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Application of NMR to study biological change Lee, Jonathan Wai Kua 1985

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A P P L I C A T I O N O F NMR T O S T U D Y B I O L O G I C A L C H A N G E by JONATHAN WAI KUA LEE B.Sc.(Hons.), The U n i v e r s i t y of B r i t i s h Columbia, 1981. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1 9 8 5 cJonathan Wai Kua Lee, 1 9 8 5 ? 8 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i A B S T R A C T The aim of this study was directed towards the application of NMR as an analytical tool to follow biological changes, by integrating imaging capabil i t ies with an analytical NMR instrument. The work described here is divided into three parts: application of 1 3 C NMR to follow biochemical transformations, evaluation of the usefulness of relaxation studies in detection of biological changes and, f i n a l l y , testing of a combination of both NMR imaging and spectroscopic techniques to study a selected model system. Spectroscopic techniques were used to study systems of interest not only to chemists but also to technologists interested in milk-souring, grape-juice fermentation, soybean germination and cartilage-degradation. Our immediate objective was to identify biological changes and to f i t them into known biochemical pathways; in the long term, this would lay down the ground work for future in-vivo studies. Relaxation techniques were used to obtain biological information and to follow biochemical changes. I n i t i a l studies involved cultured c e l l s and their NMR relaxation rates were shown to be dependent on factors affecting c e l l u l a r a c t i v i t i e s , such as growth and infect ion. Animal models of A r t h r i t i s and Multiple Sclerosis were followed by relaxation rates with moderate success. i i i F i n a l l y , a p r e l i m i n a r y study i s d e s c r i b e d , i n which a combination of s p e c t r o s c o p i c and imaging techniques was used to f o l l o w the storage and cooking of an egg. i v T A B L E O F C O N T E N T S Page A b s t r a c t i i Table of Contents iv L i s t of Tables v i i L i s t of F i g u r e s ix A b b r e v i a t i o n s x i i G l o s s a r y of Terms x i i i Acknowledgement xvi C H A P T E R I — I N T R O D U C T I O N x 1.1 Background 2 1.2 T h e o r e t i c a l B a s i s f o r Chemical A n a l y s i s 6 1.3 Methods of NMR Imaging 2 2 1.4 Sample(Tissue) P r o p e r t i e s Which A f f e c t NMR Signal(Image) 32 1.5 O r g a n i z a t i o n of t h i s T h e s i s 41 References 45 C H A P T E R I I ~ A P R E L I M I N A R Y E X P L O R A T I O N O F T H E A P P L I C A B I L I T Y O F C - 1 3 NMR FOR S T U D I E S OF B I O C H E M I C A L T R A N S F O R M A T I O N S 53 2.1 I n t r o d u c t i o n 54 2.2 Experimental Method 5 7 V Page 2.3 S o u r i n g of M i l k 57 2.4 F e r m e n t a t i o n of Grape J u i c e 58 2.5 G e r m i n a t i o n of Soybean Seed 60 2.6 D e g r a d a t i o n of C a r t i l a g e 6 6 2.7 C o n c l u s i o n and D i s c u s s i o n 7 2 R e f e r e n c e s 7 6 CHAPTER 111—RELAXATION STUDIES OF BIOLOGICAL SYSTEMS 78 3.1 H i s t o r y and I n t r o d u c t i o n 79 3.2 E x p e r i m e n t a l P r o c e d u r e s 81 Q 7 3.3 R e l a x a t i o n S t u d y of S i m p l e B i o l o g i c a l Systems (a) V i r a l I n f e c t e d C u l t u r e d A n i m a l C e l l s 8 3 (b) Rust I n f e c t e d P l a n t L e a v e s 8 6 (c) C u l t u r e d P l a n t C e l l L i f e C y c l e 8 8 3.4 R e l a x a t i o n S t u d y of P e r i p h e r a l J o i n t D i s e a s e s 9 1 3.5 R e l a x a t i o n S t u d y of A n i m a l Models of M u l t i p l e qo S c l e r o s i s (a) C h r o n i c E x p e r i m e n t a l A l l e r g i c E n c e p h a l o m y e l i t i s (EAE) i n G u i n e a P i g s 100 (b) Herpes S i m p l e x V i r u s Type 1 I n f e c t e d M i c e 1 0 3 V I Page 104 3.6 Conclusion and Discussion 108 Appendix 110 References CHAPTER IV—SPECTROSCOPIC STUDY AND IMAGING OF HEN'S EGG 1 1 6 117 4.1 Introduction 117 4.2 Experimental Procedures 119 4.3 Chemical Composition 119 4.4 The A i r C e l l 122 4 . 5 The Yolk 124 4.6 The Albumen 124 4 .7 Cooking of An Egg 4.8 Four Dimensional, Chemical S h i f t Resolved 126 Imaging !31 4.9 Conclusion 133 References CHAPTER V—SUMMARY AND DISCUSSION 1 3 5 VI 1 LIST OF TABLES CHAPTER I Methods of NMR imaging. C o r r e l a t i o n between s i g n a l i n t e n s i t y ( i m a g e b r i g h t n e s s ) and s a m p l e ( t i s s u e ) p r o p e r t i e s . E f f e c t of imaging parameters on the t i s s u e p r o p e r t i e s which c o n t r i b u t e t o the NMR images. CHAPTER II Some n a t u r a l l y o c c u r r i n g f a t t y a c i d s . CHAPTER I I I T, r e l a x a t i o n times at 100 MHz i n normal and ma l i g n a n t human t i s s u e . S p i n - l a t t i c e r e l a x a t i o n times of water i n HEp -2 c e l l s . A b s o l u t e and r e l a t i v e T, and T 2 v a l u e s i n v i r a l i n f e c t e d c u l t u r e d a n i m a l c e l l s . A b s o l u t e and r e l a t i v e T, and T 2 v a l u e s i n r u s t i n f e c t e d p l a n t l e a v e s . A b s o l u t e and r e l a t i v e T, and T 2 v a l u e s i n A n t e r i o r C r u c i a t e Ligament T r a n s e c t i o n E x p e r i m e n t a l OA. R e l a x a t i o n r a t e s as a f u n c t i o n of sample p r e p a r a t i o n and sample f r e s h n e s s . R e l a x a t i o n r a t e s change as a f u n c t i o n of water c o n t e n t . A b s o l u t e and r e l a t i v e T, and T 2 v a l u e s i n EAE i n b r e d g u i n e a p i g . A b s o l u t e and r e l a t i v e T, and T 2 v a l u e s i n HSV Type 1 i n f e c t e d mice b r a i n . v i i i Paqe CHAPTER IV 4.1a Chemical composition of egg yolk and egg white. 4.1b Fatty acids found in egg yolk. 121 4.1c The density d i s t r i b u t i o n of albumen l a y e r s . 4.Id The l i q u i d portion of albumen a f t e r f r e e z i n g and thawing. IX Page LIST OF FIGURES CHAPTER I 1.1 A nucleus can be v i s u a l i z e d as spinning about i t s own a x i s . The o r i e n t a t i o n s are s p e c i f i e d by the quantum number m and describe two cones. 1.2 P a r a l l e l and a n t i p a r a l l e l o r i e n t a t i o n s of spin 1/2 n u c l e i in the presence of a steady magnetic f i e l d . 1.3 E l e c t r o n currents around a nucleus induces a small magnetic f i e l d opposed to the applied magnetic f i e l d . 1.4 Nuclear magnetic resonance s p e c t r a l parameters. 10 12 1.5 Rotation of the magnetization M 0 in the r o t a t i n g coordinate system and a p p l i c a t i o n of 90° and 180° pulses. 1 3 1.6 Dependence of T, and T 2 r e l a x a t i o n times of protons in water on the v i s c o s i t y and c o r r e l a t i o n time of the s o l u t i o n . 1 6 1.7 Build-up of magnetization as a function of T in inversion recovery sequence. 1 9 1.8 Graphical method of T, determination. 2 0 1.9 The spin echo T 2 experiment. 2 1 1.10 Graphical method of T 2 determination. 2 3 1.11 Categorization of imaging techniques according to the volume detected per unit time. 2 5 1.12 P r i n c i p l e of p r o j e c t i o n - r e c o n s t r u c t i o n imaging technique. 2 8 1.13 P r i n c i p l e of 2D FT imaging technique. 3 0 1.14 Signal phase in the 2D FT experiment. 3 1 1.15 P r o j e c t i o n - r e c o n s t r u c t i o n method versus Fourier zeugmatography. 3 3 1.'16 Water content, s p i n - l a t t i c e r e l a x a t i o n time, and spin-spin relaxation time of various human t i s s u e s . C o r r e l a t i o n between time diagram and signal(image) i n t e n s i t y . Image contrast between normal and pa t h o l o g i c a l t i s s u e . Spin-echo and Inversion recovery images. NMR s i g n a l i n t e n s i t y as a function of flow veloc i t y . CHAPTER II NMR spectra from the forearm of a l i v e human subject. 1 3C NMR spectra of fresh and sour milk. 1 3C NMR spectra of good and fermented grape j u i c e . Conversion of glucose and sucrose to ethanol and g l y c e r o l . 'H NMR spectrum of Freon - 1 1 o i l extract of a soybean seed. High pressure l i q u i d chromatography elusion p r o f i l e of soybean o i l . High r e s o l u t i o n 1 3 C NMR spectra of l i n o l e i c a c i d and soybean o i l extract. 1 3C NMR spectra of germinating i n t a c t soybean seed. Summary of reactions in the pathway of u t i l i z a t i o n of f a t t y a c i d . Structure of c a r t i l a g e components. Proton-decoupled 1 3C NMR spectra of c a r t i l a g e components. 1 3C NMR spectra of bovine nasal septa c a r t i l a g e going through degradation. CHAPTER I I I xi T, and water content as a function of HeLa c e l l c y c l e . The growth-duplication c y c l e . L i f e c ycle of Catharanthus rosens followed by T 2, mit o t i c index, and dry weight measurements. Normal and A r t h r i t i s j o i n t s . The human brain. Free and bound water. CHAPTER IV Basic pulse sequences for volume imaging and chem i c a l - s h i f t - r e s o l v e d tomography; and structure of hen's egg. 1 3C, 'H spectra, and 4D c h e m i c a l - s h i f t - r e s o l v e d images of egg white and egg yolk. Changes in dimensions and volume of a i r c e l l in the hen's egg. Diagrams showing s i n g l e and double yolked egg. E f f e c t of heating on the v i s c o s i t y of albumen and yolk. C r o s s - s e c t i o n a l views from 3D proton NMR volume images of r e f r i g e r a t e d raw, medium b o i l e d , soft b o i l e d , and hard b o i l e d eggs. Longitudinal views from 3D proton NMR volume images of r e f r i g e r a t e d raw, medium b o i l e d , soft b o i l e d , and hard b o i l e d eggs. High r e s o l u t i o n 3 1 P spectra of raw and cooked eggs. x i i ABBREVIATIONS CP C a r r - P u r c e l l CPMG C a r r - P u r c e l l - M e i b o o m - G i l l FID Free Induction Decay MS M u l t i p l e S c l e r o s i s NMR Nuclear Magnetic Resonance OA O s t e o a r t h r i t i s RA Rheumatoid A r t h r i t i s RF Radiofrequency T, S p i n - l a t t i c e or L o n g i t u d i n a l R e l a x a t i o n Time T 2 S p i n - s p i n or Transverse R e l a x a t i o n Time T Echo Time T R e p e t i t i o n Time x i i i GLOSSARY OF TERMS A r i t h r i t i s : p e r i p h e r a l j o i n t d i s e a s e s c h a r a c t e r i z e d by s w e l l i n g , warmth, redness of the o v e r l y i n g s k i n , p a i n , and r e s t r i c t i o n of motion. Autoimmune: d i s o r d e r s caused by the body's own a n t i b o d i e s . Axonal: r e l a t e d to the s i n g l e process extending from the c e l l body of a neuron and c a r r y i n g nerve impulses away from i t . Carr-Purcell(CP) Sequence: sequence of a 90° r f pulse f o l l o w e d by repeated 180° r f pulses to produce a t r a i n of s p i n echoes; u s e f u l fo r measuring T 2. Carr-Purcell-Meiboom-Gill(CPMG) Sequence: m o d i f i c a t i o n of C a r r - P u r c e l l r f pulse sequence w i t h 90° phase s h i f t i n the r o t a t i o n frame of reference between the 90° pulse and the subsequent 180° pulses to reduce accumulation e f f e c t s of i m p e r f e c t i o n s i n the 180° p u l s e s . Suppression of e f f e c t s of pulse e r r o r accumulation can a l t e r n a t i v e l y be achieved by a l t e r n a t i n g phases of the 180° pulses by 180°. Collagen: a p r o t e i n that i s the p r i n c i p a l c o n s t i t u e n t of white f i b r o u s connective t i s s u e . I t i s r e l a t i v e l y i n e l a s t i c but has a high t e n s i l e s t r e n g t h . Demyelination: d e s t r u c t i o n or removal of the myelin sheath of a nerve or nerves. Echo Time(T ): time between middle of 90° pulse and middle of spin-echo p r o d u c t i o n . Edema: e x c e s s i v e accumulation of f l u i d i n the body t i s s u e s . Encephalomyelitis: inflammation i n v o l v i n g both the b r a i n and s p i n a l c o r d . Free Induction Decay(FID): i f t r a n s v e r s e magnetization of the s p i n s i s produced, e.g., by a 90° p u l s e , a t r a n s i e n t NMR s i g n a l w i l l r e s u l t t h a t w i l l decay toward zero w i t h a c h a r a c t e r i s t i c time constant T 2; t h i s decaying s i g n a l i s the FID. In p r a c t i c e , the f i r s t p a r t of the FID i s not observable due to r e s i d u a l e f f e c t s of the powerful e x c i t i n g r f pulse on the e l e c t r o n i c s of the r e c e i v e r . G l i o s i s : r e l a t e d to the connective t i s s u e of the c e n t r a l nervous system. Hepatoma: a malignant tumor of the l i v e r . x i v Ligament: a tough band of white f i b r o u s connective t i s s u e t h a t l i n k s two bones together at a j o i n t . Meniscus: a crescent-shaped s t r u c t u r e . Nuclear Magnetic Resonance(NMR): the a b s o r p t i o n or emission of electromagnetic energy by n u c l e i i n a s t a t i c magnetic f i e l d , a f t e r e x c i t a t i o n by a s u i t a b l e r f magnetic f i e l d . The peak resonance frequency i s p r o p o r t i o n a l to the magnetic f i e l d , and i s given by the Larmor equation. Only n u c l e i w i t h a nonzero s p i n e x h i b i t NMR. Osteophyte: a p r o j e c t i o n of bone, u s u a l l y shaped l i k e a rose t h o r n , that occurs at s i t e s of c a r t i l a g e degeneration or d e s t r u c t i o n near j o i n t s and i n t e r v e r t e b r a l d i s k s . Pathognomonic: d e s c r i b i n g a symptom or sign t h a t i s c h a r a c t e r i s t i c of or unique to a p a r t i c u l a r d i s e a s e . P o l i o v i r u s : one of a small group of RNA-containing v i r u s e s causing p o l i o m y e l i t i s , an i n f e c t i o u s disease a f f e c t i n g the c e n t r a l nervous system. P r o t e o l y s i s : the process whereby complex p r o t e i n molecules are broken down by enzymes i n t o t h e i r constituent- amino a c i d s . Repetition Time(T ): the p e r i o d of time between the beginning of a pulse sequence and the beginning of the succeeding ( e s s e n t i a l l y i d e n t i c a l ) pulse sequence. Sarcoma: any cancer of the c o n n e c t i v e t i s s u e . S c l e r o s i s : hardening of t i s s u e , u s u a l l y due to s c a r r i n g ( f i b r o s i s ) a f t e r inflammation. Synovial F l u i d : the t h i c k c o l o r l e s s l u b r i c a t i n g f l u i d that surrounds a j o i n t or a bursa and f i l l s a tendon sheath. I t i s s e c r e t e d by the s y n o v i a l membrane. S y n o v i t i s : inflammation of the membrane (synovium) that l i n e s a j o i n t c a p s u l e , r e s u l t i n g i n p a i n and s w e l l i n g ( a r t h r i t i s ) . T,: s p i n - l a t t i c e or l o n g i t u d i n a l r e l a x t i o n time; the c h a r a c t e r i s t i c time constant f o r s p i n s to tend t o a l i g n themselves w i t h the e x t e r n a l magnetic f i e l d . S t a r t i n g from zero magnetization i n the Z d i r e c t i o n , the Z magnetization w i l l grow to 63% of i t s f i n a l maximum value i n a time T,. T 2: s p i n - s p i n or t r a n s v e r s e r e l a x t i o n time; the c h a r a c t e r i s t i c time constant f o r l o s s of phase coherence among sp i n s o r i e n t e d at an,angle to the s t a t i c magnetic f i e l d , due to i n t e r a c t i o n s between the s p i n s , w i t h r e s u l t i n g l o s s of t r a n s v e r s e XV magnetization and NMR s i g n a l . S t a r t i n g from a nonzero value of the magnetization i n the XY plane, the XY magnetization w i l l decay so that i s l o s s 63% of i t s i n i t i a l value i n a time T 2. x v i ACKNOWLEDGEMENT I t i s my p l e a s u r e to thank my research d i r e c t o r , P r o f e s s o r L.D. H a l l f o r h i s constant supply of encouragement, p a t i e n c e , s t i m u l a t i n g d i s c u s s i o n s , moral support, and f o r p r o v i d i n g me an e x c e l l e n t o p p o r t u n i t y to work i n a new area of s c i e n c e , which made the c u r r e n t work so i n t e r e s t i n g and worthwhile. I am very g r a t e f u l to G. Hewitt, Drs. M. Adams, D. Van A l s t y n e , L. K a s t r u k o f f , and S. Sukumar f o r t h e i r c o o p e r a t i o n and support, f o r many h e l p f u l d i s c u s s i o n s and i n v a l u a b l e a s s i s t a n c e . F i n a l l y , although too numerous to name i n d i v i d u a l l y , I would l i k e to thank the graduate students i n t h i s l a b o r a t o r y as they c o n t r i b u t e d i n many ways to my educ a t i o n . 1 CHAPTER I — INTRODUCTION 1.1 Background 1.2 T h e o r e t i c a l B a s i s f o r Chemical A n a l y s i s 1.3 Methods of NMR Imaging 1.4 Sample(Tissue) P r o p e r t i e s Which A f f e c t NMR Signal(Image) 1.5 O r g a n i z a t i o n of t h i s Thesis References 2 1 . 1 Background In 1946 B l o c k ( l ) and P u r c e l l ( 2 ) and t h e i r c o l l e a g u e s independently p u b l i s h e d t h e i r f i r s t d i s c o v e r i e s of nuclear magnetic resonance(NMR) i n o r d i n a r y matter. The impact of t h e i r work was immediate, and a p p l i c a t i o n s of NMR have s t e a d i l y i n creased from p h y s i c s and chemistry to a s t a r t l i n g range of d i s c i p l i n e s from archeology to medicine. The importance of the d i s c o v e r y was recognized by the j o i n t award of the 1952 Nobel P r i z e f o r P h y s i c s to P r o f e s s o r s F e l i x Block and Edward P u r c e l l . The e a r l y years of NMR were mainly the province of p h y s i c i s t s and p h y s i c a l chemists. NMR was detected f o r almost a l l magnetic n u c l e i i n the P e r i o d i c Table of the elements, and i n a l l forms of matter: s o l i d s , l i q u i d s , gases, metals, semiconductors, i n s u l a t o r s , and polymers. Using NMR, nuclear p r o p e r t i e s were determined w i t h p r e c i s i o n , and magnetic f i e l d s were measured wi t h great accuracy. The e a r l y d i s c o v e r y of f i n e s t r u c t u r e i n the resonances, a r i s i n g from chemical s h i f t s and s p i n c o u p l i n g s , provided chemists w i t h one of t h e i r most v a l u a b l e s t r u c t u r a l and a n a l y t i c a l t o o l s . Nowadays, no major chemistry department i n any u n i v e r s i t y i s p r o p e r l y equipped without a h i g h - r e s o l u t i o n NMR spectrometer. I t s widespread use i n chemistry and biochemistry has caused NMR to be adopted by a number of s c i e n t i s t s i n a l l d i s c i p l i n e s and has l e d to the growth of a s u b s t a n t i a l NMR instrument i n d u s t r y i n the l a t e 1950s and e a r l y 1960s. 3 Although there had been a few e a r l y s t u d i e s of NMR i n b i o l o g i c a l systems, s i g n i f i c a n t advances awaited the development of h i g h - f i e l d , h i g h - r e s o l u t i o n , F o u r i e r - t r a n s f o r m spectrometers i n the l a t e 1960s. In one type of a p p l i c a t i o n , h i g h - r e s o l u t i o n s p e c t r a were obtained from p r o t e i n s and other biomolecules i n s o l u t i o n y i e l d i n g s t r u c t u r a l and dynamic i n f o r m a t i o n of importance t o biochemistry and b i o l o g y ( 3 ) . T h i s NMR c o n t r i b u t i o n to molecular b i o l o g y continues t o be an a c t i v e area at the f r o n t i e r of research. In the second type of a p p l i c a t i o n , NMR spectroscopy was a p p l i e d t o l i v i n g systems: f i r s t to i n t a c t blood c e l l s ( 4 - 5 ) , then to e x c i s e d and perfused t i s s u e s and organs(6-8), mainly using 3 1 P NMR, but a l s o 1H and 1 3C NMR s p e c t r o s c o p y ( 9 ) . I t was then a n a t u r a l step t o examine whole, i n t a c t organisms from b a c t e r i a t o mice, r a t s , and r a b b i t s , and f i n a l l y to man. These s t u d i e s r e q u i r e d magnets with p r o g r e s s i v e l y l a r g e r access and, i n the l a s t year or two, magnets w i t h a one-meter diameter bore have become a v a i l a b l e f o r human whole-body NMR spectroscopy. I t i s most important t o know from what anatomical part or organ the NMR spectrum o r i g i n a t e s ; t h i s i s d e l i n e a t e d e i t h e r by c a r e f u l p r o f i l i n g of the f i e l d (as i n t o p i c a l magnetic resonance)(10,11), by use of sur f a c e c o i l s ( 1 2 ) , or q u i t e r e c e n t l y by use of s o p h i s t i c a t e d m u l t i p u l s e and m u l t i d i m e n s i o n a l F o u r i e r - t r a n s f o r m techniques(13-15). The NMR s p e c t r a obtained i n t h i s way y i e l d v a l u a b l e c l i n i c a l i n f o r m a t i o n from almost a l l p a r t s of the human body(16-22). 4 In c o n t r a s t w i t h the steady onward march of NMR spectroscopy from p h y s i c s through chemistry and b i o l o g y to medicine, the second s t r a n d of NMR c l i n i c a l a p p l i c a t i o n s , namely NMR imaging, represents a d i s t i n c t l y d i f f e r e n t approach. The f i r s t NMR image of a heterogeneous o b j e c t , i . e . two tubes of water, was p u b l i s h e d by Lauterbur i n 1973(23). The impact of t h i s f i r s t image and the work which f o l l o w e d have been tremendous. W i t h i n a decade of i t s p u b l i c a t i o n , manufacturers worldwide are producing m i l l i o n - d o l l a r NMR scanners that generate h i g h - q u a l i t y images of a l l p a r t s of the human body, and e s p e c i a l l y from the bra i n ( 2 4 - 3 4 ) , heart(35-41), 1 i v e r ( 4 2 - 4 8 ) , and kidneys(49-52), and t h a t are c h a l l e n g i n g t r a d i t i o n a l m o d a l i t i e s of imaging i n c l i n i c a l p r a c t i c e . The absence of hazard compared w i t h other methods commonly used(53-56); the e x c e l l e n t t i s s u e c o n t r a s t and p a t h o l o g i c a l d i s c r i m i n a t i o n ; the a b i l i t y t o y i e l d d i r e c t t r a n s v e r s e , c o r o n a l , and s a g i t t a l images; and the ready p e n e t r a t i o n of bony t i s s u e are a l l important advantages that NMR tomography can o f f e r . Since hydrogen('H) i s the most abundant element i n a l l l i v i n g systems, the proton has been the f a v o r i t e nucleus f o r NMR imaging, but some work has a l s o been done w i t h 1 3C, 1 9 F , 2 3 N a , 3 1 P , though w i t h l e s s s e n s i t i v i t y and poorer anatomical r e s o l u t i o n . Recent development i n v o l v e s the i n t r o d u c t i o n of whole-body, h i g h - f i e l d superconducting magnets capable of both NMR imaging and i n v i v o NMR spectroscopy on the same s u b j e c t ( 1 3,1 4,57-59) . 5 In view of the importance of NMR techniques, both to NMR spectroscopy/imaging i n general and to the work of t h i s t h e s i s i n p a r t i c u l a r , a b r i e f d i s c u s s i o n of these methods i s given i n the next two s e c t i o n s . T h i s w i l l f a m i l i a r i z e the reader w i t h the ba s i c concepts and nomenclature used i n both NMR spectroscopy and imaging, which are o f t e n r e f e r r e d to i n subsequent chapters of t h i s t h e s i s . As w i l l be seen there are two d i f f e r e n t models(the c l a s s i c a l m a gnetization vector model and the quantum mechanical d e s c r i p t i o n ) which are commonly used to e x p l a i n NMR experiments; these w i l l be b r i e f l y reviewed i n the next s e c t i o n , mainly from an experimental chemist's p o i n t of view, without going i n t o mathematical d e t a i l s . Both types of nuclear r e l a x a t i o n processes, namely s p i n l a t t i c e and sp i n s p i n r e l a x a t i o n processes, and the most common techniques f o r t h e i r d e t ermination w i l l be d i s c u s s e d in o n l y s u f f i c i e n t d e t a i l s to provide a q u a l i t a t i v e overview. Of the four d i f f e r e n t types of NMR imaging t e c h n i q u e s ( i . e . s e q u e n t i a l p o i n t , s e q u e n t i a l l i n e , s e q u e n t i a l plane, and simultaneous volume methods), only planar and volume imaging methods are widely used; between them, two c o n c e p t u a l l y d i f f e r e n t ideas have evolved as w i l l be di s c u s s e d i n d e t a i l . An e v a l u a t i o n of both methods w i l l be given i n S e c t i o n 1.3 to a l l o w the reader to make an o b j e c t i v e d e c i s i o n as to which of the two methods that the reader should use. 6 F i n a l l y , i n Se c t i o n 1.4, v a r i o u s parameters a f f e c t i n g s ignal(image) c o n t r a s t w i l l be d i s c u s s e d so as to h i g h l i g h t the complexity of the NMR techniques and to f a c i l i t a t e i n t e r p r e t a t i o n of the NMR image. 1.2 T h e o r e t i c a l B a s i s f o r Chemical A n a l y s i s ( 6 0 - 6 2 ) NMR i s a phenomenon e x h i b i t e d by many atomic n u c l e i which are n a t u r a l l y abundant i n the body such as hydrogen('H), phosphorus( 3 1P) and c a r b o n ( 1 3 C ) , each of these has an odd number of e i t h e r protons or neutrons. Such n u c l e i possess quantum mechanical s p i n angular momentum which endows them w i t h a small magnetic f i e l d , or magnetic moment(Figure 1.1). When place d i n an e x t e r n a l l y a p p l i e d s t a t i c magnetic f i e l d , the energy l e v e l s f o r a spi n 1/2 nucleus are qu a n t i z e d and only two are p o s s i b l e f o r ( 2 S + 1 ) ] ; q u a l i t a t i v e l y we say that the n u c l e i are a l i g n e d e i t h e r p a r a l l e l or a n t i p a r a l l e l w i t h the a p p l i e d f i e l d . These two o r i e n t a t i o n s have s l i g h t l y d i f f e r e n t e n e r g i e s ; and the energy r e q u i r e d to induce t r a n s i t i o n s between them and thereby to ob t a i n an NMR s i g n a l i s j u s t the energy d i f f e r e n c e , AE, between the two nuclear o r i e n t a t i o n s ; t h i s i s dependent on the s t r e n g t h of the magnetic f i e l d B 0 i n which the nucleus i s p l a c e d ( F i g u r e 1.2) AE=7hB0/27r Eq. 1.1 where h i s Planck's constant (6.63X10" 2 7 erg-sec).'The Bohr c o n d i t i o n (AE=hp) enables the frequency v0 of the nuclear 7 Magnet ic Held FIGURE 1.1 (a) A nucleus can be v i s u a l i z e d as s p i n n i n g about i t s own a x i s which i s the a x i s of i t s magnetic moment. (b) O r i e n t a t i o n s that can be taken up i n an a p p l i e d f i e l d B 0 by the magnetic moment of a nucleus of s p i n 1/2. The o r i e n t a t i o n s are s p e c i f i e d by the quantum number m and d e s c r i b e two cones. 8 F i g u r e 1.2 In the presence of a steady magnetic f i e l d , B 0, some n u c l e i (e.g. 1H, 13C,> 3 1 P ) behave l i k e t i n y bar magnets and a l i g n themselves p a r a l l e l or a n t i p a r a l l e l to the d i r e c t i o n of B 0. The two o r i e n t a t i o n s , or s p i n s t a t e s , have d i f f e r i n g e nergies and t r a n s i t i o n s between these s t a t e s can be introduced by a radiofrequency magnetic f i e l d o s c i l l a t i n g at a frequency, v0. 9 t r a n s i t i o n to be w r i t t e n as *>0 = 7Bo / 2 7 T Eq. 1.2 Equation 1.2 i s oft e n r e f e r r e d as the Larmor resonance equation. The constant of p r o p o r t i o n a l i t y , 7 , i s c a l l e d the gyromagnetic r a t i o . I t i s d i f f e r e n t f o r each nuclear s p e c i e s , and consequently the NMR s i g n a l from a p a r t i c u l a r element can be observed or tuned in without i n t e r f e r e n c e from any oth e r . I f the resonance frequency of a nucleus were only d i r e c t l y p r o p o r t i o n a l t o the e x t e r n a l magnetic f i e l d , then a l l protons f o r example would absorb energy at the same frequency, and NMR would be a r e l a t i v e l y u n i n t e r e s t i n g and uninformative technique incapable of d i s t i n g u i s h i n g between protons from d i f f e r e n t atoms or molecules. However, the a p p l i e d f i e l d a l s o induces e l e c t r o n i c c u r r e n t s i n atoms and molecules, and these induced c u r r e n t s w i l l produce a s m a l l magnetic f i e l d which i s opposed t o the a p p l i e d f i e l d and a c t s to p a r t i a l l y c a n c e l the a p p l i e d f i e l d , thus s h i e l d i n g the n u c l e u s ( F i g u r e 1.3). In g e n e r a l , the induced opposing f i e l d i s about a m i l l i o n times smaller than the a p p l i e d f i e l d . Consequently, the magnetic f i e l d p e r c e i v e d by the nucleus w i l l be very s l i g h t l y a l t e r e d from the a p p l i e d f i e l d , so the resonance c o n d i t i o n of Equation 1.2 w i l l need to be m o d i f i e d t o : j>0 = 7B _./2ir=7Bo(1 -a)/2ir E<3- 1.3 err where B e f £ i s the l o c a l f i e l d experienced by the nucleus,and 0 i s a nondimensional s c r e e n i n g , or s h i e l d i n g , c o n s t a n t , and has 10 Magnetic Nucleus Electron Currents Induced Magnetic Field Figure 1.3 E l e c t r o n currents around a nucleus are induced by pl a c i n g the molecule in a magnetic f i e l d B 0. These electron currents, in turn, induce a small magnetic f i e l d opposed, to the applied magnetic f i e l d B 0. 11 values t y p i c a l l y i n the region 10~ 6-10" 3. The magnitude of o i s dependent upon the e l e c t r o n i c environment of the nucleus, and t h e r e f o r e n u c l e i i n d i f f e r e n t chemical environments gi v e r i s e to s i g n a l s at d i f f e r e n t f r e q u e n c i e s . The s e p a r a t i o n of resonance f r e q u e n c i e s from an a r b i t r a r i l y chosen reference frequency i s termed the chemical s h i f t ( 6 ) , and i s expressed i n terms of the dimensionless u n i t s of p a r t s per million(ppm) (Figure 1.4) b=(v , -v c )x1U 6/" c Eq. 1.4 sample r e f . r e f . M Equation 1.4 i m p l i e s that the chemical s h i f t i s independent of the magnetic f i e l d s t r e n g t h and t h e r e f o r e the o p e r a t i n g f requency. The above d e s c r i p t i o n of nuclear magnetization was e s s e n t i a l l y based on quantum mechanics. However, another way of viewing the NMR phenomenon i s from a c l a s s i c a l d e s c r i p t i o n . For a s p i n 1/2 nucleus i n a magnetic f i e l d of B 0, the nuclear magnetic moment w i l l precess about the Z a x i s . Some spins precess about the p o s i t i v e Z a x i s and some about the negative Z a x i s . The net r e s u l t a n t magnetization i s simply the sum of a l l the i n d i v i d u a l nuclear magnetic moments ( s p i n s ) . Since there i s a s l i g h t excess of n u c l e i o r i e n t e d with the magnetic f i e l d , the o v e r a l l sum y i e l d s a magnetization M 0 along the p o s i t i v e Z a x i s . Let us now consider w i t h the a i d of Figu r e 1.5 how an observable NMR s i g n a l can be produced. A r o t a t i n g c o o r d i n a t e system (X',Y'and Z) i s used i n which the X' and Y' axes are \2 Figure 1.4 Nuclear magnetic resonance s p e c t r a l parameters. 1 3 F i g u r e 1.5 R o t a t i o n of the magnetization M 0 i n the r o t a t i n g c o o r d i n a t e system that r o t a t e s about the Z a x i s at the nuclear magnetic resonance instrument's o p e r a t i n g frequency, (a) s p i n system at e q u i l i b r i u m i n B 0 magnetic f i e l d ; (b) a p p l i c a t i o n of ^a 90° (or 7r/2) B, p u l s e ; and (c) a p p l i c a t i o n of a 180° (or TT) B, p u l s e . 14 r o t a t i n g about the Z a x i s at the NMR instrument's o p e r a t i n g frequency. The use of such a c o o r d i n a t e system enables us to cons i d e r the e f f e c t of a p p l y i n g an RF pulse B, along the X' a x i s and observing magnetization (and t h e r e f o r e a s i g n a l ) along the Y' a x i s , i n s t e a d of being concerned about the frequency of the B, pulse and of o b s e r v a t i o n . F i g u r e 1.5a shows the magnetization M 0 at thermal e q u i l i b r i u m i n magnetic f i e l d B 0. Ma g n e t i z a t i o n w i l l be r o t a t e d i n a plane p e r p e n d i c u l a r to the a p p l i e d B, p u l s e , i . e . the Y'Z plane when B, i s along X'; of course, the RF pulse B, must be at the a p p r o p r i a t e frequency v0 as expressed by Equation 2. The angle of r o t a t i o n 8 depends on the gyromagnetic r a t i o 7 of the nucleus, the amplitude B, of the RF p u l s e , and the length of time toj the RF pulse i s a p p l i e d : 0 = 7 B , t w Eq. 1.5 F i g u r e 1.5b i l l u s t r a t e s the r o t a t i o n of M 0 by a p p l i c a t i o n of the RF pulse f o r s u f f i c i e n t time to r o t a t e M 0 by 90°(8 = ir/2 r a d i a n s ) . That pulse i s c a l l e d a 90° or 7r/2 p u l s e . A p p l i c a t i o n of the B, f i e l d f o r twice as long (6=ir r a d i a n s ) w i l l r e s u l t i n i n v e r s i o n of M 0 as shown i n F i g u r e 1.5c. The quantum mechanical analogs of 90° and 180° pulses of spi n 1/2 nucleus are as f o l l o w s : the 90° pulse produces an e q u a l i z a t i o n of p o p u l a t i o n s i n the two energy s t a t e s ; and the 180° pulse produces an i n v e r s i o n of p o p u l a t i o n s so t h a t the high energy s t a t e has a l a r g e r number of nuclear s p i n s . 15 Both types of RF pulse are applied in sequences for NMR imaging and for measuring various NMR parameters. Experimentally, the NMR s i g n a l i s detected by a tuned RF c o i l with axis perpendicular to B 0. The o s c i l l a t i n g NMR magnetization induces a voltage in the c o i l , analogous to the p r i n c i p l e of an e l e c t r i c generator. The induced s i g n a l immediately following an RF pulse i s termed a free induction decay (FID), r e f l e c t i n g the decay in the s i g n a l as nuclei dephase and eventually relax back to thermal e q u i l i b r i u m . This return to e q u i l i b r i u m i s c h a r a c t e r i z e d by two relaxation times, T, and T 2. The re l a x a t i o n processes occur by interacton of the nuclear spin with f l u c t u a t i n g magnetic f i e l d s produced by magnetic dipoles (e.g. other n u c l e i , paramagnetic ions) which are f l u c t u a t i n g due to random molecular motions, both r o t a t i o n a l and t r a n s l a t i o n a l . The nature and the rate of the molecular motions a f f e c t the T, and T 2 r e l a x a t i o n times. Molecular motions that occur at a rate comparable to the resonance frequency v0 for the nucleus are most e f f e c t i v e in promoting s p i n - l a t t i c e r e l a x a t i o n , i . e . y i e l d the lowest values for T,; T 2 values can be decreased even further as the molecular motion becomes slower than v0, but T, values w i l l begin to increase(Figure 1 . 6 ) . These r e l a t i o n s h i p s can be expressed generally as follows: 1 / T , = 7 2 H 2 [ T /1 + (2**» 0T ) 2 ] Eq. 1.6 c c and 1/T 2 = 7 2 H 2 [ T +{T /1 + (2IT V 0 T ) 2}] Eq. 1.7 c c c where H 2 i s a measure of the strength of the i n t e r a c t i o n between 1 6 Figure 1.6 Dependence of T, and T 2 relaxation times of protons in water on the v i s c o s i t y TJ of the s o l u t i o n . The c o r r e l a t i o n time T i s p roportional to the v i s c o s i t y n. (After B e a l l et al.,Data Handbook for Biomedical A p p l i c a t i o n s , Pergamon Press, New York, 1984.) 17 the nuclear s p i n and the f l u c t u a t i n g magnetic f i e l d s and r i s c the c o r r e l a t i o n time. The c o r r e l a t i o n time i s a q u a n t i t a t i v e measure of the r a t e of a molecular motion; f o r r o t a t i o n a l motion i t i s the l e n g t h of time r e q u i r e d to r o t a t e through an angle of 33°; f o r t r a n s l a t i o n a l motion, the c o r r e l a t i o n time i s the time r e q u i r e d f o r a molecule to move through a d i s t a n c e equal to i t s diameter. T y p i c a l l y , the c o r r e l a t i o n time, e i t h e r r o t a t i o n a l or t r a n s l a t i o n a l , i s 1 0 " 1 2 to 10" 1 1 second f o r s m a l l molecules, 10" 9 to 10" 6 second f o r macromolecules(or small molecules bound to macromolecules), and 10~ 6 to 10" 3 second f o r some motions i n membranes. C l e a r l y from F i g u r e 1.6 , we expect T, and T 2 to be equal f o r small molecules, and to diverge f o r macromolecules (or small molecules bound to them). F a s t e r i n t e r n a l motions can e x i s t i n a macromolecule or membrane i n a d d i t i o n to o v e r a l l r o t a t i o n a l and t r a n s l a t i o n a l motions that can make the s i t u a t i o n more complicated. S e v e r a l d i f f e r e n t pulse sequences have been u t i l i z e d to measure T, and T 2(60-62). The most common (and probably the best) technique f o r determining T, i s to employ the " i n v e r s i o n recovery" sequence (180°-r-90°). A f t e r an i n i t i a l 180° pulse which i n v e r t s the s p i n p o p u l a t i o n s , the s p i n system begins r e l a x i n g toward thermal e q u i l i b r i u m . A f t e r time T, a 90° pulse i s a p p l i e d , and the FID f o l l o w i n g the pulse i s a c q u i r e d . As r becomes lo n g e r , the magnetization more c l o s e l y approaches the e q u i l i b r i u m s i t u a t i o n r a t h e r than the i n v e r t e d m a g n e t i z a t i o n . A 90° pulse must be a p p l i e d to be able to detect any magnetization 18 because d e t e c t i o n i s p o s s i b l e only i n the t r a n s v e r s e plane. The value of T, may be determined by measuring the magnitude M of the FID as a f u n c t i o n of T using the f o l l o w i n g e x p r e s s i o n : M ( T ) = M o e [ l - 2 e x p ( - T / T 1 ) ] Eq. 1.8 where i s the magnitude of FID when r= i n f i n i t y ( i . e . at thermal e q u i l i b r i u m ) . A simple way to c a l c u l a t e T, from the measured M (r) values i s to observe the value f o r which the z s i g n a l a f t e r the 90° pulse becomes z e r o . T h i s i s r e f e r r e d to as r ... T, can be obtained from:, n u l l T n u l l - ° - 6 9 8 T ' E ( 3 - K 9 While t h i s i s a very quick way of determining T,, i t i s not u s u a l l y recommended i f an accurate T, i s d e s i r e d . N e v e r t h e l e s s , a dete r m i n a t i o n of T, from ^ helps the operator to determine the c o r r e c t p e r i o d to be used between e x c u r s i o n s ( F i g u r e 1.7). The data, M (T) determined f o r d i f f e r e n t T values can be z p l o t t e d on a s e m i l o g a r i t h m i c p l o t of [1-M (r)/M ]/2 versus 2 co r ( F i g u r e 1.8). T, i s obtained from the slope of the s t r a i g h t l i n e a c c o r d i n g to a rearranged Equation 1.8: l n { [ l - ( M (T ) /M )]/2}= - T / T , Eq. 1.10 2 0 0 Determination of T 2 i s best c a r r i e d out using a C a r r - P u r c e l l pulse sequence w i t h the Meiboom-Gill m o d i f i c a t i o n ( 6 0 - 6 2 ) . Figure 1.9 i l l u s t r a t e s the process i n the r o t a t i n g c o o r d i n a t e frame. An i n i t i a l 90° pulse r o t a t e s the magnetization i n t o the Y' 19 Figure 1.7 Build-up of magnetization as a function inversion recovery sequence. 20 1.0 CM I I N S i iZ i 0.1 .01 J L J L T (sec) F i g u r e 1.8 T y p i c a l p l o t of [1-M (T)/M ]/2 versus T f o r de t e r m i n a t i o n of T, acc o r d i n g t o the l8§°-r-9u'0 method f o r water i n t i s s u e s and c e l l s . N o t i c e the s l i g h t l y nonexponential nature of the p l o t . For pure water, such a p l o t would be a s i n g l e s t r a i g h t l i n e . The p l o t shown i s f o r an a r b i t r a r y b i o l o g i c a l system(After B e a l l et a l . , Data Handbook f o r Biomedical A p p l i c a t i o n s , Pergamon Pr e s s , New York, 1984) 21 F i g u r e 1.9 The 90°-r/2-180° T 2 experiment. The radiofrequency p u l s e s of s t r e n g t h B, are a p p l i e d along the X' a x i s i n the r o t a t i n g c o o r d i n a t e system. D e t e c t i o n i s along the Y' a x i s so a s i g n a l can be detected a f t e r the 90° pulse(b) and at formation of the s p i n - e c h o ( f ) . 22 d i r e c t i o n ( b ) . Because the B 0 magnetic f i e l d i s not g e n e r a l l y p e r f e c t l y homogeneous, the i n d i v i d u a l magnetic s p i n s w i l l have s l i g h t l y d i f f e r e n t p r e c e s s i o n a l frequencies . Consequently, the i n d i v i d u a l spins w i l l fan out i n the X'Y' plane w i t h a l o s s of phase coherence ( c ) . A f t e r a time T/2, a 180° pulse i s a p p l i e d ( d ) . Because the p r e c e s s i o n a l f r e q u e n c i e s of the i n d i v i d u a l s p i n s are unchanged ( e ) , they w i l l then achieve phase coherence at time T along the negative Y' a x i s ( f ) . This w i l l r e s u l t i n a s i g n a l , c a l l e d a spin-echo, being d e t e c t e d . The C a r r - P u r c e l l ( C P ) sequence i s i n i t i a t e d w i t h a 90° pulse f o l l o w e d by a s e r i e s of 180° p u l s e s . The Carr-Purcell-Meiboom-Gi1 1(CPMG) sequence i s e s s e n t i a l l y the same as the CP sequence, except that a phase s h i f t of 90° i s introduced i n a l l the 180° p u l s e s to e l i m i n a t e e f f e c t s due to i m p e r f e c t i o n of the ir p u l s e s . The magnitude of the echo decays e x p o n e n t i a l l y as r i n c r e a s e s , so that the echo magnitude can be expressed as: M(r)=M [ e x p ( - r / T 2 ) ] Eq. 1.11 OO where r i s the l e n g t h of time from the 90° pulse to the top of the echo. T 2 can be determined from the slope of a semilog p l o t of M(r)/M versus T ( F i g u r e 1.10) according to the f o l l o w i n g OO rearrangement of Equation 1.11: ln[M(r)/M ]=-T / T 2 Eq. 1.12 OO 1.3 Methods of NMR Imaging 23 1.0 - O 0. O o o o O Q CD T (sec) Figure 1.10 Typical plot of MT/M versus T for the Hahn spin-echo or Carr-Purcell-Meiboom-Gill sequence for a biological system. Notice the nonexponential nature of the plot; for pure water, such a plot would be a single straight line.(After Beall et a l . , Data Handbook for Biomedical Applications, Pergamon Press, New York, 1984) 24 The NMR p r i n c i p l e s discussed so far are used in NMR spectroscopy, but the output i s an average of c o n t r i b u t i o n s from d i f f e r e n t l o c a t i o n s within the sample and contains no s p a t i a l information. To obtain an image, s p a t i a l information has to be encoded into the NMR s i g n a l . There e x i s t numerous approaches to the c r e a t i o n of an NMR image. However, most share one commonality: the magnetic f i e l d i s mapped in such a manner that i t becomes position-dependent, so that nuclei r e s i d i n g in d i f f e r e n t l o c a t i o n s of the specimen resonate at d i f f e r e n t Larmor frequencies. According to Brunner and Ernst(63), NMR imaging techniques can be c l a s s i f i e d into four types, depending on the type of volume that produces the received s i g n a l used to make an image (Figure 1.11, Table 1.1). Assuming the t o t a l imaging volume to be divided into Nx, Ny and Nz volume elements (voxels) along the three s p a t i a l coordinates, then NxNyNz signa l elements are required to reconstruct a l l possible images within t h i s volume. The i n d i v i d u a l values may be obtained from N<NxNyNz experiments. In the simplest imaging experiment the s i g n a l of each voxel i s acquired independently. This method, therefore, has been termed the sequential point method, as opposed to the sequential l i n e method, where a row of voxels i s detected simultaneously. In the sequential plane method--the l o g i c a l extension of the sequential l i n e method—an e n t i r e plane of voxels i s detected at a time. F i n a l l y , in three-dimensional imaging, the signals of a l l voxels are observed at the same time. 25 F i g u r e 1.11 C a t e g o r i z a t i o n of imaging techniques a c c o r d i n g to the volume detected per u n i t time:(a) s i n g l e p o i n t ; (b) l i n e ; (c) plane; (d) volume(from documents f u r n i s h e d by General E l e c t r i c ) . 26 Table 1.1 Methods of NMR Imaging(63) 1. S e q u e n t i a l P o i n t Methods S e n s i t i v e p o i n t (SSP) (64-65) Focused nuclear resonance(FONAR) (66-67) 2. S e q u e n t i a l Line Methods S e l e c t i v e e x c i t i o n l i n e scan (68-70) S e n s i t i v e l i n e or m u l t i p l e s e n s i t i v e point(MSP) (71) 3. S e q u e n t i a l Plane Methods Two-dimensional p r o j e c t i o n - r e c o n s t r u c t ion(72-73) Two-dimensional F o u r i e r imaging(spin warp)(23,74-75) Planar imaging(76) Two-dimensional echo-planar imaging(77-78) R o t a t i n g frame imaging(79) 4. Simultaneous Method Three-dimensional p r o j e c t i o n - r e c o n s t r u c t ion(80) Tree-dimensional F o u r i e r imaging(75) M u l t i p l a n a r imaging(76) Three-dimensional echoplanar imaging(77-78) 27 I t i s probably f a i r to say that both s e q u e n t i a l p o i n t and l i n e methods have l a r g e l y been abandoned and may t h e r e f o r e be considered h i s t o r y . Both methods are r e l a t i v e l y i n e f f i c i e n t , s i n c e s i g n a l s are gathered from a r e l a t i v e l y s m a l l number of n u c l e i o n l y . Conversely, the p l a n a r and volume imaging methods, which are now favored, are both c o m p u t a t i o n a l l y more demanding. Among the g e n e r a l l y adopted planar and volume imaging techniques, two c o n c e p t u a l l y d i f f e r e n t ideas have evolved, each of which e x i s t s i n somewhat d i f f e r i n g v a r i a n t s : (a) P r o j e c t i o n r e c o n s t r u c t i o n techniques, (b) Two (three) dimensional F o u r i e r - t r a n s f o r m imaging. The f i r s t method was proposed by Lauterbur i n 1973(23). Since an NMR frequency i s d i r e c t l y p r o p o r t i o n a l to the magnetic f i e l d experienced by each nucleus, and s i n c e each response i s s h a r p l y resonant, p a r t i c u l a r l y f o r f l u i d s , the NMR spectrum of a s t r u c t u r e d specimen i n a l i n e a r f i e l d g r a d i e n t p r o v i d e s a one-dimensional p r o j e c t i o n of nuclear d e n s i t y along the d i r e c t i o n of the g r a d i e n t (Figure 1.12a). The magnetic f i e l d g r a d i e n t can be e l e c t r o n i c a l l y r o t a t e d to o b t a i n enough of these p r o j e c t i o n s and then a f i l t e r e d back p r o j e c t i o n a l g o r i t h m i s used to r e c o n s t r u c t , w i t h a computer, a c r o s s - s e c t i o n of the o b j e c t (Figure 1.12b). Any e f f e c t i v e d i r e c t i o n of the g r a d i e n t may be obtained using a system of three orthogonal g r a d i e n t c o i l s (Gx,Gy,Gz). The advantage of the p r o j e c t i o n - r e c o n s t r u c t i o n technique i s i t s r e l a t i v e s i m p l i c i t y and the use of w e l l - e s t a b l i s h e d a l g o r i t h m s f o r image r e c o n s t r u c t i o n . On the 28 [a) FREQUENCY NMR SIGNAL IN THE FREQUENCY SPACE FREQUENCY Figure 1.12 (a) Use of magnetic f i e l d gradient to obtain a pr o j e c t i o n of the sample. (b) Rotating f i e l d gradients give several p rojections from which i t i s possible to reconstruct an image of the ob j e c t ( A f t e r Descouts, Prog. Nucl. Med. 8:15,1984). 29 negative s i d e are i t s s e n s i t i v i t y to motion a r t i f a c t s and f i e l d inhomogeneity. The second method of m u l t i d i m e n s i o n a l NMR was f i r s t proposed by Kumar et a l . ( 7 5 ) . Common to t h i s c l a s s of experiments i s a succession of three d i f f e r e n t time p e r i o d s i n the pulse sequence: a p r e p a r a t i o n p e r i o d , an e v o l u t i o n p e r i o d and a d e t e c t i o n p e r i o d . During the e v o l u t i o n p e r i o d f o l l o w i n g an RF p u l s e , one of two pe r p e n d i c u l a r g r a d i e n t s , f o r example Gx, i s a c t i v e . This g r a d i e n t i s turned o f f a f t e r tx seconds and gradient Gy i s a c t i v a t e d , during which time (ty ) the FID i s c o l l e c t e d (Figure 1.13). The frequency Wy at which the magnetization precesses determines the l o c a t i o n of the re s o n a t i n g spins w i t h respect to y; whereas the magnetization f o r each l o c a t i o n x has a unique phase angle a s s o c i a t e d w i t h i t . The t r a n s v e r s e magnetization v e c t o r s , when p l o t t e d a g a i n s t the y c o o r d i n a t e , are t w i s t e d i n a s p i r a l f a s h i o n comparable i n appearance to the steps of c i r c u l a r s t a irway except, of course, that t h e i r lengths are r e l a t e d t o the proton d e n s i t y at t h e i r r e s p e c t i v e l o c a t i o n s (Figure 1.14). The image i s obtained by c o l l e c t i n g n FIDs wi t h Atx incremented from tx=0 to tx=n.Atx, where Atx i s the time increment. The r e s u l t i n g nxn data matrix i s then F o u r i e r transformed i n both dimensions. A s l i g h t l y d i f f e r e n t v e r s i o n of the method, c a l l e d the 'Spin Warp', proposed by E d e l s t e i n et a l (74), c r e a t e s the phase t w i s t by v a r y i n g the gradient amplitude d u r i n g the e v o l u t i o n p e r i o d and h o l d i n g tx constant. The e f f e c t on phase i s the same as before. The main advantage of Spin Warp over other imaging techniques i s 30 F i g u r e 1.13 P r i n c i p l e of 2D FT imaging, shown s c h e m a t i c a l l y f o r a sample c h a r a c t e r i z e d by an X and Y c o o r d i n a t e . The p r e c e s s i o n f r e q u e n c i e s d u r i n g p e r i o d s tx and ty are a f u n c t i o n of the g r a d i e n t s Gx and Gy and the sample's s p a t i a l l o c a t i o n . Data sampling occurs d u r i n g the t y p e r i o d only. Note that the s i g n a l phase i s r e l a t e d to the X c o o r d i n a t e ( f r o m documents f u r n i s h e d by General E l e c t r i c ) . 31 y Coordinate F i g u r e 1.14 S i g n a l phase i n t h e 2D FT e x p e r i m e n t of F i g u r e 1.13, showing t h e m a g n e t i z a t i o n a t t h e b e g i n n i n g of the t y p e r i o d ( d a t a a c q u i s i t i o n ) as a f u n c t i o n of t h e Y l o c a t i o n ( f r o m documents f u r n i s h e d by G e n e r a l E l e c t r i c ) . 32 the f a c t that i t i s r e l a t i v e l y t o l e r a n t to s t a t i c magnetic f i e l d inhomogeneities and, f o r c l i n i c a l a p p l i c a t i o n s , to motion a r t i f a c t s such as b r e a t h i n g , p e r i s t a l t i c motion, e t c . I t has the a b i l i t y to image r e c t a n g u l a r f i e l d s of view t a i l o r e d to body shape; and i t a l s o has some fa v o r a b l e s i g n a l - t o - n o i s e c h a r a c t e r i s t i c s . In summary, i n p r o j e c t i o n r e c o n s t r u c t i o n , a s i n g l e gradient i s r o t a t e d e l e c t r o n i c a l l y around the plane. L i n e s i n the plane are p r o j e c t e d onto p o i n t s on a l i n e from about as many d i f f e r e n t angles as there are l i n e s i n the p i c t u r e (Figure 1.15a). However, i n the two-dimensional F o u r i e r transform method, the l i n e of p r o j e c t i o n i s f i x e d ; a second g r a d i e n t , whose a x i s i s at r i g h t angles(Gx) to the g r a d i e n t that d e f i n e s the p r o j e c t e d l i n e ( G y ) ( F i g u r e 1.15c), p r o v i d e s the a d d i t i o n a l phase i n f o r m a t i o n needed to c r e a t e a two-dimensional image (Figure 1.15b). 1.4 Sample (Tissue) P r o p e r t i e s Which A f f e c t NMR S i g n a l (Image) The r e l a t i v e i n t e n s i t y of NMR s i g n a l s (or images) i s determined by v a r i o u s sample (or t i s s u e ) p r o p e r t i e s and by the experimental(imaging) parameters that are s e l e c t e d ( F i g u r e 1.16). The c o n t r a s t i s determined by:(a) the proton d e n s i t y ; (b) the T, r e l a x a t i o n time, which determines the degree of magnetization and thus the i n i t i a l resonance s i g n a l i n t e n s i t y ; (c) the T 2 r e l a x a t i o n time, determining the decay r a t e of the s i g n a l and thus the i n t e n s i t y at the moment of measuring; and (d) flow and motion e f f e c t s . S i g n a l i n t e n s i t y f o r a c o n v e n t i o n a l s i n g l e pulse 33 1 1 1 \ \ 1 1 1 1 1 1 j 1 1 1 1 t 1 1 J 1 (a) (b) r -i i -i b i i i i i i i i i j (c) F i g u r e 1.15 P r o j e c t i o n - r e c o n s t r u c t i o n method versus F o u r i e r zeugmatography. NMR s i g n a l s are c r o s s - s e c t i o n s of the f u n c t i o n F ( k x , k y ) . (a) The p r o j e c t i o n - r e c o n s t r u c t i o n method d e t e c t s the s i g n a l s along l i n e s through the o r i g i n i n the k x , k y - p l a n e , (b) F o u r i e r zeugmatography d e t e c t s the s i g n a l s along p a r a l l e l l i n e s , (c) E x c u r s i o n i n the k x , k y - p l a n e f o r d e t e c t i o n along one of the l i n e s . The path a ( p r e p a r a t i o n p e r i o d ) i s covered by t a k i n g G x negative and ( i n the case i l l u s t r a t e d ) Gy p o s i t i v e ; the d e t e c t i o n path b i s covered by t a k i n g G„ zero and G x p o s i t i v e ( A f t e r Locker, P h i l i p s T e c h n i c a l Review 41:73,1983/84). 34 S(H)(%) |-100 Brain Uver! Muscle Bone T, (ms) Brain™ I I Muscle B o n e 5 L J v e f Fat" T 2 (ms) 100 Brain Uver Muscle Figure 1.16 Water content S(H), spin-lattice relaxation time T,, and spin-spin relaxation time T 2 of various human tissues.(After Buchmann and Heinzerling(1983)GIT Lab Med 6:102) 35 proton N M R experiment i s given by combining Equations 1.8 and 1.11 a f t e r s u b s t i t u t i n g T f o r r i n Equation 1.11 and T f o r r i n E R Equation 1.8:(Figure 1.17) . S ( t ) = S ( H ) f ( v ) e x p ( - T _ / T 2 ) [ l - 2 e x p ( - T /T,)] Eq. 1.13 h i R where sample ( t i s s u e ) p r o p e r t i e s : S(H)=water content; T , = s p i n - l a t t i c e r e l a x a t i o n time; T 2 = s p i n - s p i n r e l a x a t i o n time; f ( v ) = s i g n a l i n t e n s i t y change due to flow/motion e f f e c t ; experimental parameters: T =delay time between e x c i t a t i o n and s i g n a l a c q u i s i t i o n ; E T R =delay time between s u c c e s s i v e data a c q u i s i t i o n s . The r e l a t i o n s h i p between N M R p r o p e r t i e s and N M R s i g n a l (image) i n t e n s i t y f o r a given set of experimental (imaging) parameters i s shown i n Table 1.2. In imaging, by v a r y i n g the parameters T and T , the E R r e l a t i v e weight of one of the two r e l a x a t i o n times i n the N M R tomogram can be accentuated(Table 1.3). The d i a g n o s t i c value of an N M R tomogram r e l i e s not on the image i n t e n s i t y i t s e l f , but on image c o n t r a s t , i . e . the s i g n a l d i f f e r e n c e ( b r i g h t n e s s ) between two t i s s u e s t h a t must be d i f f e r e n t i a t e d . The o b j e c t , then, of o p t i m i z i n g the parameters T and T i s to t r a n s l a t e d i f f e r e n c e s E R i n the t i s s u e p r o p e r t i e s S(H), T, and T 2 i n t o the h i g h e s t degree 36 Pulse Pulse I TR I -(Image') intensity S(H) Water content exp ' 2 V relaxation 1-lexp -V relaxation Figure 1.17 C o r r e l a t i o n between time diagram and signal(image) i n t e n s i t y . ( A f t e r Roth, NMR-Tomography and Spectroscopy in Medicine-An Introduction, Springer-Verlag Inc., B e r l i n , 1984) 37 Table 1.2 C o r r e l a t i o n between sign a l intensity(image brightness) and sample(tissue) properties.(85) Tissue property Image brightness Water content Tissue containing large amounts of water appears light Spin-lattice relaxation time T, Tissue with long T) appears dark Spin-spin relaxation time T 2 Tissue with long T 2 appears light Table 1.3 E f f e c t of imaging parameters on the which contribute to the NMR images(85) ti s s u e properties Imaging parameters Tissue properties Short T t , long T R a N(H) Short T H , short T R N(H), T, Long T L , long T K N ( H ) , T : Long T t , shortTK N(H), T , , T , Large or small relative to the corresponding relaxation time 38 of image c o n t r a s t p o s s i b l e . The determination of optimum imaging parameters i s complicated by other f a c t o r s , as w e l l . For example, many p a t h o l o g i c a l processes are a s s o c i a t e d w i t h an e l e v a t e d water content as w e l l as w i t h an increase i n both r e l a x a t i o n times. According to Table 1.2, an increased water content and a prolonged T 2 tend to i n c r e a s e image b r i g h t n e s s , while an increase i n T, tends to reduce i t . As a r e s u l t , both e f f e c t s may c a n c e l out i n c e r t a i n circumstances, and normal and diseased t i s s u e may not be d i f f e r e n t i a t e d . Thus, a d e t a i l e d knowledge of t i s s u e r e l a x a t i o n r a t e s i s e s s e n t i a l to optimize image c o n t r a s t and to d i f f e r e n t i a t e normal from diseased t i s s u e s ( F i g u r e s 1.18 and 1.19). In apparent c o n t r a d i c t i o n to what has a l r e a d y been s a i d , l a r g e v e s s e l s that c o n t a i n f l o w i n g blood r e g i s t e r "dark" on the NMR image, im p l y i n g low s i g n a l i n t e n s i t y d e s p i t e the f a c t t h a t blood i s more than 98% water. The absence of an NMR s i g n a l i n f l o w i n g blood i s based on the f a c t that i t takes a f i n i t e amount of time to r e c o r d the s i g n a l emitted by the r e s o n a t i n g n u c l e i . A f t e r the n u c l e i have been e x c i t e d i n the s e l e c t e d plane, the a c t u a l data a c q u i s i t i o n commences a f t e r an i n t e r v a l T E, and the e n t i r e d a t a - a c q u i s i t i o n process i s repeated a f t e r a w a i t i n g p e r i o d T . In a s t a t i o n a r y f l u i d , the i n t e n s i t y of the s i g n a l decays d u r i n g T by T 2 r e l a x a t i o n , and i t i n c r e a s e s d u r i n g T by T, r e l a x a t i o n . In a slow-moving f l u i d , there i s very l i t t l e 39 3 Short T t 0 TR 0 TE Time F i g u r e 1 . 1 8 The NMR proton image c o n t r a s t between normal and p a t h o l o g i c a l t i s s u e depends on d i f f e r e n c e s i n T, and T 2 r e l a x a t i o n times and, to a l e s s e r e x t e n t , on d i f f e r e n c e s i n s p i n d e n s i t y . O p t i c a l c o n t r a s t i s dependent on the p a r t i c u l a r pulse sequence t i m i n g . (a) S i g n a l i n t e n s i t y as f u n c t i o n of pulse i n t e r v a l ( r ) f o r 2 samples w i t h d i f f e r e n t T t's. I f one s e l e c t s long value f o r r, there w i l l be no d i f f e r e n c e i n s i g n a l i n t e n s i t y . I f one chooses short T, sample wi t h s h o r t e r T, w i l l have g r e a t e r i n t e n s i t y , s i n c e i t w i l l be l e s s s a t u r a t e d . Choice of c o r r e c t pulse i n t e r v a l ( r ) can l e a d to d i f f e r e n t i a l s a t u r a t i o n between adjacent t i s s u e s , causing image c o n t r a s t . (b) S i g n a l i n t e n s i t y i s p l o t t e d as f u n c t i o n of pulse intervaKr) f o r 2 samples w i t h d i f f e r e n t T 2 ' s . I f one uses short T there w i l l be no d i f f e r e n c e i n s i g n a l i n t e n s i t y . I f one chooses long T, samples wit h longer T 2 w i l l have g r e a t e r s i g n a l i n t e n s i t y than those w i t h short T 2. Choice of a p p r o p r i a t e r value w i l l y i e l d d i f f e r e n t s i g n a l i n t e n s i t i e s f o r samples w i t h d i f f e r e n t T 2's. Note that e f f e c t of long T 2 on s i g n a l i n t e n s i t y i s reverse of e f f e c t of long T,. 40 90° 180° RF- pulse f l N M R - s i g n a l -W 2 E fat m u s c l e binding t issue f i r s l s ignal So echo s ignal Si Si = SQ e T2 180° inversion 90° 180° RF-pulse magnetisation -Mr NMR s i g n a l i So** M 0 H - 2 e Til f i rst signal S 0 Figure 1.19 (a)Spin-echo imaging with enhanced T 2 contrast. (b)Inversion recovery imaging with enhanced T, contrast.(After Luiten, Diag. Imag. Clin. Med. 53:4,1984.) 41 movement of n u c l e i out of the image plane while the s i g n a l i s being r e g i s t e r e d , and the slow flow does not reduce s i g n a l i n t e n s i t y d u r i n g T . A f t e r the s i g n a l has been c o l l e c t e d a c e r t a i n p r o p o r t i o n of the n u c l e i , which are only p a r t i a l l y s a t u r a t e d , leave the region of i n t e r e s t . When the next e x c i t a t i o n pulse i s a p p l i e d , t h e r e f o r e , the image plane c o n t a i n s n u c l e i which were not p r e v i o u s l y e x c i t e d and so are s t i l l i n thermal e q u i l i b r i u m . This c r e a t e s the appearance of a s h o r t e r T, r e l a x a t i o n time, l e a d i n g t o an incr e a s e i n i n t e n s i t y . In fast-moving f l u i d s , on the other hand, the e x c i t e d n u c l e i leave the image plane d u r i n g T , and do not generate an NMR s i g n a l . High flow v e l o c i t i e s , then, l e a d t o a r e d u c t i o n of image i n t e n s i t y . These r e l a t i o n s h i p s between flow v e l o c i t y and s i g n a l i n t e n s i t y are shown g r a p h i c a l l y i n F i g u r e 1.20. The r e s o l u t i o n c u r r e n t l y a v a i l a b l e w i t h NMR tomographs permits predominantly major v e s s e l s t o be imaged. Owing to the high flow v e l o c i t i e s i n these v e s s e l s (0-100 cm/s i n the human a o r t a ) , they u s u a l l y appear dark i n the image. F i n a l l y , t i s s u e water content a l s o has a major e f f e c t on the i n t e n s i t y of p i c t u r e elements i n an NMR tomogram. Water content not only v a r i e s among d i f f e r e n t t i s s u e s , but may a l s o vary i n the same t i s s u e as a r e s u l t of n u t r i t i o n , c l i m a t e , or drug therapy. Moreover, the water content of human t i s s u e s tends to decrease wi t h age, by an average of 2.5% per decade(82). 1.5 O r g a n i z a t i o n of T h i s Thesis 42 Flow velocity (cm/s) Flow rate (ml/min) F i g u r e 1.20 NMR s i g n a l i n t e n s i t y as a f u n c t i o n of flow v e l o c i t y . As the curve i n d i c a t e s , the r e l a t i v e s i g n a l i n t e n s i t y of a f l u i d w i t h NMR p r o p e r t i e s comparable to those of blood(T,=520ms, T2=230ms) shows a marked dependence on flow v e l o c i t y . The f l u i d was contained i n a g l a s s tube w i t h a 9.6mm i n s i d e diameter(TR=500ms, Te=43ms). ( A f t e r Crooks,L, et a l , Radiology 144:843,1982.) 43 The d i s c u s s i o n s i n t h i s t h e s i s , f o r the most p a r t , are d i r e c t e d towards the a p p l i c a t i o n of NMR as an a n a l y t i c a l t o o l to f o l l o w b i o l o g i c a l changes. The work d e s c r i b e d here i s d i v i d e d i n t o three p a r t s : a p p l i c a t i o n of 1 3C NMR to f o l l o w biochemical t r a n s f o r m a t i o n s , e v a l u a t i o n of the u s e f u l n e s s of r e l a x a t i o n s t u d i e s i n d e t e c t i o n of b i o l o g i c a l changes and, f i n a l l y , t e s t i n g of a combination of both NMR imaging and s p e c t r o s c o p i c techniques to study a s e l e c t e d model system. Chapter I I of t h i s t h e s i s i s concerned w i t h an e x p l o r a t i o n of the a p p l i c a b i l i t y of 1 3C NMR f o r s t u d i e s of m i l k - s o u r i n g , g r a p e - j u i c e f e r m e n t a t i o n , soybean germination and c a r t i l a g e - d e g r a d a t i o n . These systems are of i n t e r e s t not only to chemists but a l s o to a p p l i e d s c i e n t i s t s and c l i n i c i a n s . They i n v o l v e phenomena which most of us encounter i n our d a i l y l i f e . Since recent emphasis i n the l i t e r a t u r e i s on i n - v i v o s t u d i e s of i n t a c t systems w i t h medical s i g i f i c a n c e , the u n d e r l y i n g o b j e c t i v e of t h i s chapter i s to p r o v i d e an extension of the NMR s p e c t r o s c o p i c techniques to systems of i n t e r e s t to researchers such as those i n the food and a g r i c u l t u r a l i n d u s t r i e s . Chapter I I I d e s c r i b e s i n d e t a i l the e v a l u a t i o n of the u s e f u l n e s s of r e l a x a t i o n study of systems ranging from simple s i n g l e c u l t u r e d c e l l s , t o complex animal t i s s u e s . P o s s i b l e r a t i o n a l i z a t i o n s of the experimental o b s e r v a t i o n s w i l l be given i n t h a t chapter. 4 4 Chapter IV combines both NMR imaging and s p e c t r o s c o p i c techniques and shows the u t i l i t y of such an i n t e g r a t e d study. Since NMR imaging techniques provide s p a t i a l i n f o r m a t i o n w h i l e s p e c t r o s c o p i c techniques supply biochemical d e t a i l , we b e l i e v e that t h e i r marriage w i l l f a c i l i t a t e a more complete d e s c r i p t i o n of b i o l o g i c a l processes. The f i n a l chapter of t h i s t h e s i s w i l l p r ovide a summary and commentary of the cu r r e n t s t u d i e s and t h e i r f u t u r e e x t e n s i o n . Recommendations w i l l be given as to which areas are s u i t a b l e f o r i n t a c t - s t u d i e s using NMR s p e c t r o s c o p i c techniques, and to which areas are worth pursuing w i t h imaging techniques. 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P h i l i p s T e c h n i c a l Review 41:73,1983/84. 85. NMR-Tomography and -Spectroscopy i n Medicine-An I n t r o d u c t i o n , Roth,K. S p r i n g e r - V e r l a g Inc., B e r l i n , 1984. 86. Luiten,A.L. Diagn. Imag. C l i n . Med. 53:4,1984. 53 CHAPTER I I — A PRELIMINARY EXPLORATION OF THE APPLICABILITY C-13 NMR FOR STUDIES OF BIOCHEMICAL TRANSFORMATIONS 2.1 I n t r o d u c t i o n 2.2 Experimental Method 2.3 Souring of M i l k 2.4 Fermentation of Grape J u i c e 2.5 Germination of Soybean Seed 2.6 Degradation of C a r t i l a g e 2.7 Conclusion and D i s c u s s i o n References 54 2.1 I n t r o d u c t i o n Nuclear magnetic resonance(NMR) spectroscopy, although d i s c o v e r e d i n 1946, remained l a r g e l y an a n a l y t i c a l and research t o o l i n chemistry and p h y s i c s and, to a l i m i t e d e x t e n t , i n b i o l o g y , u n t i l the beginning of the 1970s. With the development of F o u r i e r Transform NMR techniques, a new area of research emerged, namely, the a p p l i c a t i o n of NMR i n medicine. This drew a t t e n t i o n to the f a c t that NMR i s "non-invasive" and "n o n - d e s t r u c t i v e " 1 and can t h e r e f o r e be u t i l i z e d to acq u i r e u s e f u l i n f o r m a t i o n of i n t a c t l i v i n g systems without causing any known damage to those systems. The 1H, 1 3C and 3 1 P NMR sp e c t r a from a human forearm are compared i n Fi g u r e 2.1a. The 1H NMR spectrum c o n t a i n s only two s i g n a l s , a r i s i n g from t i s s u e water and the CH 2 chains i n f a t . The 1 3C NMR spectrum covers a s u b s t a n t i a l l y broader range of chemical s h i f t s and c o n t a i n s f a r more l i n e s than the 1H NMR spectrum, but a l l s i g n a l s may s t i l l be assigned t o s p e c i f i c carbon atoms that are contained i n t i s s u e f a t ( F i g u r e 2.1b). In the 3 1 P NMR spectrum, the s i g n a l s are produced by adenosine t r i p h o s p h a t e ( A T P ) , phosphocreatine(PCr), and i n o r g a n i c p h o s p h a t e ( P i ) ( F i g u r e 2.1c). The work d e s c r i b e d here concentrates on e x p l o r i n g p o s s i b l e a p p l i c a t i o n s of NMR s p e c t r o s c o p i c techniques f o r f o l l o w i n g b i o c h e m i c a l t r a n s f o r m a t i o n s . Although both 1H and 1 3C NMR sp e c t r a 1 T h i s t o p i c c o u l d be d i s c u s s e d at great l e n g t h ; i n the present c o n t e x t , we make only the c o n t r a s t w i t h the most widely used techniques of X-ray and Nuclear Medicine, both of which i n v o l v e i o n i z i n g r a d i a t i o n , o f t e n i n s u b s t a n t i a l q u a n t i t i e s . 55 (a) 200ppm 100 0 o II CHj-0-C-(CH,)„-CH, I ° 1 II CH-0-r-KH..|„-('llj (W CHi-0-C-(CH,h-CH-CH-(CH,|,-CH3 Nil, N 0 o o 1 I II ©lO-P-O-P-O-P-OCH I I I I IQI IQl IQ| e e e Yy «A,H u. o 8 NH, N^^ S—N on on Adenosine triphosphate (ATP) O i eio-p-on IQI o (Ci Inorganic phosphate (P,) e io -p -o -p -on i j I I IQI IQI o e M'\H 11/ H OH OH Adenosine diphosphate (ADP) O II eio-p-I IQI CH, I " NH-C-N-CH,-CO.0 II Nil Phosphocreatine (PC,) Figure 2.1 (a) NMR spectra from the forearm of a l i v e human subject. The peaks in the 'H-NMR spectrum a r i s e from the protons in water and in the methylene of ti s s u e f a t . The 3,P-NMR spectrum contains s i g n a l s f o r inorganic phosphate(Pi), phosphocreatine(PCr ) and the three d i f f e r e n t phosphorus atoms in adenosine triphosphate(ATP). In the 13C-NMR spectrum, a l l si g n a l s are produced by the various types of carbon atom occurring in f a t U r o m documents furnished by Oxford Research Systems), (b) Glycerin(HOCH 2-CHOH-CH 2OH) e s t e r i f i e d with p a l m i t i c , l i n o l e i c , and o l e i c a cid ( t o p to bottom), a t y p i c a l constituent of f a t . ATP, ADP, PCr, and P i . (c) 56 were obtained from the systems i n v e s t i g a t e d , i n most cases only the 1 3C NMR r e s u l t s w i l l be presented because the 1 3C NMR resonances have a l a r g e r chemical s h i f t range than t h e i r 1H c o u n t e r p a r t s , and are t h e r e f o r e much more i n f o r m a t i v e . In many c o u n t r i e s , a s u b s t a n t i a l percentage of f r e s h produce and packaged food i s not f i t f o r human consumption because e i t h e r the e x p i r y date has passed, or the food has s p o i l e d . Deeper understanding of the f a c t o r s t h a t i n f l u e n c e the r a t e s of s p o i l a g e would provide a more r e a l i s t i c e stimates of s h e l f - l i f e . Souring of milk w i l l be presented as an example. The second set of r e s u l t s r e p o r t e d here i n v o l v e d the use of 1 3C NMR to f o l l o w yeast fermentation of grape j u i c e . Throughout the c e n t u r i e s , "fermentation" has been one of the most important methods f o r p r e s e r v i n g foods. Even though the o r i g i n a l p h y s i c a l and chemical c h a r a c t e r i s t i c s of the foods may be a l t e r e d d u r i n g f e r m e n t a t i o n , t h e i r n u t r i t i v e values are u s u a l l y r e t a i n e d . Besides beer and wine, sour m i l k products, s a u e r k r a u t , p i c k l e s , v i n e g a r , and sausage are examples of important fermented foods. Soybean seed, due to i t s h i g h o i l content and n u t r i t i v e v a l u e , has a t t r a c t e d much a t t e n t i o n i n the past twenty years. S p e c i f i c a l l y , A g r i c u l t u r e Canada has expended much e f f o r t i n s e l e c t i n g f r o s t - h a r d y soybean seed that can grow i n the northern p a r t s of Canada. The c u r r e n t study i n v o l v e s the use of 1 3C NMR to f o l l o w germination of a s i n g l e i n t a c t soybean seed. 57 The l a s t set of r e s u l t s reported here were intended to i n i t i a t e a study to develop a deeper understanding about common j o i n t - d i s e a s e s , such as a r t h r i t i s . The degradation of c a r t i l a g e was to be i n v e s t i g a t e d by 1 3C NMR. 2.2 Experimental Method The 1 3C NMR experiments were performed using a home-built spectrometer(270 MHz f o r 1H, 68 MHz f o r 1 3C) based on an Oxford Instruments narrow-bore s o l e n o i d magnet(6.2T) and a N i c o l e t 1180/293B data system. A l l 1 3C NMR spect r a were obtained w i t h broadband proton d e c o u p l i n g . Due' to the small i n s i d e bore of the magnet(54mm), a l l samples s t u d i e d were placed i n 10mm NMR tubes under c o n d i t i o n as s i m i l a r as p o s s i b l e to the o r i g i n a l i n t a c t environment. I t i s hoped that f u t u r e s t u d i e s of the corresponding i n t a c t systems w i l l e v e n t u a l l y be c a r r i e d out i n the new 80 MHz N i c o l e t - O x f o r d Spectrometer-Tomograph, which has a 30cm diameter room temperature bore. Assignment of a l l 1 3C NMR resonances, unless s t a t e d otherwise, was made wit h the help of reference 1. 2.3 Souring of M i l k M i l k i s a complex mixture c o n s i s t i n g of an o i l - i n - w a t e r emulsion s t a b i l i z e d by complex p h o s p h o l i p i d s and p r o t e i n adsorbed on the sur f a c e of the f a t g l o b u l e s . I t c o n t a i n s p r o t e i n s i n c o l l o i d a l suspension, l a c t o s e i n s o l u t i o n , and numerous m i n e r a l s , 58 p a r t i c u l a r l y c a l c i u m and phosphate, f a t - s o l u b l e and wa t e r - s o l u b l e v i t a m i n s , enzymes, and v a r i o u s organic substances(2). When b a c t e r i a grow i n milk and produce a c i d , c a s e i n i s p r e c i p i t a t e d out of the s o l u t i o n at i t s i s o e l e c t r i c point(pH=4.6). Fat g l o b u l e s , which enable c a s e i n t o coal e s c e more r e a d i l y , are p r e c i p i t a t e d as w e l l . Thus, souri n g of mil k i n v o l v e s a l o s s of p r o t e i n and f a t from the s o l u t i o n . Due to the r e l a t i v e i m m o b i l i t y of the p r o t e i n molecules, t h e i r NMR resonances are very broad and co u l d not be observed under the c u r r e n t experimental c o n d i t i o n s . F i g u r e 2.2 shows 1 3C NMR s p e c t r a of both " f r e s h " and "sour" m i l k . The resonances between 60 to 110 ppm, which d i d not undergo any s i g n i f i c a n t change e i t h e r i n p o s i t i o n or i n t e n s i t y , correspond to the d i s a c c h a r i d e l a c t o s e . In c o n t r a s t , most of the f a t resonances(at 15-40,133,174ppm) disappeared d u r i n g the so u r i n g process. Therefore, monitoring the amount of f a t i n s o l u t i o n by 1 3C NMR might a l l o w an accurate d e t e r m i n a t i o n of the q u a l i t y of milk i n v o l v e d . 2.4 Fermentation of Grape J u i c e Wine i s g e n e r a l l y made from g r a p e - j u i c e , which c o n t a i n s mainly glucose and sucrose. During fermentation by ye a s t , most of these sugars are converted i n t o a l c o h o l , u n t i l the l e v e l of a l c o h o l reaches about 17%(2), at which c o n c e n t r a t i o n , the yeast i s k i l l e d . The r e s u l t a n t l i q u i d i s then "racked", or separated, from the sediment of yeast and other i n s o l u b l e s . For many wines the yeast fermentation i s f o l l o w e d by a m a l o - l a c t i c f e r m e n t a t i o n , a 01 F i g u r e 2.2 ''C NMR s p e c t r a of (a) f r e s h milk; (b) sour m i l k . Resonances, between SO to 110ppm, which d i d not undergo any s i g n i f i c a n t change both 1n p o s t i o n and I n t e n s i t y , c o r r e s p o n d to the d l s a c c h a n d e l a c t o s e ; w h i l e most of the f a t resonances) 15-40,133,174ppm) d i s a p p e a r e d d u r i n g s o u r i n g p r o c e s s e s by p r e c i p i t a t i n g out of the s o l u t 1 o n . ( S p e c t r a 1 width=+/-15000Hz; scans=2000; b l o c k s1ze=8192 p o i n t s ; p u l s e w1dth=10„sec; p u l s e delay=1 sec) 60 i n which the l a c t i c a c i d - b a c t e r i a convert malic a c i d to l a c t i c a c i d thereby reducing the t o t a l a c i d i t y . T h e r e a f t e r , the wine i s sto r e d so t hat i t can "mature"; d u r i n g that time the p r o d u c t i o n of e s t e r s and m i l d o x i d a t i o n occurs. In an experiment designed to s i m u l a t e t h i s p rocess, the j u i c e from a grape was p l a c e d i n s i d e a 5mm diameter NMR tube, and i t s 1 3C NMR s p e c t r a measured. F i g u r e 2.3 c l e a r l y shows that sucrose and glucose (60-100 ppm) were consumed soon a f t e r the a d d i t i o n of y e a s t , with the concomitant increase of both ethanol and g l y c e r o l a f t e r the a d d i t i o n of y e a s t . Two new peaks at 20, and 65 ppm c o u l d be assigned to C-2 of ethanol , and C-2 of g l y c e r o l , r e s p e c t i v e l y ( 3 - 4 ) ; w h i l e the one at 60 ppm c o u l d be assigned to C-1 of ethanol and C-1/C-3 of g l y c e r o l . As i n d i c a t e d i n F i g u r e 2.4, sucrose i s f i r s t h y drolyzed to glucose and f r u c t o s e ; the l a t t e r i s then converted v i a a s e r i e s of t r a n s f o r m a t i o n s ( 5 - 9 ) to glucose. Glucose subsequently e n t e r s the g l y c o l y t i c pathway(lO) and i s f u r t h e r converted to e i t h e r ethanol or g l y c e r o l . Although these i n t e r m e d i a t e s t a t e s were not f o l l o w e d , t h i s sample study showed the f e a s i b i l i t y of using 1 3C NMR to f o l l o w a fermentation process. Future s t u d i e s c o u l d i n v o l v e the use of NMR to measure the percentage of a l c o h o l and to study the aging of b o t t l e d wine. Both w i l l take advantage of the newly a v a i l a b l e wide bore magnet i n which an unopened b o t t l e of wine can be s t u d i e d . 2.5 Germination of Soybean Seed v/H e \V H 0 9 V ° \ *} H ''OH OH H Sucrose CHjOH b H OH n-D-Glucose /?-D-Glucose Sucrose and Glucose C, 0-Glu •» Giu lilLX (a) Glycerol C , 3 - 1 1 F i g u r e 2 t o e i t h e r 180 3 •'C NMR s u c r o s e ~1~ Ethanol (to) 160 !H0 i;o 100 80 50 40 20 0 iT" s p e c t r a of (a) good grape j u i c e ; (b) fermented grape j u i c e . Resonances i n s p e c t r u m (a) c a n be a s s i g n e d or g l u c o s e ; whereas spectrum (b) c o n s i s t s of r e s o n a n c e s of e t h a n o l and g l y c e r o l . ( S p e c t r a 1 width=+/-15000Hz; scans=1000: b l o c k slze=8192 p o i n t s ; p u l s e w l d t h =(0„sec; p u l s e d e l a y = l s e c ) Sttpl Slip 2 Step} HEIOKINASE -ATP C H . O — ( ? ) O — ® O H H ,—ATP H \ H J < ° / % „ -O H k Step 4 CH,—O— • £ THKJ5E StPp J fMOSPHATE DEHYDROGENASE SHAD red 1 CH,—o—(B > C H O H •A-o " i j - oirHcwmocLYCtiLA n OlflKMPHO-CLICERArt WNAse S* ATP • C H . - 0 - ® 1 C H O H ' c-o A-3- PHOSfHOCtKHArt Step '/ 1 C H , O H » C H - O - (E> • c - o i -:.n<osrHOCLttTXAll Step 8 c -o -HI c-o ENOLASE ^ fHOSFHOENOtTYRUVATt r c-o c-o ATP (a) C H . O H ao\l / C H , O H V H O C H , . . O . ^ 1 -(b) c=o \ H I c=o I CH, ( c ) •I40H NAD* H I H—C—0 H I CH, CH,OH 0=i NA0M «AD* (d) CH,OH HO—C—H CH,OPO,'-i-Glyc«ro( 3-photpha<« CH,OH Glycaro* F i g u r e 2.4 (a) The g l y c o l y t i c pathway f o r glucose metabolism; (b) sucrose 1s cleaved by yeast enzyme to form glucose and f r u c t o s e , the l a t t e r w i l l undergo a number of transformations(5-10) to be converted to glucose; two Intermediates of the g l y c o l y t i c pathway (c) pyruvate and (d) dihydroxyacetone phosphate are converted by yeast to ethanol and g l y c e r o l , respect 1vely. 63 (a) 14 Hz 7 Hz T 8 TMS 1 ^ 10 T 4 2 ~ 1 — r 0 PPM i L i n o l e i c Acid] CH^ ( C H 2 ) ^ C H 2 C H = C H C H 2 C H = C H C H 2 ( C H 2 )^CH 2CH 2C0UR h f i d b 0 (b) b 1 d c e H 20FA 9 ? H-COFA J v i c i n a l = 7 H z t I v i c i n a l H-COFA , = 14 HzM geminal H FA=Fatty A c i d s (c) F i g u r e 2.5 (a) High r e s o l u t i o n 1H spectrum of Freon-11 o i l e x t r a c t of a soybean seed; a l l resonances can be assigned t o d i f f e r e n t carbons of (b) l i n o l e i c a c i d , which i s the major component; both v i c i n a l and geminal c o u p l i n g constants can be c l e a r l y i d e n t i f i e d . ( S p e c t r a l width=+/-1500Hz; scans=lOO; block size=8l92 p o i n t s ; p u l s e width=8ysec; pulse delay=1 sec) 64 1 8 i 2 nominal s i z e of 10 microns » l » i f 1 s ? l l c a 9 e l »'th a Port.on of e t h e r , mode of ^ e c t f o ^ L K tlti,?™ ' 65 Table 2.1 Some naturally occurring fatty acids(lO). Symbol Structure Systematic name Common name m.p.. *C Saturated fatly acids 12:0 ai J (CH,) 1 .COOH n-Dodecanoic Laurie 44 2 14:0 auau.cooi! n-Tetradecenoic Myristic S3.9 16:0 ai,(cn,)„coon n-Hexadecanolc Palmitic 63 1 18:0 ai,(ai,)„cooH n-Octadecanoic Stearic 69 6 20:0 C H , ( C H , ) 1 1 C O O H n-Eicosanoic Arachidic 76.5 24:0 aucH^cooH n-Tetiacosanoic Llgnoceric 86 0 Unsaturated fatty acids 18 :1" ai.(cn,),cu-=ai(at,),coou Palmiloleic - 0 . 5 18 :1 " ai,(ai,i1cn=cmcu,(,cooii Oleic 13 4 1 8 : 2 " " a l , (CH, ),CH=a«CI l , C H = CHlCH,) ,CfX) l I Linoleic - 5 1 8 : 3 " " ' * a I . C H . C H — C H C H . C H — C H C H . C U — CH(CH,),COOr 1 Linolenic - 1 1 2 0 4u« ri i, CIUCIUICH—aicn , ) ,CH=cniai i) >coon Arechidonic -49.5 66 F i g u r e 2.5 shows the 1H NMR spectrum of the o i l obtained as a Freon-11 e x t r a c t of a soybean seed. Both v i c i n a l and geminal c o u p l i n g constants of the g l y c e r i d e backbone can be c l e a r l y i d e n t i f i e d . A High Pressure L i q u i d Chomatograpy study ( F i g u r e 2.6) showed at l e a s t 6 f a t t y a c i d s were present i n soybean w i t h l i n o l e i c a c i d being the major component(>50%) (Table 2.1) (11-13). Although the 1H NMR spectrum of the i n t a c t soybean seed was p o o r l y r e s o l v e d , the corresponding 1 3C NMR s p e c t r a ( F i g u r e 2.7-2.8) were q u i t e w e l l - r e s o l v e d and were used f o r subsequent s t u d i e s . Upon germination, a massive breakdown of reserve t r i g l y c e r i d e was observed; the l i p i d to sugar r a t i o decreased from 7x to 3x (Fi g u r e 2.8). The f a t was converted to carbohydrate(mainly sucrose) presumably v i a the g l y o x y l a t e c y c l e (Figure 2.9a ) (14 ) . New peaks, coresponding to malate, s u c c i n a t e , o x a l o a c e t a t e ( 1 5 ) , and other components of the g l y o x y l a t e c y c l e , were observed, as shown i n Figur e 2.8. The sucrose was subsequently converted to sugars of higher homologs(Figure 2.9b)(16). 2.6 Deqradation of C a r t i l a g e C a r t i l a g e i s an av a s c u l a r and r e l a t i v e l y a c e l l u l a r s t r u c t u r a l element of connective t i s s u e ( l 7 ) . S e v e n t y - f i v e percent of bovine n a s a l c a r t i l a g e , f o r example, i s water and the remaining 25% i s composed of equal amounts of c o l l a g e n and p r o t e o g l y c a n ( 1 8 ) . The o v e r a l l composition of bovine n a s a l c a r t i l a g e p r o t e o g l y c a n i s 86% c h o n d r o i t i n s u l f a t e , 8% p r o t e i n , 6% 67 -1 1U0 L i n o l e i c A c i ^ l C l i ^ ( C H 2 ) / ( C H = C l l C » 2 p } I = C H ( C H 2 ) ? C O O H C a r b o n ,'/ 18 13 1 2 n 10 9 1 (a) 7 3 10 '12 1 • 1 110 C D C 1 , eo 60 F i g u r e 2.7 High r e s o l u t i o n 1 3C spect r a of (a) l i n o l e i c a c i d d i s s o l v e d i n CDC1 3; and (b) soybean o i l e x t r a c t . A l l resonances can be assigned to d i f f e r e n t carbons on f a t t y a c i d chain a f t e r t a k i n g i n t o c o n s i d e r a t i o n of t h e i r p r o x i m i t y to C=C and COOR groups. Resonances between 60 and 70ppm co u l d be assigned to g l y c e r i d e . ( S p e c t r a l width=+/-15000Hz; scans=1000; block size=8l92 p o i n t s ; pulse width= 10jusec; pulse delay=1 sec) 68 Methylene of lipids C-C H0 2CCOCH 2C0 2H | j x a l a c e t i c A C T J I H0 2CCH 2CHOHC0 2H iMaile Addl H0 2CCH 2CH 2C0 2H Succinic Acidl F i g u r e 2.8 1 3C s p e c t r a of (a) i n t a c t soybean seed a f t e r soaking i n water 1 day(1ipid:sugars=7x); (b) 3 days o l d germinating soybean r o o t ( 1 i p i d : s u g a r s = 3 x ) ; (c) 5 days and (d) 7 days o l d germinating soybean seed. The i n t e n s i t y of resonances between 35 and 55ppm i n c r e a s e d , corresponding to int e r m e d i a t e s of the g l y o x y l a t e c y c l e : s u c c i n a t e , malate and o x a l o a c e t a t e . ( S p e c t r a l width=+/-15000Hz; scans=20000; block size=8l92 p o i n t s ; pulse width=1Ousec; pulse delay=1 sec) 69 z o X o FATTY ACID FATTY ACYL CoA (a) SUCCINATE- • FUMARATE MALATE- OXALACETATE J MITOCHONDRIA Nonumccnarlde AJugoee Higher hotnologe? Higher homologa? Higher homologa? Octaaaccharlde Verbucoee (TUltlnoaj) «1-6 Lolium trtsaccharlde ol-3 Seaatnose Ptanteoe* (b) UmbelllferoM al-1 t/ychnose ol-B o i l Sucrose• il-« Galactose at the glucose moiety oT-6 ol-3 Isolychnoae Galactose at the fructose moiety Octasaccnaiide F i g u r e 2.9 (a) Summary of r e a c t i o n s i n the pathway of conversion of f a t t y a c i d to sucrose, showing the i n t r a c e l l u l a r compartmentation of the three major p a r t s of the sequence; (b) scheme of the ga l a c t o s e o l i g o s a c c h a r i d e s based on s u c r o s e ( A f t e r Handler and Hopf, Occurrence, Metabolism, and Function of O l i g o s a c c h a r i d e s i n the Biochemistry of P l a n t s , P r e i s s , J . ed.; Academic P r e s s , New York, 1980, V o l . 3, p.222). 70 . . . . C i t - L - J t f - G l y - P r o - T h f protein Cor* \ " u / j | GtlNAc-G*l-AcNtu _L Caontfroltln •alph.ta I C i l I GluA I a. GalNAc a. CluA ai. SO. >. CINAc I. C.I r- »o, Hannest 1 • ••Glu (Str-Fro) • Kvtini tulphatt (a) i I** °" | Kamn-SO. j |N-aoatv>>u<aiaamiM« 1, > aalaamj 8 1.«l„ (b) (c) Figure 2.10, (a)Structure of chondroitin sulphate-protein unit; (b)repeating disaccharide units for glycosaminoglycans of cartilage; (c) structural model of bovine nasal cartilage proteoglycan complex. , core protein; , chondroitin sulfate; +++++, keratan sulfate; « X H, hyaluronic acid. o», link proteins(After Schubert and Hamerman, A Primer on Connective Tissue Biochemistry, Lea and Febiger, Philadelphia, 1968). 71 Ring co rbon* Acrtyl M« (b) P r o t e og l y c an Subun i t (c) P r o t e o g l y c o n C o m p l e x L (d) C o r t i l o g e r-. 1 7; 1 1 1 1 1 1 1 1 1 i O 20 40 60 60 100 120 140 160 180 p p m ( C S j ) F i g u r e 2.11 Proton-decoupled 1 3C NMR sp e c t r a at 25° of (a) c h o n d r o i t i n 4 - s u l f a t e ( 5 0 mg/ml), (b) proteoglycan subunit (299 mg/ml), (c) proteoglycan complex (200 mg/ml), and (d) bovine n a s a l c a r t i l a g e . Spectra a, b, c, and d were obtained a f t e r 170,000, 102,000, 124,341, 228,000 t r a n s i e n t s , r e s p e c t i v e l y . Data were recorded u s i n g a s p e c t r a l window of 5Hz w i t h 4096 data p r i n t . ( A f t e r Brewer and R e i s e r , Proc. Nat. Acad. S c i . USA, V o l . 72,3421,1975) 72 keratan s u l f a t e , and l e s s than 1% h y a l u r o n i c a c i d ( F i g u r e 2.10)(19). Recent s t u d i e s have shown that the 1 3C NMR s p e c t r a of proteoglycan and that of whole c a r t i l a g e were very s i m i l a r to that of f r e e c h o n d r o i t i n 4 - s u l f a t e c h a i n s ( F i g u r e 2.11) (20). This i n d i c a t e d t h a t the l i n k a g e of m u l t i p l e c h o n d r o i t i n s u l f a t e chains to proteoglycan core p r o t e i n and the a s s o c i a t i o n of proteoglycan with c o l l a g e n and other c o n s t i t u e n t s of c a r t i l a g e m a t r i x does not s i g n i f i c a n t l y a l t e r the s t r u c t u r e and motion of these c h a i n s . In t h i s study, d i s c s of n a s a l c a r t i l a g e , f r e s h l y obtained from the s l a u g h t e r house, were immediately s t o r e d over i c e . They were subsequently pla c e d i n s i d e a 10mm diameter NMR tube f o r 1 3C NMR measurement. New s i g n a l s ( F i g u r e 2.12) i n the 30 to 60 ppm region were observed i n heat-denatured c a r t i l a g e and the 9.5 weeks o l d sample. Those resonances arose from protonated carbons i n f l e x i b l e peptides r e l e a s e d by e i t h e r thermal d e n a t u r a t i o n or endogenous p r o t e o l y s i s ( 2 1 ) of core p r o t e i n i n the proteoglycan complex. 2 . 7 Co n c l u s i o n and D i s c u s s i o n The range of a p p l i c a t i o n s covered i n t h i s chapter t e s t i f i e s t o the general u t i l i t y of 1 3C NMR f o r s t u d y i n g biochemical t r a n s f o r m a t i o n . Although the samples a c t u a l l y s t u d i e d here were not " i n t a c t " , there should not be any major problem of c a r r y i n g out s i m i l a r experiment of the i n t a c t systems now t h a t a l a r g e r bore magnet i s a v a i l a b l e . Perhaps the most i n f o r m a t i v e s t u d i e s to be done i n the f u t u r e w i l l be those which employ, not only 1 3C, 73 F i g u r e 2.12 1 3C NMR s p e c t r a of bovine n a s a l septa c a r t i l a g e a f t e r (a) 1 day, (b) 4 weeks, (c) 5 weeks, (d) 9.5 weeks storage at room tempature; (e) 1 3C NMR spectrum of heat t r e a t e d BNS c a r t i l a g e . Both (d) and (e) look very s i m i l a r , except the r e s o l u t i o n of the former i s poorer and the l i n e w i d t h s are broader probably due to incomplete degradation. ( S p e c t r a l width=+/-15000Hz; scans=2000; block size=8l92 p o i n t s ; pulse width=lOMSec pulse delay=1 sec) 74 but also 1H and 3 1 P NMR concurrently. Each of these nuclei has advantages not shared by the others, so that i t can be a n t i c i p a t e d that the most complete d e s c r i p t i o n of the b i o l o g i c a l processes w i l l be best obtained by the combination of techniques. It i s appropriate to end t h i s section by noting that although the magnet necessary for those studies i s now a v a i l a b l e , the development of the hardware necessary for the f u l l y integrated studies r e f e r r e d to above i s not yet completed. Nevertheless, the current apparatus i s compatible with proton NMR studies of some systems and a f u l l study of one such system i s presented in Chapter IV of t h i s t h e s i s . Once such necessary technology i s a v a i l a b l e , i t w i l l be possible to scale-up the present "model" studies to f u l l s i z e . Thus, milk in cardboard cartons and p l a s t i c packages can be placed inside the instrument and studied sequentially over a period of time. Wine, in b o t t l e s , can be studied without opening them, and, given the recent scandal of Austrian wines which have been spiked with diethylene g l y c o l a n t i f r e e z e , t h i s i s of t o p i c a l i n t e r e s t . Perhaps most importantly of a l l i t w i l l be f e a s i b l e to study a r t h r i t i c j o i n t s , both in animal models as well as i n man. We do not suggest that a l l of these studies w i l l become widespread, e s p e c i a l l y , in view of the high c a p i t a l costs involved. Nevertheless, given the immense p r a c t i c a l importance of the food and a g r i c u l t u r a l i n d u s t r i e s , to say nothing of t h e i r f i n a n c i a l resourses, i t seems highly probably s u b s t a n t i a l e f f o r t s w i l l occur. With regard to a r t h r i t i s , the medical imaging 75 i n d u s t r y appears to have a n t i c i p a t e d t h i s work, and apparatus s u i t a b l e f o r s t u d i e s of human limbs i s now becoming a v a i l a b l e . 76 References 1. Johnson,L.F., & Jankowski,W.C. Carbon-13 NMR Spectra-A C o l l e c t i o n of Assigned, Coded, and Indexed Sp e c t r a . John Wiley & Sons, New York, 1972. 2. Pederson, C S . M i c r o b i o l o g y of Food Fermentat ions (2nd e d i t i o n ) , AVI P u b l i s h i n g Co., Inc., Westport, Co n n e c t i c u t , 1979. 3. den Ho l l a n d e r , J . A . , Brown,T.R., U g u r b i l , K . , and Shulman,R.G. Proc. N a t l . Acad. S c i . USA 76:6096,1979. 4. Norton,R.S. C-13 NMR Stu d i e s of I n t a c t C e l l s and T i s s u e . B u l l e t i n of Magnetic Resonance Vol.3 #1,p29-48. 5. As p i n a l l , G . O . , P e r c i v a l , E . , Rees,D.A., AND Rennie,M. IN "Rodd'S Chemistry of Carbon Compounds," Coffey,S., ed., E l s e v i e r , Amsterdam, 2nd e d i t i o n , 1967, V o l . 1, Part F,pp 596-714. 6. Neuberg,C, and Mandl,I. Enzymes, 1, Part 1,527-550,1950. 7. Go t t s c h a l k , A . In "Handbuch der P f l a n z e n p h y s i o l o g i e , " Ruhland,W., ed., S p r i n g e r - V e r l a g , B e r l i n , 1958, V o l . V I , Pp87-124. 8. Myrback,K. Enzymes(2nd e d i t i o n ) , 4:379,1960. 9. Lainpen,J.O. Enzymes(3rd e d i t i o n ) , 5:291 ,1971. 77 10. Lehninger,A.L. Biochemistry. Worth P u b l i s h e r s , Inc., New York, 1970. 11. Orr,W. A g r i c u l t u r e Canada, unpublished data, 1982. 12. S c h a e f e r , J . , Ste j s k a l ,E.O. , and Beard,CF. P l a n t P h y s i o l . 55:1048, 1975. 13. Weete,J.D., and Manley,R.C. J of Alabama Acad, of S c i . V o l . 50 #1 p35-46,1979. 14. Beevers,H. The Role of the G l y o x y l a t e Cycle i n The Biochemistry of P l a n t s , Stumpf,P.K., ed., Academic Press, New York, 1980, V o l 4, p120. 15. Coombe,B.G., and J o n e s , C P . Phytochemistry 22:2185,1983. 16. Handler,O., and Hopf,H. Occurrence, Metabolism, and Function of O l i g o s a c c h a r i d e s i n the Biochemistry of P l a n t s , P r e i s s , J . ed., Academic Press, New York, 1980, V o l 3,p.222. 17. Schubert,M., and Hamerman,D. A Primer on Connective Tissue B i o c h e m i s t r y . Lea and F e b i g e r , P h i l a d e l p h i a , Pa.,1968 18. M a l a w i s t a , I . , and Schubert,M. J . B i o l . Chem. 230:535,1958. 19. Sajdera,S.W., and Hascall,V.C. J . B i o l . Chem. 244:77,1969. 20. Brewer,C.F., and Reiser,H. Proc. Nat. Acad. S c i . USA Vol.72,3421,1975. 21. Torchia,D.A., Hasson,M.A., and H a s c a l l , V . C J . of B i o l Chem. 252:3617, 1977. 78 CHAPTER III—RELAXATION STUDIES OF BIOLOGICAL SYSTEMS 3.1 H i s t o r y and I n t r o d u c t i o n 3.2 Experimental Procedures 3.3 R e l a x a t i o n Study of Simple B i o l o g i c a l Systems (a) V i r a l I n f e c t e d C u l t u r e d Animal C e l l s (b) Rust I n f e c t e d P l a n t Leaves (c) C u l t u r e d P l a n t C e l l L i f e Cycle 3.4 R e l a x a t i o n Study of P e r i p h e r a l J o i n t Diseases 3.5 R e l a x a t i o n Study of Animal Models of M u l t i p l e S c l e r o s i s (a) Chronic Experimental A l l e r g i c E n c e p h a l o m y e l i t i s (EAE) i n Guinea Pigs (b) Herpes Simplex V i r u s Type 1 I n f e c t e d Mice 3.6 Conclusion and D i s c u s s i o n Appendix References 79 3.1 Hi s t o r y and Introduct ion The u t i l i z a t i o n of the behavior of c e l l u l a r water as a probe of the p h y s i o l o g i c s t a t e of t i s s u e s and c e l l s has a long h i s t o r y , going back to the anc i e n t Greeks. The idea that water may play a r o l e i n the cancerous process i s based on e a r l y experimental evidence. Cramer, perhaps, was the f i r s t to present evidence that the percentage of water i n tumors i s r e l a t e d to t h e i r r a t e of growth, wi t h f a s t e r growing tumors o f t e n having a higher water c o n t e n t ( l ) . McEwen et a l . l a t e r r e p o r t e d that at l e a s t part of the e l e v a t e d l e v e l s of water i n the t i s s u e s c o u l d be due to an a c t u a l uptake of water by the transformed c e l l s ( 2 ) . An e l e v a t e d water content of t i s s u e s d i s t a n t from the organ c o n t a i n i n g a tumor, termed the "systemic e f f e c t " of tumors, was observed by Schlottman and Rubenow(3). Using m u l t i p l e beam i n t e r f e r e n c e microscopy, M e l l o r s et a l . showed that i n d i v i d u a l sarcoma c e l l s contained more water than normal f i b r o b l a s t ( 4 ) . Downing et a l . ( 5 ) , using i n t e r f e r o m e t r y , were able to show that the increased water content of tumors was due to i n t r a c e l l u l a r accumulation r a t h e r than an enlargement of the e x t r a c e l l u l a r space. A l l of t h i s q u a n t i t a t i v e evidence p o i n t e d to the importance of water i n the o v e r a l l understanding of the cancerous process. F u r t h e r p u r s u i t of these o b s e r v a t i o n s seems to have been impeded by both lack of technology and theory. With the development of NMR spectroscopy, the i n t e r e s t s of b i o l o g i s t s were r e v i v e d and the i n t e r e s t s of p h y s i c i s t s were awakened to the study of water i n b i o l o g i c a l systems and i n 80 p a r t i c u l a r to water i n cancer. With t h i s technique, the c h a r a c t e r i s t i c s of hydrogen n u c l e i of t i s s u e water c o u l d be examined i n a n o n - d e s t r u c t i v e manner i n l i v i n g c e l l s . In 1971, Damadian(6) f i r s t r e p o r t e d that the NMR r e l a x a t i o n times of water protons i n Walker sarcoma and N o v i k o f f hepatomas were s i g n i f i c a n t l y longer than those of the normal t i s s u e s of o r i g i n . C o n f i r m a t i o n of these r e s u l t s f o l l o w e d r a p i d l y both i n l a b o r a t o r y animals(7-14) and i n humans(7,9,15-20). In 1973, Damadian et a l . (17-18,21) undertook an e x t e n s i v e examination(106 samples) of normal and malignant human t i s s u e s . These r e s u l t s demonstrated that T, values are e l e v a t e d r e l a t i v e to the corresponding normal t i s s u e (Table 3.1). The d i s c r i m i n a t i o n i s , however, not always as c l e a r as i t i s with the l a r g e , fast-growing experimental tumors, o f t e n used i n animal s t u d i e s , whose T, values are g e n e r a l l y e l e v a t e d w i t h respect to a l l normal t i s s u e s . For i n s t a n c e , Saryan et a l . ( 2 2 ) found f o r mice at 24.3 MHz that T, values f o r h e a l t h y t i s s u e s were between 186 and 526 msec, whereas tumor T,'s l a y i n the 593-847 msec range. Thus, i n the human case, d i a g n o s t i c problems c o u l d a r i s e i n the case of metastases(23). Frey et a l . ( 1 1 ) showed that T,'s are e l e v a t e d i n the non-tumorous organs of tumorous mice; thereby demonstrating the presence of a tumor systemic e f f e c t . T his r e s u l t means that appearently u n a f f e c t e d organs from tumorous hosts cannot be taken as r e l i a b l e c o n t r o l t i s s u e s . N e v e r t h e l e s s , recent c a r e f u l 81 s t u d i e s , u sing g r a p h i c a l , r a t h e r than n u l l T 1 f d e t e r m i n a t i o n s and employing l a r g e number of samples, have demonstrated that many human tumors can be r e l i a b l y d i s t i n g u i s h e d , f o r i n s t a n c e , breast(24-25) , g a s t r o i n t e s t i n a l ( 2 6 ) , and lung(27). There has been much s p e c u l a t i o n concerning the o r i g i n of the T , - e l e v a t i o n i n cancerous t i s s u e . Many authors b e l i e v e i t to be simply a matter of water content(8,15,22,23,28,29), whereas others c o n s i d e r that i t r e f l e c t s changes i n the macromolecular s t r u c t u r e ( 3 0 - 3 4 ) . I t has been suggested(35) that T, e l e v a t i o n i s r e l a t e d to growth r a t e , and high water contents and c o r r e s p o n d i n g l y longer T,'s have been observed i n f e t a l t i s s u e ( 2 3 ) , regenerating l i v e r ( l 5 ) , immature muscle(36), and i n growing tumors(23). E l e v a t e d T / s are a l s o seen i n a v a r i e t y of human di s e a s e s and are c e r t a i n l y not cancer s p e c i f i c ( 2 0 ) . There i s t h e r e f o r e l i k e l y to be a range of c u r r e n t l y p r e v a l e n t i l l n e s s e s which are amenable to NMR i n v e s t i g a t i o n s . 3.2 Experimental Procedures R e l a x a t i o n r a t e s were measured using a home-built 270 MHz spectrometer based on an Oxford Instruments narrow-bore s o l e n o i d magnet(6.2T) and a N i c o l e t 1180/293B data system. T, values were obtained by g r a p h i c a l method using the i n v e r s i o n recovery sequence; whereas T 2 values were obtained a l s o by g r a p h i c a l method but using the C a r r - P u r c e l l pulse sequence with the 82 Table 3.1 T, r e l a x a t i o n times at human t i ssues (1 7-1 8, 2 1 ) .a 100 MHz i n normal and malignant Probability that diderence in means arc not Tissue tumor T, normal significant Breast 1.080 ± 0.08 0.367 ± 0.079 0.52 x 10" 4 Skin 1.047 ± 0.108 0.616 ± 0.019 0.55 x 10" 4 Muscle: Malignant 1.413 ± 0.082 1.023 ± 0.029 0.50 x 10" 5 Benign 1.307 ± 0.1535 Esophagus 1.04 0.804 ± 0.108 Stomach 1.238 ± 0.109 0.765 ± 0.075 b 0.40 x 10" 2 Intestinal tract 1.122 ± 0.04 0.641 0.641 1+ 1+ 0.080 0.043 C 0.27 x 10" S Liver 0.832 ± 0.012 0.570 + 0.029 Spleen 1.113 ± 0.006 0.701 ± 0.045 Lung 1.110 ± 0.057 0.788 + 0.063 0.25 x 10" 2 Lymphatic 1.004 + 0.056 0.720 + 0.076 0.52 x 10" Bone 1.027 ± 0.152 0.554 ± 0.027 0.74 x 10" Bladder 1.241 ± 0.165 0.891 ± 0.061 0.36 x 10" 1 Thyroid 1.072 0.882 ± 0.045 . Nerve 1.204 0.557 + 0.158 Adipose 2.047 0.279 + 0.008 Ovary 1.282 + 0.118 0.989 + 0.047 Uterus: Malignant 1.393 ± 0.176 0.924 ± 0.038 Benign 0.973 Cervix 1.101 0.827 ± 0.026 Testes 1.223 1.200 ± 0.048 Prostate 1.110 0.803 ± 0.014 Adrenal 0.683 0.608 ± 0.020 Peritoneum 1.529 0.476 Malignant melanomas 0.724 ± 0.147 Tongue 1.288 Pericardial layer (mesothelioma) 0.758 Kidney 0.862 + 0.033 Brain 0.998 ± 0.016 Pancreas 0.605 ± 0.036 Heart 0.906 ± 0.046 " Probability values are reported lor series with sample size ;> 3. Errors reported are standard error of the mean (SEM). Number of cases analyzed are indicated in parenthesis. (From R. Damadian et al., Proc. Natl. Acad. Sci. U.S.A. 71, 1471 (1974).) * Small bowel. ' Colon. 83 Meiboom-Gi11 m o d i f i c a t i o n . A l l samples were packed i n e i t h e r 5mm or 10mm NMR tubes. 3.3 R e l a x a t i o n Study of Simple B i o l o g i c a l Systems (a) V i r a l I n f e c t e d C u l t u r e d Animal C e l l s Although water i s a fundamental component of b i o l o g i c a l systems, i t s r o l e i n c e l l u l a r s t r u c t u r e and f u n c t i o n i s s t i l l a matter of c o n t r o v e r s y . Nuclear Magnetic Resonance methods have been used e x t e n s i v e l y to study the p r o p e r t i e s of water s i n c e the r e l a x a t i o n t i m e s ( T 1 f T 2) are dependent on the motional freedom a v a i l a b l e to the water molecules(37). The r e l a x a t i o n times i n l i v i n g t i s s u e are s h o r t e r than those i n pure water and are a f f e c t e d both by the molecular complexity of the t i s s u e and by i t s water content(38-39). I t i s known that v i r a l i n f e c t i o n s are r e s p o n s i b l e f o r membrane changes(40-42): the v i r u s attachment on the c e l l r e c e p t o r s r e s u l t s i n a m o d i f i c a t i o n of the membrane l i p i d f l u i d i t y ( 4 3 ) . Although V a l e n s i n et a l . ( 4 4 ) r e p o r t e d the sho r t e n i n g of water T, at 90 MHz c o u l d be c l o s e l y r e l a t e d to the m u l t i p l i c i t y of i n f e c t e d HEp-2 c e l l s by p o l i o v i r u s type 1, such measurements have not been repeated at d i f f e r e n t magnetic f i e l d and, f u r t h e r , i t i s not c l e a r i f such changes are g e n e r a l l y t r u e among a l l v i r a l i n f e c t e d c e l l s . Since r e l a x a t i o n r a t e s are f i e l d dependent and r e l a x a t i o n c o n t r a s t seems to be s m a l l e r at higher f i e l d ( 4 5 ) , i t i s not known i f T, d i f f e r e n c e s between v i r a l i n f e c t e d c e l l s c o u l d be p i c k e d out at 270 MHz.1 1 A l l the r e l a x a t i o n s t u d i e s d e s c r i b e d here were completed before the 80 MHz Spectrometer-Tomograph was i n s t a l l e d i n the Department 84 In c o l l a b o r a t i o n w i t h Dr. Diane Van A l s t y n e ' s group(Department of Neurology, F a c u l t y of Medicine, UBC), we measured the r e l a x a t i o n r a t e s of v i r a l i n f e c t e d c u l t u r e d animal c e l l s , packed i n 5mm NMR tubes; observable changes were demonstrated i n both T, and T 2 r e l a x a t i o n r a t e s . Although at f i r s t s i g h t the r e s u l t s obtained by V a l e n s i n ( T a b l e 3.2a) and i n t h i s work(Table 3.2b) seem to be i n c o n f l i c t , t h i s i s not so. V a l e n s i n measured T, values of a d i l u t e suspension of v i r a l - i n f e c t e d c e l l s , whereas we were observing T, and T 2 values of a packed mass of v i r a l i n f e c t e d c e l l s . As a r e s u l t , V a l e n s i n s t u d i e d e x t r a c e l l u l a r water a f f e c t e d by v i r a l i n f e c t i o n ; i n c o n t r a s t , we were examining mainly i n t r a c e l l u l a r water. Since s u c c e s s f u l v i r a l i n f e c t i o n i n v o l v e s the attachment of v i r u s onto the host c e l l membrane and the i n j e c t i o n of v i r a l m a t e r i a l , such as n u c l e i c a c i d , i n t o the host c e l l cytoplasm, the decrease i n T, of e x t r a c e l l u l a r water a f t e r v i r a l i n f e c t i o n probably was due to a combination of increased molecular weight and s u r f a c e area w i t h i n the i n f e c t e d c e l l s . With a molecular weight i n c r e a s e , the c e l l ' s motional freedom would decrease; a bigger s u r f a c e area would imply that more water molecules c o u l d be bound; p o s s i b l y some " c r o s s - l i n k i n g " c o u l d a l s o occur. As a r e s u l t , the water environment i n the s o l u t i o n would become e f f e c t i v e l y more v i s c o u s and subsequently the water T, would decrease. Furthermore, other c e l l u l a r a c t i v i t i e s would increase a f t e r v i r a l i n f e c t i o n . Thus, i n f e c t e d c e l l s would be forced to 1 ( c o n t ' d ) of Chemistry and a l s o before the UBC H o s i p i t a l P i c k e r NMR Tomograph was i n f u l l o p e r a t i o n . 85 Table 3.2a S p i n - l a t t i c e r e l a x a t i o n times of water tn HEp-2 c e l l s ( a b o u t 2x10- c e l l / m l ) one hour a f t e r p o l i o v i r u s 1 n f e c t i o n ( 4 4 ) . f F U / c c B r , ( i ) b) 13.9 10"' 13.8 14.0 io-« 13.5 i<r» 13.2 io-' 13.0 12.5 ior« 11.2 l 10.6 10 10.0 a) *) Mean nlaet of three experiments (±5%) W This line ixfen to the cdl control Table 3.2b Absolute (at 270 MHz) and r e l a t i v e Ti and Ti values in v i r a l Infected c u l t u r e d animal c e l l s -SAMPLE T_;_ T i inf ected/T i norma 1 T t i nf ected/T > norma 1 Fresh norma 1 1 .64 sec 61 msec eel 1 s Fresh v i r a l 1nfected eel Is: Batch #1 1 .74 sec 78 msec 1 .06 1 . 28 Batch #2 1 .87 sec 84 msec 1 . 14 1 . 37 ^wo samples of each were measured 86 s y n t h e s i z e v i r a l - s p e c i f i c molecules, which i n t u r n would increase i n t r a c e l l u l a r osmotic pressure and would c r e a t e an i n f l u x of water i n t o the c e l l s . The l a t t e r i s r e s p o n s i b l e f o r the increase i n water content. Such inc r e a s e i n water content would subsequently lengthen the i n t r a c e l l u l a r water T,. (b) Rust I n f e c t e d P l a n t Leaves Rusts(46) i n c l u d e the c a u s a l organisms of many of the most important p l a n t d i s e a s e s . They are o b l i g a t e p a r a s i t e s on fe r n s or seed p l a n t s and at t a c k only the l i v i n g t i s s u e of t h e i r h o s t s . Losses i n food crops due to r u s t have been enormous s i n c e the beginning of h i s t o r y ; the Romans had a f e s t i v a l to p r o p i t i a t e the r u s t gods. Now people t r y to do i t by removing the a l t e r n a t e host, barberry t o save wheat, black c u r r a n t s t o save white p i n e , or by developing m o r e - r e s i s t a n t v a r i e t i e s f o r the e v e r - i n c r e a s i n g r u s t s t r a i n s , or by the use of f u n g i c i d e s , c l a s s i c a l l y s u l f u r , l a t e l y some of the carbamates and, i n a few cases, a n t i b i o t i c s . The c u r r e n t study was to evaluate i f NMR co u l d be used f o r the e a r l y d e t e c t i o n of r u s t i n f e c t i o n . In c o l l a b o r a t i o n w i t h Dr. Michael Shaw's group(Department of P l a n t Science, F a c u l t y of A g r i c u l t u r a l Sciences, UBC), we measured the r e l a x a t i o n r a t e s of small leaves at d i f f e r e n t stages of m a t u r i t y and i n f e c t i o n . Two to three l e a v e s , broken i n t o small p i e c e s , were packed i n s i d e a 5mm NMR tube t o give a sample-height of about 3 cm. Our r e s u l t s i n Table 3.3 showed that most young l e a v e s , normal or r u s t - i n f e c t e d , had a lower T, than t h e i r mature c o u n t e r p a r t s . On Table 3.3 Absolute (at 270 MHz) and r e l a t i v e Ti and T; values in rust i n f e c t e d p l a n t leaves Sample Normal mature 1 eaves Young normal 1 eaves Rust i n f e c t e d young leaves 0.55 sec 0.39 sec 0.44 sec 7.5 msec 6.4 msec 13 msec T i i nfected/Tinorma 1 mature Ti infected/T:normal mature oo 0.71 0.80 0.85 1 .73 88 the other hand, T 2 of r u s t - i n f e c t e d leaves showed a 70% i n c r e a s e ; whereas the corresponding normal one s u f f e r e d a s l i g h t decrease(15%). Thus, the t o t a l T 2 c o n t r a s t ( i . e . d i f f e r e n c e ) , between normal and r u s t - i n f e c t e d l e a v e s , was 85%. Such a b i g v a r i a t i o n would probably make NMR r e l a x a t i o n s t u d i e s a u s e f u l t o o l i n f u t u r e s t u d i e s of p l a n t i n f e c t i o n . In a d d i t i o n , there i s a very good p o s s i b i l i t y that NMR might be used to f o l l o w f u n g i c i d e treatment of i n f e c t e d f r u i t - t r e e s . This would r e q u i r e the development of NMR probes capable of being attached to a s u i t a b l e pruned small t r e e , p l a n t i n a pot, which c o u l d be brought i n t o the NMR l a b o r a t o r y . (c) C u l t u r e d P l a n t C e l l L i f e Cycle In 1976, B e a l l et a l . ( 4 7 ) r e p o r t e d that the HeLa c e l l c y c l e c o u l d be f o l l o w e d by T, measurement at 30 MHz. Both T, and water content (Figure 3.1) were at maxima during m i t o s i s ( i . e . when the c e l l s are a c t i v e l y r e p r o d u c i n g ) ( F i g u r e 3.2). Both parameters continued to decrease throughout the G, phase and reached t h e i r mean minimum values i n the S phase. I t was f u r t h e r shown that the water content changes c o u l d not be the only f a c t o r i n f l u e n c i n g T, , and i t was suggested that the conformation s t a t e of macromolecules should be taken i n t o c o n s i d e r a t i o n as w e l l . Their, c y c l i c p a t t e r n of T, changes appeared to be i n v e r s e l y r e l a t e d to the degree of chromatin condensation. Our study i n v o l v e d T 2 measurement(at 270 MHz) of c u l t u r e d Catharanthus rosens(48-49), a p l a n t species which produces 89 Hours F i g u r e 3.1 T, and water content as a f u n c t i o n of HeLa c e l l c y c l e . ( 0 0) T, d u r i n g the c e l l c y c l e (mean of e i g h t to ten experiments). Bars denote standard e r r o r of the mean.(• •) Water content d u r i n g the c e l l c y c l e . ( ) Actinomycin-D b i n d i n g a b i l i t y of the c h r o m a t i n . ( A f t e r B e a l l , P . T . f et a l , Science 192:904,1976.) 90 Figure 3.2 The growth-duplication c y c l e . After completion of mitosis(M), daughter c e l l s enter the G, phase. Toward the end of G,, syntheses preparing for DNA and chromosome r e p l i c a t i o n take place. At the beginning of S, DNA and histone synthesis i s i n i t i a t e d and chromosomes are r e p l i c a t e d . After DNA i s r e p l i c a t e d , the c e l l with double i t s DNA content enters the G 2 phase, a short period in preparation for chromosome condensation. At m i t o s i s , n u c l e o l i disappear and prophase begins as chromosomes condense. 91 important anti-tumor agents, such as V i n b l a s t i n e ( 5 0 ) . In c o l l a b o r a t i o n w i t h Gerry Hewitt of the B i o l o g i c a l S e r v i c e s of t h i s department, we t r a n s f e r r e d 5 ml. of Catharanthus rosens c u l t u r e i n t o each of a s e r i e s of f l a s k s c o n t a i n i n g s t e r i l i z e d B-5 medium 2. S u f f i c i e n t sample was harvested each day fo r a T 2-measurement, m i t o t i c index and dry weight measurements. The r e s u l t s showed that the T 2 values a l s o f o l l o w e d a c y c l i c p a t t e r n but was at i t s minimum during m i t o s i s ( F i g u r e 3.3). Thus, T 2 values seem to be l e s s s e n s i t i v e than T, values to changes i n water content as evidenced by t h e i r opposite behaviors throughout the c e l l l i f e c y c l e . Such c h a r a c t e r i s t i c s of T 2 would probably make i t a b e t t e r parameter than T, to be used f o r f u t u r e study of molecular changes durin g abnormal development or diseased s t a t e , both i n p l a n t s and i n humans. I f more experimental data c o u l d show t h a t T 2 values were r e f l e c t i n g f a c t o r s other than water content, then T 2 c o n t r a s t imaging would i n f a c t be a b e t t e r technique i n d i s t i n g u i s h i n g a c t u a l d i s e a s e d s t a t e from edema(or s w e l l i n g ) . 3.4 R e l a x a t i o n Study of P e r i p h e r a l J o i n t Diseases O s t e o a r t h r i t i s ( O A ) i n i t s v a r i o u s forms i s one of the commonest di s e a s e s i n man, symptomatically a f f e c t i n g about 10% of the a d u l t p o p u l a t i o n ( 5 1 ) . Despite the common l a y - and m e d i c a l - o p i n i o n , OA cannot be d i s m i s s e d as merely aging or "wear and t e a r " of j o i n t s , s i n c e comparison of "aged" j o i n t s with OA 2 see appendix 92 Figure 3 .3 L i f e cycle of Catharanthus rosens followed by ( a ) T 2 , (b)mitotic index, and (c)dry weight measurements. 93 j o i n t s shows profound metabolic and biochemical d i f f e r e n c e s ( 5 2 ) . Recent research has been d i r e c t e d both towards the pathogenesis of OA and toward t h e r a p i e s t o a l t e r the disease-course r a t h e r than t o p a l l i a t e the d i s c o m f o r t . Rheumatoid a r t h r i t i s ( R A ) , the other major type of p e r p h e r i a l j o i n t d i s e a s e , a f f e c t s about 1% of the a d u l t p o p u l a t i o n and, i f untre a t e d , the v i c t i m s are o f t e n d i s a b l e d or s e v e r e l y c r i p p l e d ( 5 3 ) . Current knowledge, assessment and therapy of RA i s somewhat more advanced than that of OA, but i s s t i l l inadequate. A c r u c i a l step i n e f f e c t i v e s t r a t e g y to t r e a t any a r t h r i t i s i s d e t e c t i o n of e a r l y disease before the process i t s e l f i s i r r e v e r s i b l e , or j o i n t damage i s profound. There must a l s o be p r e c i s e o b j e c t i v e assessment measures to evaluate therapy a c c u r a t e l y . At present, t h i s i s t o t a l l y i m p o s s i b l e f o r OA and inadequate f o r RA. NMR. imaging, due to i t s a b i l i t y t o provide good s o f t t i s s u e c o n t r a s t , o f f e r s great p o t e n t i a l t o f i l l t h i s c r u c i a l r o l e . OA i s u s u a l l y d e f i n e d p a t h o l o g i c a l l y and r a d i o l o g i c a l l y by f o c a l c a r t i l a g e l o s s w i t h osteophyte formation i n subchondral s c l e r o s i s . These are c l e a r l y l a t e m a n i f e s t a t i o n s r e f l e c t i n g severe j o i n t damage and r e p a i r . Since the c l i n i c a l f e a t u r e s i n e a r l y d i s e a s e are n o n s p e c i f i c and s i n c e there are no pathogomonic non-invasive t e s t s , the e a r l y stages of the disease are not only d i f f i c u l t t o d e f i n e c l i n i c a l l y but are a l s o d i f f i c u l t to study p a t h o l o g i c a l l y . Therefore, s e v e r a l experimental models of OA have been . devi s e d which resemble forms of the human disease 94 c l i n i c a l l y , radio-logically and p a t h o l o g i c a l l y (54) . These experimental studies, together with studies of human autopsies and s u r g i c a l materials, have demonstrated a pathogenetic sequence(54-55). This s t a r t s with an increase in the water content of c a r t i l a g e by 5-10%, a loss of c a r t i l a g e proteoglycan, a stimulation of synthesis of both an immature, d i s t i n c t proteoglycan species and synthesis of collagen in a r t i c u l a r c a r t i l a g e , meniscus and p e r i a r t i c u l a r ligaments and tendons before the appearance of s y n o v i t i s . There follows a d i s r u p t i o n of the s u p e r f i c i a l c a r t i l a g e ( f i b r i l l a t i o n ) with i n h i b i t i o n of synovial f l u i d . These changes precede c a r t i l a g e l o s s , osteophyte formation and subchondral s c l e r o s i s and represent the early diagnosis but are d i f f i c u l t to quantitate o b j e c t i v e l y c l i n i c a l l y . Unfortunately, c u r r e n t l y used imaging techniques add l i t t l e at t h i s stage. However, the profound changes in water content of c a r t i l a g e in early OA and the prominent inflammatory s y n o v i t i s in early RA suggest that NMR imaging might be used to v i s u a l i z e the early stages of these diseases. In order to assess whether t i s s u e imaging would be a l t e r e d with pathology, we induced experimental o s t e o a r t h r i t i s and excised t i s s u e s from a r t i c u l a r c a r t i l a g e , meniscus, anterior c r u c i a t e ligament stump and the synovium and j o i n t capsule(Figure 3.4), placed them in NMR tubes and assessed in a 270 MHz NMR spectrometer. The T, and T 2 values showed d i f f e r e n c e s which suggest diseased a r t i c u l a r t i s s u e s may be d i f f e r e n t i a t e d from normal t i s s u e s . The c a r t i l a g e and ligaments generally showed NORMAL JOINT ; Articular Capsule Synovial Membrane Articular Cartilage OSTEOARTHRITIS Worn Articular Cartilage New Growth of Bone of Joint RHEUMATOID ARTHRITIS Swollen Synovial Membrane Cartilage Destroyed and Joint Space Obliterated Overgrowth of Articular Surface Subluxation (partial dislocation) of Joint (a) (b) - medial vastus muscle - semimembranosus musc'e - semitenrjincsus muscie - articular cavity - articuiarxflssuiei - coDMteai fascia - gastrocnemius tendon - cartilagei - medial condyle ot the temur _ Hotfa s tat cad* — meotai meniscus; - tibia — gastrocnemius muscie - soleus muscie . lateral vastus muscle . medial vastus muscie . femur . posterior cruciare'iiqamenq . mediaHTtreniscusi . Mbiai collateral i^acc£oX . anterior cruciate'"gameriT . intercondylar eminence of the tibia . lateral E k c ^ l X . remainder ot the eoiphyseal'gartiiafle. . tiota . anterior tibial muscle F i g u r e 3 .4 h i g h l i g h t e d Normal and i n boxes) A r t h r i t i s j o i n t s ( i m p o r t a n t components are 96 i n c r e a s i n g T, and T 2 w i t h pathology while the j o i n t capsule/synovium showed a decrease(Table 3.4). Great care was taken to ensure almost i d e n t i c a l sample p r e p a r a t i o n . For example, a l l c a r t i l a g e samples were prepared i n f l a k e form and were packed i n 5mm NMR tubes. Samples were kept i n i c e before measurements to ensure minimal b i o l o g i c a l degradation and r e l a x a t i o n s t u d i e s were done w i t h i n the f i r s t 3 hours. I f such p r e c a u t i o n s were not taken, i n t e r p r e t a t i o n of NMR data was even more d i f f i c u l t ( T a b l e 3.5). A c o n t r o l study(Table 3.6) showed that T, i s more s e n s i t i v e to change i n c a r t i l a g e water content than T 2. Thus, according to both Tables 3.4 and 3.6, 5-7% i n c r e a s e i n T, of c a r t i l a g e on the operated s i d e can be e a s i l y e x p l a i n e d by a corresponding 4-6% i n c r e a s e i n water content. N e v e r t h e l e s s , the more dramatic i n c r e a s e (33-36%) i n T 2 can only be e x p l a i n e d by other f a c t o r s , such as a l o s s of macromolecules(e.g. c a r t i l a g e p r o t e o g l y c a n ) . Subsequent to t h i s study, a number df papers(55-58) have been p u b l i s h e d , which f u r t h e r show the f e a s i b i l i t y and usefulness of NMR i n examination of p e r i p h e r a l j o i n t d i s e a s e s . L i et a l . ( 5 7 ) have demonstrated the a b i l i t y of the NMR method to v i s u a l i z e the menisci and c r u c i a t e ligaments without use of c o n t r a s t agents; they a l s o showed a marked d i f f e r e n c e of NMR appearance of a r t i c u l a r c a r t i l a g e and the m e n i s c i . The c o n t r a s t was probably due to the d i f f e r e n c e s i n the c o n c e n t r a t i o n of macromolecules (such as c o l l a g e n ) and i n the water content. Turner et a l . ( 5 8 ) Table 3.4 Absolute (at 270 MHz) and r e l a t i v e Ti and Ti values in Ant e r i o r C r u c i a t e Ligament T r a n s e c t i o n Experimental OA. a Sample T i ( s e c ) Ti(msec) Tioperated/T > unoperated T ioperated/Tiunoperated a Animal(doq) #270 #271 #270 #271 #270 #271 #270 #271 Art i c u I a r c a r t 11 age Operated Unoperated 0.84 0.9G 0.80 0.91 10 14 13.3 11 1.05 1.06 1.33 1.27 Men i scus Operated Unoperated 0.83 0.83 0.66 0.83 8.5 7.8 5.5 7.2 1.26 1.00 1.55 1.08 L i gament Operated Unoperated 1.04 1.06 0.73 0.68 12.5 12.5 5.0 4.8 1.42 1.56 2.5 2.6 JOINT capsu1e/synov i um Operated Unoperated 0.89 1.01 1.30 1.01 8.5 6.0 8.5 12 0.68 1.00 1.0 0.5 Synovial f l u i d 1.59 1.71 221 209 Values expressed are r a t i o of the the unoperated s i d e ( i . e . normal) Ti or Ti values of the operated s i d e ( i . e . experimental animal model f o r OA) d i v i d e d by VD Tab le 3.5 R e l a x a t i o n r a t e s as a f u n c t i o n of sample p r e p a r a t i o n and sample f r e s h n e s s Sampl e T i Tj_ F resh bov ine nasa l s e p t a ( c a r t i 1 age) 1.06 sec 10 msec f l a k e Fresh bov ine nasal s e p t a ( c a r t i 1 age) 0.96 sec 26 msec d i s c 2 week o l d BNS d i s c 1.20 sec 32 msec VD co T a b l e 3.6 R e l a x a t i o n r a t e s change as a f u n c t i o n of water content Sample Tj^  T I X % / T I O % Tj. F resh BNS f l a k e + 0% s a l i n e ( 0 . 1 5 M NaCL) 1 .03 sec 1 .00 14 msec + 5% sa l1ne(0 .15M NaCl) 1 . 12 sec 1 .09 14 msec + 10% sa l1ne(0 .15M NaCl) 1 . 19 sec 1 . 16 14 msec + 15% s a l i n e ( 0 . l 5 M NaCl) 1 .27 sec 1 .23 14 msec + 20% s a l i n e ( 0 . 1 5 M NaCl) 1 . 28 s e c a 1 .24 14 msec a Lower than expected Ti va lues was due to s a t u r a t i o n ( i . e . BNS reached i t s l i m i t to absorb water) 99 r e c e n t l y demonstrated over 70% accuracy i n determining acute i n j u r y of the ligaments of the knee by NMR imaging techniques. 3.5 R e l a x a t i o n Study of Animal Models f o r M u l t i p l e S c l e r o s i s M u l t i p l e S c l e r o s i s i s a d i s e a s e that u s u a l l y s t a r t s i n young a d u l t s , and runs a f l u c t u a t i n g course with stepwise p r o g r e s s i o n of symptoms. Every part of the nervous system can be a f f e c t e d . There may be p a r a l y s i s or tremor. In a d d i t i o n , there i s almost always some l o s s of s e nsation together w i t h i n c o n t i n e n c e of u r i n e and sometimes l o s s of bowel c o n t r o l . V i s i o n i s commonly a f f e c t e d , so that b l i n d n e s s may occur. Inflammation i s found i n the b r a i n and s p i n a l cord and i t i s thought that d i s t u r b a n c e s of the body's immune system c o n t r i b u t e to changes i n memory, mood or behavior. I t i s not known i f the tempo of the p a t h o l o g i c a l process i s a phasic one that p a r a l l e l s the c l i n i c a l , or i s a c o n s t a n t l y developing one w i t h i n t e r m i t t e n t c l i n i c a l m a n i f e s t a t i o n s . I t i s a l s o not known i f the c l i n i c a l m a n i f e s t a t i o n s i n the r e l a p s e represents inflammation, d e m y e l i n a t i o n , or merely metabolic i n f l u e n c e s on p r e v i o u s l y demyelinated nerve f i b e r s ( 5 9 ) . Secondary f a c t o r s such as g l i o s i s or axonal degeneration may a l s o be important. Since c l i n i c a l a c t i v i t y may not be a r e l i a b l e i n d i c a t o r of a c t u a l d i s e a s e a c t i v i t y , i t thus becomes very important to have an o b j e c t i v e , s a f e , and r e l i a b l e index of p a t h o l o g i c a l a c t i v i t y . NMR may f o r the f i r s t time o f f e r such a p o s s i b i l i t y ( 6 0 ) . 100 Whether an immunologic mechanism might be r e s p o n s i b l e f o r c e n t r a l nervous system plaque development and whether i t might be d i r e c t e d toward a b r a i n - s p e c i f i c or n o n b r a i n - s p e c i f i c ( i n c l u d i n g v i r a l ) antigen are s t i l l matters of s p e c u l a t i o n . To date, evidence f o r or a g a i n s t s e n s i t i z a t i o n a g a i n s t a s p e c i f i c a n t i g e n , i n c l u d i n g m y e l i n , i n MS i s i n c o n c l u s i v e ( 6 1 - 6 3 ) . Two animal models are c u r r e n t l y under a c t i v e i n v e s t i g a t i o n by Dr. D.W. Paty's medical group at UBC: Chronic experimental a l l e r g i c e n c ephalomyelitis(EAE) i n inbred guinea p i g s (Dr. D. van A l s t y n e ) and Herpes Simplex Virus(HSV) i n f e c t i o n i n mice(64-65) (Dr. L. K a s t r u k o f f ) . Chronic EAE was developed(63) to approach questions r a i s e d i n MS by hypotheses on autoimmune demyelination and delayed h y p e r s e n s i t i v i t y ; whereas the HSV model hypothesized that MS was the r e s u l t of v i r a l i n f e c t i o n . Both animal models mimicked c l i n i c a l l y and m o r p h o l o g i c a l l y the p i c t u r e seen i n m u l t i p l e s c l e r o s i s . (a) Chronic Experimental A l l e r g i c E n c e p h a l o m y e l i t i s (EAE) i n Guinea P i g s Only one set of r e l a x a t i o n measurements was made. D i f f e r e n t s e c t i o n s of the b r a i n were separated and each s e c t i o n was placed i n a 10mm NMR tube. Almost a l l b r a i n t i s s u e s ( F i g u r e 3.5) showed decreasing T, and T 2 w i t h pathology(Table 3.7), except cerebellum which showed an increase i n T,. The l a t t e r was probably p a r t i a l l y due to an i n c r e a s e i n water content r e s u l t i n g from l o c a l edema. 101 Corpus csltosum cfrpbtai artery Hypothalamus regu'MPS bc+-+> temperature, appetite. ^ irleKPof some Sinuses if eb'a' hemisphere! govern* thought, senses. §nd movement Third vemttete Jawbone Lnrynx Esophagus Trachea Figure 3 .5 The b r a i n l i e s w e l l protected w i t h i n the r i g i d , bony case of the s k u l l . I t has three main p a r t s : the p a i r e d c e r e b r a l hemispheres; the cerebellum; and the b r a i n stem. Both the l e f t and r i g h t c e r e b r a l hemispheres are re s p o n s i b l e f o r c o n t r o l l i n g such "higher f u n c t i o n s " as speech, memory, and i n t e l l i g e n c e . Each hemisphere has a core of white matter surrounded by a la y e r of gray matter. The cerebellum i s l o c a t e d under the c e r e b r a l hemispheres. I t c o n t r o l s c e r t a i n subconscious a c t i v i t i e s , e s p e c i a l l y c o o r d i n a t i n g movement and keeping our balance. The b r a i n stem(including pons and medulla) merges i n t o the top of the s p i n a l cord and maintains the v i t a l f u n c t i o n s of the body, such as breathing and c i r c u l a t i o n . Table 3.7 Absolute (at 270 MHz) and r e l a t i v e Ti and Tj values in EAE inbred guinea p i g . Sample TiEAE/T•norma 1 T;EAE/T;normal L e f t c e r e b r a l hemisphere Normal 1.73 sec In f e c t e d 1.11 sec 63 msec 35 msec 0.64 0.56 Right c e r e b r a l hem i sphere Normal 1.86 sec In f e c t e d 1.49 sec 88 msec 53 msec 0.80 0.60 Medular and pons Normal 1.77 sec In f e c t e d 1.74 sec 80 msec 76 msec 0.98 0.95 Cerebellum Normal I n f e c t e d Spinal Cord Normal I n f e c t e d 1.87 sec 2.57 sec 1.66 sec 1.52 sec 87 msec 72 msec 225 msec 69 msec 1 . 37 0.91 0. 83 0.31 O to Table 3.8 Absolute (at 270 MHz) and r e l a t i v e T, and Ti values in HSV Type 1 i n f e c t e d mice b r a i n Sample Normal Acute i n f e c t e d 1.5 month c h r o n i c i n f e c t e d 1 year c h r o n i c 1nfected # of samp!es measured 20 5 5 Ti i n f e c t e d / T i n o r m a l Ti infected/T;norma! 1.26 sec 1.21 sec 1.14 sec 1.18 sec 29.0 msec 29.8 msec 28.8 msec 25.6 msec 0.96 0.90 0.94 1 .03 0.99 0.88 103 (b) Herpes Simplex V i r u s Type 1 I n f e c t e d Mice I n i t i a l r e l a x a t i o n measurments of se c t i o n e d mice b r a i n were made w i t h samples packed i n 5mm NMR tubes. Soft b r a i n t i s s u e s had to be cut i n t o small p i e c e s and pushed down to the bottom of narrow 5mm tubes. I n c o n s i s t e n t r e s u l t s , probably due to d i f f e r e n t amounts of water being squeezed out of the t i s s u e d u r i n g p r e p a r a t i o n , made i n t e r p r e t a t i o n of r e l a x a t i o n data i m p o s s i b l e . T h e r e a f t e r , i t was decided to repeat a l l measurements i n 10mm NMR tubes w i t h i n t a c t whole mice b r a i n s . Twenty normal mice b r a i n s were used as c o n t r o l . F i v e whole mice b r a i n s at each stage of i n f e c t i o n were prepared f o r NMR r e l a x a t i o n study. The r e l a x a t i o n data shown i n Table 3.8 i n d i c a t e that there i s a very high p o s s i b i l i t y t h a t NMR imaging can be used to i d e n t i f y i n f e c t e d from normal mice b r a i n s , and a l s o t o d i s t i n g u i s h between d i f f e r e n t stages of i n f e c t i o n . Thus, although a l l i n f e c t e d samples s t u d i e d here showed a decrease i n T,, the d i f f e r e n t extent of the T 2 changes i m p l i e s that i t might be p o s s i b l e to make a d i f f e r e n t i a t i o n between i n f e c t i o n stages by t h e i r T 2 c o n t r a s t images. Although we have no d i r e c t proof that r e l a x a t i o n changes r e s u l t e d from a c t u a l b i o l o g i c a l changes, s i m i l a r decreases i n r e l a x a t i o n r a t e s of both the EAE and HSV models appear to be much more than a c o i n c i d e n c e . C i r c u m s t a n t i a l evidences suggest that r e l a x a t i o n r a t e s are a macroscopic i n d i c a t o r of weighted averaged m i c r o s c o p i c changes. 104 3 .5 Conclusion and Discussion Some conclusions and d i r e c t i v e s w i l l now be presented in view of these and other r e s u l t s in the l i t e r a t u r e . A fundamental d i f f i c u l t y in the comparative evaluation of NMR rela x a t i o n data obtained with d i f f e r e n t NMR apparatus l i e s in the dependence of the r e l a x a t i o n times on magnetic f i e l d strength. While the T 2 values remain l a r g e l y unchanged, the T, values are m a t e r i a l l y a l t e r e d by the magnetic f i e l d strength. Thus, reported T, values are meaningful only i f the magnetic f i e l d strength i s also spec i f ied. Even i f the magnetic f i e l d strength i s known, any stage of the sample preparation which may change the water content of the samples could p o t e n t i a l l y a l t e r the re l a x a t i o n times and i s to be avoided. Fat content i s al s o important because mobile protons on l i p i d s may make a con t r i b u t i o n to NMR proton re l a x a t i o n times. When composite proton measurements are made at low f i e l d strength, an increase in the percentage of f a t t y t i s s u e , which generally has a lower T, value but a higher T 2 value than water, would decrease an observed T, value and increase an observed T 2 value and subsequently make the i n t e r p r e t a t i o n of NMR data extremely d i f f i c u l t . Although the etiology of T, and T 2 r e l a x a t i o n times of water i s well understood in theory, in p r a c t i c e T, and T 2 values can only be predicted for simple materials. Relaxation times are d i r e c t measures of the i n t e r a c t i o n s that occur between water 105 molecules and other c e l l c o n s t i t u e n t s . Due to the complex way in which water may be bound to the various c e l l components i t i s not possible to describe the re l a x a t i o n mechanisms in t i s s u e s in q u a n t i t a t i v e , t h e o r e t i c a l terms. For s i m p l i c i t y , l e t us c l a s s i f y the water molecules in t i s s u e into two types: 'free' and 'bound' water. The 'free' water i s by far the more abundant and behaves l i k e ordinary pure water. The remaining 5% to 10%, c o n s i s t s of water molecules that are bound more fi r m l y to the surface than t h e i r free counterparts(Figure 3.6). Owing to t h e i r r e l a t i v e immobility, these bound water molecules can transfer absorbed radiofrequency energy to t h e i r environment much more r a p i d l y than the free water molecules. As a r e s u l t , the T, relaxation time of bound water i s up to 1000 times shorter than that of free water. Free and bound water do not produce separate signals in the NMR spectrum, only a s i n g l e , averaged s i g n a l whose T, relaxation time i s determined by the averaged values of the free and bound water: 1/T,=f1/T,(b)]P f a+[1/T,(f)] P f Eq. 3.1 where T,(b) and T,(f) are the r e l a x a t i o n times and P^ and P^ are the percentage c o n t e n t s ( f r a c t i o n s ) of bound and free c e l l u l a r water r e s p e c t i v e l y . Because T,(f) i s much greater than T,(b), the above may be s i m p l i f i e d to an approximation: 106 Figure 3.6 The re l a x a t i o n of 'free' and 'bound' c e l l u l a r water. Due to the l i m i t e d mobility of water molecules that are bound to the various c e l l components, bound water has a shorter T, r e l a x a t i o n time than water in the free state does. 1 0 7 T,=T,(b)(1/1-P ) E q * 3 , 2 Thus, the measured proton T, value of a t i s s u e i s determined p r i m a r i l y by a t i s s u e - s p e c i f i c constant T,(b) which depends on the i n t e r a c t i o n of the 'bound' water wi t h c e l l components. T, i s f u r t h e r determined by the r e l a t i v e content of ' f r e e ' water i n the volume of i n t e r e s t . Because a r i s e of t o t a l water content mainly in c r e a s e s the r e l a t i v e abundance of f r e e water, the T, value of a t i s s u e i n c r e a s e s w i t h i t s water content. Besides water content, at l e a s t two p o s s i b l e mechanisms co u l d account f o r the observed proton r e l a x a t i o n d i f f e r e n c e between normal and diseased t i s s u e s : ( a ) a lower degree of order of the i n t r a c e l l u l a r water i n malignant t i s s u e s ; and (b) q u a n t i t a t i v e l y fewer macromolecule- bound paramagnetic i o n s , or f r e e r a d i c a l s , i n diseased t i s s u e . The l a t t e r seems not to be a s i g n i f i c a n t f a c t o r because v a r i a t i o n of paramagnetic ions i s n e g l i g i b l y s m a l l . Although i n tumors, the in c r e a s e i n T, i s probably due to fewer water molecules i n the h y d r a t i o n l a y e r , which i s i n tu r n the r e s u l t of fewer r e c o g n i t i o n s i t e s , e.g. g l y c o p r o t e i n s , on the s u r f a c e of each cancer c e l l , such an e x p l a n a t i o n may not be v a l i d i n a l l d i s e a s e d s t a t e s . Thus,unless i n f o r m a t i o n at the molecular l e v e l i s a v a i l a b l e , attempts to e x p l a i n observed change i n water r e l a x a t i o n r a t e s w i l l probably f a i l . N e v e r t h e l e s s , an expermental knowledge of r e l a x a t i o n r a t e s i s e s s e n t i a l i n o p t i m i z i n g the c o n t r a s t of NMR images and that alone w i l l j u s t i f y f u r t h e r s t u d i e s i n t h i s a rea. 108 A P P E N D I X B-5 Medium O.L. Gamborg and D.E. Eveleigh, Can. J. Biochem., «t£, *»17, 1968. Ingredient mg/1 NaH2POu- HjO 150 KN03 2500 (NH 4) 2S0 | + 134 MgSOu 7H20 250 CaCl 2 2H20 150 Iron* 28 KI 0.75 Micronutrients* 1.0 ml VitaminsJ 10-0 ml Sucrose 20.0 g Final pH 5.5 Stock solution. Dissolved in 100 ml water: 1 g MnSD^-r^O, 300 mg H 3B0 3, 300 mg ZnSO^f^O, 25 mg NajMoO^ • 2H20, 25 mg CuS0,j 5 H 2 ° 25 mg CoCl 2-6H 20. Stock solution. Dissolved in 100 ml H20: 10 mg nic o t i n i c acid 100 mg thiamine, 10 mg pyridoxine, l g myoinositol. Dry Weight and Mitotic Index Determinations There are several parameters for measuring growth of cultured cells: cell number, packed cell volume, fresh and dry weight, total nitrogen, etc. None of them reflects growth in all its facets which include cell division, elongation and specialization (differentiation). Measuring growth by dry weight determination has the advantage of being a method which is simple, used quite commonly, and gives acceptable assessment of the overall synthetic activity of the cells. DRY WEIGHT A. EQUIPMENT 1. Miracloth* discs (dried in vacuum oven and stored in dessicator. 2. Millipore* filter holder. 3. Water aspirator. 4. Vacuum oven. 5. Forceps. 6. Balance. 7. Petri dishes. B. MATERIAL Any cell material grown on solid and in liquid medium. C. PROCEDURES 1. Callus grown on agar media. Free callus carefully from residual agar, place it on a pre-weighed Miracloth* disc and transfer the material into a petri dish. Dry callus inside the petri dish in vacuo at 60°C for at least 12 h (overnight). 2. Cells grown in liquid medium. Collect cells on a pre-weighed Miracloth" disc in a MiUipc^* filter holder with application of a water aspirator. Wash the cells with water. Transfer disc with packed cell mass into petri dish. Dry the cells in vacuo at 60°C for at least 12 h (overnight). 3. Dry weight determination. Determine dry weight and subtract dry weight of Miracloth* disc. Deter-minations should be carried out in replicates and averaged. D. COMMENT The relation between dry weight and fresh weight of cultured cells generally stay; linear at fresh weight values below 500 mg. MITOTIC INDEX The mitotic index of a given cell culture may be used as a parameter for growth and in particular for cell cycles. A. EQUIPMENT 1. Pipettes, 5 ml, wide mouthed. 2. Pasteur pipettes. 3. Vials, 10 ml, capped. 4. Slides and cover slips. 5. Burner with pilot flame. 6. Roller. 7. Counter. 8. Fixative: glacial acetic acid:ethanol (1:3). 9. Stain: Carbol-fuchsin solution (see Chap. 10). B. MATERIAL -o Any cell material grown on solid or in liquid medium. vo C. PROCEDURES 1. Sample S ml of cell suspension and mix it with I ml of fixative for 30 min. 2. Transfer a sample of fixed cells onto a slide and add an equivalent amount of stain. 3. Wait for a few minutes, cover preparation with a cover slip and heat the slide until small bubbles appear. 4. Cover slide with paper tissue and squash the preparation gently with a roller.. Note: Nuclei are stained red, there should be only a faint background staining.' 5. Count 2000 nuclei and determine the number of nuclei in mitosis (prophase-telophase). 6. Mitotic Index (Ml) - number of nuclei in mitosis x l Q Q total number of nuclei observed D. COMMENT Rapidly growing cell populations will show MI of 3-5% on average. 110 Ref reneces 1. Cramer,W.J. P h y s i o l . 50:322,1916. 2. McEwen,H.D., Haven,F.L. Cancer Res. 1:148,1941. 3. 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Lancet 8255,1981. 61. Paterson,P.Y. J . Chron. D i s . 26:119,1973. 62. Raine,C.S. Experimental a l l e r g i c e n c e p h a l o m y e l i t i s and r e l a t e d c o n d i t i o n s . In Zimmerman,H.M., ed. Progress i n Neuropathology, New York, Grune and S t r a t t o n , 1976, V o l . 3, p225. 63. Raine,C.S., and Stone,S.H. J . of Med. 1693,1977. 64. Kastrukof f , L. , Long,C, Doherty ,P.C. , Wroblewska , Z . , and Koprowski,H. Nature 291:432,1981. 65. Kastrukof f ,L. , Hameda,T., Schumacher ,U. , Long,C, Doherty,P.C., and Koprowski,H. J . of Neuroimmun. 2:295,1982. 1 1 6 CHAPTER IV—SPECTROSCOPIC STUDY AND IMAGING OF HEN'S EGG 4.1 I n t r o d u c t i o n 4.2 Experimental Procedures 4.3 Chemical Composition 4.4 The A i r C e l l 4.5 The Yolk 4.6 The Albumen 4.7 Cooking of An Egg 4.8 Four Dimensional, Chemical S h i f t Resolved Imaging 4.9 Conclusion References 1 17 4.1 I n t r o d u c t i o n Because the o v e r a l l s t r u c t u r a l f e a t u r e s of hen's eggs have been so thoroughly documented(1-3), such eggs now provide an i d e a l model-system f o r e v a l u a t i n g v a r i o u s nuclear magnetic resonance imaging measurements. In 1968, Jackson et a l . f i r s t p u b l i s h e d NMR data from eggs; i n 1977, Krebs and W i l l i a m s s t u d i e d mobile a v i a n p r o t e i n ( 4 ) . In t h i s chapter we report b r i e f l y the use of three dimensional proton nuclear magnetic resonance imaging (3-D NMRI) to map the gross i n t e r n a l compartmentalization of the egg white w i t h a s p a t i a l accuracy of ca. 2mm. This study a l s o serves to demonstrate the f u t u r e p o t e n t i a l of combining NMR spectroscopy and imaging, p r e v i o u s l y a l l u d e d t o i n t h i s t h e s i s . 4.2 Experimental Procedures The method used i s based on the "spin-warp" technique(5-8), which i s summarized i n F i g u r e 4.1. The measurements were made w i t h a home-built spectrometer-tomograph based on an Oxford instruments 1.9 T e s l a , 30cm. bore s o l e n o i d that g i v e s 1 p a r t i n 10 7 homogeneity f o r a 7cm. diameter sphere. The console i s adapted from a N i c o l e t NT-300 system and uses a N i c o l e t 1280 computer and a 293c pulse programmer, running software developed at UBC. Both s l o t t e d r esonator(9) and helmholtz c o i l s were used and were found i n t h i s study t o give s i m i l a r r e s u l t s . The images d e s c r i b e d i n the f o l l o w i n g s e c t i o n were t y p i c a l l y a c q u i r e d as a Figure 4.1 (a) Basic pulse sequence for 30 voTume imaging; (b) basic pulse sequence for 40 chemlea 1-shtft-resolved tomography; (c) diagram showing the structure of the hen's egg at the time of laying [adapted from ( 1 ) ] ; (d) diagram showing the structure of 1 week old hen's egg with noticeable air ce l l . 1 1 9 32x32 data matrix and z e r o - f i l l i n g was used to give a. f i n a l image di s p l a y s i z e of 128x128. The t o t a l data a c q u i s i t i o n time was t y p i c a l l y 25 minutes. 1 The natural-abundance carbon-13 NMR spectra were run at 68 MHz using a home b u i l t spectrometer based on a N i c o l e t 1180 computer and the proton NMR spectra were run using the same system operating at 270 MHz. 4.3 Chemical Composition It i s well known that on a weight-percentage basis an egg c o n s i s t s of albumen, ca. 60%; yolk, ca. 30%, and s h e l l , ca. 10%. Table l a summarizes the p r i n c i p a l components of both egg-yolk and albumen and Table l b gives the f a t t y - a c i d components in egg-yolk. The natural abundance carbon-13 NMR spectra given in fig u r e 2 r e f l e c t those data. Thus the egg-white (Figure 4.2a) shows a spectrum l a r g e l y - reminiscent of that of hen-egg white proteins(11). In contrast, that of egg-yolk (Figure 4.2b) shows p r i n c i p a l l y the resonance of the mobile l i p i d components, the proteins g i v i n g no d i s c e r n i b l e resonances. The analogous proton NMR spectra are given in fig u r e 4.2c and d. Both are dominated by the water-signal. 4.4 The Air_ C e l l 1 subsequent images acquired as a 64x64 data matrix showed s l i g h t improvements in d i g i t a l r e s o l u t i o n s but no added d e t a i l s were observed. lipid water ^4 o T c m F i g u r e 4.2 H l g h - r e s o l u t Ion 1'C NMR s p e c t r a ( 6 8 MHz ) o f ( a ) egg w h i t e (* of a c q u i s 111 on = 50,000 ) and ( b ) e g g y o l k (# o f a c q u i s ! t I o n s - 1 0 0 ) . E x p e r i m e n t a l p a r a m e t e r s : s p e c t r a l w i d t h * * / - 1 5 0 0 0 Hz. b l o c k size»8K. p u l s e w 1 d t h = 1 0 „ s e c . a c q u i s i t i o n t1me=135 msec; r e l a x a t i o n d e l a y ! s e c ; H i g h - r e s o l u t i o n 'H NMR s p e c t r a ( 2 7 0 MHz) o f ( c ) egg w h i t e and ( d ) egg y o l k . E x p e r i m e n t a l p a r a m e t e r s : s p e c t r a l width=8 „ s e c . a c q u i s i t i o n t1me=»1.3 s e c , r e l a x a t i o n d e l a y * 1 s e c : 4D c h e m i c a l - s h 1 f t - r e s o l v e d Images of ( e ) egg w h i t e and (f) egg y o l k ( c r o s s - s e c t i o n ) . 4096 s 1 g n a l s ( 1 6 x 1 6 x 1 6 ) were a c q u i r e d f o r eachm o f t h e c o m b i n a t i o n s o f Gx, Gy and Gz v a l u e s . E x p e r i m e n t a l p a r a m e t e r s a s p e c t r a l w1dth( f 6)=*/-1000 Hz. b l o c k s l z e ( 6 ) * 5 1 2 p o i n t s . I • 16 . X 1 =Y I =Z 1 =0.042 G/cm( 180 Hz/cm), p u l s e w i d t h = 30 »sec. tx = t y t z = 1 msec. scan=1. r e l a x a t i o n d e l a y * 1 see. 121 Table 4.1a Chemical composition of egg yolk and egg white(l) Components Egq yolk Egg whi Water 48.7% 87.9% Proteins 16.6% 10.6% Lipids 32.6% trace Carbohydrates 1.0% 0.9% Minerals 1 .1% 0.6% Table 4.1b Fatty acids found in egg yolk(1,10) Fatty ac ids Amount in egg yolk Oleic 50% Palmitic 27% Li n o l e i c . 27% Stearic 6% Palmitoleic 6% Linolenic Clupansdonic Myristic Table 4.1c The density d i s t r i b u t i o n of albumen layers(15-16) Layers of albumen Density (di1) Outer l i q u i d 1 .032 Middle dense 1 .036 Inner l i q u i d 1 .040 Chalaziferous 1 .045 Table 4.Id The l i q u i d portion of albumen after freezing and thawing(17) Temperature Control -3°C -16°C Liquid portion of albumen 42% 50% 67% 1 2 2 The egg, at the moment i t i s l a i d , contains no a i r c e l l (Figure 4.3). It i s generally thought (1) that the formation of the a i r c e l l i s the r e s u l t of d i f f e r e n t rates at which the s h e l l and the egg contents contract as the new-laid egg cools from the temperature of the hen's body (Figure 4.1c and d). In composition, the gas within the a i r c e l l i s a mixture s i m i l a r to a i r ( l 2 ) . The normal position of the a i r c e l l , between the inner and outer shell-membranes, at the blunt end of the egg appears to help supplying a i r to the embryo at the time when pulmonary r e s p i r a t i o n i s i n i t i a t e d . Thus, the head of the embryo l i e s d i r e c t l y beneath the a i r c e l l ; furthermore, the pores in the eggshell are more numerous at that end of the egg than at the other(13). As the egg ages, the a i r c e l l grows larger and the egg contents evaporate(l) ; thus the volume of a i r increases to ca. 1 ml at 16 days and to 6 ml at 100 days(l4). Figure 4.4b and Figure 4.4c are NMR images which show the progressive increase in the size of the a i r c e l l as a f r e s h l y l a i d egg was stored in a f r i d g e . 4.5 The Yolk Immediately a f t e r an egg i s l a i d , the yolk, which i s of greater s p e c i f i c gravity than the albumen, tends to sink below the centre of the egg. As the egg ages the albumen becomes more concentrated because of loss of water both to the yolk and to the surrounding atmosphere; and the yolk then tends to r i s e . Since 123 HOLDING PERIOD COAtS) F i g u r e 4.3 Changes i n ( a ) , dimensions, and (b) volume of a i r c e l l i n the hen's egg hel d at approximately 28 C and 82% r e l a t i v e h u m i d i ? y . ( A f t e r Romlnoff and Romanoff, The Avian Egg, John Wiley & Sons, Inc., New York,1949) 124 the yolk can r o t a t e about the egg's long a x i s , the yolk of a r e f r i g e r a t e d raw egg, s i t t i n g on a cushion of much denser albumen, always appears on the top (Figure 4.4). Double yolked eggs tend to be longer, l a r g e r , and p r o p o r t i o n a l l y narrower; the two ends are apt to be n e a r l y the same s i z e and rounded rather than pointed (Figure 4.4a). 4.6 The Albumen The whole body of the albumen i s disposed i n four l a y e r s (Figure 4.1c); an inner l i q u i d l a y e r ; a middle dense l a y e r ; an outer l i q u i d l a y e r and the chalazae which composes part of the ligamentum albumenis, a network of filamentous p r o t e i n which extends through the albumen from the outer membrane of the yolk to the inner membranes of the s h e l l . The percentage of p r o t e i n i n c r e a s e s i n albumen from the outermost to the innermost l a y e r , as does the percentage of m i n e r a l s ( 1 5 ) , and the d e n s i t y of albumen tends to increase s l i g h t l y from the outermost to the innermost l a y e r (Table 4.1c). Figure 4.4b and 4.4c show that when albumen i s frozen at any temperature (or kept i n a f r i d g e ) and then thawed, there i s an increase i n volume of the l i q u i d p o r t i o n and a corresponding decrease i n the dense part ( i . e . the white p o r t i o n of the image becomes smaller upon r e f r i g e r a t i o n ) . This agrees wi t h Moran's 1925 o b s e r v a t i o n ( 1 7 ) , as summarized i n Table Id. 4.7 Cooking Of An Egg 125 Figure 4.4 Diagram showing a s i n g l e yolked egg(LEFT) and a double yolked e g g ( r i g h t ) ; 3D proton NMR volume images of (b) f r e s h s i n g l e yolked raw egg; (c) 2 month o l d s i n g l e yolked raw egg with large a i r c e l l ; (d) double yolked raw egg kept at room temperature; and (e) double yolked raw egg kept i n r e f r i g e r a t o r (white area decreases i n s i z e upon r e f r i g e r a t i o n ) . 1024 s i g n a l s (32x32) were acquired for each of the combinations of Gx and Gy values, which a f t e r 3D F o u r i e r t r a n s f o r m a t i o n gave the i n t e r p o l a t e d 128x128 images. Experimental parameters: s p e c t r a l width(f6)=+/-8000 Hz, block size(6)=5l2 p o i n t s , i=32, Xi=Yi=0.042 G/cm( 180 Hz/cm), pulse width=30 *isec, tx = ty=1 msec, scan=1, r e l a x a t i o n delay=1 sec. 126 The albumen i s an e l a s t i c , shock-absorbing, i n s u l a t i n g s e m i - s o l i d l a r g e l y composed of water. The high s p e c i f i c heat of water enables i t to absorb the heat l i b e r a t e d by the v i t a l a c t i v i t i e s of the developing egg, or heat from an o u t s i d e source, and by minimizing r a p i d temperature f l u c t u a t i o n s thereby p r o t e c t s the c h i c k embryo. The coagulation-temperature of the albumen i s s l i g h t l y lower than that of the y o l k ; thus when an egg i s soaked i n b o i l i n g water, the albumen coagulates f i r s t . According to Payawal, Lowe, and Steward(18)(Figure 4.5), between 60° and 70°C, the v i s c o s i t y of the albumen c o n t i n u a l l y decreases whereas that of the yolk i n c r e a s e s . Since the albumen l a y e r s are more f l u i d - l i k e when the egg i s heated, the albumen l a y e r s tend to extend themselves to e n c i r c l e the yolk (Figure 4.6a-d), thus pushing the yolk to the r e a r - c e n t r e of the egg (Figure 4.7a-d). Thus even under these unnatural c o n d i t i o n s the albumen continues to optimize the i s o l a t i o n of the yolk (and of the chick embryo) from the d e l e t e r i o u s o u t s i d e environment. 3 1 P NMR s p e c t r a (Figure 4.8) showed a l l observable mobile phosphorus s p e c i e s , namely i n o r g a n i c phosphate and egg yolk phosphorous storage p r o t e i n s ; the l a t t e r were e i t h e r immobilized or destroyed by heat. 4.8 Four Dimensional, Chemical Shi f t Resolved Imaging 127 0.0u ' 1 1 -1 »800 L ' 1 J « i • . 60 62 64 66 68 70 60 62 64 66 68 70 TEMPERATURE (•<:.> Figure 4.5 E f f e c t of heating on the v i s c o s i t y of ( a ) , albumen, and ( b ) , y o l k . ( A f t e r Romanoff, Food Research 8:286,1943) 1 2 8 Figure 4.6 C r o s s - s e c t i o n a l views from 3D proton NMR volume images of r e f r i g e r a t e d (a) raw egg, (b) medium b o i l e d egg, (c) s o f t b o i l e d egg, and (d) hard b o i l e d egg. Experimental parameters: same as i n Figure 4.4. 129 Figure 4 .7 L o n g i t u d i n a l views from 3D proton NMR volume images of r e f r i g e r a t e d (a) raw egg, (b) medium b o i l e d egg, (c) so f t b o i l e d egg, and (d) hard b o i l e d egg. Experimental parameters: same as i n Figure 4 . 4 . 130 egg yolk phosphorous storage inorganic phosphate (b) ' ' I 1 1 1 1 I 1 -1000 -1500 Hz F i g u r e 4.8 High r e s o l u t i o n 3 1 P NMR spectra (32.5 MHz) of (a) raw egg, " (b) cooked egg. 131 It is clear from the composition data given in Table 4.1a and 4.1b, and the proton NMR spectra of Figure 4.2, that the proton-containing content of the yolk and albumen are s ignif icant ly different ; par t i cular ly , over 90% of the l i p i d is contained in the yolk. It follows from this , that an image derived from the proton resonance signal of fat should largely show the position of the yolk. Demonstration of this fact using the four dimensional data acquisition sequence(l9) summarized in Figure 1b, which encodes in addition to the three spatial coordinates (x,y and z) , the entire chemical shif t ranges (6); is summarized in Figure 4.2e and f; the fact that these two images are essentially featureless indicated that the detailed internal structure visualized in the "composite" water-plus-fat proton images shown in the other figures stem from other in t r ins ic differences in the molecular properties of the water in the egg. This point is under further investigation. 4.9 Conclusion Although this is only a preliminary study, several points merit comment. F i r s t l y , i t is clear that proton NMR imaging methods appear to provide a simple, non-invasive means for following storage-degradation and cooking of hen's eggs; i t seems reasonable to surmise that studies of other fresh produce may be worthwhile. Secondly, any major abnormality is c learly shown (in this case the double yolk) . In the overall context of this thesis , i t is important to note that this is the f i r s t 1 3 2 o p p o r t u n i t y to expand from the s t u d i e s of i n - v i t r o samples using a small bore magnet to the i n t a c t systems. Future study w i l l probably i n v o l v e observation of the embryonic development of a hen-chick by NMR imaging methods . 133 References 1. Romanoff,A.L., and Romanoff,A.J. The Avian Egg, John Wiley & Sons, Inc. , New York, 1949. 2. Romanoff,A.L., The Avian Embryo, MacMillan Publishing L t d . , New York, 1960. 3. Pattern,B.M., Early Embryology of the Chick, F i f t h edit ion, McGraw-Hill Book Co. , New York, 1971. 4. Jackson,J .A. , and Langham,W.H., Rev. S c i . Instrum. 39:51 0, 1968. 5. Krebs , J . , and Will iams,R.J .P . Academic Press, London, pp348-349,1977. 6. Edelstein,W.A., Hutchinson,J.M.S., Johnson,G., and Redpath,T. J . Phys. Med. B i o l . 25:751,1980. 7. Johnson,G., Hutchinson,J.M.S., and Eastwood,L.M., J . Phys. E: S c i . Instrum. 15:74,1982. 8. H a l l , L . D . , and Sukumar,S. J . Magn. Reson. 56:314,1984. 9. Kumar,A., W e l t i , I . , and Ernst,R.R. J . Magn. Reson. 18:69,1975. 10. H a l l , L . D . , Sukumar,S., and Lee,J .W.K., to be published. 11. H a l l , L . D . , Rajanayagam,V., and Sukumar,S. J . Magn. Reson. 61 : 188, 1 985. 134 12. H a l l , L . D . , Marcus,T. , N e a l e , C , Powell ,B. , S a l l o s , J . , and Talagala ,S.L. J . Magn. Reson. 62:525,1985. 13. Riemenschneider,R.W., E l l i s , N . R . , and Titus,H.W. J . B i o l . Chem. 126:255,1938. 14. Baker ,CM.A. , The Proteins of Egg White. In Egg Quality- a Study of the Hen's Egg, Chapter 3, C a r t e r , T . C . , e d . , Oliver & Boyd, Edinburgh,1968. 15. Romijn ,C , and Roos,J. Physiol . 94:365,1938. 16. von Wittich,W. Z. Wiss. Zool, 3:213,1851. 17. Fronda, and Clemente,D.D. Philippine Agr. 25:191,1936. 18. Romanoff,A.L. Food Research 8:286,1943. 19. Romanoff,A.L. Food Research 5:291,1949. 20. Moran,T. Proc. Roy. Soc. (London), 98(B):436,1925. 21. Payawal,S.R., Lowe,B., and Stewart,G.F. Food Research 11:246,1946. 22. Maudsley,A.A., H i l a l , S . K . , Perman,W.H., and Simon,H.E. J . Magn. Reson. 51:147,1983. 23. P y k e t t , I . L . , Rosen,B.R. Radiology, 149:197,1983. CHAPTER V—SUMMARY AND DISCUSSION 1 3 6 The l a s t f i f t e e n years has witnessed many changes and advances i n the a p p l i c a t i o n s of NMR to study b i o l o g i c a l changes. From the i n i t i a l use of narrow bore, electro-magnets and l i q u i d u s samples, t o whole-body tomographs and i n - v i v o spectroscopy, there have emerged many new techniques and new instruments. U n f o r t u n a t e l y , most of these developments have taken place i n in d u s t r y and are not widely p u b l i s h e d . This i s e s p e c i a l l y true i n NMR imaging where not enough d e t a i l s have been given i n the l i t e r a t u r e to allow other researchers to repeat many of the experiments, unless they have purchased a s p e c i a l i z e d NMR instrument; even then, that instrument w i l l probably perform e i t h e r spectroscopy or imaging, but not both. The work presented i n t h i s t h e s i s was undertaken as part of a team e f f o r t i n P r o f e s s e r H a l l ' s Laboratory to bridge the t e c h n i c a l gap which prevented s p e c t r o s c o p i c and imaging inform a t i o n t o be obtained from the same NMR instrument. Given that most p r a c t i c i n g chemists have r e s t r i c t e d access t o t h i s type of s o p h i s t i c a t e d NMR instruments, we a c c o r d i n g l y s t a r t e d o f f with a v e r t i c a l , narrow-bore, h i g h - r e s o l u t i o n superconducting magnet. While Dr. S. Sukumar was developing imaging techniques a p p l i c a b l e to our 270 MHz spectrometer, t h i s author was e v a l u a t i n g a number of b i o l o g i c a l systems so as to determine i f t h e i r changes could be f o l l o w e d by NMR; that work formed the b a s i s f o r t h i s t h e s i s . Chapter I I d e a l t w i t h an e x p l o r a t i o n study of the use of h i g h - r e s o l u t i o n NMR to monitor b i o l o g i c a l t r a n s f o r m a t i o n s . The main t h r u s t of that Chapter was to determine which changes were 1 3 7 observable and to f i t those changes into known biochemical pathways; we believe that t h i s forms a firm basis for future in-vivo studies. Although a 30 cm. h o r i z o n t a l bore magnet i s now available(80 MHz for 'H, 20 MHz for 1 3 C ) , many a d d i t i o n a l technical developments, such as radiofrequency(RF) probe construction, noise reduction, and s i g n a l l o c a l i z a t i o n , have to be explored before many studies w i l l be possible. For example, due to the absence of a capacitor in the tuning c i r c u i t , "resonator" c o i l s 1 g i v e s the largest homogeneous volume and highest signal-to-noise r a t i o . Thus, resonator c o i l s designed for s p e c i f i c tasks are a mandatory s t a r t i n g point; however, constructing such a c o i l which i s resonant at 20 MHz for carbon-13 studies i s a n o n - t r i v i a l task: the fact that the two f l a p s of the capacitor have to be so long and the Teflon d i e l e c t r i c so t h i n , makes the construction very d i f f i c u l t . Furthermore, because at 20 MHz both signal-to-noise r a t i o and res o l u t i o n are poor, noise reduction and resolution enhancement w i l l require a major e f f o r t . Localized spectroscopy w i l l also be necessary i f we want to obtain NMR signals only from c e r t a i n regions of the sample, as in the study of a r t h r i t i s . Chapter III represented a discussion of the a p p l i c a t i o n of proton NMR relaxation measurements to follow microscopic changes. 1 The resonator usually consists of a s i n g l e copper f o i l wrapped around a P l e x i g l a s c y l i n d i c a l former with two flaps which project normally from the circumference of the c y l i n d e r . These are separated by a sheet of Teflon, sandwiched between two P l e x i g l a s plates, and provide the capacitance required for tuning the probe. The resonator i s inductively coupled to the transmitter v i a a single copper ring attached to the end of a coaxial c a b l e ( H a l l et a l . , J . Magn. Reson. 62:525,1985.). 1 3 8 Although relaxation rate changes were observed in most of the systems investigated, there was no single theory or equation that could be used to explain a l l the differ e n c e s observed. Even though many of the changes were unpredictable, our main concern was only to e s t a b l i s h "contrast" and to singl e out the parameter best used to describe c e r t a i n phenomena; such information w i l l f a c i l i t a t e our future imaging experiments for the same systems and w i l l provide us with the optimal parameters to obtain high-contrast and top-quality images. The fact that many b i o l o g i c a l changes manifest themselves in macroscopic changes in relaxation rates i s s u f f i c i e n t l y encouraging to j u s t i f y pursuit of t h e i r p o t e n t i a l a p p l i c a t i o n s . Our r e s u l t s , and others described in the l i t e r a t u r e , demonstrate that relaxation rates are intimately connected to the nature of the signal-producing molecules and t h e i r binding state. As a r e s u l t , NMR appears to have the a b i l i t y to provide information on the processes occurring inside l i v i n g c e l l s . Relaxation rates can provide information about both the water and fat content; they can r e f l e c t changes in the concentration of macromolecules and t h e i r conformations; they can be related to the growth rate and to d i f f e r e n t stages of c e l l u l a r a c t i v i t y , such as degradation and i n f e c t i o n . Chapter IV highlighted our e f f o r t s to integrate both NMR spectroscopy and imaging into the same NMR instrument. While spectroscopic data helped to i d e n t i f y major components in the egg-white and the egg-yolk, and t h e i r associated properties, 1 3 9 imaging data gave us the oppor t u n i t y to v i s u a l i z e changes o c c u r r i n g during storage and cooking. The s i m p l i c i t y and e f f e c t i v e n e s s of such i n t e g r a t e d , complementary techniques w i l l , i n the long term, y i e l d much i n f o r m a t i o n , and i t i s c l e a r that t h i s approach w i l l be a very d e s i r a b l e non-invasive a n a l y t i c a l and c l i n i c a l d i a g n o s t i c t o o l . At the time the author s t a r t e d the s t u d i e s d e s c r i b e d i n t h i s t h e s i s , i t was u n c e r t a i n whether the combination of both sp e c t r o s c o p i c and imaging c a p a b i l i t i e s , at the same magnetic f i e l d s t r e n g t h , would be t e c h n i c a l l y f e a s i b l e . Although t h i s i n t e g r a t i o n of methods i s s t i l l not widely used, t h i s work has c l e a r l y demonstrated i t s p o t e n t i a l , which appears to have many u s e f u l p r a c t i c a l advantages. These techniques s t i l l remain to be f u l l y explored, and f u r t h e r s t u d i e s w i l l c e r t a i n l y y i e l d knowledge p r e v i o u s l y u n a v a i l a b l e . 

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