A NON-INVASIVE DIAGNOSIS OF MALIGNANT HYPERTHERMIA USING 3'P

NMR SPECTROSCOPY

Baldev S. Ahluwalia Department of Medical Biophysics

A thesis submitted in partial fulfillment of the requirements for the degree of

Masrers of Science

Fanilty of Graduate Studies

The University of Western Ontario

London, Ontario

December 1997

Baldev S. Ahluwalia 1998

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ABSTRACT

Malignant Hyperthermia (MH) is a potentially fatal skeletal muscle disorder

induced by certain anaesrhetics. The present method of diagnosis entails a six cm

muscle biopsy and subsequent in vitro contracture tests. The purpose of this study was

to develop a non-invasive test to diagnose MH based on differences in muscle

metabolism exposed during an exercise protocol and monitored with phosphoms

nuclear magnetic resonance spectroscopy CIP MRS).

Enrolled in this study were 11 controls (3513 years) and 25 M H susceptible

patients diagnosed by muscle biopsy: 9 patients with an HCK-MH diagnosis (39+4

years), and 16 from a less susceptible (mixed-MH) group (43 13 years). The exercise test

consisted of wrist flexion at a progressive work rate which continued to volitional

fatigue, o r when the phosphocreatine ( K r ) concentration was reduced by 80% from

resting values. The post exercise recovery kinetics of PCr and p H were modeled with a

mono-exponential funcrion and characterized by time constants rPCr and rpH

respectively. The calculation for T ~ H started from the minimum value of pH post

cessation of exercise with the delay noted as the pH lag time.

The HCK-MH group had a longer pH lag tirne than controls (6917 seconds

verses 45 16 seconds, p < O.O5), and a longer rPCr (1621 14 seconds verses 9415

seconds, p < 0.05) when corrected for end exercise pH. There were no significant

differences between the less severe M H group and controls during recovery.

Discriminant analysis between the HCK and control subjects lead to complete

delineation between the groups (ANOVA, p~0.001) . The use of "P MRS as a

screening tool for MH susceptibility could reduce the number of patients who require a

muscle biopsy and thus decrease the morbidity associated with diagnosing the disease.

ACKNO WLEDGMENTS

Completion of this thesis was o d y possible with the cooperation, and assistance

of a number of people. I am indebted to my primary supervisor Dr. Terry Thompson

for his unending patience and support throughout this project, especidly during the

writing of this manuscript. Also, I thank him for providing an atmosphere where there

are no 'black boxes", and where the answer ro any question can be pursued. I greatly

appreciate the help of my CO-supervisor Dr. Gregory Marsh, and the rernaining

memben of my thesis advisory cornmittee, Dr. Dick Drost, and Dr. Stephen Dain.

I am grateful to dl the Malignant Hyperthermia susceptible patients who

volunteered for this study. Their infectious enthusiasm helped drive this projea dong.

Special rhanks are extended toward John Potwarka for his humour, friendship,

and expert cornputer skills, ail of which helped make this project manageable.

Ir is wirh sincere gratitude chat 1 thank my girlfriend, Cynthia Maier, and my

parents. Their encouragement, patience, and emotional support helped me through to

the completion of this degree.

TABLE O F CONTENTS

. . CERTIFICATE OF EXAMINATION .................... .......... .................................... 11

TABLE OF CONTENTS ....................................................................................... v

. . ................................................................. LIST OF FIGURES ................... ..... vil

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

LIST OF ABREVIATlONS ................................................................................... x

CHAPTER 1

INTRODUCTION .................................................................................................... 1 1.1 Pathophysiology of MH and Elective Diagnosis ..................................................... 2

1.1.1 The in vitro Caffeine Halothane Contracture Test ......................................... 4 1.1.3 A Genetic Link ............................................................................................... 6

........................................................................................... 1.2 Ca" Regulation in M H 8 1.3 Phosphorus Nuclear Magnetic Resonance Spectroscopy:

a Diagnostic Tool .................................................................................................. 11 1.4 Research Goal ....................................................................................................... -14 1.5 Thesis Outline ...................................................................................................... 1 5

CHAPTER 2

NMR PRINCIPLES AND APPLICATIONS ............................................................ 16 2.1 Introduction .................................................................................................... 16 2.2 NMR Principles ................................................................................................. 1 6

2.2.1 Quantum Phenornena .................................. ....... ........................................... 17 . . 2.2.2 Classical Description ...................................................................................... 18 . . 2.2.3 Spin Exciracion ................... ..... ................................................................ -20 2.2.4 Relaxation ...................................................................................................... 21 2.2.5 C hemicd S hift and Spin-Spin Coupling ........................................................ -22 2.2.6 Determination of pH ...................................... .. ............................................. 25 2.2.7 Imaging: Spatial information .......................................................................... 25

2.3 NMR in Practice .................................................................................................... 26 2.3.1 Surface Coils ................................................................................................... 26 . . 2.3 -2 Adiabatic Excitation ................,................................................................ 27

CHAPTER 3

METHODS ............................................................................................................ 1 3.1 Wnst Mode1 ........................................................................................................... 31 3.2 Exercise Ergorneter ................................................................................................ 31 3.3 Experimental Methods .......................................................................................... .32

........................................................................................... 3.3.1 Exercise Protocoi 33 .................................................................................... 3.3.2 NMR data acquisition 34

......................... ...................*................-...-............*......... 3.3.3 Data Andysis ..... 35

CHAPTER 4

RESULTS ................................................................................................................... -38 4.1 MRI Guided Positioning ....................................................................................... 38 4.2 "P Spectroscopy ................................................................................................... -39

4.2.1 At Rest and During Exercise .......................................................................... 39 .................................................................................................... 4.2.2 In Recovery 42

4.2.3 Corrections to d'Cr ....................................................................................... 47 . . . 1.3 Discriminant Analysis ............ ... .......................................................................... 48

DISCUSSION. SUMMARY. AND FUTURE WORK ............................................. 50 3.2 Summary ............................................................................................................. - 5 7 5.3 Future Work .......................................................................................................... 58

APPENDIX A ............................................................................................................... 60

TRANSORMATION OF A TMR MAGNET iNTO A CLINICAL IMAGING UNIT .................................................................................. -60

...................................................................... ................................ APPENDIX B ... 65

.............................................. IMPROVEMENTS IN SIGNAL TO NOISE RATIO 65

BIBLIOGRAPHY ........................................................................................................ 66

VITA .............................................................................................................................. 75

LIST OF FIGURES

Page Number

Figure 1-1: Excitation contraction coupling and Ca2+ regdation during muscle

contraction ......................................................................................................... 9

Figure 2-1:

Figure 2-2:

Figure 2-3:

Figure 2-4:

Figure 2-5:

Figure 2-6:

Figure 2-7:

Figure 3-1 :

Figure 3-2:

Figure 4-1:

Figure 4-2:

Figure 4-3:

Figure 4-4:

Figure 4-5:

Figure 4-6:

Figure 4-7:

Figure 4-8:

Figure 4-9:

The energy states and precessiond frequency of a spin-% nucleus ................. 18

Spin population distribution .......................................................................... 19 . . . . ........................................................... Classical descnption of spin excttarion 20

....................... Example of discrete Fourier transform ...................... .... 23

Chemical structure of ATP showing the a. P and y phosphorus nuclei ........ 24

Effective magnetic field dunng adiabatic excitation .................................... 28

Cornparison of signal intensities between the 3ms secldtan adiabatic

pulse and optimized 30 ps hard pulse ............................................................ 29

Exercise ergometer ........................................................................................ 3 2

Experimetal setup for phosphonis spectroscopy ............................................ 34

Axial gradient echo proton image of forearm flexor muscles ........................ 38

Exarnple of "P speara acquired at rest and end of exhaustive exercise .......... 40

Typical changes in log(Pi/PCr) and pH as a funaion of

work rate ........................................................................................................ 41

Typical changes in pH as a funaion of rime throughout the

exercise protocol ........................................................................................... -43

Typicai pattern of change in the recovery kinetics of Pi .................... .. ...... 4-4 Typical pattern of change in the recovery kinetics of PCr ............................ 45

Kinetics of ADP concentration post exercise ................................................. 47

Correlation between end exercise p H and recovery time

constant of PCr .............................................................................................. 48

Two parameter discriminant analysis for group comparisons ....................... 49

Figure A-1: Field mapping trajeaory and gradient uniformity plots ............................... 62

Figure A-2: Gradient echo images of a cylindrical grid phantom .................................... 63

vii

........................................................... Figure A-3: D-O Gradient localized spectroscopy 63

.................................... Figure A4: High resolution magnetic resonance image of f iger 63

.................................................. Figure B-1: Speara showing reduaions in sysrem noise 65

LIST O F TABLES

Page Number

Table 1-1: Sensitivity and specificity of the in vitro Caffeine Halothane

............................................................................................ Contracture Test 5

Table 4-1: Phosphate metabolite concentrations and intracellular pH

.............................................................................. of resting skelerd muscle 39

Table 4-2: Summary of changes in phosphate metabolites and pH during exercise ..... 41

Table 43: Summary of the merabolic state of muscle at the end of exercise ................ 46

.......... Table M: Summary of the metabolite and pH recovery kinerics post exercise 16

LIST O F ABREVIATIONS

ADP adenosine diphosp hare

adenosine triphosphate ATP

ANOVA

applied natic magnetic field

oscillating magnetic field generated by a radio frequency pulse

effective magnetic field

muscle contracture caffeine the CHCT increased

CHCT in vitro caffeine halothane contracture test

creatine kinase

caffeine specific concentration CSC

CSC-H caffeine specific concentration in the presence of 1 O/O halot hane

dihydropyridine receptor

discriminant value

FCU

FDP

flexor carpi ulnaris

flexor digitorum profundus

flexor digitorum superficidis

free induction decay

FDS

FID

FOV field of view

full width at half maximum

Increased muscle contracture to Halothane

hydrogen or proton

Plank's constant

Plank's constant divided by 271

spin angular momentum qUanNm number

inositol 1-4,5 triphosphonate

increased muscle contracture to caffeine and halothane

J k

K

M

MH

MHS

m MRS

NMR

nOe

PCr

PDE

PH

PH 1%

Pi

PME

PPM

RF

RYR

SNR

T,

T2

T',

X' y '2'

BI

joules

Boltzmann's constant (1.38 x 10'~ J/K)

Kelvin (unit for absolute temperarure)

net magnetization of the spin system or

magnitude of the transverse component

Malignant Hypenhennia

Malignant Hyperthermia susceptible

sample

of M

nuclear magnetic resonance

nuclear magnetic resonance

nuclear magnetic resonance

nuclear Overhauser effea

p hosp ho rus3 1

p hosp hocreatine

p hosp hodiesters

spect roscopy

concentration ot protons expressed as -log [H']

tirne to minimum pH pon cessation of exercise

inorganic phosphate

p hosp homonoesters

parts per million

radio frequency

the ryanodine receptor calcium channel

signal to noise ratio

longitudinal, or spin-lanice relaxation

transverse, or spin-spin relaxation

tirne constant. of signal decay in an FID

rotating frame of reference co-ordinate system

concentration of metabolite X

gyromagnetic ratio

tip angle

chernicd shift in PPM

the post exercise time constant of PCr] recovery

the pon exercise t h e constant of pH recovery

the post exercise time constant of pi] recovery

frequency

Larmor frequency

angular frequency

Larmor angular frequency

anguiar frequency of B, field

INTRODUCTION

General anaesthetics and muscle relaxants are required for most surgical

procedures. For the 1 in 15 000 people affected with the hereditary disorder

Mdignant Hypert hermia, the administration of t hese agents c m have life-t hreatening

rather than life-saving consequences. The exposure of a Malignant Hyperthermia

(MH) susceptible patient to a triggering anaesthetic can cause uncontrolled skeletal

muscle rigidity, accompanied by tachycardia, respiratory and metabolic acidosis, and

hypenhermia wenborough, 19621. This situation is potentially fatal if not quickly

recognised and treated. However, it c m be avoided with the use of alternative non-

triggering anaesthetics if susceptibility to MH is known prior t o surgery. There are

few, if any, day-to-day symptoms of the disease which could forewarn the

anaesthesiologist of a possible M H episode. Identification of susceptible patients

depends upon knowledge of previous adverse anaest hetic reactions in family history ,

and accurate diagnosis. At present, the most widely used and clinically trusted

method of diagnosis requires a k m to 7cm muscle biopsy, usudly taken from the

thigh, and subsequent in vitro contracture tests. The biopsy leaves the patient with

long term muscle weakness and an unsighrly scar. The ~rocedure also requires one to

two days of hospitalisation, up to a week's absence from work, and carries with it the

risks, costs, and inconvenience of any surgical procedure [Britt 19911. Though there

is rnorbidity and risk associated with this method of elective diagnosis, the procedure

has undoubtedly saved many lives and continues to be ~er formed f r equen t l~ with 150

patients being tested in Ontario last year alone [Loke, 19971.

To reduce the morbidity associated wirh this merhod of M H diagnosis,

alternative noninvasive techniques for detecring the disease have been explored. The

majority of proposed alternatives have been based on a simple blood test to search for

erythrocyte abnormalities [Lee 19851, serum protein concentration imbalances

penborough 19701, o r hereditary mutations in genetic code WcLennan 19921. T o

date these methods have had linle success in nearing the sensitivity of the in vitro

contracture test. In vivo methods of monitoring skeletal muscle function through

electromyography monno 1974, Konno 19761 or twitch testing [Quinlan 19891 have

also not proven valuable for diagnosis.

The skeletal muscle me~abolism of M H susceptible patients has previously

been examined during an exercise protocol with phosphonis nuclear magnetic

resonance spectroscopy CIP MRS) [Webster 19901. The advanrage of in vivo "P MRS

is that it can provide a nrar continuous monitor of bioenergetics without affecthg

the tissue being observed. In Webster and CO-workers' study, significant differences

were found between a group of normal controls and M H susceptible individuals

(diagnosed by the in vitro contracture test) during and after exercise. Their work

served well to prove that M H could be detected using "P MRS, but unfortunately,

the NMR technology that was implemented at that tirne limited the exam's further

application as a diagnostic tool.

In the present study, 1 have implemented improved methods of MRS data

acquisition and data analysis to quantify the changes in metabolism. In addition, 1

have used a modified exercise protocol to bener elucidate the physiological

differences previously re~orted. If this "P MRS test is successful in differentiating

between controls and an M H population, it can then be used to reduce the morbidity

associated with diagnosing the disease by reducing the number of patients who

require the muscle biopsy.

1.1 Pathophysiology of MH and Elective Diagnosis

Most of what is known about the pathophysiology of M H has resulted from

work on animal models. In 1966, Hall reponed that an MH episode was induced in

certain breeds of pigs by administering halothane or succinylcholine - the most

potent of triggering agents. The porcine and human syndromes are virtually

identical with regards to changes in vital signs, metabolism, acid-base balance, and

muscle tone. Using the animal mode1 as a basis, it was derermined that the skeletal

muscle rigidity of an M H episode stems from an inability to control intracellular

calcium homeostasis [Gronert 19771. Free intracellular calcium is an intrinsic

regulator of muscle contraction, and it is thought that a triggering anaesthetic causes

calcium to continually difhise from its intracellular storage organelle, the

sarcoplasmic reticulum. Continual flooding of the cytoplasm with calcium causes a

dramatic increase in muscle metabolism due to the constant contractions. When the

body cannot meet the resultant oxygen and energy demands, the situation leads

towards lactic acidosis and eventud ce11 membrane breakdown wit h subsequent

hyperkalemia and ventricular fibrillation. During this reaction, the constant

urilization of energy can cause the body temperature to rise one degree Celsius every

fifteen minutes, with some case reports recording a final body temperature in excess

of 44 degrees [Gronert 19941.

Until the mid 1970s, the anaesthesiologist was limited to treating an MH

attack by trying to alleviate the symptoms of an episode. Many methods were

attempted in the pressures of the operating room with the most frequent being the

application of ice packs to lower the body temperature and use of vasodilators to

expedite heat loss. This method of treatmenr resulted in an 80% death rate [Britt

19911. With the advent of sodium dantrolene in 1975 an M H patient's prognosis

drarnaticall~ im~roved, as did our understanding of the disease [Harrison, 19733.

Sodium dantrolene reduces the flux of Ca" from the sarcopiasmic reticulurn in both

the normal and M H episode States. Initially the drug was available only in an oral

form and was administered prior to surgery as a preventative measure.

Unfortunately, the side effects of administering oral sodium dantrolene at a dose that

could prevent an M H episode included nausea, severe headaches, muscle weakness,

and vomiting [Wedel, 19951. This p ractice of pre-operative administration was

discontinued in the early 1980s when sodium dantrolene became available in an

intravenous form. Presently, its main use is in the operating room where it is the

only known antidote to an M H episode.

By the lare 1980s, fewer chan 10% of M H susceptible individuals died from

anaesthetic complications, and the present-day rate of rnortality is even less [Britt

19911. Along with the advent of sodium dantrolene, the main reasons for the

decrease in the number of anaesthetic related deaths are improved pharmacology and

pharma~olo~ical practices, and equally importantly, knowledge of an individual's

su~ceptibil i t~ to M H through family history and elective diagnosis.

Awareness of a patient's susceptibility to MH is paramount to avoiding

anaesthetic complications, unfortunately, there is currently no "gold standardn for

diagnosis. Even the absence of an M H episode during triggering anaesthesia does not

preclude the possibility of having the disorder because some patients who have

undergone as many as thirteen operations without an incident have subsequently had

a reaction [Puschel 19781. To date, most research direned toward improving M H

diagnosis has been focused in two areas: either on improving the in vitro caffeine

halothane contracture test (CHCT), or searching for a genetic link in the disease.

Z.I. 1 The in vitro Caffeine Halothane Contracture Test

The development of the CHCT was initiated by Britt [1971, 19681 and

colleagues [Ellis 1974, Moulds 19741, and has been used extensively to diagnose M H

since adopted by the North American Malignant Hypenhermia Association in 1974.

This ~a infu l and stressful method of diaposis requires a large muscle biopsy and

subsequent in vitro contracture tests. The following is a brief summary of the

CHCT method. Under a local anaesthetic, a 5cm-7cm saxnple is removed from a

large rnixed type I/type II fibre muscle, usudly from the gracilis o r pectoralis major

muscle. The evcised muscle is dissected into small strips (fascicles) which are rhen

individually secured to an isometric force transducer. Each fascicle is given an initial

tension of 10-20 mN and then stimulated with an electrode induced pulse every 5

seconds. Caffeine, an agent known to inhibit calcium uptake into the sarcoplasmic

reticulum [Caputo 19661, is added directly to the muscle bath in increments from 0.5

to 32 mM. The fascicle contracture level is recorded four minutes after altering the

caffeine concentration. The procedure is repeated starting at O mM caffeine in the

presence of 1% Halothane. The dose of caffeine required to increase the contracture

by 10 m N is known as the caffeine-specific concentration (CSC). When performed in

the presence of halot hane, the required caffeine

The C H C T differentiates patients into

concentration is known as the CSC-

three categories of increasing MH

su~ce~tibility: K, CK, and HCK. The H represents increased contracture in the

presence of 1% halothane alone, C represents a lower than normal CSC and CSC-H

but no halothane contracture, and in the K diagnosis subjects have a lower than

normal CSC-H value. The categories of M H susceptibility are only used to delineate

possible phenotypes of the disease and patient management in the operating room is

independent of whether the result was a K, CK, or HCK.

The sensitiviry and specificity of the CHCT were evaiuated by Brin Cl9911 in

a study comparing the results from a sample of M H susceptible individuals and

normal controls. MH-positive individuals were subjects who had had a reaction to

an anaesthetic characterised by fever, skeletal muscle rigidity, tach~cardia, and

metabolic acidosis. Normal controls were those individuals who have never had a

M H reaction to ii triggering anaesthetic, nor were aware of any in their family

history. The test characterisation was based on a population of 275 individuais (170

controls and 105 MHS) and the results are summarised in Table 1-1.

Specificity Sensitivity TP FN FP TN - - - - . - - . - - -- -

HCK 1 O0 17.5 20 85 O 170

CK 98.8 53.3 56 49 2 168

K 3 5.9 97.1 102 3 1 09 61

Table 1-1: Characteristics of the Caffeine Halot hane Contracture Test: TP = true positive, TN = true negative, FP = faise positive, F N - faise negative. Specificity - TN/TN+ FP, Sensitivity - TP/TP + F N [revised from Britt 19911

Britt's study showed that an HCK diagnosis is very specific (lOOO/o) and a

positive response leaves no doubt of an patient's susceptibility to MH, however, i t is

not very sensitive ( 1 7 ~ ~ ) and many subjects who have the disease would report a

negative result. At the other extreme is the K category which has a high sensitivity

(97%) but approximately 50% of the individuals in this category are false positives.

The caffeine concentration for a positive K diagnosis was set to provide a high

sensitivity at the expense of reduced specificity because: 1) failure to detect M H may

result in potentially severe or fatal outcornes 2) ability to detect the disease leads t o

effective prevention or treatment, and 3) false-positive results do not produce

sipificant emotional or economic harm to the patient [Larach 19931. The

consequence of a positive diagnosis is that surgery is limited to major medical centres,

ernergency care can be compromised, and anaesthesia in combination with

respiratory or cardiac ~roblerns is more complicated.

The reason that the C H C T characteristics Vary so substantially among

diagnostic caregories stems from rhe heterogeneity of the disease. M H is thought to

be an "umbrella" diagnosis containing many different myopathies which present

themselves in the same manner. Even though the test is the most sensitive method of

detecting M H to date, great care must taken when using it as the "gold standardn for

developing alternative methods of diagnosis due to the possible high number of false

positive individuals. Nevercheles, because it is the most accurate of the existing

diagnostic techniques, the CHCT has been widely used as a standard to test

alternative techniques and also to probe the pathophysiology of the disorder. The

resulting improved understanding of the pathophysiology of M H has narrowed the

scope of search for possible genetic mutations by pointing t o specific proteins

involved in the myopathy.

1.1.2 A Genetic Link

The first research into the hereditary nature of M H was reported by

Denborough El9601 in a paper titled "Anaesthetic deaths in a family". After tracing

the medical history of a family, Denborough considered M H to be due to an

autosomal dominant gene (or genes). Unfortunately, a full family history could not

be completed because the only method of diagnosis at the time was the observation

of a MH reaccion during or following surgery. Consequently, a number of

susceptible individuals probably escaped detection. With the use of the CHCT, Britt

et al. Cl9681 detailed the hereditary patterns of the disorder in a French-Canadian

family and confirmed Denborough's findings.

Furcher advances in the genetic research of M H stemmed from the porcine

model. Biochemical studies into the hereditary nature of the disease led McLennan et

al. [1992] to ~ u b l i s h a breakthrough paper in which they reported the discovery of a

mutation in the gene which transcribes the ryanodine recepror (RYR) (Figure 1-1).

The RYR is a channel on the sarcoplasmic reticulum which regulates Ca" efflux into

the cytosol in response to neuromuscular signalling. The discovery of this mutation

provides a definitive diagnosis in MH susceptible swine; however, when a similar

mutation was discovered in the human syndrome, it was present in only 3O/0-7% of

susceptible individuals [McLennan 19921. The present lack of sensitivity of molecular

genetics as a preoperative screening test for MH limits its use to a few well-

characterised families. Although it is highly probable that additional mutations

causative for M H will be identified, including additional problems with the RYR

[Fruen 19951, the heterogeneity of the disease will eventually determine the extent

that genetic tests play as an alternative to the CHCT.

Biochemistry- and physiology-based analyses of human M H provide valuable

information for the development of alternatives to the invasive CHCT while helping

direct the search for additional generic defects. Unfonunately, progress has been

hampered by the limited amount of available material from human muscle biopsies.

Difficulties in performing membrane preparations and biochemical measurements on

the small amounts of tissue obtained, and the poor quality of the samples, have also

added variability to the results. Additional problems arise from the apparent

heterogeneit~ of the human disorder and high false positive rate of the CHCT, both

of which are absent in the porcine model. Despite these difficulties, there have been

several significant differences found when comparing M H and control populations.

The studies important to my work show that there is insufficient regulation of Ca2+

from the sarcoplasmic rericulum even in the absence of triggering anaesthetics. These

results help explain why M H could be diagnosed using "P MRS.

1.2 Ca2+ Regulation in MH

The calcium concentration within skeletal muscle varies dramatically between

the qnosol (O. lmM), intracellular organelles# , and extracellular space (1 mM) . Increases in the cytosolic free calcium concentration dictate the tension and duration of

muscle contraction and chus are an intrinsic regulator of ce11 metabolism. Muscle

concraction results from the conversion of chemical potential energy, stored in the

form of adenosine triphosphate (ATP), to mechanical motion. Under normal

physiologic conditions, contraction is initiated when an action potential from the

central nervous syaem propagares dong the sarcolemma and into the T-tubules where

it causes t he voltage de pendent t ransmem brane dihydrop yridine @HP) recept ors to

change state (Figure 1-1). Coupling from the DHP channels briefly opens the RYR

channels on the sarcoplasmic reticulum. Once open, the RYR channels allow Ca" to

diffuse down its concentration gradient into the cytosol where the ion binds to the

aain-bound protein troponin-C. Only when CaL+ is bound to troponin-C can there be

a crossbridge formation between the actin and myosin filaments. The globular heads of

the myosin filament have an enzymatic site for binding and h~drolysing ATP. The

energy released from ATP hydrolysis is used to torque the myosin head while it is in

contact with actin, chereby increasing the overlap between the two filaments and

causing mechanical motion [Huxley 19711. Dissociation of the two filaments requires

the reformation of the myosin-ATP cornplex. Cycling between cross bridge

formation, movement, and dissociation continues until the cytosolic free [Ca"] is

decreased to resting Ievels mainly via active transport into the sarcoplasmic reticulum

by Ca2+-ATPase. The high density of Ca2+-ATPase channels which lie adjacent to the

myofibrils make the sarcoplasmic reticulum the most important regulator of

int racellular calcium homeostasis.

Mean intncdular Ca2* concenvauon is 1.5 mM with majority bound to protebu or containcd in orgaaelIes.

excitation excitation Ca'+ movement

relaxation Troponin- C A&

w

Figure 1- 1 : Major regdators of cytosok calcium during excitation and reIaxation. Couyling [rom the voltage dependent dihydropyridine recepton (DIT) cause Ca" to diffuse through the ryanodine receptor channeis of the sarcoplamsic reticulum. Active transport of Cal' back inco the sarcoplasmic reticulum is via G2'-ATPase.

In the human M H population there is Iittle evidence t o indicate a significant

incidence of Ca2+-ATPase defects. The first study to examine isolated M H

susceptible (MHS) and normal human sarcoplasrnic reticulum preparations reported

no statistical difference in Ca" uptake [Kalow 19701, however, a subsequent article

from the same group reported that MHS Ca" uptake was 60% of that of controls

[Britt, 19731. Nelson et al. [1987] examined CaL+ uptake into the sarcoplasmic

reticulum in a large number of MHS and normal humans and found no significant

difference between the two groups with regard to their sequestering ability o r CaL+-

ATPase content. Other reports of a decreased rate of Ca" transport into the SR

[Mabuchi 1978, Allen 19861 did not distinguish arnong a decreased rate of pump

activity, an increased membrane leakiness, or an increased rate of Ca2+ eMux through

the Ca2+ release channel. A review article by Michelson [1996] suggests that most

reports of a significantly decreased rate of Ca" uptake activity into sarcoplasmic

reticulum of human M H are due to preparation-induced artifaas.

The release of free calcium from the sarcoplasmic reticulum via the RYR is

not only regulated by the DHP receptor (Figure LI), but also by two second

messenger systems: one involving free Ca", and the other inositol 1,4,5-

triphosphonate (IPJ. Nor every RYR is directly coupled to a DHP receptor and it is

thought that these second messenger systems are responsible for opening the

uncou~led channels. Endo et al. [1983] demonstrated that single fibers prepared from

an MHS patient had a greater chan normal rate of RYR Ca2+ release in the presence

of varying st imulatory Ca2+ concent rations. The half maximal sr imulating

concentration for Ca"-induced-Ca" release from these fibers was 3.0 pM and 5.4 pM

for MHS and normal fibers respectively. The rate of single-fiber Ca2+-induce-

Ca2+release has since been examined in an 84 patient study [Kawana, 19921. Kawana

et al. reported that the rate of release was strongly correlated wirh the severity of the

MH symptoms incurred; patients with accelerated Ca2+ release had a significantly

greater fever during anaesthesia and a significantly lower arterial p H than subjects

with lower release rates. 0rding [1991] reported that above a certain concentration,

free cytosolic Ca2+ inhibits rather than stimulates its release from the RYR. The

threshold for inhibition is higher in the M H ~ o ~ u l a t i o n than for normal controls,

which may explain why an anaesthetic induced episode can become malignant.

The evidence favouring IP, as a second messenger in CaL+ release from the

sarcoplasmic reticulum is two fold. Firstly, its concentration in the cytosol increases

rapidly after stimulation of the muscle fibre, and secondly, the application of very

low concentrations to the sarcoplasmic reticulurn cause free Ca" release. Micro

injection of IP, increased cytosolic Ca" in intact skeleral muscle from MHS swine

with higher potency and efficacy than in muscle from non-susceptible swine [Lopez,

19951. Omission of extracellular Ca" did not modify the responses, but sodium

dantrolene was able to prevent the IP, induced reactions. When examined in a 34

patient human study, the IP, concentrations were significantly higher in MHS muscle

than in normal controls [Wappler, 19961, which implicates it in the pathophysiology

of MH.

The in vitro studies on the second messenger systems of Ca2+ release from the

RYR were al1 performed in the absence of any anaesthetics in the sample bath. A

continuation of these myopathies in vivo could result in malfunctions of both

contraction and metabolism of M H skeletal muscle outside of the operating room.

These rnalfunctions may manifest themselves as the transient symptorns experienced

by some M H patients, and include an increased susceptibility to: exertional hear

stroke, muscle cramps, undue fatigue, and spontaneous muscle twitches [Britt 19911.

To determine if disniprions to calcium homeosrasis was the b a i s of these symptoms,

Lopez [1990, 19941 performed single and multi-ce11 microelectrode analyses on intact

muscle biopsies to mesure the cytosolic Ca" concentration. He revealed an

increased resting concentration of cytosolic free calcium in a group of M H

susceptible individuals as compared to normal controls. Since Ca" regulation is

intrinsically tied to metabolism, the evidence for increased resting cytoplasmic free

Ca" levels together with the aforementioned symptorns indicate that an assessrnent

of skeletal muscle metabolism could provide a basis for an elective diagnostic

technique. Such a technique, if noninvasive, could replace the C H C T and would

result in greatly decreased patient morbidiry from MH diagnosis.

1.3 Phosphoms Nuclear Magnetic Resonance Spectroscopy: a Diagnostic Tool

The study of normal and abnormal muscle metabolism, or bioenergetics, is

mainly concerned with the biochemical pathways involved in the supply and

utilization of energy for muscle contraction and ion transport. Phosphoms nuclear

magnetic resonance spectroscopy ("P MRS) can noninvasively monitor muscle

bioenergetics by con~inuously measuring the relative concentration of the

intracellular metabolites: ATP, inorganic phosphate (Pi), and phosphocreatine ( K r ) ,

as well as intracellular pH. The free energy from the hydrolysis of ATP is used for

muscle contraction and active ion transport, Pi is an end product of ATP hydrolysis,

and PCr is an energy resenre against ATP depletion. Collectively these metabolites

are referred to as the high energy phosphates (HEP), and changes in their

concentration reflect the metabolic state of muscle. A "P MRS spectnim can be

acquired in less than a minute and thus serial measurements of skeletal muscle HEPs

during rest, exercise and recovery provide a means to monitor varying degrees of

muscle metabolism. During moderate intensity exercise, the concentration of ATP

does not change because ir is replenished through aerobic and anaerobic respiration

with the extent of anaerobic respiration dictating the largesr changes in intracellular

pH. There is also a work load dependent decrease in the concentration of PCr wirh a

concomitant increase in Pi. The Pi/PCr ratio is an indicator of the energy state of

tissue [Chance, 19861 and a rise in this ratio signifies a reduced capacity for aerobic

p hosphorylation.

To date, only four groups of investigators have used in-vivo "P NMR

spectroscopy to noninvasively srudy MH. Each group used the C H C T as rheir "gold

standard" method of diagnosis although there were slight variations in the type of

categories used. Each group reported some success, yet there are inconsistencies

among their results as outlined beiow. The first experiments were performed by

Olgin et al. [1988] who monitored the forearm flexor muscles during rest, graded

exercise, and recovery in a t hree-step, incremental exercise protocol. The protocol

consisted of wrist flexion of 0.5 second duration once every 5 seconds. Each subject

exercised at 20°/0, 1090, and then 60°% of their maximum voiuntary torque for six

minute periods at each level. A "P NMR spectmm was acquired at rest, during the

last two minutes of each exercise level, and at one minute intenrals for the first three

minutes post exercise. Olgin reported two significanr results in his study: a higher

resting Pi/PCr ratio, and a slower recovery of Pi/PCr posr exercise in M H

susceptible patients compared to normal controls. It was concluded that the changes

in the Pi/PCr ratios were due to a metabolic abnormality and not a metabolite

deficiency. While investigating the resting metabolite concentrations in the

gastrocnemious muscle, Payen et al. [WU] also observed a significantly higher resting

Pi/PCr ratio. Webster et al. [1990] were not able to reproduce the differences in

resting metabolite concentrations demonstrated by Olgin or Payen when observing

the forearm flexor muscles. They did, however, observe significant differences in

HEP and pH kinetics during a two-stage exercise protocol. The exercise protocol

consisted of manometer bulb squeezing at a 0.5 Hz repetition rate with bulb

pressures of 100 mmHg and then 300 mmHg. The NMR spectrum acquisition time

was 72 seconds during exercise and 36 seconds during the first two minutes of

recovery. Webster reported a significantly larger drop in the M H susceptible group's

pH and PCr/(Pi+PCr) ratio during the initial portion of the exercise protocol.

Contrary to Olgin's results there was a delayed recovery of pH post exercise and no

difference in PCr recovery kinetics in the M H susceptible group.

In 1993, Bendahan and CO-workers reported similar results to Webster when

monitoring the forearm flexor muscles during intense finger flexion at a constant

a-ork rate. With an NMR spectral sampling time of one minute, they reported an

early rise in the Pi/PCr ratio, an increased level of acidosis in the early stages of

exercise, prolonged recovery of p H post exercise, and no significant differences in

metabolite concentrations at rest for the M H group as compared to controls.

However, unlike Webster, Bendahan did confirm one of Olgin's results and reported

a significantly slower recovery of PCr post exercise in the M H population.

Al1 of the aforementioned exercise protocols and data acquisition schemes

were able to detect significant differences between normal controls and a group of

MH susceptible individuals, although the differences they found varied considerably.

Unfonunately, none of these studies monirored either the HEPs or pH at a high

enough temporal sampling frequency to accurately monitor the expected changes,

and thus better the observed differences. The changes in the concentration

of Pi and PCr post exercise follow a near mono-exponentid curve, and the time

constants for recovery in normal controls have been reported in the literature to be

between 30 and 90 seconds for both metabolites [McCreary 1996, Marsh 1993, Taylor

19861. However, the highest temporal resolution of the studies which examined M H

metabolism was once every 36 seconds, with the majority of studies acquiring spectra

only once per minute. Moreover, the rate of PCr recovery is dependent upon the

end exercise p H WcCreary 19961 and end exercise decrease in PCr concentration

[Taylor 19861. None of the previous M H exercise protocols had patient-normalized

work rates, and consequently rhere was a dispersion of recovery times independent of

any myopathy. Even Olgin's three step graded-exercise protocol cannot be

accurarely normalized for each patient because the work rates were based on the

maximum voluntary contraction of each individual and each step was of a fiied

duration.

ability to

each step

In

A maximum voluntary contraction test does nor correlate well with the

perform aerobic o r anaerobic work warsh , 19931, and the fixed duration of

cannot guarantee the same metabolic end point for each patient.

addition to drawbacks in their exercise protocols, al1 of the previously

described studies investigating M H with NMR used metabolite quantification

techniques which may be inaccurate and suffer from operator bias [van den Boogaart,

19951. Moreover, none of these studies used magnetic resonance imaging (MRI) to

localize the acquired spectra. Without the use of MRI for proper positioning of the

targeted muscle group in the spectroscopic setup, it is unknown how much of the

spectrum was contaminated by signal originating from non-working muscle

[Fleckenstein 19891.

An improved protocol would entail: M N to guarantee proper positioning of

the working muscle groups for spectroscopy localization; a progressive resistance

exercise regime which would slowly increase the work rate to a uniform state of

fatigue and thus maintain a consistent metabolic end point among subjects; an

accurate and unbiased method of spectrum analysis; and an increased spectral

sampling frequency over previous studies which would enable better modeling of the

HEP and p H kinetics.

1.4 Research Goal

The goal of my thesis was to develop a noninvasive method to diagnose MH.

If successful, this test could then be used as an alternative to the in vitro contracture

test and thus reduce the morbidity presently associated with diagnosing the disease.

To reach my goal and to clarify some of the existing controversy presented in the

15

literarure concerning HEP and pH kinetics, 1 developed a system to monitor the

metabolic response of forearm skeletd muscle to dynamic exercise using phosphorus

nuclear magnetic resonance spectroscopy. I then compared a group of normal

controls and M H susceptible individuals who tested positive using the CHCT.

Included in this thesis project was considerable NMR system characterization and

optirnization. Careful attention to system quality control was necessary in order t o

achieve the performance levels required for an in vivo spectroscopy and magnetic

resonance imaging examination.

1.5 Thesis Outline

The remainder of the thesis is comprised of four chapters as outlined below.

In Chapter Two, 1 d l first introduce the basics of NMR and its application to in

vivo phosphonis spectroscopy, and then briefly discuss some of rhe quality control

experiments involved in setting up a new system to perform high resolution imaging

m d spectroscopy. The system development work was presented as "Transformation

of a Small Bore Topical Magnetic Resonance System to a Clinical

Imager/Spectroscopy Unit", by B.S. Ahluwalia, K.S. St.Lawrence, T-Y, Lee, and R.T.

Thompson, at the Fourth Scientific Meeting of the Society of Magnetic Resonance in

Medicine, New York, NY, May 1996. Chapter Three contains the experimental

methods used to invesrigating MH. Details include the exercise apparatus which 1

designed and builr, and an outline of the cornputer program which 1 wrote t o

automate the data analysis. In Chapter Four the results from my investigation into

M H are presented. These results were also presented as: "Diagnosis of Malignant

Hypertherrnia Susceptibility Using in-vivo "P-MRSn, B.S. Ahluwalia, R.T.

Thompson, G.D. Marsh, and S.L. Dain, at the Fifth Scientific Meeting of the Society

of Magnetic Resonance in Medicine, Vancouver, British Columbia, May 1997.

Chapter Five discusses my results and compares them to previous studies. The thesis

closes wirh a summary of my work and how it can positively impact the MH

population.

c h a p t e r Z

NMR PRINCIPLES AND APPLICATIONS

2.1 Introduction

The experimental foundations for NMR were set by Feliv Bloch and Edward

Purcell in 1946, work for which they shared the Nobel p i l e for physics in 1952. Since

that rime, NMR has been developed to deterrnine molecular structure (Pake, 19481 and

concentration [Shaw, 19521, to classify tissues for medical applications parnadian,

19711, and to obtain spatial information via imaging [Lauterbur, 19731. The first NMR

study looking at phosphom merabolites and intracellular pH in working muscle was

performed by Hoult CI9741 when he exarnined an excised rat hind-leg muscle. Human

in vivo applications soon followed at Oxford University in 1981 moss 1981, Gadian

198 11.

This chapter gives a brief description of the basics of NMR and is divided into

two sections: NMR principles, and praaical application. The first section describes the

basic processes involved in NMR, starting from quantum mechanics and progressing to

a classical representation of the resonance phenornenon. The second section describes

sorne of the erperimental and practical aspects of in vivo ''P NMR which needed to be

addressed before proceeding in the MH population.

2.2 NMR Principles

Nuclear magnetic resonance, as the name suggesrs, is a consequence of the

properties of nuclear partides: protons and neutrons. In general rerms, an NMR

experiment consists of rhree phases 1) preparing the sample by placing it in a strong

magnetic field, 2) perturbing the sample by applying an oscillating radio frequency

magnetic field (BJ, and 3) dereaing a signal from the sarnple. Strictly speaking, NMR

can be descnbed completely only in terms of quantum mechanics; however, when a

large ensemble of nuclei is considered, a classical description is helpful in developing a

more intuitive understanding. With this in mind, a few quantum principles will be

introduced to describe the effea of a magnetic field on a nucleus with spin, then

classical mechanics will be used to describe the net effect of magnetic field pemirbations

on a nuclear system.

2.2. I Quantum Pbmomma

Nuclear spin is a quantum mechanical property but it can be considered

concepmally as an angular momentum resulting from the rotationai motion of an

electrically charged nucleus about its own axis. This rotation defines two collinear

vector quantities which are key to NMR, namely the spin angular momentum 1, and

the magneric moment p. The magnitude of 1 is proporrional to Plank's constant h, (h

= W2a) and the spin quantum number '1':

The dimensionless spin quantum number 1 is limited to positive multiples of M and

depends on the number of protons and neutrons in the nucleus. The magnetic moment

vector p is linearly proportional to 1 and classically, we can think of it as a small magnetic

dipole field at the center of the nucleus. The strength and direction of this field is:

where y is the gyromagnetic ratio and a property of the nuclear system (units

radians/Tesla second). This discussion will be limited to spin 1/2 (I- 1/2) systems

because the two relevant nuclei midied in this work CH and "P) are members of this

group.

The first s e p in an NMR experiment is to place the sarnple containing the nuclei

of interest in a large uniform magnetic field Bo, which by convention is oriented dong the

z-direction. The torque on from Bo will cause each spin-% nucleus to have two energy

eigen States, either spin up and parallel to Bo, or spin down and anti-parallel to Bo. (Figure

2-1) The two spin orientations have quantized zcomponents of magnetization given by:

with an energy difference of:

The expectation value for the magnetic moment C W also precesses about B, with

frequency v,, known as the Larmor frequency.

v, = yB,/k (2-5)

For most clinical systems, 1 Bo 1 ranges between 0.15 and 2.0 Tesla, Ieaving the Larmor

frequency in the radio frequency (rf) range (v, = 6 - 84MHz).

spin up (1= -'A) Energy = - H hvo

spin down (1 =%) Energy = 'irhv,

Figure 2-1: The energy stares and precessiond frequency of ri spin M nucieus.

2.2.2 Chsical Description

If we place a collection of identical non-interacting spin-% nuciei in the Bo field,

and mechanisms exist by which they can anain thermal equilibrium, there will be a

population difference between the two energy States. In thermal equilibrium, if we let N-

equal the number of spins in the lower energy state (parallel with BJ, and N+ equal the

number of spins in the higher energy nate (anti-parallel with BJ, the population

difference determined by the Boltzmann distribution is

where AE is the energy difference between states, k is the Boltzman constant (units:

Joules/OK), and T is the absolute temperature. For a magnetic field strength of 2 Tesla,

N- z1.OOOOl *N+ at room temperature. The signal chat we observe in NMR results from

the absorption or emission of energy when we alter the population distribution berween

the two energy states. Because the population difference is so small, the NMR signal is

inherently weak. To alter the population distribution, we nipply AE quanta of energy co

the spins by a~plying a small magnetic field (BJ oscillating at the appropriate frequency.

The vector sum of d l nuclear moments in the sarnple will result in a net

macroscopic magnetic moment M,

Since the azimuthal angle of p for each nucleus is random and evenly disrnbuted from

O+Zn, there will be no net xy component of magnetization. We can then safely represent

the net equilibnum magnetization as a vector aligned with the magnetic field (Figure 2-2)

The behavior of this system can be described u ing classical mechanics and any

perturbation to the system can be represented as a perturbation to M. Thus we can

change from the quantum mechanical rreatment of spins to the macroscopic motion of

the net magnetization vector M.

Figure 2-2: Spin population chribution and resulting net magnetic moment at thermal equilibrium.

2.2.3 Spin

At

20

Excitation

thermal equilibrium, M is parallel to the magnetic field and cannoc be

measured directly. The technique of NMR is to tip M away from the z-axis using the

principle of resonance. This is achieved by applying a small magnetic field, B,, which

rotates in the transverse plane with the same frequency and in the sarne direction as the

individual spins. M will experience a torque due to Bo and B,, and move in a

downward helisphere away from the z-axis (Figure 2-3 b). The tip angle (0) between M

and the z-awis at the end of rhe rf pulse is:

0=y1B,I t

where t is the duration of B,. Since B, is in the MHz or radio frequency range, and irs

duration ranges from 0.05 - 5 ms, it is oken referred to as an rf pulse. In practice, B, is

created by passing an aiternaring current through an inductor, or probe, which surrounds

the sarnple. When we turn off the rf field while there is an xy component of M, the

tip angle 8

Induced signal (FID) I

Figure 2-3: Classicd mechanic description of spin excitation: a) net magnetic moment at thermal equilibrium, b) application of the B, rotating magnetic field and tnjeccory of M, c) precession of magnetic moment, and ci) induced voltage and received NMR signal.

precwion of M will induce an oscillating voltage in the nirrounding inductor (Figure 2-

3). The induced signal is cdled a free induction decay @D) and how it changes in time

reflecu the molecular or environment characteristics of the sample, as will be explained.

2.2.4 Relaxation

The signal received after an rf pulse will not last forever - recdl that it results

frorn the veaor sum of a large number of spins which are initially phase coherent after

the rf pulse. As time progresses, phase coherence is lost between isochromats due to

small variations in the magnetic field experienced by each nucleus which change their

insrantaneous precession frequencies. These srnall variations arise from interactions

between neighbouring spins which lead to a loss of phase coherence and hence net

signal. This process was referred to by Bloch as spin-spin relaxation because the

energy from one spin is transferred to another. For liquids, the probability for this

process is constant in time; hence the rate of signal loss due to molecular interactions is

exponential and characterized by the time constant, denoted T2.

The observed rate of signal decay after an excitation pulse differs from T,.

Inhomogeneities in Bo cause variations in the precession frequency across the sample,

resulting in an additional loss of phase coherence to T2 mechanisms. The observed rate

of signal decay is called 'T, starn where TL* I T2. This undesired source of signal loss

a a s to reduce chernical resolution and signal intensity.

Another type of relaxation is called spin-lattice or T, relaxation, and describes

the regrowrh of magnetization dong the z-axis after an rf pulse. The energy given to

the spin system by the B, field is eventually transferred to the 'lattice" or molecular

framework. Y, relaxation transfers energy to the thermal bath and increases the

thermal motion of the spins. This energy transfer is primarily induced by the molecular

motion of neighbouring magnetic dipoles which create transverse (x-y) fluctuations of

an externai magnetic field near the resonant frequency of the affected nucleus. These

fluctuations provide a mechanism for energy transfer, similar ro the way B, fluctuations

at the resonant frequency give energy to the spins. Again, for liquids, the probability

for this process is constant and thus can be characterized by an exponential tirne

constant T,.

If we excite the sample (8 5 a/2)

pulse-acquire again without waiting, the

This is due to the fact that the firsi

22

with an rf pulse, acquire the signal and then

second signal will be smaller than the fint.

pulse altered the arnount of longitudinal

magnetization and we have perturbed the system with another pulse before the system

could regain equilibriurn through T, relaxation. At this time the system is partidly

saturated. Consequently, T, determines how frequently we can pulse the systern and

still measure a reasonable amount of signal, while T1* limits the duration of the signal

after a pulse.

To display the frequency components of a sample, the time domain signal is

converted to the frequency domain using a discrete Fourier transform (FT) pracewell

19781. Figure 2-4 shows a FID and corresponding FT, or spectrum. The area under each

resonant peak is proportional to the concentration of the nucleus it represents. The

following section descnbes how spectra contain information about the chernicd nature

of a sample's composition.

2.2.5 Chernical Shift and Spin-Spin Coupling:

The electron clouds which mediate the chemical bonds between atoms interact

with any externally applied magnetic field. This electron cloud interaction results in a

nucleus experiencing a magnetic field which subtly differs from the applied magnetic

field. There is a consequent change in the Larmor frequency which depends on the

exrent of interaction. The change in frequency is referred to as the chernical shift of

the nucleus. Since the chemical shift of a nucleus depends on the behaviour of the

elearons around it, two atoms of the same nuclear species in different chemical

environments will have different chemical shifts. Consequently, the received NMR

signal from a cornplex molecule will be composed of many different resonant

frequencies; when the time domain signal is Fourier-transformed and displayed dong a

frequency axis, the result is an NMR spectrum (Figure 2-4).

The frequency axis of the speamm is the chemical shift, denoted by 6, and is

measured in the dimensionless units of parts per million (PPM). Each resonant peak is

measured from a reference frequency, and for in vivo "P the reference frequency

corresponds to the PCr resonant frequency.

23

6 = K 0 - 0 ,&mc, )/ %mncel x 1 Ob (2-9)

Since the chemical shift is a result of electron interactions with the external magneric field,

the absolute frequency dispersion of peaks in the NMR spectrurn scales with the field

strengh, but the chemical shift in PPM remains the same.

FID (acquisition domain)

Figure 24: Exampie of discrete Fourier transformeci FTD showing the correspondence b e m n the time (acquisition) domain and frequency domain signais.

Figure 2-5: Chernical structure of ATP showing the a, P phosphorus nudei.

A single nuclear position in a given molecule may

a d Y

produce two or more

associated peaks. The magnetic moment of a nucleus creates a strong field in its local

environment which polarizes the electron cloud around it. One nucleus can convey its

spin orientation (either up or down) and indirectly act on its neighbouring nuclei via

electrons shared through chemical bonds. This indirect interaction is referred to as spin-

spin or J coupling and causes the lines in the NMR spectrum to have fine structure

[Harris 19921. That is, the resonant frequency of a nucleus may depend on the relative

spin orientation of its neighbours. As a nile of thumb, J coupling is limited to less than

3 chemical bonds and the number of peaks associated with a single spin-% nucleus is Zn-',

where n is the number of coupled chernical-~hifr-e~uivalent nuclei. Unlike chemical

shift, the degree of ~ e a k splining due to J coupling is independent of magnetic field

strength and is measured in Hertz. The "P nucleus in the P phosphate group of ATP

(Figure 2-5) is coupled to both the a and y phosphate groups, hence the peak will be

split twice resulting in a quadntplet. However, since the Ja8 and J,, coupling constants

are similar, the two middle lines are so closely spaced that the P ~ e a k appears as a

triplet. The a and y peaks in the ATP spectrum are doublets because each is coupled to

the p phosphate group but are too distant to be coupled to one another.

2.2.6 D e t m ination of pH

In addition to monitoring the relative concentration of phosphorus metabolites,

"P NMR c m also determine p H by using the chemical shift of the inorganic phosphate

(Pi) peak. Dihydrogen phosphate (H,PO,-) is a weak acid located at 3.8 PPM, and

monohydrogen phosphate (HPO,") is its conjugate base located at 5.0 PPM. The rate of

proton exchange between these two metabolites is fast on the NMR time scale (< IFS)

and rather than observing two resonant ~ e a k s at 3.8 PPM and 5 PPM respectively, we

receive a single time-averaged peak whose position depends on the concentration of the

two species. Using well documented titration curves, the pH can be calculated from the

chemicd shift of the Pi peak.

pH = 635 + log[(S - 3.27) / (-5.69 - 2Qj (2- 1 O)

where 6 is the chernical shift in PPM between the Pi and PCr resonant peaks p o o n 1973,

Taylor 19831.

2 2.7 haging: Spatial in fomtarzon

Magnetic resonance imaging (MRI) is spatial spectroscopy. Rather than a

nucleus' chemical environment determining its position in a spectmm, its physical

position determines its location on the frequency axes. By using three orchogonal

magnetic field gradients (G,, Gy, GJ which linearly change the Larmor frequency as a

hnction of position, the spatial distribution of a nuclear species c m be determined in

two or three dimensions. Almost al1 biological MRI is based on the proton signal

because of its abundance and relative high sensirivity. A hiil description of MRI is

beyond the scope of this thesis and 1 point the reader to an excellent book for furcher

explanation mishimura 19961.

2.3 NMR in Practice

An NMR synem has many components which must be finely tuned and

integrated to ensure that the inherently weak nuclear signal is not drowned in thermal

or environmental noise. In the year 1 staned my MSc. prograrn, a new Surrey Medical

Imaging Systems (SMIS) NMR console and imaging hardware were coupled to Our

existing magner. The previous system was designed for smdl volume spectroscopy of

the forearm and the goal of the upgrade was to include high resolution imaging. Prior

to investigating Mdignant Hypenhermia, 1 spent considerable energy irnproving the

system to maximize the signal-to-noise ratio (SNR) and ensure proper performance. In

vivo "P audies demand scrutiny in this respect because the concentration of rnost

phosphates is below 10 rnM and thus produce a signal much smaller in comparison to

that from the 100 M concentration of 'H used for imaging. The problem is

compounded by the reduced NMR sensitivity of phosphorus ( ~ 5 % of 'H). Irnproving

SNR increases the accuracy of metabo lice concentration measurements or decreases

acquisition cime by reducing the number of averages for one spectrum - both of which

are important in measurements of changing metabolism. In addition, my

characterization and development of the system included: improving Bo homogeneity,

improving the grounding planes, calibration of the irnaging gradients to ensure that

they were within ~~ecificarions, writing and testing imaging sequences, developing a

technique to monitor room vibrations, and developing tests for monitoring phase

stability. Some of this additional work is presented in the appendices: Appendix A

includes the poster which I presented at the Fourth Scientific Meeting of the Society of

Magnetic Resonance in Medicine, New York, NY, May 1996; and Appendk B shows

the improvements in SNR. The rernainder of this chapter bnefly describes one of the

methods I used to improve spectrum SNR, narnely the implementation of adiabatic rf

pulses.

2.3.1 Surface Coils and Adiabatzc Excitation

The purpose of an NMR probe (or coil) is to transmit the radio frequency BI

field, and receive the induced signal. The proximity of the coil to the sample, f i l h g

factor, coil circuits., and coil geometry are integral in determining SNR [Chen 19891.

The NMR probe for this experiment contained concentric "P and 'H surface coils with

4 cm and 8 cm diameters respectively. As the narne suggests, surface coils are single loop

solenoids which lie on the surface of the sample. Their shape and proximity to the

sample provide excellent signal reception and inherent localization. The trade-off of

these benefits, however, is that the radiation field is non-uniform and thus there is a

distribution of tip angles throughout the sample when using traditional hard rf pulses

[Evelhoch 19831. The distribution c a w s the tip angles of some volume elements to be

near zero degrees while others are tipped by 720 degrees or more. Neither condition

contributes to xy magnetization, hence there is a loss in signal intensity. Additional

signal loss results from regions of the sample which have been tipped between 180-360

degrees, 540-720 degrees, or similar ranges because they will contribute negatively to the

signal. Also, when using hard pulses to monitor multiple metabolites, the region of the

sample which contributes the majoriry of the signal differs depending upon the Tl of

each metabolite. For example, when using a 4 cm diameter surface coil with the tip

angle calibrated to 90 degrees at the center of the coil, the effective distance to the center

of the excited volume is 2.6 cm for PCr (T,,,, 7 sec) and just 2.1 cm for Pi (Tl,, = 3

seconds) when the interpulse delay (TR) is one second. To combat these problerns 1

used adiabatic rf pulses which modulare the amplitude and frequency of the BI field to

provide a uniform tip angle in regions of 10 fold B, inhornogeneity [Garwood, 19921.

2.3.2 Adiabatic Excitaion

Adiabatic excitation relies on the following theorem: if a single magnetic field B

moves with angular velocity Q, the net magnetization vector M will follow (stay

parallel to) B provided

Rather than moving the main magnetic field, adiabatic excitation moves an

effective field which is a vector mm of B, and Bo. The effective field staru parallel to

Bo and M, and ends ~ a r d l e l to B, - thus ~roviding a 90 degree tip angle. The motion of

M in the presence of a magnetic field B is calculated using the Bloch equation,

where X is the cross product, B is the vector sum of B, and Bo, and we assume that the

pulse duration is much les than the T, or T, of the sample. Since adiabatic pulses are

both amplitude- and frequency- modulated, the process is best explained from a frame of

reference which has the instantaneous angular frequency a,(t) of the rf pulse and the same

plane of rotation. That is, the z'-axis of the rotating fame of reference is collinear with

the z-axis of the stationary frarne of reference. In the rotating frame of reference B,(t) has

a constant orientation and is arbitrarily chosen dong the x'-axis. The change of reference

frames requires the gened equation of motion,

where dA,h is the tirne rate of change of vector A in a stationary frarne of reference, and

SA/& is the tirne rate of change of A in a frame of reference with angular velocity R. The

substitution of equation 2-12 into equation 2-13 with Q equaling o, leaves,

S M / & = yM X (BI (t)i + (a, - o,(t))/y k) (2- 14)

where i, j, and k are unit vectors, and the last expression is the effective field in the

rotating frame of reference (Figure 2-6a). M precesses about the effective magnetic field in

the sarne way that it precesses about the main magnetic field when it has a transverse

component (Figure 2-3c). At thermal equilibrium, the effective field is essentially colinear

with Bo and M if w, c <o, and IB,I O (Figure 2-6b). If o(t) slowly approaches

Figure 2-6: Precession of M about an effective magnetic field during adiabatic excitation in the rotating frame of reference. a) decomposition of the effmive field, Bdf b) precession of M about Bd c) spin excitation, movement of M to x'y' plane

resonance and 1 B, 1 increases from zero while satisfying the adiabatic theorem, B, will

rotate to the xYy'-plane with M in tow. At resonance, there will be no z'component of

the effective magnetic field, M will precess about B,, and the magnetization will lie near

the ~ ~ - ~ I a n e , thus completing a 90" tip angle (Figure 2-6c). The tip angle will be sirnilar

over a large range of 1 B, 1 because the magnitude of the effective field has no maximum

bound making it ideal for surface coi1 excitation.

There are many combinations of amplitude and frequency modulated pulses

which satisfy the adiabatic criteria [Garwood 1992, Urgurbil 1988, de Graaf 19971. 1

have used a 3 ms sech/tanh (B, arnplitude/frequency) modulated rf ~ u l s e because it has a

sufficiently wide excitation bandwidth and thus excites al1 metabolites with sirnilar

intensity. When comparing the performance of this adiabatic pulse with a 30 ps

Frequency (Hz)

Figure 2-7: Cornparison of signai intensities between the 3ms sechltanh adiabatic pulse (diamond symbols, norrnalized to 100) and optimized 30 ps hard pulse (open square symbols). A forearm spectrurn is overlain on the graph to illustrate the bandwidth of inrerest .

optimized hard pulse, the average gain in SNR is approximately 2Soh over the

bandwidth of interest (Figure 2-7). One drawback of the adiabatic pulse is that it does

not have a uniform excitation bandwidth over al1 frequencies. This occurs when the

adiabatic condition fails and care m m be taken to ensure that there is no selecrive peak

suppression, and rhat phase is linear over the bandwidth of interest [Bansal 19931.

METHODS

3.1 Wrist Mode1

The bioenergetics of many human muscle groups have previously been

examined using "P-NMR spectroscopy WcCreary 1996, Marsh 1993, Martin 19911.

The forearm flexor muscles were used in this midy because our experimenral semp is

well suited to it in terms of main field geometry, magner availability, and patient

cornfort. But more imponantly, in previous studies with controls and patients our

group has shown that vital pathophysiology can be illuminated using this muscle group

warsh 1993, Thompson 19961. Of the forearm exercises, wrist flexion was used rather

than finger flexion or bulb squeezing because it involves a larger muscle mas. In wrist

flexion, the dorsal to volar movement of the hand is controlled by the anterior forearm

muscles, specifically, the flexor digitorum profundus (FDP), flexor digitomm

superficialis (FDS), and flexor carpi ulnaris (FCU). A benefit of exercising a larger

muscle mass is a reduction in the spectral sarnpling rime and a smaller likelihood of

observing non-working muscle Ueneson 19931. The forearm flexor muscles are not so

large, however, that isolated exercise will stress the cardiovascular or respiratory

system and add to the complexity of the ex~erirnental results.

3.2 Exercise Ergorneter

I designed and built the wrist flexion exercise ergometer based on the rope and

pulley system of Marsh and et al. [1991] (Figure 3-1). The ergometer allowed isotonic

contraction of the forearm flexor muscles with the work rate dependent upon the

frequency of contraction, mass of the weight being lifted, and the distance of Mt. The

exercise consisted of pressing a lever through an arc about the wrist while the forearm

was supported motionless in a w o r n made cradle. The hand grip was f i e d off-axis

between two circular plates which

through a nylon brace and pulley.

were themselves connened by a rope to a weight

Structural incegrity of the ergometer derived from

three circular Wn thick acrylic bulkheads, two of which were separated by ?An

duminum rods and the other by a 1" thick acrylic platform. The platform supported

the forearm bed and rotating hand grip while the duminum rods acted as runners for

an oil impregnated nylon brace. The brace was used to stabilize the rope and weight

during contraction. The NMR robes were in the base of the forearm bed and the

diaance from the probes to the hand grip was adjustable to ensure proper positioning

of the FDP,

20" flexion)

magner bore

FCU, and FDS.

which lifted the

was adjustable to

Wrist flexion was limited to 70" (from 50" extension to

weight IO cm.

improve patient

The angle of the ergomecer inside the

comfort and muscle positioning.

Figure 3-1: The exercise ergorneter: typical placement of the hand and f o r m in preparation for wrist flexion exercise. Black rurow indicates position of the concentric 'H and "P surface coils of 8 cm and 4 cm diameter respeaively. White arrow shows position of pulley and rope connected to water pail.

3.3 Experimental Methods

This experiment was approved by the University of Western Ontario's human

ethics cornmittee and informed written consent was obtained from each subject.

Earolled in the smdy were 11 normd controls (4 female, 7 male, meankSEM 3513

years old) and 25 M H susceptible patients deemed positive via the CHCT. The M H

patients were divided into two caregories: the first contained 9 subjem (3 female, 6

male, 39k3 years old) who were diqnosed as HCK; and the second group, labeled as

mixed-MH, contained 16 subjects (6 female, 10 male, 43 + 3 years old). Within the

mixed-MH group were patients diagnosed as either CK (3 male, 41 t 3 years old), K (4

female, 3 male, 44+ 7 years old), or positive (2 female, 4 male, 43 * 3 yean old). The

positive subjects could not be properly cacegonzed because their complete contraccure

results were not available, but they were listed in the North American Mdignant

Hyperthermia Registry of susceptible individuals. Normal conrrols were those

individuals who have never had an MH reaction, nor were aware of any in their family

history. Al1 subjects were screened to ensure that they were not using medications or

had any other myopathies which may influence the MRS results, and each was asked to

refrain from consuming caffeine and performing strenuous exercise for the two days

prior to testing.

3.3.1 Exmcise Pro tocol

Prior to positioning in the magnet, each subject stretched the forearm muscles

of their dominant hand for 5 minutes. The average maximum voluntary grip strength

was subsequently measured from three independent contractions using a grip

dynamometer (manufacnired by: Takei Kiki, Kogyo Japan).

Each patient performed the exercise protocol lying supine on a bed with their

dominant arm extended at right angles to their body and into the magnet bore (Figure

3-2). The exercise protocol consisted of wrist flexion at 0.5 Hz with the work rate

starting at 0.50 Watts and increased at 0.13 Warts/min. until volitional fatigue o r when

the PCr resonant reached 20% of its resting amplitude. Exercise cadence was

rnaintained via audio stimulation from an elearonic metronome. The work rate was

increased by pumping water at a rate of 275 ml/min. into an aluminum bucket which

was suspended from the ergorneter rope outside the magnet bore. A maximum cutoff

point of an 8O0/0 reduction in PCr concentration was used because further decreases can

cause a drop in [ATP] and a consequent increased recovery time of K r , Pi, and pH

back to resting levels [Radda 19871.

Figure 3-2: Positioning the patient in the 1.89 TesIa magnet in preparation for esramination.

3.3.2 NMR data acqrrzsztion

Al1 'H magnetic resonance images and "P spenra were acquired in a 1.89 Tesla

smdl bore (20 cm diameter) magnet coupled to a SMIS console (a complete description

of the sysrem is provided in Appendix A). Before staxting the exercise protocol, each

subjects' arm was imaged using a multislice gradient echo sequence to ensure proper

positioning of the FDP, FDS, and FCU above the phosphom surface coil. The proton

signal was rhen used to shim the magnet homogeneity and improve spectral resolution.

While shimming, the 8cm diameter surface coil was used for adiabatic rf transmission,

while the 4cm coil was used for signal reception. Homogeneity was adjusted until the

FWHM of the water peak was less chan 0.4 PPM and the peak was Lorentzian in shape.

Aker tuning the receiver to the PCr resonant frequency, spectra were collected at rest,

throughout exercise, and during the first 20 minutes of recovery. Al1 spectra were

acquired using a 3ms adiabatic 90 degree rf pulse, 12 ps delay time, 3.33 kHz receiver

bandwidth, and 2048 complex data points. Al1 previous in vivo "P MRS studies

exarnining M H used hard pulses of less than 50 ps for spin excitation. Six resting

spectra were collecred in total: two with

prior to narting exercise. The fim high

high SNR, and four to establish a baseline just

SNR' spectmm (PCr SNR265) was an average

of eight acquisitions and used a 30 second repetition time to eliminate any T, saturation

effeas (longest T, is PCr = 6 seconds). The second spectrum was an average of the final

40 acquisitions of a 19 acquisition spectmm, with the sarne 3 second TR as used during

the exercise prorocol (PCr SNR1.60). The initial nine excitations were used to establish

steady aate T, saturation. Al1 spectra collected during baseline, exercise, and recovery

were the sum of eight acquisitions which reduce the sampling cime to 24 seconds (PCr

peak SNR=26), and again, prior to the firn spectmm, nine scans were used to achieve

neady state saturation.

3.3.3 Data Analyszs

Two computer programs were custom written for the data analysis: one for

metabolite quantification, and the other for cdculating HEP and p H kinetics.

Metabolite was performed in the time (acquisition) domain by fining the

data to a sum of components. Each component corresponded to a resonant frequency

in the specrrum and was modeled with an exponentially damped sinusoid which could

be va+d in amplitude, phase, delay time, darnping constant, and frequency. The

quantification software was developed by fellow graduate students John Pocwarka

[1995] and Rob Banha [1995] and used a-prion knowledge with the Levenberg-

Marquardt aigorithm marquardt 19631 co iteratively reduce the difference between the

data and ex~onential model. The sofiware required little operator intervention and al1

spectra were fit to the same template aker apodization with a 2 Hz Lorentzian filter.

The first 1.5 msec of data was not used in order to eliminate the very broad

(FWHM > 1OOHz) phosphorus components originating from regions with large

inhomogeneities or bone. The area of each peak in the frequency domain, and thus its

corresponding relative concentration, was taken as the amplitude of the exponential

model funnion at tirne equal zero.

dculated as the ratio of peak ampiitude ro srandard deviation of noix in a region of the rpecuum void of NMR s i g n a

Once al1 the spectra for a single patient had been quantified, the high energy

phosphate and pH kinetics were cdculated using software that 1 developed. The high

S N R spectra were used to determine the resting metabolite concentrations and T,

saturation correction coefficients. The correction coefficients were only used when

calculating the ADP concentration and did not influence any other result. There is

evidence to show that the free cytosolic [ADP] is an important regulator of oxidative

phosphorylation [Chance 1985, Taylor 19861; however, ADP is difficult to detecr using

NMR and its concentration must be determined in an indirect manner. The

calculation for the [ADP] followed the methods of Arnold [1984] (equation 3-1) and

assumed that the creatine kinase (CK) reaction (equation 3-2) was always at

equili b rium:

[ADP] = [ATP] n o t a 1 CreatineJ/[PCr] -1) / K, m + ]

PCr + ADP + H+ -ATP + Creatine (3-2)

[ATP] was calculated assurning that the resring concentration was 8.2 mmole/l ce11 water

and its change throughout exercise was proportional to the area of the P A T P ~ e a k ;

cytosolic [PCr] (mmole/l ce11 water) was assumed linear with its ~ e a k area and was

normalized to the resting signal intensiry ratio of PCr/PATP; the intracellular w+] was

calculated using the chernical shift between the Pi and PCr resonant frequencies as

described in Section 2.2.6; and it was assumed that the total creatine pool D o t a l

Creatine]=42.5 mmole/l ce11 water) and K, (l.66+10-~ mole-') were constant. The [ADP]

was monitored at ren, end exercise, and for the first five points in recovery. The data

points were averaged within each group and a repeated meanires ANOVA was used ro

determine if there were significant differences @ < 0.05) between each group.

During exercise the work rate for the onset of intracellular acidosis and

accompanying change in phosphorylation potenrial were calculated from the break

point in the pH and log(Pi/PCr) respectively. Each break point was at the intersection

of two best-fit lines which modeled the data. The slope and intercept of each line was

optimized using piecewise linear regression p i e t h 19891.

In recovery, there were two methods employed to monitor the phosphate and

pH kinetics: either by fitting the changes to a mode1 equation, or by performing a

point-by-point andysis. The recovery kinetics of Pi and PCr, were each fit t o a three

parameter monoexponential w hich produced time constants rPi (equation 3-3) and

rPCr (equation 3-4) respectively.

pi(t)] = A exp(-t/rPi) + C (3-3)

The rPi and rPCr were calculated starting from the first data point poa cessation of

exercise and the parameten were O ptimized using the Levenberg-Marquardt algo rit hm.

To characterize pH recovery, three meanires were used: the "delta (A) pH", "pH time

lag", and rpH. The ApH was the difference between the end exercise pH and the

minimum pH post cessation of exercise; the pH time lag was the duration of this

continued decrease; and the r p H was the rime constant from a mono-exponential fit of

the recovery data calculated starring from the minimum pH value.

The time constant for the recovery of PCr is linearly proportional to the end

exercise pH WcCreary 19961. The lower the pH, the longer rPCr. To reduce within-

group variations caused by different end exercise p H values, rPCr was corected for pH

following the methods described by McCreary [1996]. Furrher merhods are explained

when the data is presented in the next chapter.

Statisrical cornparisons of the mean recovery pararneters between the normal

control and MH groups were performed using an ANOVA with a Tukey post hoc test.

A pS0.05 was considered significantly different.

c h a p t e r 4

RESULTS

4.1 MRI Guided Positioning

A magnetic resonance image with the proper positioning of the forearm flexor

muscles on the phosphonis surface coi1 is shown in Figure 4-1. The axial slice image

was positioned 7 cm distal to the medial epicondyle of the humerus and was concentric

with the main magnetic field and r.f. surface coils. A contour plot showing the change

in B, field intensity (and hence receptive sensitivity woult , 1979D as a hinaion of

position for rhe "P coill is superimposed on the proton image to illustrate that only a

small portion of the NMR signai originates frorn outside the FDP, FDS, or FCU.

Flexor C Radialis

Figure 4-1: Axial gradient echo proton image of forearm f h o r musdes (256x256 pixeis, TE - Ems TR- Zûûrns, FOV = km, slice thickness - 3rnrn). The profde of the dculated B, field of the "P surface coil is superimposed o n the image, contour lines are normalized to 100% at the center of the coil and each Iine represents a decrease of 10% in B, magnitude.

' Caiculared using the Biot-Savan law and assumes no r-f. attenuation in tissue.

4.2 "P Spectroscopy

4.2.2 At Resrand D ~ n n g Exerczse

Representative spectra acquired at rest and at end exercise are shown in Figure

4-2. In the spectrum acquired at resr, the chemical shift of the Pi resonant peak was

4.85 ppm, and from Equation 2-10 this corresponded to an intracellular pH of 7.04.

The mean metabolite concentrations and pH for each group of subjects are listed in

Table 4-1; rhere were no significanc differences in the resting conditions between

groups. As expected during exercise there was a decrease in pH, as evident by the

change in chemical shift of Pi, and a drop in @?Cr] with a concomitant rise in [Pi]. The

pattern of changes in the log(Pi/PCr) and the pH with respect to work rate for a single

patient are illustrated in Figure 4-5. Both data series displayed two distinct phases: an

initial phase where the rare of change was slow, followed by a second phase with a

rapid rate of change. The pH and log(Pi/PCr) transition points, as calculated by

piecewise linear regression and indicated by arrows on the figure, were correlated

within each group (p < 0.05). There were also no significant differences in the average

position of the breakpoints between groups (Table 4-2).

At Rest 1 Controls HCKXH mzxed-MH

Grip Strength (N)

Pi (mM/l ce11 water)

PCr ( d l ce11 water)

Pi/PCr

ADP (pM/l ceIl water)

PH

O. 13 * .O1 0.11*.01 0.12&.01

29 *4 23+4 23k2

7.03 * -01 7.03 .O1 7.03 + .O1

Table 41: Maximum voluntary grip nrength, and metabolite concentrations and pH during the restmg state. Values represented as mean* SEM. There were no signifiant differences between the groups

ATP

End Exercise Spectnun

Specvum Quantification

15 10 5 O -5 - 10 - 15 -20 -25

PPM

Figure 4-2: Sarnple resting (a) and endaetcise (b) spectra acquired in 147 and 24 seconds respectively. The results of the quantification s o b e have been overlain on the end cxercise spectrum and details of the quantikation mode1 are shown in (c).

0.5 1 1.5 2

Power (Watts)

Figure 4-3: Typical changes of Iog(Pi/PCr) and pH during exercise as a hnction of output power for one control subject. The resting vdues are shown at O Watts. The biphasic solid Iine overlain each chta set is the result from the piecewise linear regression with the intracellular threshold indicated with an arrow. In this example the Iog(Pi/PCr) threshold occurred at 4.02 and 1.42 Watts, and for pH the coordinates were 6.97 md 1.41 Watts.

Table 4-2: Piecewise linear regression analyses of log(Pi/PCr) and pH for al1 subjects. Values are represented as mean + SEM. There were no significant differences between

P-Line Analysis

log(Pi/PCr) threshold (W)

log(Pi/PCr) value at thres hold

pH work rate threshold (W)

pH value at threshold

the groups.

Con trois HCK-MH mzxed-MH

1.35+0.15 1.22+0.18 1.23 rt O. 10

-0.2010.11 -0.2OiO.lI O.O*O.lO

1.24 + 0.08 1.20*0.12 1.28&0.11

6.97 * 0.02 6.94î 0.04 6.96 * 0.02

The metabolic state of muscle was characterized at the end of exercise by

observing the changes of PCr], [ATP], and p H from resting values (Table 43) . There

were no significant differences between the groups with respect to the mean percentage

decrease in PCr] or [ATP], however, the end exercise pH for the mixed-MH group

was significantly higher than the values for both the normal controls and HCK-MH

subjects (ANOVA p < 0.05).

4.2.2 In Recovery

Representative changes in intracellular p H throughout the exercise prorocol are

plotred for a single subject in Figure 4-4. The three parameters for characrerizing the

p H recovery kinetics have been included on the graph ( A ~ H , pH lag time, and mono-

eaponentid mode1 with characteristic time constant rpH). The continued decrease in

p H post exercise is due to the production of protons from the resynthesis of PCr via

the reverse CK reaction (Equation 3-2). Examples of Pi and PCr recovery kinetics are

shown with their mono-exponential models in Figures 4-5 and 4-6 respectively, and the

mean values of the recovery kinetics for each group are lisred in Table 4-4. There were

no significant differences in the recovery kinetics of the HEP, rpH or ApH between

the three categories of subjects, however, the p H lag tirne for the HCK-MH group was

significantly longer than for normal controls.

The time course of mean [ADP] for each group post cessation of exercise

shown in Figure 4-7. There were no significant differences in the concentration or race

of change of [ADP] between the three groups.

Rarnped Exemise

5 10 15 20 25 30 35 tirne (minutes)

Figure U: Typical changes in pH as a funaion of tirne throughout the exercise protoc01 for a single subjecc. Shown are the: intracellular threshold during the ramp exercise; post exercise decrease in pH, ApH-0.13; duration of pH deche, pH tirne 1% = 48 seconds; and the mono-exponential mode1 of pH recovery, T ~ H - 376 seconds.

Re s iduals

Time into recovery (minutes)

Figure 4-5: The top chart shows the typicd recovery kinetics of F i ] as a function of time post cessation of exercise. The solid line is the rnono-exponential fit to the &ta which had a characteristic time constant of rPi - 78 seconds. The lower chart shows the difference between the mode1 and data with the sucter of points about zero.

O .006T Res iduals

Time into recovery (minutes)

Figure 4-6: The top chan: shows the tppicai recovery kinetics of FCr] as a function of time post cessation of exercise. The solid Iine is the mono-exponential fit to the data. which had a characteristic time constant of rPCr = 130 seconds. The lower c h m shows the ciifference between the mode1 and data with the scatter of points about zero.

End Exercise

Maximum Work Rate

Oh decrease in PCr

O/* decrease in ATP

PH

-

Con troh HCK-MH mixed-MH

Table 4-3: Maxùnum work rate during ramp exercise, and end exercise percent difference in HEP and pH frorn resting Ieveis. Values expressed as meanIsem. The mixed-MH group h d a & d k n t l y higher end exercise than both the HCK-MH or controi groups (' ANOVA p < 0.05)

In Recovery

rPCr (s)

TPH (4

Con trds HCK-MH mîxed -MH

45+6 6917 * 6 0 t 9

0.12 t 0.03 0.15*0.04 0.15+0.04

751 12 74+_11 5014 p-0.095

96+7 156129 92+ 10

2471 15 302 * 27 222 + 23 1

Table 4-4: Recovery kinetics for the three groups of subjects. Values expressed as rneanksern with significant differences from normal controls idenrified with an * (ANOVA p < 0.05). Listeci p value is frorn a cornparison to normal controls.

Time into recovery (s) -100

Figure 4-7: Concentration of ADP as a huiction of tirne post cessation of exercise. The data points for the mixed-MH md control groups have been slighdy naggered in tirne to better view the error bus (k 1 stmdvd dwiation). The data points positioned in 'negative" tirne are the vdues at the end of exercise. There were no signifiant ciifferences between the groups.

4.2.3 Cowections ro rPCr

An exarnple of the dependence of rPCr on end exercise pH is shown in Figure

4-8. The dope of the best fit line to the data is used to correct the sPCr for each subject

to a cornrnon pH of 6.50 using the following equation:

corrected rPCr = rPCr + dope * 16.50 - end exercise p w

A common pH of 6.50 was used because it is within the range of mean end exercise pH

for the normal controls (6.471 0.04) and HCK (6.51 10.07) subjects. Each group was

corrected individuall~. Regression analyses produced a slope of -107 s p H unit?

(RL=0.43, p-0.03) for the controls, and -333 s pH units" (R2=0.76, p=O.OOî) for the

HCK group. The resulting rnean (I sem) corrected sPCr values were 94 f 5 and 162 * 14

seconds for the control and HCK groups respectively. There was a significant difference

between the control and HCK groups (ANOVA pc0.01). There was no correlation

between the end exercise pH and rPCr for the mked-MH group and thus corrections to

rPCr were not calculated.

I4O T

End exercise pH

Figure 4-8: tPCr ris a funaion of end exercise pH for the normal concrois. The soIid line is the result from regression analysis (R2=0.43, p-0.03).

4.3 Discriminant Analysis

To determine if the results from this study could be used t o delineate between

the control and M H population, a discriminant analysis was performed. In essence, the

procedure generates a funcrion based on a linear combination of the measured

parameters with the coefficients of each parameter optimized to provide the greatest

significanr difference between the groups. The function is generated for a sample of

cases for which membership is known, and then it is applied to new cases to predict

unknown members. Since there is a strong possibiliry chat the mixed-MH group

contains subjects with fdse positive diagnosis, the discriminant analysis was based

solely on the results from the normal controls and the HCK-MH susceptible patients.

The uncorreaed rPCr was used for the analysis because in future the application of this

test, knowledge of diagnosis would not be known and thus the appropriate correction

factor could noc be applied. The discriminant funcrion was:

where DV is the discriminant value. The means (*standard deviations) of the D V were

125c25 and 230+43 for the conrrol and HCK-MH groups respectively (ANOVA

p<O.OOl) (Figure 4-8). The maximum DV for the control group was 176 and the

minimum for the HCK group was 184. Applying the same discriminant hinaion to the

mixed-MH group produced a DV of 141 t 37 with a maximum of 197 and a minimum of

74. Three of the mixed-MH subjects have a DV which is higher than the maximum DV

for the concrol group, and of these three, one has a DV within the range of the HCK

population.

HCK-MH

Figure 4-9: Two patameter discriminant andysis cdculated between the normal controls and HCK- MH groups using T P C ~ and pH cime hg. h d y s i s was nibsequently applied to the mixed MH group. Mean t lsd (minimum/maxirnum) were: Control 125 + 25 (93/176), HCK-MH 230 + 43 (1 84/3 l9), and mixd-MH 14 1 I 37 (71/ 1%).

c h a p r e r 5

DISCUSSION

The results of the present study show that the recovery kinetics of PCr and pH

post cessation of progressive wrist flexion exercise are slower in a sub-population of

patients with MH than in normal controls. The HCK-MH subjects had a significantly

longer corrected sPCr and longer pH Iag tirne than conrrols, however there were no

significant differences between the mixed-MH group and controls in recovery. Other

research groups have also found significant differences between an MH and control

populations when comparing resting phosphate concentrations payen 199 1, 1996,

Olgin 19891, pH kinetics Webster 1991, Bendahan 19931, and PCr kinetics [Bendahan

1993, Olgin 1989, Olgin 19931, however, there was no specific pattern or abnormaiity

present among al1 research groups. The lack of consistent results may be due to the

apparent heterogeneity of the disease, but variations in experimental procedures cannot

be discounted.

None of the previous studies which used "P NMR as a tool to investigating MH

implemented magnetic resonance imaging (MRI) to guide spectroscopie localization.

The use of MRI in my study provided an initial qualitative assessment of the

contribution of different skeletd muscle groups of the forearm to the sensitive volume

of the surface coil and thus aided proper coil positioning. Signal contributions from

inactive muscle attenuate the observed metabolic response of working muscle and cause

errors in bioenergetic measurements. Fleckensrein [1989] showed that errors in

positioning of a surface coil over wrist flexors from typical ~ a i ~ a t i o n could result in a

wide range of measured metabolite responses to the same exercise: Pi increases ranged

from 1.5 to 5.7 times chat of resting values, while PCr levels decreased from between

goh and 42%. Improper positioning of the surface coil aiso increased the coefficient of

variance (standard deviatiodmean) of HEP metabolite responses by greater than four

fold in a series of finger flexion exercises due to the signal contamination from non-

working muscle Ueneson, 19931. Jenesson's LI9931 and Flechenaein's [1989] studies

stress the imponance of using imaging to remove effecrs of coi1 mispositioning from

true metabolic differences.

There were no significant differences among the three categories of subjects in

this nudy with regard to any resting meanirement. This is consistent with the results

of Webster [IWO] and Bendahan [1993], but contrary to the findings of Olgin [1989,

19911 and Payen [1989, 19961- The variability arnong research groups may stem from

the heterogeneity of the disease itself. When smdying a population of 27 MH

susceptible patients, Olgin et al. Cl9911 reported that 17 of the 27 had an elevated

resting Pi/PCr ratio, and 11 of the 24 patients who finished the exercise protocol had a

slower PCr/Pi recovery rate post exercise compared to normal controls. Only two

M H susceptible patients had both an increased resting ratio and recovery rate. Al1 of

the MH subjects in Olgin's study originated from a pool of four families and the

specific NMR abnormality was consistent within each lineage, thus lending evidence

that M H may be a spectrum of hereditary diseases. The lack of a difference in the

resting Pi/PCr values of our MH populations may be a result of not observing the

same phenotype observed by other groups rather than due to differences in

experimental setup or procedure.

In addition to confirming the results of Olgin et al [1989,1991], Payen and co-

workers [1996] examined the pathology of muscle tissue and found no significant

difference in fibre type distribution between MH and normal controls. An increased

proportion of type 1 fibres would increase the Pi/PCr ratio [Meyer, 19851. Neither

Olgin nor Payen provide further biochemical or physiological data ro support their

results.

Perhaps an alternate explanation arises [rom the fact that each group which

found an increased resting Pi/PCr ratio used frequency domain metabolite

quantification methods afcer appl~ing a convolution difference, manually phasing the

spectra, and then correcting for baseline distortions. The convolution difference

weights the area of each peak with its corresponding T2* by removing the broad

spectral components from the initial portion of the FID. In the frequency domain this

corresponds to removing the broad hump underlying the Pi and PCr resonant peaks.

This can potentially lead to errors as the hump can be substantially larger in area chan

either the PCr o r Pi peaks. The reported differences in metabolite ratios might not

only be due to actual concentration differences but also differing T, relaxation rimes.

Al1 other groups investigating MH with "P MRS used frequency domain spectral

quantification and neglected to describe details of their firting methods thus limiting

hr ther cornparison. The use of manuai phasing and baseline correction schemes add

operator bias and inaccuracies to spectral quantification [van den Boogart, 19961. The

method of spectral analysis employed in this study overcomes these flaws by being

operator independent and modeling the signds in the tirne domain potwarka, EV] .

During progressive exercise, both the log(l?i/PCr) and pH showed a biphasic

response to increasing work rate (Figure 4.3). The breakpoint berween the two phases

has been taken to indicate a transition in the relative contribution of giycolytic and

oxidative pathways to ATP production [Chance 19861. At work rates below the

intracellular threshold, oxidative phosphorylation provides sufficient energy. At work

rates higher than the intracellular threshold, the energy demands exceed the aerobic

capacity for ATP production and energy production relies more heavily on anaerobic

glycolysis and the forward creatine kinase reaction [Chance 1986).

Cornparisons of the log(Pi/PCr) and p H kinetics during exercise revealed no

significant differences among the three classifications of subjeccs in this snidy. No

previous study investigaring M H had sufficient rime resolution, o r an appropriate

exercise protocol to monitor the HEP and p H kinetics in this manner; nonetheless, the

lack of a signifiant difference among the subject classifications c m be considered as

contrary to results published by Webster [1990] and Bendahan [1993], but consistent

with Olgin [1989,1991]. Both Webster and Bendahan reporred a significant increase in

the rate of PCr hydrolysis and acidosis during exercise for the M H group. This would

have corresponded to lower work rate values at the intracellular thresholds in my

study. The inconsistencies are probably due to the exercise protocols. Both Webster

and Bendehan used a fixed, high-intensit~, shortduration (53 minutes) finger flexion

exercise regime. In Bendehan's protocol, each patient lifted a 6 kg weight at a 1.5

second interval. This equates to a work rate of approximately 6 Watts - almon double

the peak work intensity in this srudy (rnean peak intensiry 93.5 Watts). Webster's

protocol used 300 mmHg manometer bulb squeezing at 2 Hz for 2.3 minures as the l a s

step of a two step exercise. The work rate cannot be accurately calculated in Webster's

nudy, but the rapid drop in pH and PCr at the start of the 300 mmHg step reveals that

this work rate is well above the intracellular threshold for both M H and controls. In

contras, Olgin's [1989, 19911 wrist flexion exercise regime was 18 minutes in duration

and consisted of three incremental steps in work rate at 20% 40%, and 60°/0 of each

subjects' pre-exercise maximum voluntary contraction. During exercise Olgin et al.

acquired a single spectrurn over the last two minures of each sep, thus ensuring they

were monitoring steady state exercise. The average duration of exercise in my study

was 13 minutes and the slow rate of work increase and high NMR spectral sarnpling

frequency (1/24 seconds) provided a near steady state monitor of HEP and p H kinetics.

The quick onset of intense exercise in Bandahan's and Webster's studies would illicit

different muscle recruitment patterns, haemodynamic responses, and substrate

utilization than the controlled slow increase in work rates used in Olgin's and my

exercise protocols Uuel 19971. The differenr types of exercise stresses rnay contribute to

the inconsistent results reported by the various researchers.

Monitoring the decrease in PCr concentration as a method of ensuring the sarne

metabolic end point among subjects was successful for the HCK-MH patients and

controls, but not for the mixed-MH subjects (Table 4-3). The rnean end exercise pH

for the mixed-MH subjens was significantly higher than for the other two groups even

though the percent decrease in PCr from resting levels was not significantly different.

This result is surprising because Webster CI9901 reponed that the change in p H was

excessive for MH susceptible subjects compared to controls for similar PCr reductions.

The higher end exercise pH in the mixed-MH group could indicate either a reduced

capacity for anaerobic glycolysis or increased ability to remove protons o r lactate from

the cell. The latter reason may be discounted since there was no significant difference

in the recovery kinetics of p H post exercise in the mixed MH population compared to

controls, but funher studies are required to resolve this issue.

The recovery kinetics of Pi and PCr have been discussed in a number of articles

and shown to follow a near mono-exponential hnction [Arnold 1984, Ioni 1993, Kemp

19931. Marsh and CO-workers LI9931 compared a single exponentiai to a double

exponential when modeling Pi and PCr recoveries and found that the second order

kinetic did not significantly improve the accuracy of the fit. Other investigators

however, have reported a biphasic recovery of Pi and PCr, but it was dependent upon a

low end exercise pH (< 6.3), significant reductions in [ A m ] (> 15O/0), and an end

exercise @?Cr] below 15% of resting values [Arnold 1984, Mole 1985, Taylor 19861.

Since the subjects in our study did not reduce their [ATP], PCr ] , o r intracellular p H to

such levels, a mono-exponential function was believed to be a sufficient mode1 for

recovery analysis.

The recovery of Pi post exercise is mainly dependent upon the rate of its

absorption by the mitochondria potti 1993, Ioni 19961. Pi absorption is driven by the

p H g-adient across the inner mitochondrial membrane through a symport with H'.

The hdf tirne for recovery of Pi from moderate intensity forearm flexor muscle

exercise has been reported between 30 [Taylor 1986, Arnold 19841 and 72 seconds

[Argov 19961 for normal controls. This corresponds to a rPi between 43 and 104

seconds. There were no significant differences in the sPi among the three categories of

subjects in this study (Table 1-9, and al1 mean values were within the range of

previously published data.

The end exercise p H and ATP concentration have a considerable effect on the

recovery tirne constant of PCr [Argov 1996, Kemp 19931. The link between rPCr,

ATP, and pH is via the reverse CK reaction. Decreases in pH and ATP change the

equilibrium position of the reaaion and thus reduce the rate of PCr resynthesis. The

ex~onential recovery kinetics of PCr afrer dynamic exercise in the forearm flexor

muscles have been previously reporced for untrained and myopathy free individuals to

range between 81 and 103 seconds Bemp 1993, Taylor 1986, Arnold 19841. In these

studies the end exercise p H was near 6.4 and there was no reduaion in the ATP

concentration. The rPCr of the control group in this experiment was (mean+SEM)

96 t 7 seconds which was consistent with previous midies.

The significant increase in the corrected T P C ~ of the HCK group compared to

controls is due to a metabolic defect rather than differences in the end exercise

metabolic state of the muscle. Since the end exercise pH, and percent drop in PCr and

ATP concentrations were not different between the two groups, any increase in rPCr

must originate in either reduced creatine phosphorylation or increased PCr

breakdown. With the presence of CK and creatine in the cytosol, any excess ATP is

converred to PCr during recovery. Also, glycogenolysis is not active in the absence of

contraction in the normal population, and thus oxidative p hosp horylation is

responsible for the resynthesis of PCr [Taylor 19861. In vitro studies of mitochondria

from normal muscle tissue have shown that the rate of oxidative ph~s~hory la t ion is

regulated by the availability of ADP [Chance 19851. In a similar in vitro study which

exarnined the mitochondria of MH susceptible patients, Cheah et al. [1981] concluded

that there was linle evidence for abnormalities in respiratory activity. This resulr is

confirmed in my study because there were no significant differences in the rate of

recovery or concentration of ADP among subject groups.

It is important to note rhat a decrease in the perfusion of muscle could also

increase rPCr by limiting ~ h e availability of substrates for oxidative phosphorylation

[Toussaint 19961. However, this myopathy is probably not a factor in MH because in a

96 patient study comparing pathology and positive CHCT results, only one person

showed microvascular abnormalities Figarella, 19911.

An increase in corrected sPCr in the absence of any mitochondrial dysfunction

indicates an increased rate of ATP hydrolysis. The M H myopathy of an elevated

cytosolic Ca" concentration in the absence of triggering anaesthetics could increase

ATP hydrolysis in three ways: firstly, by binding to troponin and activating

actomyosin ATPase; secondly, through active transport of ~ a " back into the

sarcoplasmic reticulum via Ca2+-~TPase; and thirdly, via active transport of Ca" from

the ce11 at the sarcolemma. This third mechanism would also decrease the intracellular

pH because the protein exchanges two protons for each Ca" removed from the cell.

The importance of the process has yet to be determined, but it is ex~ected to play a

minor role in Ca" homeostasis due to the high density of C a 2 + - ~ T ~ a s e o n the

sarcoplasmic reticulum and low cytosolic Ca" concentration in the absence of

contraction [Carafoli 19851.

The processes controlling the efflux of H+ from the ceil remains to be defined.

It is likely however that intracellular pH recovery is accomplished largely by secondary

active transport mechanisms on the sarcolemma uuel 19973. The cause of the

significant increase in the p H lag tirne for the HCK-MH group verses controls cannot

be determined from this study, but similar results were reporred by Webster et al.

and Bendehan et al. [1993]. Both groups of researchers found a significant delay

in the pH recovery of the M H population. It is difficult, however, to make direct

cornparisons arnong al1 the studies due to the different exercise regimes and methods of

data analysis. Both Webster and Bendehan monitored point-by-point the absolute

values of p H throughout recovery. Since the M H groups in their studies had a

significantly lower end exercise pH than the controls, their method of analysis would

not be able to distinguish differences in the rate of recovery between patient groups.

The same problem is overcome in this study by modeling the data with a mono-

exponential function and comparing tirne constants. The results of this project suggesc

that there is no difference in the rate of pH recovery arnong the patient groups because

once the majority of PCr has been resynthesized (Le. afier the pH lag time) the T ~ H

values were not significantly different (Table 4-4). In addition, the significant delay in

pH recovery found by Websrer [1991] and Bendehan [1993] cannot be distinguished

between a myopathy from reduced proton or lactate efflux from the cell, o r a

myopathy which caused the MH group to have a significantly lower end exercise

(Kr]. Their srudies also had insufficient spectral temporal resolution to determine the

p H lag time (Webster et al. 36 seconds, Bendahan et al. 60 seconds) as compared to this

study (24 seconds).

The use of discriminant analysis to more accurately differentiate the MH

population from controls is, to my knowledge, unique for this population study.

Olgin et al. [1991] used individual parameters to distinguish between the patient

groups, but did not use a common parameter for the entire population. The results

from the discriminant analysis show conclusively that we can diagnose a sub-

population of M H susceptible individuas who have been diaposed as positive with the

CHCT. Al1 of the patients in the HCK-MH (n=9) group had a higher DV than the

maximum value from the controls. If the threshold for a positive result was generated

by using the mavimum discriminant value of the control group, then the application of

the discriminant function to the mixed-MH group (n= 16) would successfully diagnose

a further 3 subjects. Of these three individuals, 2 were diagnosed as CK, and the third

was assumed positive after her son died from an M H reaction and her husband tested

negative with the CHCT. Of the 150 patients in Ontario who had the muscle biopsy

performed 1st year, 35 were diagnosed as HCK, and a further 80 were diaposed as

either CK or K b o k e 19971. Since our patient population was weighted more heavily

with HCK's than is present in the general population, the results from this study

project that we could reduce the number of people who would proceed to a biopsy by

approximately 35%. It should be recognized however, that for the discriminant value

statistic to be tmly tested, it musr by applied to another M H population.

As a screening tool this "P MRS exarn would be used on the patient population

which has traditionally been sent for muscle biopsy. Ideally, if a positive "P diagnosis

was recorded then the patient would be assumed to be M H susceptible and not need

the invasive biopsy; and ideally of course, if the "P test result was negative it would

not be necessary to have the biopsy and the patient would by assumed noc MH

susceptible. More redistically however, because of the potential dangers of a false-

negative diagnosis, those who test negative by the "P exam would then proceed to a

muscle biopsy.

In this project I have implemented a number of techniques co optimize the "P

NMR rneasurernent of forearm skeletal muscle bioenergetics which had previously not

been used in other studies investigating MH. The use of MRI to guide spectroscopie

positioning improved the accuracy of HEP and p H kinetics by reducing the signal

from non-workmg muscle. The implementation of an adiabatic rf excitation pulse

provided a uniform flip angle over the region of interest. This increased the signal to

noise ratio by =250h and ennired that al1 metabolites were being monitored from the

same volume. By using tirne domain spectral analysis 1 improved the accuracy of

metabolire quantification and eliminated operator intervention. The implementation

of these techniques in conjunction with exercising and observing a larger volume of

working muscle allowed spectra to be acquired at a temporal sampling frequency which

was greater than two rimes that of most previous studies. This increase in time

resolution improved the accuracy of monitoring HEP and pH kinetics. With the use

of a ramped exercise protocol and a maximum limit of metabolic aaivity, I reduced the

patient-to-patient variability of recovery kinetics. Finally, by rnodeling the HEP and

p H kinetics with previously tested hnctions, especially in recovery, I reduced the

error associated with the point-by-point analysis implemented in previous studies. In

combining the improved temporal resolution results with a discriminant value analysis,

I have shown that it is potentially possible to reduce the need of muscle biopsy in 30-

40°h of the presently tested population.

5.2 Future work

Since 1 have confirmed that the increase in p H lag time and corrected rPCr post

exercise for the M H susceptible ~at ients are the mosr usehl rneasurements, work

towards even better time resolution is warranted. By increasing the B, field strengh

(Le. 3 or 4 Tesla) a linearly proportional gain in SNR can be achieved. Since the SNR

is proporrional to the root of the spectrum acquisition time, a factor of two gain in

SNR can be translated into a factor of four reduction in acquisition time. Sorne

addirional gain in SNR c m be realized by using a narrow band receiver rather that the

broad band used in the present study. The NMR system developed for this study is

also capable of implementing both decouphg and nOe, both of which have potential

to increase SNR [Harris 1986bl and thus reduce the acquisition time.

It has been hypothesized in the literature that the changes in 'H TL which occur

with exercise are dependent on the changes in intraceliular pH [Ploutz-Snyder 19971. If

this proves t o be tme, then the potential of using very fast irnaging techniques to

mesure T2 could provide a more useful monitor of the pH lag time post exercise.

Regardles of these potentid improvements, the results from my work indicate

that even with the present setup, a substantial reduction in the required number of

muscle biopsies is possible. A blinded study must be undertaken to veriS the results of

rny present work, determine the appropriate discriminant vdue threshold, and thus

derermine the beneficial impact of this ''P MRS exam.

a p p e n d i x A

Contained in this appendix is the poster which was presented at the Fourth

Scientific Meeting of the International Society of Magnetic Resonance in Medicine. Of

the work presented, I was responsible for ensuring that the gradient coils met the

uniformity standards, testing image spatial integrity, develo~ing the phosphorus

spectroscopy channel, and for writing clinicd imaging pulse sequences. Keith St.

Lawrence was res~onsible for designing and implementing the projection presaturation

method of D 2 0 localization, as well as the development of clinical imaging pulse

sequences and software.

TRANSORMATION OF A TMR MAGNET INTO A CLINICAL IMAGING

UNIT

Introduction

Topicd Magnetic Resonance (TMR) magnet/spectrometersl were specifically desiped to acquire spectra from a localized volume of the forearm. Localization is accomplished by niperimposing large scale axial Bo inhomogeneities outside the region of interest.

Our 1.89 Tesla TMR magner has a 4 cm diameter volume of high homogeneity whch is offset from the magnet center and located just 30 cm from the fluted entrance - making the sweet spot easily accessible. (Clear bore diameter of 32 cm.)

To date, the main application of the syaem has been "P surface coi1 spect roscopy.

Goal

To upgrade Our presenr system to include high resolution clinical imaging of the hand, wrist, and lower arm whde expanding Our research capabilities.

Research topics would include simuitaneous multinuclear acquisitions, diffusion measurements, and gradient localized spectroscopy.

Requirements

Expand main magnetic field homogeneity to meer imaging requiremenu over an 8 cm diameter volume.

Add three orthogonal magnetic field gradient coils for imaging applications and for performing localized spectroscopy over volumes l e s than 4 cm in diarneter.

Upgrade electronics to nippon the desired system.

The Transformation

passive shirnrning: By strategicdy placing Icml sofi iron chips in the bore of the magner, TMR large scale inhomogeneities can be nippressed to a level which is corremable via resistive shims. Suppression of large 22 and 2 4 inhomogeneities was critical.

resistive shimming: A room temperature 16 coi1 shim insert was N n o m built by Magnex Scientific to replace the TMR's onginal 12 shim insen. A Resonance Research precision current source (MHU-P24) with computer interface allows automated controi of shim values if desired.

Adding Magnetic Field Gradimr Coils

The gradient coils were custom-made by Magnex Scientific and designed to produce an uniform gradient field over a 12 cm diameter spherical volume. hner diarneter of gradient insert is 21 cm.

The water-cooled, self-shielded coils are capable of generating gradient strengchs of 100 mT/m in approximately 150 ps at efficiencies: 0.57, 0.55, and 0.58 mT/m/A for X, Y, and Z respectively.

Each channel is driven by a Techron amplifier controlled by a gradient management unit (Magnex E3500MK.m). The management unit provides eddy current compensation and adds the DC offset of the linear shim with the corresponding g-adient input line.

The previous console was replaced with a personalsomputer based Surrey Medicd Imaging Systems console (SMIS MultiSpec-N2).

The system contains two broadband transmit/receive channels for simultaneous multinuclear experimenu.

A number of custom-built instruments were dso required, including a 16 mng quadrature high-pass birdcage rf coi12.

Testing Gradient uniformity

Gradient uniformity was measured by moving a small NMR probe through a helis~hericd trajectory3 of radius 6.25 cm. Effem of main field inhomogeneities were eliminated by uing the difference in frequency field plots obtained from positive and negarive gradient coi1 currents.

The percent standard deviation (o) of the gradient field was normdized to the radius of the mapping sphere4 as shown below.

O-= 1

n best fit dope- mapping radius

Field Mapping Trajectory G , Field Linearity m

radius = 6 . 2 5 cm

G y Field Linearity

Relative Frequency ( H z )

a = I 2 . 4 %

G , Field Linearity BI

Relative Frequency (Hz) Relat ive Frequency (Hz)

a = = 1.7% a = i 1.4%

Figure A&l: Field mapping trajeccoxy and gradient uniformity plots showing exellent linearity. a - 1 L4%, -t 1.7%, and r 1.4% for the X,Y, and Z gradients respectively.

System Characterization

a cylindrical grid phanrom was w d to show spatial distortions due to remaining main field inhomogeneities.

Figure A-6-2: Gradient echo images (TE = 16rns TR = 1500ms FOV= 12 cm, slice thickness 3mm) of a cylindrical grid phantom. A) axial image at 2-0, B) coronai image at y=O. The grid spacing is 1.6 cm and an 8 cm diameter circle has been superimposed on the images to demonstrate effeas of main field uniformity within our derirecl region of interest.

Figure A-3: Gradient echo image ol localized region in a D20 flood phantom

using a 2dirnensiond excitation Gaussian profile, 8 ms 6-spd pulse, FOV- lOcm, ROI- lcm, 128x128 pixels, required G = 100mT/m, solid Iines are image intensity through center of ROI

Figure A4: 1, weighted sagittd image if index fmger (TE=23ms, TR==600ms, Slice thickness Zmm, NEX-2, FOV = k m , 256x56 pixels)

Spect roscopy

For an H,O 4cm diameter sphencal volume (DSV) FTUHM = 2.5 Hz, FWZOOh = 9.8 Hz. For an 8cm DSV FWHM = 6.6 Hz, FWIOO/f = 78 Hz.

Since the transformation, the acquisition tirne for a "P muscle spectrurn has reduced by a factor of one-third while maintaining signai to noise ratio for the sarne 4cm diameter rf coil. (refer to Figure 4-2 in body of thesis)

An example of gradient localized spearoscopy using projection presaturation on a D,O phantom is shown in Figure A-3

Discussion

Figures A & B show rhat the magnetic field inhomogeneities are more pronounced in the z direction than in the x-y plane: a consequence of uncorrected axial inhomogeneities, specifically 28.

Within the 8cm diameter volume, inhornogeneities are negligible and images of iesser diameter suffer only minor disto nions.

spatial resolurion of 160 Fm x 160 p m is achieved in clinically relevant scanning rimes (4.5 minutes).

With the strong gradients, easily accessible main field, and upgraded synem, we can now acquire high resolurion clinical images of the hand, wnst, and lower arm (figure C & D) while ~roviding much greater patient cornfort over w hole-body systems.

The upgraded Topical Magnetic Resonance magnet is now responsible for al1 hand, wrist, and lower arm imaging in the Dept. of Nuclear Medicine at St. Joseph's Healrh Care Centre. (patient load ' 370 per year)

We have greatly expanded Our areas of research with the ability ro perform gradient Iocalized spectroscopy, a major improvement over shim localized spect roscopy .

Re fer ences:

1. Gordon, R.E., Hanley P.E. and Shaw D., Prog. in Nuc. Mag. Res. Spec., 15:1, 147, 1982. 2. Enzo Barberi, RF Coi1 Development Facility, Dept. of Radiology, University Hospital, London, Ontario, Canada. 3. Work completed by Dr. P. Starewia, Resonance Research Incorporated. 4. Punchard, W.F.B, P rivate Communication, Resonance Research Incorpo rated. 5. Pauly, J., Nishimura D., and Macovski A., J Mag Res. 81, pp 43-56,1989.

a p p e n d i x B

REDUCTIONS IN SYSTEM NOISE

The received signal in an NMR experiment is inherently very weak and can

easily be masked by electronic noise emanating [rom either the local detecting system

or distant sources [Chen 19881. The purpose of this appendix is to show the

improvemenu in S N R between the initial srate of the system when I srarted my

project and the final state just prior to investigating MH. To gauge improvements in

the semp of the SMIS system, a 2.5 cm diarneter spherical "P phantom was used t o

monitor SNR. The search for noise sources was pedantic and required considerable

"trouble shootingn. Improvements resulted from changes to the system grounding

scheme, ph~sical isolation of power supplies, installing rf shielding on the magnet bore,

and changes to the ~reamplifier. The results shown below do nor indicate the

improvements in magnet homogeneity.

Figure M 3 : Example of ciifference in SNR between pre- and post- system developrnent work (top and bottom trace respectively). Top trace S N R = 8.5, bottom trace SNR = 30.5 (dculated via {Pi peak amplitude}/{std dw. of lm 16 points in spectra}). Gch spectrum was acquired with the same acquisition panmerers

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