weights (a.k.a. calculating “threshold doses”) in the PTZi test. If latencies, rather

than “threshold doses” are examined, neither KD appears to have anticonvulsant effects.

Taken together, we concluded that the KD may not be anticonvulsant in rats when

the PTZi seizure test is used, and that the numerous recent findings of Bough et al. may

that the KD produces high levels of acetone in humans, but not in rats (Likhodii &

Burnham, 2004). If the acetone hypothesis is correct, and if acetone is low in rats, then

one would not expect the KD to have anticonvulsant effects in rats. It, therefore, became

necessary to measure acetone levels in our preparation.

An initial question was whether it would be valid to measure acetone after seizure

testing — the most convenient time. Therefore, we designed an experiment to determine

whether seizures affect acetone levels (Experiment 3). We found that they do.

We then ran a small study to determine acetone levels in rats fed the KD. We

found that acetone levels are indeed low in our preparation. We then inhibited the

metabolism of acetone by administering the cytochrome P450 2E1 (CYP2E1) inhibitor,

diallyl sulfide. This caused acetone concentrations to increase to ~0.6mM, which is still

likely sub-therapeutic. Further studies are currently being conducted to determine

whether DAS can elevate acetone to therapeutic levels in rats fed the KD.

Taken together, this research has revealed critical problems with a generally accepted,

commonly used rat preparation for modeling the anticonvulsant actions of the KD. At the

same time, it generally supports the idea that acetone plays a role in the anticonvulsant

actions of the KD.

106

FUTURE EXPERIMENTS

1. Should we be using “threshold dose” calculations or “seizure latencies”?

As demonstrated in Experiments 1 and 2, the KD’s ability to elevate “seizure

threshold” is dependent on how one measures threshold elevation. If one uses “threshold

dose” calculations, then the 6.3:1 KD appears to significantly elevate seizure threshold in

pups. If one uses “seizure latencies”, then the 6.3:1 KD appears to significanthydecrease

seizure threshold (1.e., it has significantly proconvulsant effects). There are no present

pharmacokinetic data available on PTZ distribution using an i.v. preparation. We plan to

study PTZ concentrations in fat tissue, muscle tissue, and brain tissue in animals after

PTZi testing. We are currently developing an HPLC assay to measure PTZ in these

tissues. If PTZ concentrations are similar in the brain tissue of heavy animals and light

animals after a standard dose, then “seizure latencies” should be used, and not “threshold

dose” calculations. If, however, PTZ concentrations are different in the tissue of light and

heavy animals after a standard dose, then “threshold dose” calculations should be used,

and not “seizure latencies”.

2. Do seizures affect in vivo acetone levels?

As in Experiment 3, this study would examine whether seizures decrease blood-

acetone levels in rats. As in Experiment 3, we would employ the scPTZ and MES seizure

tests. Unlike Experiment 3, we would use a sub-therapeutic dose of acetone (e.g. 1mM).

This would not protect animals from full scale seizures. We hypothesize that full scale

seizures will have an even greater impact on blood-acetone levels, than the sub-maximal

seizures observed in Experiment 3. Measurements will involve several difference

107

concentrations. If the drop is always ~15-20%, then this drop could be used as a

“correction factor” in future studies that examine blood-acetone levels post-seizure.

3. Does chronic administration of DAS further elevate blood-acetone levels?

Experiment 4 showed that DAS could be used to elevate blood-acetone levels.

Acute administration of DAS, however, did not elevate acetone into what is thought to be

the “therapeutic range” for experimentally induced seizures (1-e22<4mM, see Likhodii et

al., 2003). It is unknown, however, whether chronic administration of DAS would further

elevate blood-acetone levels in rats fed a KD. This experiment would be performed to

determine whether blood-acetone levels could be pushed into the “therapeutic range” in

rats fed a 4:1 KD. We hypothesize that chronic DAS administration may elevate blood-

acetone levels beyond those seen after acute administration of DAS. If we can push

acetone into the “therapeutic range”, we could then seizure test the animals to determine

whether they have elevated seizure thresholds.

4. DAS supplementation in patients on the KD

DAS could be given to children on the KD as a food additive, without having to

go through the various phases of drug development. DAS could be used for two reasons.

It could push acetone into the “therapeutic range” in patients that haven’t otherwise

developed therapeutic levels of acetone. It could also be used in patients who have

developed therapeutic levels of acetone, potentially allowing them to consume a less

rigorous diet (e.g. a higher protein, lower fat diet). This experiment could be performed in

conjunction with the clinical study (see below).

108

5. Does acetone correlate with seizure protection in children on the KD?

We hypothesize that plasma and urine acetone concentrations will correlate highly

with seizure protection in children on the KD. Toronto’s Hospital for Sick Children hosts

Canada’s largest ketogenic diet program for children. Meetings have taken place between

our lab and the head of the ketogenic diet program. The program’s head is excited about

collaborating on a study examining the acetone hypothesis. Children are required to

attend monthly check-ups where a physician takes blood and urine samples. These blood

samples would be used to determine acetone levels in blood and urine. Guardians of

patients on the KD are asked to keep detailed log books, recording the frequency and

severity of the patient’s seizures. Acetone levels obtained from urine and plasma samples

would be correlated with seizure frequency data given by the guardians. The Ketogenic

Diet Program at Toronto’s Hospital for Sick Children sees approximately 25-30 new

children every year. These children, upon admittance to the program, are required to stay

on the KD for 6 months unless a medical emergency requires them to quit the KD. As

such, experimental attrition would not be a large concern with this present study.

6. What is acetone’s anticonvulsant mechanism of action?

It is unclear how acetone exerts its anticonvulsant effects. We plan to study

acetone’s effects on ion flux in vitro. First, we would need to demonstrate that acetone is

“anticonvulsant” in vitro. To determine this, we would examine acetone’s ability to

suppress “kindled” seizures in repeatedly tetanized hippocampal slices. If acetone is

shown to have anticonvulsant properties in vitro, we would continue to study acetone’s

effects on ion channels by examining extracellular and intracellular electrophysiological

recordings from rat hippocampal brain slices during perfusion with acetone.

109

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