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The history of muscular dystrophy Holst, Holst 1989

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"THE HISTORY OF MUSCULAR DYSTROPHY" By DENICE LAUREEN HOLST B.Sc., Simon Fraser U n i v e r s i t y , B.C., 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1989 ® Denice Laureen H o i s t 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 P h y s i o l o g y The University of British Columbia Vancouver, Canada Date A p r i l 28, 1989 DE-6 (2/88) i i ABSTRACT The muscular dystrophies, Duchenne muscular dystrophy being the most common type, are a group for which there is no apparent pathology to the spinal motor neurons concomitant to progressive muscular degeneration. After this fact was established during the nineteenth century through postmortem examination, Charcot divided muscular disease into the "Great Classes" of myopathy and neuropathy. Erb's study of the histopathology brought a further division between the two before death and therefore the muscle biopsy became a tool for d i f f e r e n t i a l diagnosis. He also discovered that the response of muscle in myopathy and neuropathy to the application of electric current differed. While the response of myopathic muscle was progressively diminishing and equally so to the application of either galvanic or alternating current, neuropathic muscle maintained its ability to contract upon the application of galvanic current during the course of the disease. As well as the animating power of the nervous system by way of the anterior nerves, its trophic effect upon the muscle was evident by loss of volume upon interruption of i t s influence. Even though the absence of a lesion involving the spinal motor neurons or descending motor tracts was a constant in muscular dystrophy, there remained some reluctance to accept myopathy as being independent of the nervous system. According to the maintenance of the i i i c o n t r a c t i l e response to g a l v a n i c c u r r e n t i n neuropathy Erb suggested that there was a nerve centre other than that of the a n t e r i o r cornua of the s p i n a l c o r d which s u p p l i e d the t r o p h i c i n f l u e n c e . When he found h i s t o l o g i c a l f e a t u r e s which were t y p i c a l o f myopathy i n p o l i o m y e l i t i s he was c o n v i n c e d t h a t m u s c u l a r d y s t r o p h y was t h e r e s u l t o f a t r o p h i c d i s t u r b a n c e . H o w e v e r , t h i s t h e o r y was n o t s u s t a i n a b l e because there was no anatomical evidence f o r a s p e c i a l t r o p h i c c e n t r e . In 1970 McComas a g a i n p r o p o s e d t h a t a n e u r o g e n i c phenomenon was r e s p o n s i b l e f o r the p a t h o g e n e s i s of t h e m u s c u l a r d y s t r o p h i e s . I t was t h e r e - e m e r g e n c e o f a n e u rogenic h y p o t h e s i s f o r muscular dystrophy which was the purpose of my e x e r c i s e . In order to answer the q u e s t i o n as to why a t r o p h i c theory reappeared I f o l l o w e d the r e s e a r c h and theory regarding muscular dystrophy over time. The p o w e r f u l e f f e c t of the s o m a t i c i n n e r v a t i o n upon m u s c l e m e t a b o l i s m as d e t e r m i n e d by c r o s s i n n e r v a t i o n e x p e r i m e n t s d u r i n g t h e 1 9 6 0 ' s , s e t t h e s t a g e f o r t h e r e a s s e r t i o n of a t r o p h i c d i s t u r b a n c e i n muscular d y s t r o p h y . In a d d i t i o n , the d i v i s i o n between myopathy and neuropathy had become l e s s . d i s t i n c t by 1970 i n terms of h i s t o l o g y , s e r u m e n z y m e s a n d t h e i n t r a m u s c u l a r i n n e r v a t i o n . H i s t o l o g i c a l f e a t u r e s c o n s i d e r e d to by t y p i c a l of myopathy were seen i n the b i o p s i e s of Charcot-Marie-Tooth d i s e a s e and K u g e l b e r g - W e l a n d e r s p i n a l m u s c u l a r a t r o p h y . As w e l l , iv abnormally elevated serum levels of creatine phosphokinase which was characteristic of muscular dystrophy, were measured in these neuropathies. Changes in the intramuscular innervation of myotonic dystrophy and the animal model for muscular dystrophy also brought into question the myopathicity of muscular dystrophy. By 1970 the types of muscular dystrophy had been classified according to clinical and genetic criteria and were thus known to be genetically distinct diseases. A unifying hypothesis is always desireable and therefore, mental deficiency according to c l i n i c a l assessment in Duchenne and myotonic dystrophy, the latter being an "impure" dystrophy, were considered to be supportive of the neurogenic hypothesis. "Hypertrophic paraplegia of infancy of cerebral origin" was the original t i t l e of what became known as Duchenne dystrophy. The frequent occurrence of mental retardation was the foundation of the name but with knowledge concerning the profound influence of the nerve upon the metabolism of the muscle, the alleged cerebral defect in Duchenne dystrophy favoured the neuropathicity of dystrophy even more. TABLE OF CONTENTS ABSTRACT . . i i : INTRODUCTION 1 MUSCULAR DYSTROPHY DEFINED 5 Pseudohypertrophic P a r a l y s i s . . . . 6 P r o g r e s s i v e Muscular Atrophy 13 P r o g r e s s i v e Muscular Dystrophy 16 Myopathy versus Neuropathy 20 The T r o p h i c Theory of Erb 23 MUSCULAR DYSTROPHY AND MUSCLE METABOLISM . . 27 C r e a t i n e and C r e a t i n i n e 28 The O r i g i n of U r i n a r y C r e a t i n i n e 29 C r e a t i n u r i a 42 The S y n t h e s i s of C r e a t i n e 47 The P h y s i o l o g y of C r e a t i n e 53 Muscular Dystrophy as an Endocrinopathy 58 Muscular Dystrophy as a Neuropathy . . . . . . . . 73 Vitamin E and the F i r s t Animal Model 83 DUCHENNE MUSCULAR DYSTROPHY, AN X-LINKED RECESSIVE TRAIT: NEUROPATHY? 98 The X-linkedness of Duchenne Dystrophy . . . . . . 99 The C e r e b r a l Defect 110 The D i f f e r e n t i a l D i a g n o s i s o f M y o p a t h y v s . Neuropathy 120 T h e P a t h o l o g y o f t h e I n t r a m u s c u l a r M o t o r I n n e r v a t i o n 129 Tr o p h i c I n f l u e n c e s of Nerve Upon Muscle . . . . . 133 The Neurogenic Hypothesis 139 The C a r d i a c Defect 152 The Membrane Defect 155 Met a b o l i c Consequences of a Membrane Defect . . . 156 The Question of the C e r e b r a l Defect 160 B i b l i o g r a p h y 163 1 INTRODUCTION Muscular dystrophy i s a term which a p p l i e s to a group o f d i s o r d e r s which a r e d i c t a t e d by d i f f e r e n t d e f e c t i v e genes, but which a l l manifest degeneration of the s k e l e t a l muscle w i t h no apparent pathology to the p e r i p h e r a l nerves or s p i n a l c o r d . Duchenne mu s c u l a r d y s t r o p h y , the most common ty p e o f muscular d y s t r o p h y , i s i n h e r i t e d as an X-l i n k e d r e c e s s i v e t r a i t . T h e r e f o r e , e x c e p t i n r a r e i n s t a n c e s , i t i s e x c l u s i v e t o m a l e s . The on s e t o f t h e di s e a s e normally occurs b e f o r e age four and never l a t e r than age seven, at which time g a i t d i s t u r b a n c e s begin to pre s e n t themselves. A posture w i t h a protuberant abdomen i s adopted by the boy, who walks with o s c i l l a t i n g h i p s , who i s i n c l i n e d to t r i p and p o s s i b l y f a l l as a r e s u l t of such c l u m s i n e s s . These symptoms are the m a n i f e s t a t i o n o f muscular weakness and wasting which commences i n the i l i o p s o a s , quadriceps and g l u t e u s muscles and descends upon the lower e x t r e m i t i e s t o i n v o l v e the a n t e r i o r t i b i a l group r e l a t i v e l y e a r l y i n the d i s e a s e . M u s c u l a r e n l a r g e m e n t , b e g i n n i n g w i t h t r u e h y p e r t r o p h y f o l l o w e d by pseudohypertrophy due to f i b r o s i s and f a t t y r e p l a c e m e n t i n the c a l v e s and sometimes t h e q u a d r i c e p s , p r e c e d e s t h e e v e n t u a l a t r o p h y o f the lower l i m b s . While bipedalness i s f a i l i n g , the c o s t a l o r i g i n of the p e c t o r a l i s major, the l a t i s s i m u s d o r s i , b i c e p s , t r i c e p s and b r a c h i o r a d i a l u s begin t o become i n v o l v e d i n the degenerative 2 p r o c e s s . Muscular involvement i s symmetrical and at f i r s t s e l e c t i v e , but i t i s o n l y the f a c i a l muscle which i s u s u a l l y spared i n the f i n a l a n a l y s i s . The muscles of m a s t i c a t i o n w h i c h may show s l i g h t w e a k n e s s o f t e n show i n i t i a l enlargement as does the tongue and the d e l t o i d s , i n c o n t r a s t to o t h e r muscles of the upper l i m b s . C o n t r a c t u r e s develop i n the c a l f muscles f i r s t to r e s u l t i n p l a n t a r f l e x i o n and then i n the h i p f l e x o r s . C o n t r a c t u r e s i n the l a t t e r a r e accentuated with confinement to a wheelchair which occurs on or about the tenth year of l i f e . T h e c o u r s e o f t h e d i s e a s e t h e n i s one o f r a p i d p r o g r e s s i o n and death i s i n e v i t a b l e u s u a l l y by the second or t h i r d decade of l i f e . The f u n c t i o n of the diaphragm remains q u i t e normal but the i n t e r c o s t a l s are very much i n v o l v e d i n the l a t e r s t a g e s of the d i s e a s e and t h e r e f o r e r e s p i r a t o r y f u n c t i o n i s g r e a t l y d e p r e s s e d , m a k i n g t h e l u n g s v e r y v u l n e r a b l e . C a r d i a c a b n o r m a l i t i e s are a constant but o n l y about t e n p e r c e n t d i e as a r e s u l t of c a r d i a c f a i l u r e . In the m a j o r i t y , death i s due to an i n t e r c u r r e n t d i s e a s e such as pneumonia or some other r e s p i r a t o r y i n f e c t i o n . The c l i n i c a l syndromes of muscular d i s e a s e which a r e secondary to p a r a t h y r o i d i n s u f f i c i e n c y or r e n a l d i s e a s e i n c h i l d h o o d and t h a t o f Kugelberg-Welander s p i n a l muscular a t r o p h y , which i n v o l v e s the p r o g r e s s i v e l o s s of s p i n a l motor neurons, may i m i t a t e Duchenne muscular dystrophy. However, t h e d i a g n o s i s i s q u i t e e a s i l y c o n f i r m e d by p h y s i c a l 3 e x a m i n a t i o n , m u s c l e b i o p s y , EMG and an e l e v a t e d serum c r e a t i n e phosphokinase. Family h i s t o r y o f the d i s e a s e i s a l s o among the d i a g n o s t i c c r i t e r i a , but a p p r o x i m a t e l y one t h i r d o f cases are new mutations. T h e r e f o r e , the mutation r a t e of the gene r e s p o n s i b l e f o r the d i s o r d e r i s h i g h , being t w i c e t h a t o f h a e m o p h i l i a , an X - l i n k e d r e c e s s i v e t r a i t as w e l l . Duchenne m u s c u l a r d y s t r o p h y i s i n f a c t the most common of a l l X - l i n k e d r e c e s s i v e d i s e a s e s with an i n c i d e n c e as high as one i n every 2500 l i v e male b i r t h s . In Canada, i n c i d e n c e among neuromuscular d i s e a s e s i s second only to myotonic dystrophy, a g e n e t i c a l l y determined d i s o r d e r as w e l l , f o r which the d i c t a t i n g gene i s harboured by chromosome 19. L i k e muscular dystrophy i t i n v o l v e s the d e g e n e r a t i o n o f t h e s k e l e t a l m u s c l e unaccompanied by a l e s i o n o f the s p i n a l c o r d , but i t i s a d i f f u s e s y s t e m i c d i s e a s e i n which a myriad of symptoms may accompany muscular wasti n g and the p e r s i s t e n c e of muscular c o n t r a c t i o n a f t e r v o l u n t a r y e f f o r t has ceased. Symptoms i n c l u d e c a t a r a c t s , f r o n t a l b a l d n e s s i n the male, g o n a d a l a t r o p h y , i m p a i r e d pulmonary v e n t i l a t i o n , , m i l d endocrine d i s t u r b a n c e s , c a r d i a c d i s e a s e and bone changes, which i n Duchenne dy s t r o p h y are c o n s i d e r e d to be the r e s u l t of d i s u s e . Mental d e f i c i e n c y a c c o r d i n g t o c l i n i c a l assessment i s a l s o f r e q u e n t but not c o n s t a n t i n both Duchenne and myotonic d y s t r o p h y . U n l i k e Duchenne dystrophy the age of onset and s e v e r i t y of myotonic d y s t r o p h y a r e v e r y v a r i a b l e . The s i m i l a r i t i e s and 4 d i f f e r e n c e s between the two w i l l u l t i m a t e l y be r e s o l v e d by the molecular a n a l y s i s of each which has been made p o s s i b l e by DNA technology. During the ni n e t e e n t h c e n t u r y W i l l i a m Erb put f o r t h a theory concerning the pathogenesis of muscular dystrophy as a whole, which proposed t h a t a neurogenic phenomenon was r e s p o n s i b l e f o r the degeneration o f the s k e l e t a l muscle. In 1970 A l l a n McComas again proposed a neurogenic h y p o t h e s i s f o r muscular d y s t r o p h y which was a l s o a p p l i e d t o myotonic d y s t r o p h y . The q u e s t i o n I have sought t o answer i n the account that f o l l o w s i s : "Why d i d a neurogenic h y p o t h e s i s re-emerge and, as i t were, meet i t s demise?" In attempting t o answer the q u e s t i o n I w i l l be p r e s e n t i n g a h i s t o r y o f re s e a r c h and theory regarding the pathogenesis and a e t i o l o g y o f muscular d y s t r o p h y , the emphasis of the account b e i n g p l a c e d upon Duchenne dystrophy. I t c o n s t i t u t e s the bulk of cas e s and any paper devoted to muscular dystrophy must, of n e c e s s i t y , f o c u s upon t h e most common t y p e . I w i l l u l t i m a t e l y a d d r e s s the q u e s t i o n o f t h e myogenic v e r s u s n e u r o g e n i c f o u n d a t i o n of the d i s o r d e r and w i l l s p e c u l a t e a b o u t i t s s p e c i f i c p a t h o g e n e s i s i n view o f i t s u n i q u e b i o c h e m i c a l b a s i s . PART I MUSCULAR DYSTROPHY DEFINED F o l l o w i n g the f o r m u l a t i o n and e n u n c i a t i o n of Newton's l a w s o f m o t i o n i n t h e s i x t e e n t h c e n t u r y , m u s c u l a r c o n t r a c t i o n was b e l i e v e d t o be b r o u g h t a b o u t by t h e g a l v a n i z a t i o n of a r a r e e l a s t i c s p i r i t which pervaded the u n i v e r s e . T h i s s p i r i t was thought to i n h a b i t the nerves and upo n m e n t a l e f f o r t w o u l d p u l s a t e a l o n g t h e n e r v e t o communicate with the same s p i r i t c o n t a i n e d i n the muscle, the s w e l l i n g of which was the essence of i t s c o n t r a c t i o n . I t was always obvious t h a t the b r a i n was the seat of v o l i t i o n , f r o m w h i c h e n e r g y somehow p r o c e e d e d t o t h e s k e l e t a l muscle v i a the s p i n a l c o r d and n e r v e s . However, the b r a i n a l s o r e c e i v e d i n f o r m a t i o n regarding s e n s a t i o n by way of the s p i n a l c o r d , from which nerves emanated from both the a n t e r i o r and p o s t e r i o r a s p e c t s . P a r a l y s i s was the i n e v i t a b l e outcome o f the i n t e r r u p t i o n o f the n e r v o u s i n f l u e n c e o r i g i n a t i n g i n the c e n t r a l systems as a r e s u l t of i n j u r y or trauma. In such an event there was u s u a l l y a l o s s of s e n s a t i o n with the l o s s of muscular power. Whether the same nerve was r e s p o n s i b l e f o r both s e n s a t i o n and movement o f a p a r t i c u l a r p a r t o f the anatomy was an i n e s c a p a b l e q u e s t i o n . C h a r l e s B e l l p r o v i d e d the answer i n 1830 by c u t t i n g the p o s t e r i o r r o o t s of the s p i n a l nerves s u p p l y i n g the limbs of a r a b b i t . When th e c r e a t u r e was s t i l l a b l e to c r a w l he c o n c l u d e d t h a t i t was the a n t e r i o r r o o t s o f the s p i n a l nerves and the a n t e r i o r column of the s p i n a l cord composed 6 o f grey substance that had power over the muscular system. The a n t e r i o r nerves were confirmed to convey the i n f l u e n c e of the w i l l to bestow the power of muscular motion and thus they were the u l t i m a t e governors of the s k e l e t a l muscle. As w e l l as the l o s s of movement caused by the i n t e r r u p t i o n of the nervous i n f l u e n c e there was a r e d u c t i o n i n the volume of the muscle which i n d i c a t e d that the n u t r i t i o n of the muscle was dependent upon nervous agency. The term atrophy which means "no n u t r i t i o n " was a p p l i e d to the d e p r i v a t i o n of the n e r v o u s i n f l u e n c e . I t was a l s o a p p l i c a b l e t o t h e d i s c o n t i n u e d a r r i v a l of n u t r i t i v e m a t e r i a l v i a the a r t e r i a l blood system but i n any event, became the e q u i v a l e n t of l o s t volume. Pseudohypertrophic P a r a l y s i s A p a r t i c u l a r p a t i e n t of Meryon's (1852) was a young man who had been a seemingly h e a l t h y c h i l d , robust and heavy as an i n f a n t but he walked l a t e r than other c h i l d r e n . When he f i n a l l y d i d walk, he d i d so without a "healthy e l a s t i c i t y " . He had d i f f i c u l t y ascending a s t a i r c a s e and was i n c l i n e d to t r i p . At age n i n e he was put on a s t r e t c h i n g board by a s u r g e o n c a l l e d Mr. T. whose d i a g n o s i s was a " d e f i c i e n t e l o n g a t i o n of the s p i n a l c o r d " . Two years l a t e r he c o u l d not walk, nor c o u l d he s t a n d without grave d i f f i c u l t y and h i s c o n d i t i o n brought the i n t e r v e n t i o n of another surgeon, who d i v i d e d the A c h i l l e s tendons as w e l l as the tendons of 7 four other muscles of the l e g s . He walked with braces f o r a time f o l l o w i n g t h i s i n t e r v e n t i o n , but soon t h i s a b i l i t y was l o s t and before long much of the power i n h i s arms was gone. Although the a b i l i t y of the muscle to develop f o r c e was l o s t there was an i n i t i a l g a i n i n f l e s h . When the young man succumbed to some kind of f e v e r at a g e t w e n t y - f o u r h i s b o d y was o u t w a r d l y e m a c i a t e d c o r r e s p o n d i n g t o the d e g e n e r a t i o n of the v o l u n t a r y muscle that was seen under the microscope. The integument of the i n d i v i d u a l f i b r e s was l o s t , f a t g l o b u l e s and g r a n u l a r matter t a k i n g t h e i r p l a c e ; t h e r e f o r e i t seemed that a metamorphosis from muscle to f a t had o c c u r r e d . Because the pathology of t h e m u s c l e was not l i k e t h a t o f " o r d i n a r y " p a r a l y s i s r e s u l t i n g from i n j u r y or trauma to the nervous system, and by v i r t u e of the normal appearance of the b r a i n and s p i n a l c o r d a t a u t o p s y , M eryon c o n c l u d e d t h a t t h e m u s c u l a r d e g e n e r a t i o n was not a consequence of the m i s a p p r o p r i a t i o n of nervous agency. I n s t e a d , he a t t r i b u t e d the " f a t t y and g r a n u l a r d e g e n e r a t i o n o f t h e v o l u n t a r y m u s c l e " t o a d e f e c t i v e n u t r i t i o n o f t h e m u s c l e r e s u l t i n g f r o m t h e i n e f f i c a c y of i t s a s s i m i l a t i n g p r o c e s s e s . In 1861, a f t e r g a t h e r i n g i n f o r m a t i o n from t h i r t e e n " l i t t l e p a t i e n t s " Duchenne wrote of what he c o n s i d e r e d to be a n o v e l d i s e a s e , but as i t w i l l be seen he was m i s t a k e n . The d i s e a s e was the a n t i t h e s i s of muscular atrophy i n t h a t a c c o r d i n g to Duchenne t h e r e was p a r a l y s i s of muscles th a t 8 were a t h l e t i c i n a p p e a r a n c e , m u s c l e s w i t h " H e r c u l e a n " p r o p o r t i o n s . When Meryon wrote o f t h e d i s e a s e o f t h e v o l u n t a r y muscle i n 1852 the l e g s o f t h e t h r e e year o l d b r o t h e r o f h i s f i r s t c a s e were b e c o m i n g t h i c k - - an o b s e r v a t i o n that made him very s u s p i c i o u s . In the same way, one o f Duchenne's p a t i e n t s , who were a l l young boys, was s a i d to have thickened l e g s as e a r l y as s i x months. F i v e of the t h i r t e e n boys were " m e n t a l l y backward"; most n o t a b l y speech was slow and o f t e n m i s a r t i c u l a t e d . For t h i s reason Duchenne s u s p e c t e d a c e r e b r a l b a s i s to the d i s e a s e which appeared to commence d u r i n g i n f a n c y . P a r a l y s i s of the body was g e n e r a l i n some, but i n most the arms were s t i l l moving. On the b a s i s of the aforementioned o b s e r v a t i o n s the d i s e a s e was c a l l e d " h y p e r t r o p h i c p a r a p l e g i a of i n f a n c y of c e r e b r a l o r i g i n " . F u r t h e r study demonstrated that p a r a l y s i s i n v a r i a b l y s p r e a d t o the upper l i m b s which u n l i k e the lower became t h i n , and t h e r e f o r e " p a r a p l e g i a " was not a p p l i c a b l e . From what Duchenne c o u l d gather through i n t e r v i e w i n g parents of f u r t h e r cases and o t h e r s who observed the morbid phenomenon i n the e a r l i e s t p e r i o d , i t was i n f a c t a d i s e a s e of i n f a n c y , but the s i g n s and symptoms d i d not always draw the a t t e n t i o n of a p e r s o n who was u n a c q u a i n t e d w i t h the c o n d i t i o n . A c e r e b r a l o r i g i n was not c o m p a t i b l e w i t h the f i n d i n g s o f Eulenberg and Conheim i n 1863 who found the b r a i n and s p i n a l cord to be normal at autopsy i n an i n d i v i d u a l a f f l i c t e d by 9 " h y p e r t r o p h i c p a r a p l e g i a of i n f a n c y of c e r e b r a l o r i g i n . " Thus " h y p e r t r o p h i c " was the o n l y a s p e c t of the t i t l e with c r e d i b i l i t y . The muscle c o u l d of course be examined post mortem but by t h i s time, j u s t as the gain i n f l e s h was l o s t i n Meryon's c a s e , t h e h y p e r t r o p h y was e i t h e r i n c o n s p i c u o u s o r t r a n s f o r m e d t o a t r o p h y . In order t o s o l v e the r i d d l e of muscular hypertrophy one needed to look at the muscle while the d i s e a s e was s t i l l i n p r o g r e s s and when G r i e s i n g e r had h i s f i r s t encounter w i t h the d i s e a s e i n 1865 he took f u l l advantage of i t i n view of i t s b e l i e v e d r a r i t y . A p e a - s i z e d p i e c e of muscle from the d e l t o i d was s u r g i c a l l y removed under a n a e s t h e t i c from a boy of f o u r t e e n whose lower limbs were h y p e r t r o p h i c while the upper were a t r o p h i c . P r o f e s s o r B i l l r o t h , t h e esteemed f r i e n d of G r i e s i n g e r , committed h i m s e l f to a h i s t o l o g i c assessment of the muscle which was s a i d t o be so f a t t y as to be mistaken f o r adipose t i s s u e by one with no knowledge of i t s o r i g i n . However, metamorphosis of muscle to f a t , as suggested by Meryon, d i d not seem to be an adequate e x p l a n a t i o n s i n c e a r e d u c t i o n i n the number of m u s c l e f i b r e s d i s p e r s e d w i t h i n t h e f a t c o u l d n o t be d i s c e r n e d . G r i e s i n g e r o r i g i n a l l y intended to use something c a l l e d the M i d d l e d o r f harpoon for the e x t r a c t i o n of muscle t i s s u e , but d e c i d e d a g a i n s t i t because he thought that i t would not p r o v i d e a l a r g e enough s p e c i m e n . Duchenne c l a i m e d the 10 i n v e n t i o n of an instrument which he c a l l e d the t i s s u e punch and with which he could remove t i n y amounts of muscle from h i s " l i t t l e p a t i e n t s " . These t i n y amounts s u f f i c e d f o r the second muscle b i o p s y which Duchenne performed i n 1865 as w e l l , the p a r t i c u l a r s of i t being reported i n 1868 i n the c l a s s i c a l d e s c r i p t i o n of the d i s e a s e d e s t i n e d to bear h i s name. The t i s s u e punch c o u l d be used without a n a e s t h e t i c and he a d v i s e d that i f the s k i n was p u l l e d t a u t l y and the in s t r u m e n t was c o n c e a l e d from h i s " l i t t l e p a t i e n t s " , the pa i n would be minimal. He d i d not i n d i c a t e the part of the anatomy upon which he employed h i s t i s s u e punch nor the stage of the d i s e a s e , but these two parameters must account f o r the d i f f e r e n c e s v between h i s o b s e r v a t i o n s and t h o s e o f G r i e s i n g e r and B i l l r o t h . I t was not the p r o f u s i o n of f a t which was the outstanding f e a t u r e of the biopsy, but the l a v i s h n e s s of the c o n n e c t i v e t i s s u e i n the i n t e r s t i c e s of the muscle f i b r e s . Although f a t globules were a l s o present Duchenne judged the accumulation of f a t to be a fe a t u r e of the l a t e r stages of the d i s e a s e . In agreement with Duchenne, G r i e s i n g e r noted the m u l t i p l i c a t i o n of the co n n e c t i v e t i s s u e and suggested t h a t i t s g r o w t h had come b e f o r e t h e b u i l d u p o f f a t . G r i e s i n g e r s t a t e d that there was some s w e l l i n g of the muscle t i s s u e i t s e l f . T h i s point was compatible with the o p i n i o n of Duchenne who concluded that a congested c o n d i t i o n of the m u s c l e was an e a r l y e v e n t w h i c h c o n t r i b u t e d t o t h e 11 e n l a r g e m e n t . As f o r t h e c o m p l i m e n t o f m u s c l e f i b r e s Duchenne d i d not commit h i m s e l f but quoted H e l l e r who was c e r t a i n t h a t some of the f i b r e s d i s a p p e a r e d . He reasoned t h a t the bulk of the muscle would be f a r g r e a t e r i f t h e r e was s i m p l e a d d i t i o n of other t i s s u e without s u b t r a c t i o n of the normal elements. Now t h a t Duchenne had seen what was beneath the s k i n of the "monstrous" l i m b s he o f f e r e d two new t i t l e s f o r the d i s e a s e . He h e s i t a t e d to f a s t e n " p r o g r e s s i v e " to the t i t l e b e c a u s e a l t h o u g h i t was a d i s e a s e w h i c h c u l m i n a t e d i n complete i m m o b i l i t y b e f o r e the r e l e a s e unto death by some i n t e r v e n i n g d i s e a s e , he b e l i e v e d he had cured one such case by e l e c t r i c c u r r e n t , " h y d r o p a t h y a n d s h a m p o o i n g " (hydrotherapy and massage?). " P r o g r e s s i v e " was t h e r e f o r e i n h i s v i e w a d e s i g n a t i o n o f i n e v i t a b l e d e a t h . " P s e u d o -h y p e r t r o p h i c p a r a l y s i s " was i n o p p o s i t i o n yet complimentary to " m y o s c l e r o t i c p a r a l y s i s " which denoted the p r o l i f e r a t i o n of the c o n n e c t i v e t i s s u e . " P s e u d o h y p e r t r o p h i c p a r a l y s i s " became the t i t l e of Duchenne's d i s e a s e p o s s i b l y because i t r e f l e c t e d t h a t seen by the unaided r a t h e r than the a i d e d eye. I n i t i a l l y , Duchenne c o u l d not comprehend the weakness of the muscle when he l e a r n e d t h a t the nerve centres were f r e e o f p a t h o l o g y i n p s e u d o h y p e r t r o p h i c p a r a l y s i s . G r i e s i n g e r conceived of a c e r t a i n n e u r a l c o n t r i b u t i o n to the d i s e a s e a c c o r d i n g to t h e i n v o l u n t a r y spasms, which a r e 12 q u e s t i o n a b l e , but which the s u b j e c t of h i s biopsy a p p a r e n t l y e x p e r i e n c e d a few y e a r s p r i o r t o t h e i r meeting. The rose c o l o u r e d hue of the s k i n c o v e r i n g h y p e r t r o p h i e d muscle i n the boy was a l s o thought to be a p o s s i b l e m a n i f e s t a t i o n of sympathetic nerves which a l t e r e d blood f l o w . G r i e s i n g e r was d e t e r m i n e d not t o t a k e s i d e s as r e g a r d s the i n e v i t a b l e q u e s t i o n concerning the myogenic versus neurogenic b a s i s of the d i s o r d e r ; however, he l i s t e d towards the myogenic view i n the end. Duchenne f i n a l l y conceded th a t the f e e b l e n e s s of movement was caused by the i n v a d i n g c o n n e c t i v e t i s s u e w h i c h w o u l d s e p a r a t e and damage t h e m u s c l e , t h e r e b y d e s t r o y i n g i t s a b i l i t y t o c o n t r a c t . The e s s e n t i a l l e s i o n was t h e r e f o r e a s c r i b e d to the c o n n e c t i v e t i s s u e but what caused i t s p r o l i f e r a t i o n Duchenne was unable to say. The l a c k of changes to the s p i n a l motor c e l l s was a constant and the i n t a c t s e n s i t i v i t y to touch and c o n t r o l of the s p h i n c t e r s were a l s o c o n s i s t e n t w i t h a non-nervous o r i g i n . The absence of a primary l e s i o n of the s p i n a l motor neurons was confirmed i n Duchenne's twenty-eighth c a s e , who was m e n t a l l y d e f i c i e n t , "though not an i d i o t " . Gowers, ( 1 8 8 6 ) who g a v e t h e f i r s t E n g l i s h a c c o u n t o f pseudohypertrophic p a r a l y s i s i n 1879, found mental d u l l n e s s i n some but the m a j o r i t y of h i s p a t i e n t s were i n t e l l e c t u a l l y s o u nd and o f t e n a d v a n c e d o f u n a f f l i c t e d i n d i v i d u a l s . T h e r e f o r e , the c e r e b r a l d e f e c t which sometimes accompanied the m u s c u l a r changes i n p s e u d o h y p e r t r o p h i c p a r a l y s i s was 13 d i s m i s s e d as a " c o m p l i c a t i o n " . C o n t r a r y t o G r i e s i n g e r , D u c h enne b e l i e v e d t h a t p s e u d o h y p e r t r o p h i c p a r a l y s i s was a v e r y common d i s o r d e r w h i c h a p p e a r e d t o be more p r e v a l e n t i n males t h a n i n f e m a l e s . A c c o r d i n g t o Gowers (1886) the male to female r a t i o was seven to one and the f a c t t h a t males f u r n i s h e d most of the cases seemed to be r e l a t e d to a e t i o l o g y . Both the male p r e p o n d e r a n c e and the f a m i l i a l tendency o f the d i s e a s e are w e l l - i l l u s t r a t e d by Meryon's (1852) c a s e , a l l of whose b r o t h e r s were a f f l i c t e d w h i l e the s i x g i r l s o f the f a m i l y were q u i t e h e a l t h y . Duchenne accepted the f a m i l i a l tendency of the d i s e a s e as evidence of i t s h e r e d i t a r y nature but Gowers b e l i e v e d t h a t h e r e d i t y was not a constant i n view of the f a c t t h a t i t was o f t e n c o n f i n e d to one g e n e r a t i o n . D o c t o r P o o r e , who gave the f i r s t E n g l i s h t r a n s l a t i o n o f Duchenne's works i n 1883, p r e d i c t e d t h a t one day t h e embryologist would s o l v e the mystery of the d i s e a s e . Progressive Muscular Atrophy " P r o g r e s s i v e m u s c u l a r a t r o p h y " was i n t e n d e d t o d e s i g n a t e t h e e v o l u t i o n o f a d i s e a s e d e s c r i b e d by Aran ( 1 8 5 0 ) , who t h o u g h t t h a t i t was c l o s e l y a l l i e d t o t h e " u s u a l " atrophy consequent t o a n e u r a l or v a s c u l a r l e s i o n but was d i f f e r e n t i n a number of ways. The musculature of a whole member o r l i m b was p a r a l y z e d i n t h e " o r d i n a r y " p a r a l y s i s whereas i n t h i s new c o n d i t i o n some muscles were 14 attacked, others spared. As a pioneer of "localised electrisation", which tested the contractile response of muscle to the application of electric current, Duchenne collaborated in a number of Aran's cases by verifying the identity of the particular muscles involved according to this procedure. In the eight examples of the disease described by Aran proximal muscles of the upper extremities were commonly affected in those whose occupations demanded that they l i f t heavy things. Muscles distant from the shoulder girdle were affected in some while the hands alone were affected in the only woman of the group. The general health of a l l subjects was good and while muscular weakness and wasting proceeded along a limb, sensitivity to touch remained unaffected. Some did, however, show fasiculations or involuntary contractions which occurred with greater frequency in those suffering from general atrophy involving the whole of the muscular system and therefore the lower limbs. Postmortem examination revealed that the muscle had been transformed into a "greasy texture" which seems to equate with the "fatty and granular degeneration" in Meryon's case. In any event, the pathology of the muscle did not resemble that of ordinary atrophy in which fat was deposited exterior to the muscle fibre that had maintained the integrity of the sarcolemma. The autopsy of a young man in his late teens disclosed no pathology within the nervous 15 system and t h e r e f o r e Aran b e l i e v e d t h a t p r o g r e s s i v e muscular a t r o p h y was the r e s u l t o f a d i s e a s e p r o c e s s w h i c h was indigenous to the muscle. O r i g i n a l l y , some thought t h a t the p s e u d o h y p e r t r o p h i c p a r a l y s i s was a s p e c i a l v a r i a n t o f p r o g r e s s i v e m u s c u l a r a t r o p h y because the muscular impotence of one approximated the o t h e r . Controversy concerning the nature of p r o g r e s s i v e muscular atrophy f o l l o w e d i t s i n t r o d u c t i o n i n t o the world of medical s c i e n c e i n p a r t because of c o n f l i c t i n g p a t h o l o g i c a l r e p o r t s regarding the nervous system. A l s o , many were not w i l l i n g to accept the s e p a r a t i o n between the muscle and i t s animator when at times no p a t h o l o g i c a l changes were t o be found i n the b r a i n or s p i n a l c o r d . The lack of d e g e n e r a t i v e c h a n g e s i n t h e a n t e r i o r c o r n u a o f p s e u d o h y p e r t r o p h i c p a r a l y s i s u n d o u b t e d l y p e r s u a d e d Duchenne t o adopt t h e m y o g e n i c v i e w o f A r a n c o n c e r n i n g p r o g r e s s i v e m u s c u l a r a t r o p h y . F r e i d r e i c h b e l i e v e d that the s p i n a l changes that had been r e p o r t e d f o r the d i s o r d e r were s e c o n d a r y t o a d i s e a s e p r o c e s s w h i c h was p r i m a r y t o the m u s c l e . When succeeding r e p o r t s c o n s i s t e n t l y demonstrated a s p i n a l l e s i o n the view of C h a r c o t , who h e l d the c h a i r of n e u r o l o g y i n France (1877), which was that the muscular changes were the d i r e c t r e s u l t o f t h e s p i n a l changes became the a c c e p t e d v i e w . As a r e s u l t " s p i n a l muscular a t r o p h y " became the a l t e r n a t e t i t l e . 16 P r o g r e s s i v e M u s c u l a r D y s t r o p h y For a p e r i o d of time a f t e r the consensus as regards the nature of p r o g r e s s i v e muscular atrophy had been reached, a l l f o r m s o f m u s c u l a r a t r o p h y were b e l i e v e d t o be t h e consequence o f a s p i n a l l e s i o n by v i r t u e o f the n e r v e ' s t r o p h i c power. However, when Leyden (1879), Erb (1884) and, Landouzy and Dejerne (1884) documented muscular a t r o p h i e s of a p r o g r e s s i v e nature f o r which pathology was c o n f i n e d to the s k e l e t a l muscle, Charcot was l e f t with no other c h o i c e but t o d i v i d e m u s c u l a r d i s e a s e i n t o the "Great C l a s s e s " of s p i n a l and n o n s p i n a l o r p r i m a r y and s e c o n d a r y . I n a c c o r d a n c e w i t h t h e a p p a r e n t n o n - n e r v o u s o r i g i n o f p s e u d o h y p e r t r o p h i c p a r a l y s i s i t could not be excluded from Charcot's "Great C l a s s " of primary muscular d i s e a s e . G o w e r s ( 1 8 8 6 ) o r i g i n a l l y t h o u g h t t h a t p s e u d o -h y p e r t r o p h i c p a r a l y s i s was an e n t i t y unto i t s own and was t h e r e f o r e c o m p l e t e l y d i s t i n c t from t h e o t h e r forms of primary muscular d i s e a s e . Atrophy p r e s i d e d over hypertrophy i n E r b ' s " j u v e n i l e " f o r m , the onset of which was always before twenty y e a r s , but the c a l v e s r e t a i n e d t h e i r g i r t h and there was at times hypertrophy of the shoulder as there was i n p s e u d o h y p e r t r o p h i c p a r a l y s i s . T h e r e were d e f i n i t e a n a l o g i e s between the d i s e a s e s of Erb and Duchenne as there were between each and the form d e s c r i b e d by Leyden (1879) w h i c h i n v o l v e d an a t r o p h i c a s c e n d i n g p a r a l y s i s o f t h e p r o x i m a l m u s c u l a t u r e . C h a r c o t thought i n i t i a l l y t h a t the 17 involvement of the zygomatic musculature of the face i n the " f a c i o s c a p u l o h u m e r a l " form of Landouzy and Dejerne (1884) was u n i q u e , so as t o s e t i t a p a r t from the r e s t . A f t e r f u r t h e r c o n s i d e r a t i o n he d e c i d e d t h a t i f the f a c e was d i s r e g a r d e d i t was much l i k e Erb's j u v e n i l e form and at the same t i m e t h e r e was o v e r l a p w i t h p s e u d o h y p e r t r o p h i c p a r a l y s i s . The i n d i v i d u a l forms of " i d i o p a t h i c " muscular di s e a s e d e s c r i b e d by E r b , Leyden, Landouzy and Dejerne, and Duchenne were d i s t i n c t yet there were common f e a t u r e s among them, the involvement of the proximal musculature being the most i m p o r t a n t . I n d i v i d u a l c a s e s were a l s o o f t e n " i n t r a n s i t i o n " between two of the types which e f f e c t i v e l y f used the c l i n i c a l v a r i e t i e s to make them seemingly i n s e p a r a b l e . More i m p o r t a n t l y , Erb (1895) forbade t h e i r s e p a r a t i o n a c c o r d i n g t o t h e h o m o g e n e o u s n a t u r e o f t h e i r h i s t o p a t h o l o g i e s . They were considered to be a f a m i l y , not of d i s e a s e s , but o f one d i s e a s e . S i n c e t h e i r e s s e n t i a l p a t h o l o g i e s were s i m i l a r i t was assumed t h a t the same m o r i b u n d p r o c e s s gave r i s e t o t h e d i f f e r e n t c l i n i c a l e n t i t i e s . E r b c a l l e d the d i s e a s e " p r o g r e s s i v e m u s c u l a r d y s t r o p h y " , t h e f o u n d a t i o n o f t h e name b e i n g t h e m u l t i p l i c a t i o n of the co n n e c t i v e t i s s u e , which he determined through h i s study of the h i s t o p a t h o l o g y , to concur with the eventual disappearance of the muscle f i b r e s . There appeared to be a s t r u g g l e f o r s u r v i v a l , a " d i f f i c u l t n u t r i t i o n " or dystrophy. Although the name u n i f i e d the group of c l i n i c a l 18 v a r i e t i e s , c l a s s i f i c a t i o n was c o n v e n i e n t and was o f d i a g n o s t i c s i g n i f i c a n c e . The most popular c l a s s i f i c a t i o n scheme used i n f u t u r e years was very simply based on the c l a s s i c a l d e s c r i p t i o n s of the vari o u s types and t h e r e f o r e on the c l i n i c a l syndromes: 1) pseudohypertrophic (Duchenne) 2) facioscapulohumeral (Landouzy and Dejerne) 3) j u v e n i l e (Erb) For some reason the form d e s c r i b e d by Leyden d i d not p e r s i s t but these three were always members of any c l i n i c a l l y based c l a s s i f i c a t i o n scheme. In keeping with Duchenne's o r i g i n a l c o n c l u s i o n , the pseudohypertrophic type was a very common d i s o r d e r . Because i t was the most common type of muscular d y s t r o p h y , f u t u r e study r e g a r d i n g the pa t h o g e n e s i s o f the d i s e a s e , employed subjects with the pseudohypertrophic form more o f t e n than not. Unlik e the pseudohypertrophic v a r i e t y , both sexes were a f f e c t e d without bi a s i n the other r a r e r forms of muscular dystrophy. Aside from the females who manifest d i s e a s e with slower course and l a t e r o n s e t , pseudohypertrophic muscular dystrophy was one of c h i l d h o o d while the age of onset was very v a r i a b l e i n the remaining forms, the range being two to s i x t y y e a r s . For t h i s r e a s o n Erb (1895) a t t e m p t e d t o c l a s s i f y the d i s e a s e a c c o r d i n g to age of o n s e t . D e s p i t e these d i f f e r e n c e s i n sexual p r o c l i v i t y and age of on s e t , the his t o p a t h o l o g y of the muscle which was undeprived of n e u r a l 19 i n f l u e n c e and the f a m i l i a l tendency of a l l were the common d e n o m i n a t o r s . The r e m a r k a b l e p r o p e n s i t y o f m u s c u l a r dystrophy to a f f e c t f a m i l i e s , a p r o p e n s i t y which was r a r e l y r e s t r i c t e d to one g e n e r a t i o n i n the l e s s common forms of the d i s e a s e , was immediately recognized as being of a e t i o l o g i c a l s i g n i f i c a n c e . However, because the pseudohypertrophic type o c c u r r e d i n f a m i l i e s with no previous h i s t o r y of the d i s e a s e h e r i t a b i l i t y was r u l e d o u t . Mendel had by t h a t t i m e c h a l l e n g e d the view of i n h e r i t a n c e which was b e l i e v e d to be a "blending" of elements contained i n the sperm and ovum but h i s theory of " p a r t i c u l a t e " i n h e r i t a n c e would remain on the pages o f h i s m a n u s c r i p t u n t i l a f t e r t h e dawning o f t h e t w e n t i e t h c e n t u r y . T h e r e f o r e , when muscular dystrophy was d e f i n e d i n terms o f t h e h i s t o l o g i c a l changes o f m u s c l e u n a s s o c i a t e d with any changes i n the s p i n a l c o r d , g e n e t i c s was not yet a s c i e n c e . A " c o n g e n i t a l tendency" which c o u l d m a n i f e s t i t s e l f at any time of l i f e was the only d e f i n i t e l i n k to the cause of the d i s e a s e . Gowers (1902) coined the term a b i o t r o p h y to denote an i m p e r f e c t v i t a l endurance of t i s s u e s which caused t h e i r n u t r i t i o n to f a i l , muscular dystrophy, p r o g r e s s i v e muscular a t r o p h y , and premature baldness being i l l u s t r a t i o n s of such a d e f e c t i v e n u t r i t i o n . He c o n s i d e r e d a b i o t r o p h y to be a more c o m p l e t e d e s c r i p t i o n o f the m u s c u l a r malady t h a n d y s t r o p h y . I t appeared t o be, as Meryon (1852) suggested f o r the pseudohypertrophic type s p e c i f i c a l l y , the r e s u l t of 20 a d e f e c t i v e n u t r i t i o n r a t h e r than j u s t a d i f f i c u l t one i n which the muscle was l a c k i n g i n an i n h e r e n t h a r d i n e s s and c o u l d t h e r e f o r e not e n d u r e . W h i l s t a b i o t r o p h y was an acceptable and convenient term and was used with respect to the muscular malady i n f u t u r e , muscular d y s t r o p h y was t o endure as the t i t l e of the d i s e a s e . Myopathy ve r s u s Neuropathy Charcot f u r t h e r s u b d i v i d e d h i s "Great C l a s s " of s p i n a l muscular d i s e a s e i n t o that which was due to a primary l e s i o n of the s p i n a l motor neurons and that a r i s i n g from a l e s i o n which was s e c o n d a r y . "Amyotrophic l a t e r a l s c l e r o s i s " was the t i t l e g i v e n to the l a t t e r w h i l e " p r o g r e s s i v e muscular atrophy" was r e s e r v e d f o r the former, but they were o f t e n i n d i s t i n g u i s h a b l e i n the event of the d i a g n o s i s of a l i v i n g i n d i v i d u a l . L i k e w i s e , d i f f e r e n t i a t i o n between neurogenic and myogenic types of muscular d i s e a s e was o f t e n d i f f i c u l t i n t h a t s e n s i t i v i t y t o t o u c h was i n t a c t i n b o t h w h i l e p r e s e n t i n g s i g n s o f p r o g r e s s i v e m u s c u l a r weakness and w a s t i n g . T h e c o n f l i c t i n g r e p o r t s f r o m p o s t m o r t e m e x a m i n a t i o n o f those d i a g n o s e d w i t h p r o g r e s s i v e muscular atrophy were then very l i k e l y the r e s u l t of i n d i s t i n c t i o n and m i s d i a g n o s i s . The o r i g i n a l r e p o r t of Aran i n 1850 i s a c t u a l l y c o n s i d e r e d to be an e a r l y c i t a t i o n o f m u s c u l a r d y s t r o p h y , b u t I s u g g e s t t h a t h i s s u b j e c t s were a c o m b i n a t i o n o f n e u r o g e n i c and myogenic t y p e s , those who 21 e x h i b i t e d f a s i c u l a t i o n s and i n v o l u n t a r y spasms b e i n g a f f l i c t e d by the neurogenic t y p e . D i f f e r e n t i a l d i a g n o s i s c o u l d be accomplished a c c o r d i n g to the presence of f a s i c u l a t i o n s and/or the excess of the knee jerk which were the s i g n s of a neural l e s i o n , but these signs were not always present i n neuropathy. As a r e s u l t , other d i a g n o s t i c i n d i c a t i o n s were sought. The a f f l i c t i o n of more than one f a m i l y member was e v e n t u a l l y c o n s i d e r e d to be one of the most important noninvasive d i a g n o s t i c i n d i c a t i o n s of m u s cular d y s t r o p h y or myopathy. However, i t was the h i s t o l o g i c a l f e a t u r e s o f t h e muscle which i n the f i n a l a n a l y s i s brought a d i v i s i o n between myopathy and neuropathy p r i o r to d e a t h . A l t h o u g h p o s t mortem e x a m i n a t i o n o f i n d i v i d u a l s d e s c r i b e d by Aran r e v e a l e d a homogeneous p a t h o l o g y , i t i s s t i l l p o s s i b l e t h a t some were s u f f e r i n g from n e u r o g e n i c atrophy. The h i s t o l o g y of the muscle i n the t e r m i n a l stages of d y s t r o p h y and c h r o n i c d e n e r v a t i n g d i s e a s e i s q u i t e i n d i s t i n c t . With the a d v e n t o f the m u s c l e b i o p s y t h e h i s t o p a t h o l o g i e s of the two c o u l d be s t u d i e d and compared as the d i s e a s e progressed which i s p r e c i s e l y what Erb d i d . In doing so he d i s c o v e r e d that a specimen of d y s t r o p h i c m u s c l e p r e s e n t e d a v a r i a t i o n i n f i b r e s i z e s i n c l u d i n g s m a l l e r t h a n n o r m a l , n o r m a l and sometimes l a r g e r t h a n n o r m a l . H y p e r v o l u m i n o u s f i b r e s were f o u n d i n t h e ps e u d o h y p e r t r o p h i c and i n Erb's j u v e n i l e type and i n order 22 t o s i m p l i f y c l a s s i f i c a t i o n E r b s u g g e s t e d t h a t m uscular d y s t r o p h y be d i v i d e d i n t o two g r o u p s : H y p e r t r o p h i c , i n which f i b r e s were sometimes hypervoluminous i n the e a r l i e r stages and a t r o p h i c which would be s u b d i v i d e d i n t o with and without f a c i a l involvement. Regardless of the volume of the i n d i v i d u a l f i b r e i t was r o u n d e d i n s h a p e , n u c l e i had m i g r a t e d t o w a r d s t h e c e n t r e f r o m t h e p e r i p h e r y , t h e c o n n e c t i v e t i s s u e had p r o l i f e r a t e d and f i b r e s were o f t e n s p l i t . The f i b r e s of neurogenic d i s e a s e l o s t volume while r e t a i n i n g t h e i r shape and t h e i r c o n t a c t w i t h n e i g h b o u r i n g f i b r e s , thereby m a i ntaining a mosaic. Degenerative changes were d i s c o v e r e d t o be a f e a t u r e of the l a t e s t a g e s and t h e r e f o r e , t h e r e was not the ongoing r e d u c t i o n i n numbers as t h e r e was i n p r o g r e s s i v e m u s c u l a r d y s t r o p h y i n w h i c h d e g e n e r a t i v e changes were a s a l i e n t f e a t u r e . The muscle b i o p s y became the major means of d i f f e r e n t i a t i n g between n e u r o p a t h y and myopathy and was t h e r e f o r e paramount t o d i a g n o s i s o f m u s c u l a r d y s t r o p h y , e s p e c i a l l y when an i n d i v i d u a l case d i d not conform to any p a r t i c u l a r c l i n i c a l syndrome or e x h i b i t any symptoms of a n e u r a l l e s i o n . Gowers (1902) e n c o u n t e r e d a c o n d i t i o n , w h i c h i n c o n t r a s t to the syndromes which formed the b a s i s of muscular dystrophy i n v o l v e d the d i s t a l musculature. Nonetheless, he b e l i e v e d i t t o be a form of the d i s e a s e i n view of the l a c k o f any symptoms a t t r i b u t a b l e t o a n e u r a l l e s i o n and a d y s t r o p h i c h i s t o l o g y . For the same r e a s o n , B a t t e n (1909) 23 i n s i s t e d t h a t amyotonia congenita and myotonia a t r o p h i c a be i n c l u d e d among the types of muscular d y s t r o p h y . Myotonia a t r o p h i c a e v e n t u a l l y became known as my o t o n i c d y s t r o p h y a c c o r d i n g t o the d y s t r o p h i c h i s t o l o g y of muscle which was a p p a r e n t l y undeprived of n e u r a l i n f l u e n c e but the myotonia would d i s t i n g u i s h i t from the "pure" d y s t r o p h i e s . L i k e w i s e , the f l a c c i d i t y of the muscle i n amyotonia congenita which, as the name i n d i c a t e s i s present at b i r t h , d i d not apply t o " t r u e " muscular dystrophy. Myopathy and muscular dystrophy were o r i g i n a l l y e q u i v a l e n t and, although myopathy became a more i n c l u s i v e term over time, the h i s t o l o g i c a l f e a t u r e s of d y s t r o p h i c muscle as d i s c o v e r e d by Erb became the f e a t u r e s which were con s i d e r e d to be d i a g n o s t i c of myopathy. The T r o p h i c T h e o r y o f E r b The e l e c t r i c i r r i t a b i l i t y of muscle was a l s o d i s c o v e r e d t o p r o v i d e a means f o r d i s c e r n i n g between myopathy and neuropathy. I r r i t a b i l i t y i n general was p r o p o r t i o n a l to the degree of wasting i n both, but the responses to g a l v a n i c and a l t e r n a t i n g c u r r e n t d i f f e r e d . The response of d y s t r o p h i c muscle to g a l v a n i c c u r r e n t remained equal to the response t o a l t e r n a t i n g c u r r e n t a t a l l s t a g e s o f t h e d i s e a s e . In c h r o n i c neurogenic atrophy there was an i n c r e a s i n g l y s m a l l e r r e s p o n s e t o a l t e r n a t i n g c u r r e n t w i t h the r e t e n t i o n and sometimes i n c r e a s e d r e s p o n s e t o g a l v a n i c c u r r e n t . E r b b e l i e v e d t h a t t h e d i f f e r e n t i a l r e s p o n s e s o f c h r o n i c 24 denervating d i s e a s e to a l t e r n a t i n g and g a l v a n i c c u r r e n t was p o s s i b l e evidence that the s k e l e t a l muscle r e c e i v e d another t y p e o f i n n e r v a t i o n w h i c h s i m p l y s u p p l i e d a t r o p h i c i n f l u e n c e t o t h e m u s c l e and was not i n v o l v e d i n i t s a c t i v a t i o n . Even a f t e r s t u d y i n g the h i s t o l o g y of d y s t r o p h i c muscle and d i f f e r e n t i a t i n g i t from t h a t of neuropathy Erb s t i l l had h i s r e s e r v a t i o n s a b o u t the i n d e p e n d e n c e o f muscular dystrophy from nervous i n f l u e n c e : "I cannot a v o i d the s u s p i c i o n that a f t e r a l l the a f f e c t i o n may be dependent upon the nervous system. I t i s tempting to suppose, as I f o r m e r l y e x p r e s s e d i t , t h a t we have to do w i t h a k i n d of t r o p h o - n e u r o s i s having i t s o r i g i n i n the t r o p i c c e n t r e s of the cord — a d i s t u r b a n c e of the f u n c t i o n of these c e n t r e s which f i n d s i t s e x p r e s s i o n i n the very c o m p l i c a t e d muscle changes of the d i s e a s e . A f u r t h e r r i c h e x p e r i e n c e , much thought expended upon the q u e s t i o n , and the above d e s c r i b e d anatomic f i n d i n g s (he r e f e r s t o myopathic changes i n the muscles observed i n p o l i o m y e l i t i s i n c l u d i n g h y p e r t r o p h i e d f i b r e s , f i b r e s p l i t t i n g and c e n t r a l n u c l e a t i o n ) have served to confirm me i n my s c r u p l e s . "1 Such a t h e o r y which proposed th a t muscular d y s t r o p h y was t h e r e s u l t o f t h e m i s a p p r o p r i a t i o n o f a t r o p h i c i n f l u e n c e was e v e n t u a l l y abandoned because t h e r e was no a n a t o m i c a l e v i d e n c e t o s u p p o r t a s p e c i a l t r o p h i c c e n t r e Quoted by Bramwell (1925). 25 a p a r t f r o m the s o m a t i c n e r v e s . C h a r c o t d e s i g n a t e d t h e s o m a t i c n e r v e s as t h e s e c o n d a r y t r o p h i c u n i t s o f t h e s k e l e t a l m u s c l e , the p r i m a r y c e n t r e s b e i n g s u p r a s p i n a l . Although the way i n which the nerve a c t i v a t e d the muscle was unknown Gowers s u g g e s t e d t h a t the s t r u c t u r a l c o n t i n u i t y b e t w e e n t h e two i n v o l v e d some s o r t o f " m o l e c u l a r i n t e r a c t i o n " which s e r v e d to n o u r i s h both the muscle and c o n n e c t i v e t i s s u e . In the same way that the p r o l i f e r a t i o n of the g l i a or the i n t e r s t i t i a l t i s s u e of the nervous system was i n i t i a l l y b e l i e v e d t o be t h e p r i m a r y e v e n t i n a m y o t r o p h i c l a t e r a l s c l e r o s i s , t h e a b u n d a n c e o f t h e c o n n e c t i v e t i s s u e i n muscular dystrophy d i d not escape the c u r s o r y assignment of the e s s e n t i a l l e s i o n . However, the degree o f c o n n e c t i v e t i s s u e p r o l i f e r a t i o n v a r i e d i n t h e d i f f e r e n t types of the d i s e a s e , a pronounced degree being a t t a i n e d i n t h e p s e u d o h y p e r t r o p h i c f o r m , moderate and i n c o n s p i c u o u s d e g r e e s b e i n g s e e n i n t h e o t h e r f o r m s . Regardless of the degree of connective t i s s u e p r o l i f e r a t i o n or the i n i t i a l changes i n the s i z e o f the muscle f i b r e , d e g e n e r a t i o n was the f a t e of a l l . Thus the d i s e a s e was c o n s i d e r e d to be the r e s u l t of a " q u a l i t a t i v e " d e f e c t of the muscle which caused i t to p e r i s h prematurely and c o n n e c t i v e t i s s u e m u l t i p l i e d i n response to the d e g e n e r a t i o n of the muscle f i b r e . In summary, muscular dystrophy was d e f i n e d as a d i s e a s e which i n v o l v e d the p r o g r e s s i v e degeneration of the s k e l e t a l 26 muscle to g i v e a t y p i c a l h i s t o l o g i c a l p i c t u r e but with no concomitant pathology t o the s p i n a l motor neurons. I t thus p r e s e n t e d a h i s t o l o g i c a l p i c t u r e which was d i s t i n g u i s h a b l e f r o m m u s c u l a r d i s e a s e c o n s e q u e n t t o a n e u r a l l e s i o n . A l t h o u g h a v a r i e t y o f c l i n i c a l s y n d r o m e s , t h e p s e u d o h y p e r t r o p h i c t y p e b e i n g by f a r the most common, conformed to t h i s d e f i n i t i o n , they were c o n s i d e r e d to be v a r i a n t s of the same d i s e a s e process and t h e r e f o r e muscular d y s t r o p h y was a p r o t e a n d i s e a s e . " P r o g r e s s i v e m u s c u l a r d y s t r o p h y " was then a g e n e r a l d e s i g n a t i o n f o r a group of c l i n i c a l syndromes b u t b e c a u s e p a r t i c u l a r c a s e s were sometimes i n d i s t i n c t , the absence of symptoms i n d i c a t i n g a n e u r a l l e s i o n and a d y s t r o p h i c h i s t o l o g y were s u f f i c i e n t f o r the d i a g n o s i s of muscular dystrophy. On the whole there was a b i a s t o w a r d s t h e m a l e s i m p l y b e c a u s e t h e r e was a p r e d i l e c t i o n f o r the male i n the pseudohypertrophic type but f a m i l i a l t e n d e n c y was the most s a l i e n t and c o n s i s t e n t f e a t u r e and was the o n l y known c o n n e c t i o n to the cause of t h e d i s e a s e . I m m e d i a t e f u t u r e s t u d y p e r t a i n i n g t o pathogenesis and a e t i o l o g y , while o f t e n employing those w i t h the p s e u d o h y p e r t r o p h i c type as s u b j e c t s f o r s t u d y , d i d not d i s c r i m i n a t e between the c l i n i c a l v a r i e t i e s with r e s p e c t to r e s u l t s . PART I I MUSCULAR DYSTROPHY AND MUSCLE METABOLISM 27 A f t e r the n e u r o l o g i s t and p a t h o l o g i s t had p r o v i d e d a q u a l i t a t i v e d e f i n i t i o n of muscular d y s t r o p h y d u r i n g the n i n e t e e n t h c e n t u r y , the approach to the study of the d i s e a s e became one with a p h y s i o l o g i c a l - c h e m i c a l p o i n t of view. As a r e s u l t , r e s e a r c h concerning muscular dystrophy c o n t r i b u t e d to the knowledge and understanding of muscle metabolism, i n p a r t i c u l a r , with r e s p e c t to c r e a t i n e and c r e a t i n i n e . The f i r s t chapter of P a r t II deals with c r e a t i n e and c r e a t i n i n e g e n e r a l l y , t h e i r r e l a t i o n s h i p to the metabolism of muscle and t h e i r a b n o r m a l i t i e s i n muscular dystr o p h y which gave r i s e to t h e o r i e s concerning p a t h o g e n e s i s . The remainder of Part II d e a l s with muscular dystrophy per s e . Although the p h y s i o l o g i c a l - c h e m i c a l approach was predominant i n what I have c a l l e d the "metabolic phase of muscular dy s t r o p h y " the a n a t o m i c a l - p a t h o l o g i c a l a s p e c t s of the d i s e a s e were not ignored i n t o t a l i t y . Such an approach was i n v o l v e d i n the p r o p o s a l of a t r o p h i c theory b r i d g i n g t h a t of Erb and McComas. Aside from t h i s p a r t i c u l a r t h e o r y , most of which i s presented i n Part II i s i m p e r t i n e n t to the q u e s t i o n posed at the outset of t h i s paper. I t i s presented as a matter of i n t e r e s t and i t i s hoped that i t w i l l g i v e the reader a g r e a t e r knowledge of the h i s t o r y of muscular d y s t r o p h y and t h e p h y s i o l o g i c a l r e s e a r c h i n v o l v e d i n a t t e m p t i n g t o u n d e r s t a n d t h e d i s e a s e . I t i s a l s o d e m o n s t r a t i v e o f t h e o n g o i n g d i f f e r e n t i a t i o n between 28 myopathy and n e u r o p a t h y w i t h i n t h e c o n t e x t o f m u s c l e metabolism. C r e a t i n e a n d C r e a t i n i n e C r e a t i n e i s a b i o l o g i c a l molecule which i s s y n t h e s i z e d by the l i v e r , kidney and pancreas a c c o r d i n g to the f o l l o w i n g s y n t h e t i c pathway: A r g i n i n e + g l y c i n e •* guanidine a c e t i c a c i d then, guanidine a c e t i c a c i d + methionine -*• c r e a t i n e S i m p l y s a i d , t h e s y n t h e s i s o f c r e a t i n e i n v o l v e s t h e a m i d i n a t i o n of g l y c i n e at the expense of a r g i n i n e f o l l o w e d by the m e t h y l a t i o n of guanidine a c e t i c a c i d at the expense o f methionine. C r e a t i n e i s r e l e a s e d i n t o the blood from i t s p l a c e of f o r m a t i o n and conveyed to the s k e l e t a l muscle, the heart and b r a i n . Both membrane t r a n s p o r t and i n t r a c e l l u l a r t r a p p i n g a r e i n v o l v e d i n the uptake of c r e a t i n e by the s k e l e t a l muscle ( F i t c h et a l . , 1968) which c o n t a i n s 98% of the t o t a l body c r e a t i n e , and i n which the c o n c e n t r a t i o n of c r e a t i n e i s a b o u t t w i c e t h a t i n the c a r d i a c muscle and b r a i n . Once c r e a t i n e has entered the c e l l s of the v a r i o u s t i s s u e s i t i s phosphorylated to become phosphocreatine which a c t s as a phosphate donor i n the r e a c t i o n : phosphocreatine + ADP -»• ATP c a t a l y z e d by the enzyme, c r e a t i n e phosphokinase. Phosphocreatine t h e r e f o r e serves t o m a intain the ATP supply w h i c h i s a b s o l u t e l y e s s e n t i a l f o r the c o n t r a c t i o n and 29 r e l a x a t i o n of the muscle. In the b r a i n , and elsewhere, i t f u n c t i o n s i n the maintenance of membrane p o t e n t i a l . A f t e r d o n a t i n g of the phosphate c r e a t i n e i s converted no n e n z y m a t i c a l l y to c r e a t i n i n e which d i f f u s e s from the c e l l and i s e x c r e t e d . Because most u r i n a r y c r e a t i n i n e o r i g i n a t e s i n the s k e l e t a l muscle, output r e f l e c t s the f u n c t i o n a l mass of the m u s c l e . Output thus g r a d u a l l y d e c l i n e s w i t h the p r o g r e s s i v e muscular d e t e r i o r a t i o n i n muscular d y s t r o p h y . The e x c r e t i o n of c r e a t i n e , or c r e a t i n u r i a , i s a l s o a f e a t u r e of d y s t r o p h y and i s c o n s i d e r e d t o be a c o n s e q u e n c e o f i n e f f e c t i v e i n t r a c e l l u l a r t r a p p i n g r e s u l t i n g from muscular d e g e n e r a t i o n . T h e r e f o r e , the magnitude of c r e a t i n u r i a a l s o i n c r e a s e s with p r o g r e s s i o n of the d i s e a s e . The O r i g i n of U r i n a r y C r e a t i n i n e By the end of the 19th c e n t u r y c r e a t i n i n e had become known as an end product of mammalian metabolism, a r e g u l a r c o n s t i t u e n t o f u r i n e l i k e t h e b e t t e r known n i t r o g e n c o n t a i n i n g s u b s t a n c e , u r e a . A r e l a t i o n s h i p between c r e a t i n i n e o u t p u t and body w e i g h t was n o t e d , a g r e a t e r output c o i n c i d i n g with a g r e a t e r weight. A lowered output was documented i n cases of p r o g r e s s i v e muscular atrophy and a d i m i n i s h e d o u t p u t was r e p o r t e d i n an advanced case of p s e u d o h y p e r t r o p h i c muscular d y s t r o p h y i n 1877 w i t h a l e s s r a d i c a l l y d i m i n i s h e d output i n a m i l d e r c a s e . W h i l s t the values f o r c r e a t i n i n e output i n these cases were undoubtedly 30 subnormal, they were based on the rather i n a c c u r a t e method of p r e c i p i t a t i o n by z i n c c h l o r i d e which e n t a i l e d a number of s t e p s . Each s u c c e s s i v e step i n t r o d u c e d an a d d i t i o n a l e r r o r t o g i v e v a l u e s t h a t were u n d e r e s t i m a t i o n s . The procedure was a l s o very time consuming which discouraged the study of c r e a t i n i n e as a f a c t o r of metabolism. C o l l s (1897) spoke of a new and more e f f i c i e n t method u s i n g m e r c u r i c c h l o r i d e to p r e c i p i t a t e c r e a t i n i n e . Such a method again r e q u i r e d a s i m i l a r procedure and i s c e r t a i n to have p r o v i d e d a n a l y s i s with no g r e a t e r degree of accuracy or b r e v i t y . In one o f C o l l s ' e x p e r i m e n t s normal u r i n e was t e s t e d w i t h p i c r i c a c i d which gave c o l o u r a t i o n due to i t s r e a c t i o n w i t h c r e a t i n i n e . T h i s t e s t was a "very d e l i c a t e one f o r c r e a t i n i n e " , but i t was on l y a q u a l i t a t i v e one. At the b e g i n n i n g of the t w e n t i e t h c e n t u r y F o l i n made u s e o f t h e c o l o u r r e a c t i o n b e t w e e n p i c r i c a c i d and c r e a t i n i n e i n order to q u a n t i f y c r e a t i n i n e . He employed the c o l o u r i m e t e r which measures the a b s o r p t i o n of l i g h t by a f l u i d l i k e u r i n e c o l o u r e d by p i c r i c a c i d and c r e a t i n i n e . The amount of l i g h t which passes through w i l l be i n v e r s e l y p r o p o r t i o n a l t o the c o n c e n t r a t i o n of the c o l o u r e d p r o d u c t . I n s t e a d of hours to days needed f o r p r e c i p i t a t i o n by z i n c c h l o r i d e e t c e t e r a an a c c u r a t e c r e a t i n i n e d e t e r m i n a t i o n c o u l d be made i n l e s s than f i f t e e n minutes. F o l i n (1905) i n c l u d e d h i s new method i n the a n a l y s i s of normal u r i n e t o determine c o m p o s i t i o n s c o r r e s p o n d i n g t o a 31 standard d i e t . D i e t s which d e v i a t e d from the standard were used i n a s e p a r a t e i n q u i r y w h i c h , r a t h e r than comparing t o t a l n i t r o g e n i n t a k e of the food with output i n the u r i n e , was devoted to d i s t r i b u t i o n of u r i n a r y n i t r o g e n among u r e a , ammonia, u r i c a c i d and c r e a t i n i n e . U r i n a r y c o m p o s i t i o n corresponding to a p r o t e i n r i c h d i e t was compared with t h a t f o r a p r o t e i n poor one c o n s i s t i n g of cream and arrow r o o t . The purpose of t h i s " s t a r c h and cream" d i e t was to minimize the p r o t e i n m e t a b o l i s m , and with i t s r e d u c t i o n the t o t a l u r i n a r y n i t r o g e n was found to f a l l as much as t h r e e f o l d . The c o n t r i b u t i o n made by urea to the t o t a l n i t r o g e n f e l l c o r r e s p o n d i n g l y , w h i l e t h a t of c r e a t i n i n e r o s e . However, t h e a b s o l u t e q u a n t i t i e s o f c r e a t i n i n e were u n c h a n g e d , r e g a r d l e s s of the d i e t , i r r e s p e c t i v e of the t o t a l amount of ni t r o g e n i n the u r i n e or volume of the u r i n e . The n i t r o g e n of the u r i n e was p a r t i t i o n e d between a v a r i a b l e and a c o n s t a n t component, u r e a and c r e a t i n i n e r e p r e s e n t i n g them, r e s p e c t i v e l y . The laws g o v e r n i n g the amounts and r e l a t i v e amounts of these nitrogenous substances i n u r i n e must s u r e l y , F o l i n thought, r e f l e c t the p r o t e i n c a t a b o l i s m o f t h e human o r g a n i s m . At t h e end of t h e n i n e t e e n t h c e n t u r y t h e r e were two p r e v a i l i n g t h e o r i e s p e r t a i n i n g to p r o t e i n metabolism, one of which h e l d that the c a t a b o l i s m of p r o t e i n to g i v e urea would occur "under the i n f l u e n c e o f the l i v i n g protoplasm" but without u n i t i n g with the l i v i n g protoplasm or the c e l l . T h i s seems to mean that 32 p r o t e i n c a t a b o l i s m o c c u r r e d i n the b l o o d or e x t r a c e l l u l a r f l u i d b a t h i n g the t i s s u e s . The other theory proposed t h a t p r o t e i n t e m p o r a r i l y became p a r t o f t h e c e l l w h i c h was b e l i e v e d to be necessary f o r o x i d a t i o n i n the p r o d u c t i o n of u r e a . The whole i s s u e of metabolism was confused because i t was not c l e a r l y understood that metabolism occurs w i t h i n the c e l l which i s the b u i l d i n g block of t i s s u e . The m i s u n d e r s t a n d i n g i s e v i d e n t i n the new t h e o r y advanced by F o l i n which d i v i d e d p r o t e i n m e t a b o l i s m i n t o endogenous and exogenous or e s s e n t i a l and n o n e s s e n t i a l . The unchanging u r i n a r y c r e a t i n i n e was, a c c o r d i n g to F o l i n , the r e s u l t o f the d a i l y decomposition of the l i v i n g p rotoplasm which was a process i n d i s p e n s a b l e to l i f e and was something w h i c h d i s t i n g u i s h e d l i v i n g f r o m dead c e l l s . F o l i n ' s endogenous or e s s e n t i a l metabolism was a c t u a l l y a s i m p l i f i e d v e r s i o n of the dynamic e q u i l i b r i u m of the c e l l i n t h a t a s i n g l e s u b s t a n c e , namely c r e a t i n i n e , was the end p r o d u c t . Urea, on the other hand, was b e l i e v e d to f l u c t u a t e a c c o r d i n g to changes i n the exogenous metabolism, the source of which was s i m p l y the p r o t e i n i n f o o d . Along with t h i s new theory of p r o t e i n metabolism F o l i n c h a l l e n g e d t h e p o s i t i o n o f p r o t e i n as t h e p r e f e r r e d s u b s t r a t e f o r o x i d a t i o n by the m u s c l e s . The a c t i o n o f muscles r e q u i r e d much oxygen but he noted that the s p l i t t i n g of nitrogenous groups from p r o t e i n d i d not need oxygen. A molecule of water, r a t h e r , would s u f f i c e but such a p r o c e s s 33 d i d not r e l e a s e a great deal of heat which was b e l i e v e d to p r o v i d e the energy f o r muscular c o n t r a c t i o n . U r e a , which c o u l d form from these nitrogenous groups of the p r o t e i n , was a l s o not to be found i n the muscle. I t was reasoned th a t i f the product of the u n l i n k i n g of n i t r o g e n was not to be found and h y d r o l y s i s d i d not p r o v i d e s u f f i c i e n t energy, p r o t e i n c a t a b o l i s m would not be necessary f o r muscular work, nor would i t be e f f i c i e n t . F o l i n suggested that the removal of n i t r o g e n u l t i m a t e l y t o g i v e u r e a was t h e e x o g e n o u s , n o n e s s e n t i a l p r o t e i n m e t a b o l i s m t h a t s e r v e d to make the r e m a i n i n g c a r b o n , o x y g e n and h y d r o g e n a v a i l a b l e f o r o x i d a t i o n , as i n the combustion of carbohydrates and f a t . A c c o r d i n g to F o l i n then, c r e a t i n i n e was not a s s o c i a t e d with any s p e c i f i c t i s s u e , and the f a c t t h a t an obese person e x c r e t e d l e s s than a l e a n i n d i v i d u a l was thought to support the r e l a t i o n s h i p between c r e a t i n i n e and a c t i v e or l i v i n g t i s s u e . The c o l o u r i m e t r i c method brought a sweeping change t o u r i n e a n a l y s i s b u t males m o n o p o l i z e d t h e s t u d y of c r e a t i n i n e as a f a c t o r of metabolism i n i t i a l l y . The female became the focus of Benedict and Myers (1907) f o r the sake of comparison and f o r whom c r e a t i n i n e c o e f f i c i e n t s , which expressed output as a f u n c t i o n of body weight, were f o r the most p a r t lower than f o r men. In keeping with the g r e a t e r p r o p o r t i o n o f i n e r t s u b c u t a n e o u s f a t i n f e m a l e s t h e c o n n e c t i o n between c r e a t i n i n e and " a c t i v e p r o t o p l a s m i c t i s s u e " was a g a i n s u p p o r t e d . However, i t was not t h e 34 p r e s e n c e of adipose t i s s u e which a f f e c t e d the magnitude of c r e a t i n i n e e x c r e t i o n , f o r women with w e l l developed muscular systems gave c r e a t i n i n e outputs comparable to men. I n v i e w o f t h e s w i f t n e s s and s i m p l i c i t y o f t h e c o l o u r i m e t r i c method and t h e i n s i g h t t h a t c r e a t i n i n e e x c r e t i o n i n d i s e a s e might provide to the p h y s i o l o g y o f the p r o c e s s , S p r i g g s (1907a) s e i z e d upon the o p p o r t u n i t y t o examine the p a r t i c u l a r s of c r e a t i n i n e e x c r e t i o n i n a man showing signs of pseudohypertrophic muscular d y s t r o p h y . The p a t i e n t was e v e n t u a l l y p l a c e d on a " s t a r c h and cream d i e t " but whether on i t or an o r d i n a r y d i e t , the q u a n t i t i e s of u r i n a r y c r e a t i n i n e were much below those of one of F o l i n ' s normal u r i n e s from a man approaching the same weight. The p r o p o r t i o n a l i t y between c r e a t i n i n e output and muscular mass, r e c o g n i z e d b e f o r e the advent of the c o l o u r i m e t r i c method, was v e r i f i e d . C r e a t i n i n e appeared to hold a s p e c i a l p l a c e i n the metabolism of muscle. Spriggs b e l i e v e d that " c r e a t i n i n e i s connected w i t h the n u t r i t i o n a l m e t a b o l i s m of the muscle f i b e r and i s not a substance formed i n the act of c o n t r a c t i o n . I f we l i k e n the m u s c l e t o a machine, c r e a t i n i n e as a waste p r o d u c t would s t a n d i n r e l a t i o n to the s t r u c t u r e o f the machine and not the f u e l which the machine uses." S h a f f e r (1908) d i d not commit h i m s e l f t o e i t h e r S p r i g g s or F o l i n , as regards the s p e c i f i c t i s s u e that c r e a t i n i n e was a s s o c i a t e d w i t h , when he s t a t e d t h a t the c r e a t i n i n e producing process was a measure 35 of muscular or g e n e r a l c e l l u l a r e f f i c i e n c y . However, he expected output to vary w i t h muscular a c t i v i t y but he was d i s a p p o i n t e d t o f i n d t h a t output was the same whether h i s s u b j e c t was working or r e c l i n i n g . D i m i n i s h e d output was confirmed i n muscular dystrophy and low c o e f f i c i e n t s were d i s c o v e r e d i n o t h e r c o n d i t i o n s , such as d i a b e t e s m e l l i t u s and e x o p h t h a l m i c g o i t r e which tended to e m a c i a t e . When S h a f f e r c o u l d not r e c o n c i l e the lowered c r e a t i n i n e output of exophthalmic g o i t r e w i t h the i n c r e a s e d endogenous m e t a b o l i c r a t e i n t h i s h y p e r t h y r o i d s t a t e , i n accordance with F o l i n ' s theory of the endogenous metabolism, he g r a v i t a t e d towards the view of S p r i g g s . He p r o p o s e d t h a t c r e a t i n i n e was the p r o d u c t o f a n o r m a l c e l l u l a r p r o c e s s which became s l u g g i s h i n e x o p h t h a l m i c g o i t r e . I f t h i s undefined c e l l u l a r process slowed down i n muscular d y s t r o p h y the muscle might f a l l i n t o a s t a t e of poor n u t r i t i o n and the "machine" then would have a reduced c a p a c i t y to f u n c t i o n . Without a muscle-nerve connection the muscle was s t i l l a sound p i e c e of machinery, but i f the machine i t s e l f was damaged, as i n myopathy, S h a f f e r b e l i e v e d t h a t o n l y then w ould c r e a t i n i n e e x c r e t i o n be l o w e r e d . W i t h t h i s he u n c o n s c i o u s l y r e i n f o r c e d the b e l i e f i n the myogenic nature of muscular d y s t r o p h y . He was, i n f a c t , q u i t e wrong i n th a t subnormal o u t p u t s were c o n f i r m e d i n neuropathy by S p r i g g s ( 1 9 0 7 b ) . A l t h o u g h he e x c l u d e d n e u r o p a t h y , S h a f f e r d i d 36 emphasize tha t a "low e x c r e t i o n of c r e a t i n i n e i s found i n a r e m a r k a b l y l a r g e number o f p a t h o l o g i c a l s u b j e c t s , r e p r e s e n t i n g a v a r i e t y of c o n d i t i o n s , and the e x c r e t i o n of an abnormally small amount of t h i s substance i s by no means p e c u l i a r t o any one d i s e a s e . " The d i m i n i s h e d o u t p u t i n muscular d y s t r o p h y , i n p a r t i c u l a r , and neuromuscular d i s e a s e i n g e n e r a l was s t i l l v e r y much i n f a v o u r o f a u n i q u e a s s o c i a t i o n between c r e a t i n i n e and s k e l e t a l muscle. C r e a t i n i n e was a c t u a l l y d i s c o v e r e d when c r e a t i n e was f i r s t e x t r a c t e d from meat and subjected to e v a p o r a t i o n with h y d r o c h l o r i c a c i d i n 1835 . Upon a n a l y s i s o f the "new n i t r o g e n c o n t a i n i n g substance" i n u r i n e H e i n t z found that he had i s o l a t e d c r e a t i n e and t h e r e f o r e b e l i e v e d that i t was the substance i n u r i n e which was p r e c i p i t a t e d by z i n c c h l o r i d e . L e i b i g (1847) who had p r o v i d e d the c h e m i c a l f o r m u l a f o r c r e a t i n e which H e i n t z used i n h i s a n a l y s i s , proved H e i n t z wrong by showing that i t was not c r e a t i n e t h a t p r e c i p i t a t e d w i t h z i n c c h l o r i d e , but c r e a t i n i n e . P e t t e n k o f e r , who had i s o l a t e d the new n i t r o g e n c o n t a i n i n g s ubstance from u r i n e t h e same y e a r , but i n a d i f f e r e n t l o c a t i o n from H e i n t z s u b j e c t e d " h i s " substance to elemental a n a l y s i s and r e s u l t s gave an " i n between" f o r m u l a . L e i b i g a t t r i b u t e d the c o m b i n e d f o r m u l a t o c o n t a m i n a t i o n by c r e a t i n e i n c o n c e n t r a t e d u r i n e samples and t h e r e f o r e he b e l i e v e d t h a t c r e a t i n e was a l s o normally found i n the u r i n e . Heintz took h i s t u r n i n p r o v i n g L e i b i g wrong by d e m o n s t r a t i n g t h a t 37 procedures used i n l i b e r a t i n g c r e a t i n i n e from z i n c c h l o r i d e r e s u l t e d i n the c o n v e r s i o n of c r e a t i n i n e to c r e a t i n e and t h e r e f o r e c r e a t i n e was normally absent from u r i n e . That c r e a t i n e was a major and p l e n t i f u l c o n s t i t u e n t o f s k e l e t a l and heart muscle i n a l l higher animals was L e i b i g ' s s u s p i c i o n a f t e r e x t r a c t i n g i t from the f l e s h o f c e r t a i n mammals, b i r d s and f i s h e s . His s u s p i c i o n was confirmed by 1 8 5 3 , b e f o r e w h i c h t i m e S c h l o s s b e r g e r s u c c e e d e d i n e x t r a c t i n g i t from human muscle. I t was a l s o found i n the b r a i n , but because the bulk of the t o t a l body amount was c o n t a i n e d by t h e s k e l e t a l m u s c l e , and i t was h i g h l y c o n c e n t r a t e d t h e r e , the f u t u r e study of c r e a t i n e was focused upon the s k e l e t a l muscle. By v i r t u e of the very d e f i n i t e chemical r e l a t i o n s h i p between c r e a t i n e and c r e a t i n i n e i t was taken f o r granted by most during the ni n e t e e n t h century th a t the c r e a t i n e of muscle was the pr e c u r s o r of the c r e a t i n i n e of u r i n e . T h i s was the o r i g i n a l f o u n d a t i o n of the s p e c i a l p l a c e of c r e a t i n i n e i n the metabolism of muscle. Although c r e a t i n i n e was not a c o n s t i t u e n t of the u r i n e f o r the normal a d u l t male, the t r a n s f o r m a t i o n of c r e a t i n i n e to c r e a t i n e was l a t e r d i s c o v e r e d to take p l a c e with r e l a t i v e ease i n u r i n e specimens that were l e f t s t a n d i n g . Even when u r i n e was preserved i n c h l o r o f o r m and thymol some c r e a t i n i n e was known to be transformed to c r e a t i n e which made immediate a n a l y s i s e x p e d i e n t . C r e a t i n e d i d not g i v e a col o u r r e a c t i o n 38 w i t h p i c r i c a c i d and thus c r e a t i n e c o u l d be underestimated i f some co n v e r s i o n had o c c u r r e d . F o l i n ' s method was e a s i l y adapted t o measure u r i n a r y c r e a t i n e by h e a t i n g t h e specimen w i t h h y d r o c h l o r i c a c i d which would r e v e r t any c r e a t i n e present t o c r e a t i n i n e , t h a t c o u l d then be q u a n t i f i e d c o l o u r i m e t r i c a l l y . However, the p r e s e n c e of c r e a t i n e was not only due t o t r a n s f o r m a t i o n . P r e f o r m e d c r e a t i n e was d e m o n s t r a t e d i n the u r i n e o f a s t a r v i n g man. Benedict and Diefendorf (1907) analyzed the u r i n e of a f a s t i n g woman and found a constant but very low c r e a t i n i n e o u t p u t , a c i d o s i s a c c o r d i n g t o c a l o r i e - n i t r o g e n r a t i o s near the end of the f a s t and most n o t a b l y , a r i s e i n pr e f o r m e d c r e a t i n e which g r a d u a l l y d e c l i n e d as the f a s t c o n t i n u e d . L a r g e e x c r e t i o n s were t h e n e n c o u n t e r e d i n d i a b e t e s m e l l i t u s , e x o p h t h a l m i c g o i t r e and m u s c u l a r d y s t r o p h y ( S h a f f e r , 1908) which, i f c r e a t i n e to c r e a t i n i n e c o n v e r s i o n was a p h y s i o l o g i c a l p r o c e s s , appeared t o be r e l a t e d t o the d i m i n i s h e d o u t p u t o f c r e a t i n i n e i n a l l c o n d i t i o n s . As w e l l as the s e p a r a t i o n between c r e a t i n i n e and muscle as a s p e c i f i c t i s s u e i n v o l v e d i n i t s p r o d u c t i o n F o l i n i n t r o d u c e d e v i d e n c e a g a i n s t a p h y s i o l o g i c a l r e l a t i o n s h i p between c r e a t i n e and c r e a t i n i n e . Some of the c r e a t i n e t h a t was a d m i n i s t e r e d by mouth appeared i n the u r i n e unchanged but the accompanying c r e a t i n i n e d i d not match the m i s s i n g amount. I t appeared th a t c r e a t i n e had been r e t a i n e d as some 39 k i n d o f "food" and F o l i n d i d not b e l i e v e that i t c o u l d be the p r e c u r s o r of a waste product as w e l l . The experiments o f Levene and K r i s t e l l e r (1909) were embarked upon with a very d e f i n i t e premise, that there was a mechanism of c r e a t i n e c a t a b o l i s m . A p r e c o n d i t i o n f o r the acceptance of s u b j e c t s to be used i n t h e i r i n v e s t i g a t i o n was e i t h e r the abnormal q u a n t i t i e s of c r e a t i n i n e or the presence o f c r e a t i n e i n the u r i n e . The u r i n e of a l l t h o s e w i t h muscular dystrophy c o n t a i n e d abnormally low q u a n t i t i e s of c r e a t i n i n e a s w e l l as p r e f o r m e d c r e a t i n e , a n d by m a n i p u l a t i n g the d i e t the c r e a t i n i n e / c r e a t i n e r a t i o s were seen t o change. The r a t i o was 1/2 when s u b j e c t s were on a c e r e a l d i e t , becoming s m a l l e r when on a hig h p r o t e i n d i e t d e v o i d o f c r e a t i n e and s m a l l e r s t i l l when c r e a t i n e was added. A f t e r a l l r e s u l t s f o r a l l muscular a f f l i c t i o n s were reviewed the c o n c l u s i o n was that there was a d e c e l e r a t i o n i n the chemical c o n v e r s i o n of c r e a t i n e to g i v e c r e a t i n i n e which would e x p l a i n the e x c e s s of u n c o n v e r t e d c r e a t i n e w i t h a r e d u c t i o n o f c r e a t i n i n e i n t h e u r i n e . The r a t e o f c a t a b o l i s m was t h e r e f o r e concluded to be c o n t r o l l e d by the muscle but the muscle was accepted as being only one of the r e g u l a t i n g f a c t o r s . T h a t c r e a t i n e was t h e p h y s i o l o g i c a l p r e c u r s o r of c r e a t i n i n e was l o g i c a l and t h e a s s o c i a t i o n b e t w e e n c r e a t i n i n e and m u s c l e h i n g e d on t h i s v e r y p o i n t . The c o n v e r s i o n of c r e a t i n e to c r e a t i n i n e i n i s o l a t e d muscle was 40 a p p a r e n t l y demonstrated d u r i n g the n i n e t e e n t h c e n t u r y and was l a t e r confirmed (Myers and F i n e , 1915). However, t h i s d i d not prove that the c r e a t i n e of muscle was the p r e c u r s o r of the c r e a t i n i n e of u r i n e . F o l l o w i n g the c h a l l e n g e r e g a r d i n g the r e l a t i o n s h i p b e t w e e n c r e a t i n e and c r e a t i n i n e , f e e d i n g e x p e r i m e n t s c o n t i n u e d t o a d d r e s s the i s s u e . For example, Rose and Dimmit (1916) reported that c r e a t i n i n e that was i n g e s t e d by normal men appeared i n the u r i n e unchanged. They a l s o found a very p e r c e p t i b l e r i s e i n c r e a t i n i n e output upon i n g e s t i o n of c r e a t i n e , b o t h r e s u l t s s u p p o r t i n g a p h y s i o l o g i c a l l y i r r e v e r s i b l e c o n v e r s i o n of c r e a t i n e to c r e a t i n i n e . D e s t r u c t i o n -by a l i m e n t a r y b a c t e r i a was i n i t i a l l y thought by some to be r e s p o n s i b l e f o r the "incompleteness" of c r e a t i n e to c r e a t i n i n e formation i n f e e d i n g experiments. T h e " l o s s " o f a c e r t a i n a m o u n t w i t h p a r e n t e r a l a d m i n i s t r a t i o n d i s p r o v e d such a t h e o r y . That c r e a t i n e was a " f o o d " was d e m o n s t r a t e d by i n c r e a s e s i n muscle c r e a t i n e f o l l o w i n g a d m i n i s t r a t i o n . Myers and F i n e (1913) found th a t a p o r t i o n of the a d m i n i s t e r e d amount was accounted f o r by i n c r e a s e s i n muscle c r e a t i n e . A n o t h e r p o r t i o n matched i n c r e a s e s i n c r e a t i n i n e o u t p u t , but not a l l c o u l d be accounted f o r . E x t r i n s i c c r e a t i n e c o u l d never be accounted f o r i n t o t a l i t y , simply because the t r a v e l s of the i n g e s t e d amount c o u l d not be f o l l o w e d , as i t were. 41 The q u e s t i o n of c r e a t i n e - c r e a t i n i n e c o n v e r s i o n and the o r i g i n of u r i n a r y c r e a t i n i n e remained u n r e s o l v e d f o r many yea r s a f t e r F o l i n ' s f i r s t feeding experiment. In 1932 Magee found that the t o t a l c r e a t i n i n e c o e f f i c i e n t , which i n c l u d e d both c r e a t i n i n e and c r e a t i n e , was lowered more so than the c r e a t i n i n e c o e f f i c i e n t i n a n a d v a n c e d c a s e o f p s e u d o h y p e r t r o p h i c muscular d y s t r o p h y . She i n t e r p r e t e d t h e s e r e s u l t s t o mean t h a t t h e r e was a d e f e c t i n the c r e a t i n e t o c r e a t i n i n e c o n v e r s i o n , but even t h e n , the i n v i v o r e a c t i o n had yet to be proven. The i n t r o d u c t i o n and a v a i l a b i l i t y of i s o t o p e s i n the l a t t e r p a r t of the t h i r d decade f i n a l l y proved that u r i n a r y c r e a t i n i n e o r i g i n a t e d i n the muscle and that c r e a t i n e was i t s p r e c u r s o r . B l o c h e t a l . (1941a) found t h a t a f t e r f e e d i n g c r e a t i n e l a b e l l e d with i s o t o p i c n i t r o g e n f o r s i x days to e x p e r i m e n t a l a n i m a l s , l a b e l l e d c r e a t i n i n e appeared i n t h e u r i n e i n i n c r e a s i n g c o n c e n t r a t i o n s t h a t reached a maximum. L a b e l l e d output corresponded to the decrease i n l a b e l l e d body c r e a t i n e . When a l l t h r e e n i t r o g e n s were l a b e l l e d t h e r e were n e g l i g i b l e c o n c e n t r a t i o n s of u r e a and ammonia bearing the l a b e l . U n l i k e a r g i n i n e which exchanged l a b e l l e d f o r u n l a b e l l e d amidino groups, c r e a t i n e was a very s t a b l e m o l e c u l e , i t s o n l y f a t e b e i n g c o n v e r s i o n t o c r e a t i n i n e . Borsook and Dubnoff (1947) l a t e r accounted f o r the f a c t t h a t most of the c r e a t i n e of the muscle was phosphorylated 42 w h i c h F i s k e and S u b b a r r o w d i s c o v e r e d i n 1927. They e x t r a c t e d p h o s p h o c r e a t i n e from r a b b i t muscle and of the t h r e e r e a c t i o n s t h a t o c c u r r e d i n t h e t e s t t u b e : P h o s p h o c r e a t i n e •* c r e a t i n e + i n o r g a n i c p h o s p h a t e and c r e a t i n e •* c r e a t i n i n e , t h e r a t e of the f i r s t r e a c t i o n accounted f o r d a i l y c r e a t i n i n e o u t p u t , assuming an enzyme was not i n v o l v e d . The f a c t t h a t a l o w e r e d c r e a t i n i n e o u t p u t was not r e s t r i c t e d to myopathy i n the event of muscular d i s e a s e was v e r i f i e d by M i l h o r a t and Woolf (1937, 1938). C r e a t i n i n e c o e f f i c i e n t s corresponded to the degree of muscular wasting i n both myopathy and neuropathy. C r e a t i n u r i a M o s t t h e o r i e s a b o u t c r e a t i n u r i a as a g e n e r a l phenomenon, f o l l o w i n g i t s d i s c o v e r y , t r e a t e d c r e a t i n e with complete d i s r e g a r d f o r c r e a t i n i n e . Although c r e a t i n u r i a was known to be a f e a t u r e of carcinoma of the l i v e r , because the a b n o r m a l i t y was a f e a t u r e of c o n d i t i o n s which i n v o l v e d the l o s s of muscle mass, such as d i a b e t e s m e l l i t u s , exophthalmic g o i t r e , i n a n i t i o n and m u s c u l a r d y s t r o p h y , c r e a t i n e was thought t o be l i b e r a t e d i n the p r o c e s s of muscle t i s s u e breakdown, and was t h e r e f o r e endogenous to the muscle. In 1907 C a t h c a r t r e p o r t e d the r e l i e f of c r e a t i n u r i a consequent to f a s t i n g by the a d m i n i s t r a t i o n of c a r b o h y d r a t e . M e n d e l a n d R o s e (19 1 1 ) , f o u n d t h a t t h e c o n t i n u e d 43 administration of a soluble starch suspended in water eliminated creatine output in starvation completely, thereby confirming Cathcart's report. Both creatine and glucose were excreted in diabetes mellitus. Dogs treated with phlorizin, which simulated the glucose nonutilization of diabetes mellitus also excreted creatine. That there was a definite relationship between creatine and carbohydrate was evident. Carbohydrate was believed to spare the protein of the muscle which might be obligated to provide an energy source in the event of a disturbance in carbohydrate metabolism. In doing so, the muscle would liberate the creatine it contained. Even when Rose (1911) discovered the presence of creatine in the urine of healthy children who had not reached puberty, he suggested that a peculiar carbohydrate metabolism during growth and development was responsible. Folin and Denis (1912) reasoned that i f this were true c r e a t i n u r i a would be more pronounced at times of carbohydrate deprivation such as during the night. However, they discovered that the nocturnal urine of children contained much less creatine. Powis and Raper (1916) confirmed this finding in a study of output in a normal child and one suffering from amyotonia congenita. The excretion in the latter was higher overall but the diurnal and nocturnal variations paralleled those of the normal child. 44 A f t e r F o l i n and Denis (1912) f i r s t brought a t t e n t i o n to the d a i l y p a t t e r n of c r e a t i n u r i a i n c h i l d r e n they o f f e r e d an e x c e s s i v e p r o t e i n consumption which was necessary to n o u r i s h the growing body as an e x p l a n a t i o n f o r c r e a t i n u r i a d u r i n g t h i s p e r i o d of l i f e . Denis and Kramer (1917) s t u d i e d the i n f l u e n c e of p r o t e i n f e e d i n g upon c r e a t i n e e x c r e t i o n i n c h i l d r e n . W h i l s t on a low p r o t e i n d i e t the d i u r n a l and n o c t u r n a l outputs were compatible with previous s t u d i e s , but on a h i g h p r o t e i n d i e t daytime and n i g h t t i m e outputs were e q u i v a l e n t . I f p r o t e i n intake was r e s t r i c t e d to a minimum whi l e s t i l l m a i n t a i n i n g c a l o r i c i n t a k e i t was p o s s i b l e to make the u r i n e c r e a t i n e - f r e e . P h y s i o l o g i c a l c r e a t i n u r i a which appeared to be r e l a t e d to the menstrual c y c l e was a l s o d iscovered i n a d u l t females. Denis and Minot. (1917) succeeded i n producing c r e a t i n u r i a i n women by f e e d i n g them h i g h p r o t e i n , c r e a t i n e - f r e e d i e t s w h i l e men were r e s i s t a n t to such d i e t a r y i m p o s i t i o n s . C r e a t i n u r i a o f i n d i v i d u a l s s u f f e r i n g from e x o p h t h a l m i c g o i t r e seemed to be r e l a t e d to a p p e t i t e , the more v o r a c i o u s the a p p e t i t e the g r e a t e r the degree of c r e a t i n u r i a . T h i s o b s e r v a t i o n l e d D e nis (1917) t o t e s t the i n f l u e n c e o f p r o t e i n i n t a k e upon h y p e r t h y r o i d s u b j e c t s i n w h i c h c r e a t i n u r i a was p a t h o l o g i c a l r a t h e r than p h y s i o l o g i c a l . J u s t as i n s t a t e s f o r which c r e a t i n u r i a was p h y s i o l o g i c a l the q u a n t i t y o f the p r o t e i n i n t a k e had an u n m i s t a k a b l e e f f e c t upon c r e a t i n e e x c r e t i o n . 45 That a p o r t i o n of the p r o t e i n consumed i n the food was converted to c r e a t i n e which was t r a n s p o r t e d to and absorbed by t h e m u s c l e became t h e w o r k i n g h y p o t h e s i s . The c r e a t i n u r i a o f c h i l d h o o d , e x o p h t h a l m i c g o i t r e , muscular d y s t r o p h y and o t h e r p a t h o l o g i c a l s t a t e s c o u l d thus be expla i n e d by a low s a t u r a t i o n p o i n t of the muscle which l e d to the r e f u s a l and e x c r e t i o n o f c r e a t i n e . The f a c t t h a t c r e a t i n u r i a o c c u r r e d with the amputation of a limb (Cameron and G i b s o n , 1922) was a p o i n t very much i n support of the h y p o t h e s i s . G i b s o n and M a r t i n (1921) r e p o r t e d t h a t e x t r i n s i c c r e a t i n e was promptly and completely e l i m i n a t e d by a case of ps e u d o h y p e r t r o p h i c muscular d y s t r o p h y . However, i t c o u l d not be s a i d whether c r e a t i n e was being absorbed and not r e t a i n e d or simply not absorbed by the muscle. Although the degree of c r e a t i n u r i a was g e n e r a l l y l e s s i n n e u r o p a t h y , no sharp d i s t i n c t i o n between myopathy and neuropathy was made i n terms of output. M i l h o r a t and Woolf (1937, 1938) d i d d i s c o v e r , though, that c r e a t i n e t o l e r a n c e , t h a t i s , t h e p e r c e n t r e t e n t i o n o f i n g e s t e d c r e a t i n e , d i f f e r e n t i a t e d between the two. Myopathic i n d i v i d u a l s were much l e s s t o l e r a n t than n e u r o p a t h i c , both r e t a i n i n g l e s s when g l y c i n e was administered s i m u l t a n e o u s l y . When the l i v e r was determined to be one of the s i t e s of c r e a t i n e s y n t h e s i s (Borsook and Dubhoff, 1940), the source of c r e a t i n u r i a i n carcinoma of the l i v e r was ob v i o u s . The exogenous v e r s u s endogenous o r i g i n o f c r e a t i n u r i a i n 46 muscular dystrophy was, on the other hand, s t i l l u n r e s o l v e d . Was c r e a t i n e l e a v i n g the muscle or was i t p a s s i n g by without g a i n i n g a c c e s s ? The experiments of Roche et a l . (1952) and B e n e d i c t e t a l . (1955) were p r e d i c a t e d on the assumption tha t c r e a t i n e d e r i v e d from a d m i n i s t e r e d g l y c i n e c o n t a i n i n g i s o t o p i c n i t r o g e n would be e q u i v a l e n t t o c r e a t i n i n e i n i s o t o p i c c o n c e n t r a t i o n i f c r e a t i n e leaked from the muscle. I f the muscle was capable of removing only a small f r a c t i o n of the b l o o d c r e a t i n e , i s o t o p i c c r e a t i n e generated by the l i v e r would be r i c h e r than c r e a t i n i n e d u r i n g the i n t e r v a l f o l l o w i n g f e e d i n g . The l a t t e r s i t u a t i o n o c c u r r e d when i s o t o p i c g l y c i n e was given to i n d i v i d u a l s i n advanced stages of muscular dystrophy. In 1968 F i t c h et a l . determined that h a l f times f o r the d i s a p p e a r a n c e of a C14 l a b e l i n u r i n a r y c r e a t i n i n e d e r i v e d from a d m i n i s t e r e d c r e a t i n e were s h o r t e r than normal. In c o n t r a s t t o the e a r l i e r s t u d i e s , t h e y c o n c l u d e d t h a t c r e a t i n e was e n t e r i n g the muscle f i b r e but i n t r a c e l l u l a r t r a p p i n g was i m p a i r e d . L i k e those i n v o l v e d i n the f i r s t s t u d i e s , though, t h e i r r e s u l t s were i n t e r p r e t e d to mean that the c r e a t i n u r i a of muscular dystrophy was not pathognomonic, not t h e p r i m a r y e x p r e s s i o n o f a d e f e c t , but a p r o c e s s secondary t o muscular d e g e n e r a t i o n . They a l s o found that h a l f times f o r neurogenic a t r o p h y were l o n g e r than normal i n d i c a t i n g reduced membrane t r a n s p o r t of c r e a t i n e i n t o the 47 m u s c l e f i b e r , b u t l i k e t h a t f o r m u s c u l a r d y s t r o p h y , c r e a t i n u r i a was the consequence of d i s e a s e . T h e S y n t h e s i s o f C r e a t i n e When McCollum and Steenbock (1912) induced c r e a t i n u r i a i n growing p i g s they found that i t was not n e c e s s a r i l y the q u a n t i t y o f p r o t e i n t h a t d e t e r m i n e d t h e d e g r e e o f c r e a t i n u r i a but the cha r a c t e r of the p r o t e i n . For example, when on a d i e t i n which ni t r o g e n was d e r i v e d from corn meal c r e a t i n e was on l y an o c c a s i o n a l and minor c o n s t i t u e n t of the u r i n e . When g l u t e n meal which c o n s i s t e d p r i n c i p a l l y of the p r o t e i n s from the co r n endosperm was the n i t r o g e n s o u r c e , t h e c r e a t i n e o u t p u t r o s e m a r k e d l y . The s e q u e n t i a l a r r a n g e m e n t o f amino a c i d s was by t h e n a w e l l known s t r u c t u r a l aspect of the p r o t e i n m o l e c u l e . As a r g i n i n e was the o n l y amino a c i d which possessed a guanidino group, which was a l s o conspicuous i n c r e a t i n e , i t was the h i g h l y favoured candidate as the pre c u r s o r of c r e a t i n e . The c r e a t i n u r i a o f muscular dystrophy was s u b s t a n t i a l and b e c a u s e i t was v e r y e a s i l y i n f l u e n c e d by e x t r i n s i c p r o t e i n , i n d i v i d u a l s w i t h muscular dystrophy were employed f o r t h e t e s t i n g o f t h e • " c r e a t o g e n i c i t y " o f d i e t a r y p r e c u r s o r s . A r g i n i n e was, very unexpectedly, not one of the s u b s t a n c e s w h i c h had an a p p r e c i a b l e e f f e c t upon t h e c r e a t i n u r i a of the d i s e a s e . Gibson and M a r t i n (1921) and B r a n d e t a l . (1929) f o u n d t h a t g u a n i d i n e a c e t i c a c i d 48 produced very s i g n i f i c a n t i n c r e a s e s i n o u t p u t . Brand et a l . (1929) a l s o found that g l y c i n e enhanced c r e a t i n u r i a . With t h e p o s s i b i l i t y t h a t g u a n i d i n e a c e t i c a c i d was an i n t e r m e d i a t e i n the s y n t h e s i s of c r e a t i n e which reacted w i t h g l y c i n e t o f o r m c r e a t i n e , G i b s o n and M a r t i n ( 1 9 2 1 ) a d m i n i s t e r e d b e n z o i c a c i d which would combine with g l y c i n e t o d i v e r t i t as h i p p u r i c a c i d . There was no r e s u l t a n t change i n c r e a t i n u r i a , which seemed not to f a v o u r t h e p a r t i c i p a t i o n of both g l y c i n e and guanidine a c e t i c a c i d as s y n t h e t i c i n t e r m e d i a t e s . I n c o n t r a s t t o o t h e r s , B e a r d and B a r n e s ( 1 9 3 1 ) c o n s i d e r e d p a t i e n t s w i t h m u s c u l a r d y s t r o p h y t o be i n a p p r o p r i a t e s u b j e c t s f o r t h e i n v e s t i g a t i o n o f t h e c r e a t o g e n i c e f f e c t s o f d i e t a r y p r e c u r s o r s b e c a u s e c r e a t i n u r i a was a l r e a d y p r e s e n t . Assuming that c r e a t i n e was the p r e c u r s o r of c r e a t i n i n e , c r e a t i n i n e output i n response to the a d m i n i s t r a t i o n of amino a c i d s was used as an index of c r e a t o g e n i c i t y . A d u l t r a t s and men were used i n order to a v o i d b o t h p h y s i o l o g i c a l and p a t h o l o g i c a l c r e a t i n u r i a . G l y c i n e d i d , i n t h i s i n s t a n c e , cause the g r e a t e s t i n c r e a s e s i n c r e a t i n e e x c r e t i o n . In o p p o s i t i o n t o the f i n d i n g s of G i b s o n and M a r t i n ( 1 9 2 1 ) , H a r r i s and Brand (1931) f o u n d c r e a t i n u r i a of d y s t r o p h i c i n d i v i d u a l s t o d e c r e a s e upon a d m i n i s t r a t i o n of b e n z o i c a c i d owing to c o n j u g a t i o n w i t h g l y c i n e . 49 The a b e r r a n t c r e a t i n e - c r e a t i n i n e m e t a b o l i s m was the o n l y o t h e r known a b n o r m a l i t y a s i d e from t h e m u s c u l a r d e g e n e r a t i o n o f m u s c u l a r d y s t r o p h y . A l t h o u g h t h e b i o c h e m i c a l s y n t h e s i s o f c r e a t i n e was not yet e s t a b l i s h e d g l y c i n e u n d o u b t e d l y had a s p e c i a l and s i g n i f i c a n t r e l a t i o n s h i p to t h i s aspect of metabolism. For t h i s reason M i l h o r a t , Techner and Thomas (1932) t r e a t e d i n d i v i d u a l s d i a g n o s e d with p s e u d o h y p e r t r o p h i c muscular d y s t r o p h y w i t h g l y c i n e and d i s c o v e r e d t h a t while c r e a t i n e e x c r e t i o n rose i n i t i a l l y i t f e l l w i t h c o n t i n u e d t r e a t m e n t . There was a corresponding g a i n i n c r e a t i n i n e output which was c o n s i s t e n t w i t h c l i n i c a l a m e l i o r a t i o n o f the c o n d i t i o n . M u s c u l a r s t r e n g t h was g r e a t e r and the a c t i v i t i e s of d a i l y l i f e c o u l d be c a r r i e d o u t w i t h g r e a t e r e a s e and e f f i c i e n c y . Improvement was so remarkable i n some that p h y s i c a l f e a t s , i n c l u d i n g c l i m b i n g s t a i r s , r i s i n g from the f l o o r , w a l k i n g c o n s i d e r a b l e d i s t a n c e s and even r i d i n g a b i k e , were a g a i n p o s s i b l e . Recovery was so amazing that suggesting a p l a c e f o r g l y c i n e i n the pathogenesis of the d i s e a s e c o u l d not be a v o i d e d . F o l l o w i n g treatment with g l y c i n e M i l h o r a t (1933) found b i o p s i e s which were composed of normal, p a r t i a l l y a t r o p h i e d and c o m p l e t e l y a t r o p h i e d f i b r e s p r i o r to treatment to be comprised o n l y of normal and c o m p l e t e l y a t r o p h i e d f i b r e s . M u scle i n the advanced s t a g e s of d e g e n e r a t i o n showed no i m p r o v e m e n t a f t e r g l y c i n e t h e r a p y and r e s u l t s were 50 i n t e r p r e t e d as being very much i n support o f the a b i o t r o p h i c nature of the d i s e a s e , t h a t i s , g l y c i n e c o u l d only r e t r i e v e those f i b r e s having a "weak molecular energy" which had not gone p a s t a p o i n t o f no r e d e m p t i o n . i n t h i s c a s e , i f g l y c i n e was given at an e a r l y enough stage of the d i s e a s e , i t was b e l i e v e d that i t would provide a c u r e . On the b a s i s that muscular dystrophy i n v o l v e d a g l y c i n e d e f i c i e n c y F r e i b e r g and West (1933) compared h i p p u r i c a c i d output i n response to the a d m i n i s t r a t i o n o f benzoic a c i d i n t h r e e w e l l advanced c a s e s o f p s e u d o h y p e r t r o p h i c muscular d y s t r o p h y w i t h t h a t o f t h r e e o r t h o p e d i c c a s e s . H i p p u r i c a c i d e x c r e t i o n was the same f o r both groups i n d i c a t i n g e qual abundance o f g l y c i n e i n both c o n d i t i o n s and d i s p r o v i n g a g l y c i n e d e f i c i e n c y i n m u s c u l a r d y s t r o p h y p a r t i c u l a r l y . F u r t h e r study r e g a r d i n g treatment with g l y c i n e was, s t i l l , c o n s i d e r e d to be w a r r a n t e d by some i n c l u d i n g M e t t e l and Slocum (1933) who found two of t h r e e s u f f e r i n g from the p s e u d o h y p e r t r o p h i c form o f dystrophy to improve f o l l o w i n g g l y c i n e t h e r a p y . Boothby (1932) had s u c c e s s w i t h g l y c i n e upon m y a s t h e n i a g r a v i s , w hich a c c o r d i n g to the l a c k o f p a t h o l o g y t o the p e r i p h e r a l nerves or s p i n a l c o r d , was c l a s s i f i e d by some as a m y o p a t h y . A c t u a l l y , among n e u r o m u s c u l a r d i s e a s e s i t was m y a s t h e n i a g r a v i s and p s e u d o h y p e r t r o p h i c m u s c u l a r d y s t r o p h y t h a t showed t h e g r e a t e s t b e n e f i t from g l y c i n e , although i t was q u i t e c l e a r t h a t t h e y were a e t i o l o g i c a l l y d i s t i n c t . C r e a t i n u r i a , i f 51 p r e s e n t at a l l , was u s u a l l y s l i g h t i n myasthenia g r a v i s . Treatment s t u d i e s e v e n t u a l l y confirmed that g l y c i n e was not c u r a t i v e i n muscular dystrophy nor any other neuromuscular d i s e a s e but because b e n e f i t had been very d e f i n i t e i n some i t became "a new and n o n o f f i c i a l remedy" f o r m u s c u l a r dystrophy and myasthenia g r a v i s i n 1935. When Beard and Barnes (1931) monitored changes i n the c r e a t i n e c o n t e n t of muscle i n young r a t s as an index of c r e a t i n e formation from e x t r i n s i c p r e c u r s o r s they found that w h i l e a measurable i n c r e a s e i n c r e a t i n e c o n t e n t was the response to a l l amino a c i d s , glutamate gave the g r e a t e r percent i n c r e a s e s . G l y c i n e , i n c o n t r a s t to i t s c r e a t i n u r i a enhancing a b i l i t y , gave increases that were among the lower of the amino a c i d s and guanidine a c e t i c a c i d , i n keeping w i t h i t s e f f e c t upon c r e a t i n u r i a , c aused the g r e a t e s t p ercent in c r e a s e s of a l l e x t r i n s i c substances. In view of the r e s u l t s r e g a r d i n g g l u t a m a t e i t was deemed to have p o t e n t i a l as a t h e r a p e u t i c agent. T r i p o l i et a l . (1934) o b s e r v e d improvement i n b o t h m u s c u l a r d y s t r o p h y and m y a s t h e n i a g r a v i s upon a d m i n i s t r a t i o n of g l u t a m a t e . A l t h o u g h r e s u l t s o b t a i n e d by o t h e r s f o r the a b i l i t y of glutamate to c l i n i c a l l y ameliorate the c o n d i t i o n s were not c o n s i s t e n t , g l u t a m a t e , which was not as e x p e n s i v e as g l y c i n e , a l s o became a new and n o n o f f i c i a l remedy o f muscular dystrophy and myasthenia g r a v i s . 52 On the b a s i s of the r e s u l t s o b t a i n e d by Beard and Barnes concerning the a b i l i t y of amino a c i d s to promote the f o r m a t i o n o f muscle c r e a t i n e , T r i p o l i and Beard (1934) proposed a theory for the i n v i v o s y n t h e s i s of c r e a t i n e : A r g i n i n e •*• guanidine b u t y r i c a c i d •* g u a n i d i n e a c e t i c a c i d -> c r e a t i n e a c c o u n t i n g f o r 70% of t o t a l c r e a t i n e f o r m a t i o n . Seventy percent of muscle c r e a t i n e was d e r i v e d from a r g i n i n e w h i l e , a c c o r d i n g to the p e r c e n t i n c r e a s e s i n muscle c r e a t i n e i n r e s p o n s e to t h e i r a d m i n s t r a t i o n , g l y c i n e , g l u t a m a t e and v a l i n e accounted f o r 15%, 21% and 34% of muscle c r e a t i n e , r e s p e c t i v e l y . The remainder of the amino a c i d s s t i m u l a t e d s p e c i f i c dynamic a c t i o n c r e a t i n e f o r m a t i o n through some unknown mechanism. A c c o r d i n g to t h i s scheme i t was a l s o p roposed t h a t m u s c u l a r d y s t r o p h y was t h e r e s u l t o f an amino a c i d d e f i c i e n c y stemming from e i t h e r an i n s u f f i c i e n t i n t a k e of p r o t e i n , a d e f e c t i n d i g e s t i o n or i n u t i l i z a t i o n . The l a c k of amino a c i d s was thought to be r e s p o n s i b l e f o r the l o s s of c r e a t i n e , the r o l e of which by t h a t time, although i t had always been recognized that c r e a t i n e was concentrated i n the muscle f o r a purpose, had been e l u c i d a t e d . T h e r e f o r e i n c o n t r a s t t o t h e o r i e s w h i c h h e l d c r e a t i n u r i a t o be a consequence of muscular degeneration, a general amino a c i d 53 d e f i c i e n c y which l e d to a c r e a t i n e l o s s was b e l i e v e d to be the cause of muscular d e g e n e r a t i o n . In view of the f a c t that c r e a t i n e was being formed, by v i r t u e of i t s abundance i n the u r i n e , i t seems strange that T r i p o l i and B e a r d (1934) t h o u g h t t h a t a d e f i c i e n c y o f p r e c u r s o r s a n d / o r a d e f i c i e n c y o f amino a c i d s w h i c h s t i m u l a t e d formation was r e s p o n s i b l e f o r i t s l o s s . However, u n l i k e s t u d i e s w h i c h i n v o l v e d t h e m a n i p u l a t i o n o f c r e a t i n u r i a by d i e t a r y s u b s t a n c e s , t h e i r s t u d i e s supported the r o l e of a r g i n i n e i n c r e a t i n e s y n t h e s i s . A g a i n , l i k e the o r i g i n o f u r i n a r y c r e a t i n i n e and the p r e c i s e o r i g i n o f u r i n a r y c r e a t i n e i n v a r i o u s c o n d i t i o n s , i t was i s o t o p e s that enabled the d e l i n e a t i o n of the s y n t h e t i c pathway of c r e a t i n e (Bloch et a l . , 1941b). The P h y s i o l o g y of C r e a t i n e D y s t r o p h i c i n d i v i d u a l s were " d i a b e t i c " as r e g a r d s c r e a t i n e i n t h a t t h e y c o u l d not r e t a i n t h a t which was i n g e s t e d . Chanutin (1926) drew an analogy between glucose and c r e a t i n e by n o t i n g that both substances were normally r e t a i n e d and s t o r e d but when the body could not accommodate e i t h e r i t was e x c r e t e d . G l u c o s u r i a e v i n c e d g l u c o s e i n t o l e r a n c e a s c r e a t i n u r i a was a s i g n o f c r e a t i n e i n t o l e r a n c e . Because the p o l y s a c c h a r i d e , g l y c o g e n , was the s t o r a g e form of glucose i t was suggested that p o l y c r e a t i n e 54 was a p l a u s i b l e s t o r a g e f o r m o f c r e a t i n e , but s u c h a s u g g e s t i o n , although imaginable, was f a r from r e a l i t y . I t was r e c o g n i z e d t h a t c r e a t i n e c o u l d not be p a r t of the p r o t e i n s t r u c t u r e of the muscle s i n c e i t was e a s i l y e x t r a c t e d with water, as was phosphate. F i s k e and Subbarrow (1927) d e v e l o p e d a means of q u a n t i f y i n g the phosphate i n p r o t e i n - f r e e muscle f i l t r a t e s u s i n g a m i n o - n a p h t h o s u l f o n i c a c i d to produce a blue r e d u c t i o n product of phosphomolybdic a c i d which c o u l d be q u a n t i f i e d c o l o u r m e t r i c a l l y . Using t h i s method they found that maximum c o l o u r p r o d u c t i o n was delayed even when a l l of the i n o r g a n i c phosphate was f i r s t removed by p r e c i p i t a t i o n ; thus i t appeared that phosphate was being l i b e r a t e d from an organ i c compound through h y d r o l y s i s . The f a c t t h a t the phosphate c o n t e n t i n c r e a s e d when muscle was s t i m u l a t e d to the poi n t of f a t i g u e and resumed the o r i g i n a l c ontent f o l l o w i n g a p e r i o d of r e s t supported the e x i s t e n c e o f a c o m p o u n d c o n t a i n i n g a " l a b i l e " p h o s p h a t e . P h o s p h o c r e a t i n e was d e t e r m i n e d to be the s o u r c e o f the l a b i l e phosphate according to the one to one s t o i c h i o m e t r y between c r e a t i n e and phosphate p r e c i p i t a t e d from f i l t r a t e s d u r i n g a p a r t i c u l a r time i n t e r v a l . The f a c t t h a t phosphate i n c r e a s e d and c r e a t i n e was no longer p r e c i p i t a b l e by copper f o l l o w i n g c o n t r a c t i o n , p l u s the reduced c a p a c i t y of f a t i g u e d muscle t o r e t a i n both c r e a t i n e and phosphate a c c o r d i n g t o p e r f u s i o n and d i a l y s i s e x p e r i m e n t s , made the p l a c e f o r 55 p h o s p h o c r e a t i n e i n the c h e m i s t r y of muscular c o n t r a c t i o n r a t h e r o b v i o u s . D u r i n g t h e n i n e t e e n t h c e n t u r y one of the s u g g e s t e d r o l e s o f c r e a t i n e as a b a s i c s u b s t a n c e was t h a t o f n e u t r a l i z i n g the l a c t i c a c i d produced w i t h c o n t r a c t i o n . C r e a t i n e was t hen, l i k e l a c t i c a c i d , a substance a t t r i b u t e d to f a t i g u e but heat generated by the p r o d u c t i o n of l a c t i c a c i d was u l t i m a t e l y b e l i e v e d to p r o v i d e the energy f o r muscular c o n t r a c t i o n . With the d i s c o v e r y of phosphocreatine the p o s i t i o n of l a c t i c a c i d i n the c h e m i s t r y of muscular c o n t r a c t i o n was a l t e r e d . In 1930 Lundsgaard2 proved that l a c t i c a c i d p r o d u c t i o n was not n e c e s s a r y f o r m u s c u l a r c o n t r a c t i o n . M u s c u l a r c o n t r a c t i o n s t i l l o c c u r r e d f o l l o w i n g the a p p l i c a t i o n of i o d a c e t a t e but l a c t i c a c i d f a i l e d t o be p r o d u c e d . The e x o t h e r m i c r e a c t i o n i n which the phosphate i s s p l i t from p h o s p h o c r e a t i n e o c c u r r e d and t h e r e f o r e t h i s r e a c t i o n was t h e r e a f t e r b e l i e v e d to p r o v i d e the immediate energy f o r c o n t r a c t i o n . Muscles p o i s o n e d with i o d a c e t a t e c o n t r a c t e d f o r a l i m i t e d p e r i o d of time f o l l o w i n g which a " c u r i o u s " form of c o n t r a c t i o n s e t i n . Because l a c t i c a c i d was not b e i n g p r o d u c e d nor p h o s p h o c r e a t i n e being r e s t o r e d i t was concluded th a t the heat d e r i v e d from l a c t i c a c i d p r o d u c t i o n 2C i t e d by Lehninger (1975). 56 was used i n exchange f o r the endothermic r e c o n s t i t u t i o n of phosphocreatine. I n 1 9 3 2 L o h m a n n3 d i s c o v e r e d t h a t A T P a n d p h o s p h o c r e a t i n e were i n v o l v e d i n a r e a c t i o n i n which a p h o s p h a t e was e x c h a n g e d . H o w e v e r , u n d e r a n a e r o b i c c o n d i t i o n s p h o s p h o c r e a t i n e c o n c e n t r a t i o n f e l l by more than twenty p e r c e n t w h i l e ATP f e l l by l e s s than t e n p e r c e n t . Such changes appeared to be c o n s i s t e n t w i t h the r o l e of phosphocreatine as the energy source behind c o n t r a c t i o n and the r o l e of ATP, l i k e t h a t of l a c t i c a c i d p r o d u c t i o n , was thought to be i n the r e p h o s p h o r y l a t i o n of c r e a t i n e . As c o n t r a c t i o n o c c u r r e d under a n a e r o b i c c o n d i t i o n s , o x i d a t i o n was f o r a t i m e t h o u g h t by some t o p a r t i c i p a t e i n t h e "aftermath" of c o n t r a c t i o n i n the r e s y n t h e s i s of g l y c o g e n . The s t a t u s of muscular c o n t r a c t i o n and the knowledge of the processes s u b s e r v i n g i t set the stage f o r examination of these p r o c e s s e s i n human muscular d i s e a s e s . Nevin (1934) a n a l y z e d t h e m u s c l e f r o m p s e u d o h y p e r t r o p h i c m u s c u l a r d y s t r o p h y , myasthenia g r a v i s and m y o t o n i c d y s t r o p h y f o r changes i n phosphorous c o n t a i n i n g compounds before and a f t e r e l e c t r i c a l l y s t i m u l a t e d c o n t r a c t i o n . T h e r e was an a b n o r m a l l y s m a l l c h a n g e i n t h e c o n c e n t r a t i o n o f p h o s p h o c r e a t i n e i n m y o t o n i c d y s t r o p h y w h i l e a p p r o p r i a t e c h a n g e s i n t h e c o n c e n t r a t i o n s o f b o t h A T P a n d 3C i t e d by Lehninger (1975). 57 phosphocreatine occurred w i t h c o n t r a c t i o n and r e l a x a t i o n i n m y a s t h e n i a g r a v i s and muscular d y s t r o p h y . That i s , they f e l l by t e n and t w e n t y p e r c e n t , r e s p e c t i v e l y , w i t h c o n t r a c t i o n f o l l o w i n g which they were r e s t o r e d . T h e r e f o r e , t h e mechanism of m u s c u l a r c o n t r a c t i o n was d e m o n s t r a b l y normal i n muscular dystrophy. U n l i k e myasthenia g r a v i s and myotonic d y s t r o p h y , the r e s t i n g c o n c e n t r a t i o n s o f t h e t o t a l a c i d s o l u b l e phosphorous, and ATP and phosphocreatine, i n d i v i d u a l l y , were g r e a t l y diminished i n an advanced case of pseudohypertrophic muscular d y s t r o p h y , c o n c e n t r a t i o n s being d i m i n i s h e d to a s m a l l e r e x t e n t i n a l e s s advanced case. There was a l s o a d i s p a r i t y between the c o n c e n t r a t i o n s of the "phosphagens", ATP and p h o s p h o c r e a t i n e , i n both the e a r l i e r and advanced c a s e s , the c o n c e n t r a t i o n of phosphocreatine being much lower than ATP. Reinhold and K i n g s l e y (1938) found that c r e a t i n e and g l y c i n e a d m i n i s t r a t i o n f o r up to one year r e s u l t e d i n higher c o n c e n t r a t i o n of phosphorous c o n t a i n i n g compounds i n d y s t r o p h i c m u s c l e , but t h e i r d i s t r i b u t i o n was unchanged. R e g a r d l e s s o f t h e s t a g e o f the d i s e a s e , even b e f o r e f u n c t i o n a l i m p a i r m e n t o c c u r r e d , t h e r e w e r e l o w c o n c e n t r a t i o n s of c r e a t i n e i n c l u d i n g both phosphorylated and u n p h o s p h o r y l a t e d f o r m s . As the d i s e a s e p r o g r e s s e d t o t a l c r e a t i n e became p r o g r e s s i v e l y l o w e r , but the changes i n p h o s p h o c r e a t i n e , s p e c i f i c a l l y , were o f t e n g r e a t e r . A c r e a t i n e d e f i c i e n c y was i n i t i a l l y i n d i c a t e d i n t h e 58 p a t h o g e n e s i s o f the d i s e a s e a c c o r d i n g t o t h e c h e m i c a l c o m p o s i t i o n o f t h e m u s c l e , but a l a s , when a s i m i l a r c o m p o s i t i o n was seen i n amyotrophic l a t e r a l s c l e r o s i s , a c r e a t i n e d e f i c i e n c y as the cause of muscular d e t e r i o r a t i o n i n m u s c u l a r d y s t r o p h y was a b a n d o n e d . A l t h o u g h t h e d i s t r i b u t i o n o f phosphorous c o n t a i n i n g compounds was u s u a l l y normal and the o v e r a l l changes l e s s profound i n neuropathy i n g e n e r a l , no s h a r p d i s t i n c t i o n between myopathy and neuropathy i n terms of chemical composition was c o n s i d e r e d to be p o s s i b l e i n the f i n a l a n a l y s i s . Muscular Dystrophy as an Endocrinopathy A f t e r F o l i n c h a l l e n g e d the view t h a t p r o t e i n was the major s u b s t r a t e of energy metabolism, glucose d i s p l a y e d i t s a b i l i t y to r e v i t a l i z e muscle th a t was exhausted by e x e r c i s e . Hypoglycemia was a l s o found to c o i n c i d e with the f a t i g u e of m u s c l e . By 1910, t h e l i t e r a t u r e c o n c e r n i n g d i a b e t e s m e l l i t u s i n w h i c h h y p e r g l y c e m i a p r e v a i l e d , was q u i t e v o l u m i n o u s . H y p o g l y c e m i a was t h e n a known f e a t u r e of A d d i s o n ' s d i s e a s e , w h i c h a c c o r d i n g t o p o s t m o r t e m examination, was due to adrenal gland d y s f u n c t i o n . P o r g e s ( 1 9 1 0 )4 d e m o n s t r a t e d t h a t h y p o g l y c e m i a a c c o m p a n i e d by g l y c o g e n d e p l e t i o n of the l i v e r was the r e s u l t of a d r e n a l gland e x t i r p a t i o n i n experimental a n i m a l s . C i t e d by U n d e r h i l l and F i n e (1912). 59 Frank and Isaac (1911)5 found t h a t the a d m i n i s t r a t i o n of phosphorous produced the same e f f e c t . U n d e r h i l l and F i n e ( 1 9 1 2 ) d e m o n s t r a t e d t h a t h y d r a z i n e a n t a g o n i z e d t h e h y p e r g l y c e m i a p r o d u c e d by e x t i r p a t i o n of the p a n c r e a s . Knowing tha t hydrazine had the same e f f e c t as phosphorous upon blood glucose and l i v e r glycogen s t o r e s , they o f f e r e d a t e n t a t i v e t h e o r y t o e x p l a i n the way i n which the b l o o d glucose was normally r e g u l a t e d . Although not u n i v e r s a l l y accepted, the hormone t h e o r y , which held that substances were rel e a s e d by endocrine glands i n t o the blood to t r a v e l to target t i s s u e s and exert t h e i r e f f e c t , was w e l l entrenched i n the minds of p h y s i o l o g i s t s . The c o n d i t i o n s consequent to the e x t i r p a t i o n of a gland i n e x p e r i m e n t a l a n i m a l s , which were demonstrable i n blood and u r i n e , c o u l d t h e r e f o r e be a t t r i b u t e d to " f a c t o r s " of a g l a n d . The same a l t e r a t i o n s i n b l o o d and u r i n a r y c o n s t i t u e n t s i n the human i n f e r r e d a glandular d y s f u n c t i o n . U n d e r h i l l and Fine (1912) proposed that f a c t o r s of the a d r e n a l and pancreas served to m a i n t a i n a c o n s t a n t b l o o d g l u c o s e l e v e l . The a d r e n a l f a c t o r would l i b e r a t e glucose from g l y c o g e n s t o r e s i n the l i v e r w h i l e the p a n c r e a t i c f a c t o r would promote the a s s i m i l a t i o n of g l u c o s e by the l i v e r . However, h y p o g l y c e m i a was a l s o a f e a t u r e o f p i t u i t a r y d i s t u r b a n c e s and t h e r e f o r e , such a symptom c o u l d 5C i t e d by U n d e r h i l l and Fine (1912). 60 i m p l i c a t e t h e p i t u i t a r y g l a n d i n i t s r e g u l a t i o n o f carbohydrate metabolism. In 1916 McCrudden and Sargent a s c r i b e d the hypoglycemia measured i n an i n d i v i d u a l diagnosed with muscular dystrophy t o a d e f e c t i v e c a r b o h y d r a t e metabolism. An i n s u f f i c i e n t s u p p l y o f g l u c o s e f o r muscular e f f o r t was b e l i e v e d to be r e s p o n s i b l e f o r muscular weakness and wasting i n muscular d y s t r o p h y and was t h e r e f o r e the u n d e r l y i n g c ause of the d i s e a s e . The r e l a t i o n s h i p between c r e a t i n e and carbohydrate was i n evidence and the presence of c r e a t i n e i n the u r i n e was t h o u g h t t o s u p p o r t a d i s t u r b a n c e i n c a r b o h y d r a t e metabolism. L i k e w i s e , a f a l l i n blood glucose f o l l o w i n g a s h o r t f a s t was c o n s i s t e n t w i t h a d e f e c t i n c a r b o h y d r a t e metabolism. In order to demonstrate that hypoglycemia was not the r e s u l t of i m m o b i l i t y , b l o o d glucose was measured i n p a t i e n t s with a r t h r i t i s . As expected, hypoglycemia was not c o i n c i d e n t to the d i s u s e of muscle i n such c a s e s . Blood c o n c e n t r a t i o n s of a substance c a l l e d " c h o l e s t r i n " were a l s o lower than normal i n the case of McCrudden and S a r g e n t . " C h o l e s t r i n " had been d e t e c t e d i n the e f f e r e n t b l o o d of the a d r e n a l g l a n d . With the a s s o c i a t i o n between hypoglycemia, muscular weakness and d i s t u r b a n c e s i n a d r e n a l and p i t u i t a r y f u n c t i o n , a drenal and p i t u i t a r y e x t r a c t s were p r e s c r i b e d as t r e a t m e n t f o r t h e d i s o r d e r . The f a t t y i n f i l t r a t i o n of the muscles i n muscular dystrophy was a l s o s u g g e s t e d t o be e x p l i c a b l e on t h e b a s i s of an e n d o c r i n e 61 d i s t u r b a n c e e x e m p l i f i e d by P o r g e s1 e x p e r i m e n t s . Upon e x t i r p a t i o n of the a d r e n a l gland the l i v e r l o s t glycogen but gained f a t . Since the s k e l e t a l muscle a l s o s t o r e d g l y c o g e n , the same s i t u a t i o n was c o n c e i v a b l y the consequence o f an a l t e r e d c a r b o h y d r a t e metabolism r e s u l t i n g from e n d o c r i n e gland d y s f u n c t i o n . A c o n s e n s u s had been r e a c h e d c o n c e r n i n g the bone c h a n g e s c o i n c i d e n t t o m u s c u l a r w a s t i n g i n m u s c u l a r d y s t r o p h y , t h a t i s , they were r e c o n c i l e d as b e i n g t h e consequence of d i s u s e . Timme (1917) departed from t h i s view when he d i s c o v e r e d p i n e a l "shadows" i n i n d i v i d u a l s whose bones were more s l e n d e r than normal, had a t r o p h i e d a l o n g w i t h the muscle t h a t moved them. These i n d i v i d u a l s were members a f a m i l y with a h i s t o r y of what was c a l l e d muscular, dystr o p h y . I t extended through three s u c c e s s i v e g e n e r a t i o n s and resembled the c l i n i c a l syndrome of Erb's j u v e n i l e form but was a d u l t i n onset and u n u s u a l l y slow i n i t s c o u r s e . There was s a i d to be a g e n e r a l l a c k of muscle development accompanied by c o n t r a c t i o n s or t i g h t e n i n g of some muscles w h i c h l i m i t e d movement. O c u l a r m u s c l e i m p a i r m e n t was s u p p o s e d t o be t h e r e a s o n f o r the w e a r i n g o f g l a s s e s . A l t h o u g h p i n e a l " s h a d o w s " were f r e q u e n t i n n o r m a l i n d i v i d u a l s they were known to be e v i d e n c e o f c a l c i f i e d b o d i e s i n o l d e r p e r s o n s and t h r e e o f the f o u r p a t i e n t s examined and X-rayed by Timme were under twenty-four years o f a g e . A c c o r d i n g t o o s s e o u s c h a n g e s , o c u l a r m u s c l e 62 impairment and c o n t r a c t u r e s e a r l i e r r e p o r t e d i n d i s e a s e r e s u l t i n g from p i n e a l tumours, muscular dystrophy appeared t o be an e n d o c r i n o p a t h y . L i k e muscular d y s t r o p h y p i n e a l tumours were r e p o r t e d to be more frequent i n males than i n females. Only two of the nine d y s t r o p h i c cases of Janney et a l . (1918) d i s p l a y e d p i n e a l "shadows". However, a l l showed r a r e f a c t i o n of long bones, which, i f muscular dystrophy were an e n d o c r i n o p a t h y , i m p l i c a t e d the t h y r o i d , p a r a t h y r o i d , p i t u i t a r y and gonads. S k e l e t a l changes were a s s o c i a t e d with the d y s f u n c t i o n o f these g l a n d s . Although t h e r e were no known changes d e m o n s t r a b l e i n b l o o d or u r i n e , s t r u c t u r a l c h a n g e s c o u l d o n l y be t h e r e s u l t o f a l t e r n a t i o n s i n metabolism because the endocrine glands were the r e g u l a t o r s of metabolism. Metabolism and endocrinology were t h e r e f o r e c o n s i d e r e d to be i n s e p a r a b l e . O r i g i n a l l y , t h e y were c a l l e d t h e d u c t l e s s g l a n d s because they were b e l i e v e d to d i s c h a r g e substances i n t o the c i r c u l a t i o n , r a t h e r than i n t o an a d j o i n i n g c o n d u i t . The term e n d o c r i n e , a Greek c o n j u n c t i o n of " w i t h i n " and " t o s e p a r a t e " , e v e n t u a l l y c r e p t i n t o the l i t e r a t u r e . One wonders whether t h e name was i n t e n d e d to encompass the interdependence which had suggested i t s e l f by the time Timme (1917) o f f e r e d an e x p l a n a t i o n f o r reputed cures i n muscular d y s t r o p h y . He s u g g e s t e d t h a t t h e c l a i m e d c u r e s o f d y s t r o p h i c females, which were seemingly timed to the f i r s t 63 p e r i o d of menstruation, were brought about by an i n t e r a c t i o n between the o v a r i e s and the p i n e a l . C r e a t i n u r i a and hypoglycemia were fe a t u r e s of a l l nine of Janney et a l . ' s (1918) s e r i e s . In a d d i t i o n to being the metabolic f e a t u r e s of Addison's d i s e a s e and h y p o p i t u i t a r i s m they were a l s o r e p o r t e d i n hypothyroidism or myxedema. The e n d o c r i n e s t a n d p o i n t f o r m u s c u l a r d y s t r o p h y was t h u s s t r e n g t h e n e d . F i v e of the nine excreted glucose f o l l o w i n g t h e a d m i n i s t r a t i o n o f g l u c o s e , t h e r e b y i n d i c t i n g t h e p a n c r e a s a l o n g w i t h t h e o t h e r g l a n d s . W i t h m u l t i p l e c a n d i d a c y Janney et a l . were f o r c e d to d i v i d e f a u l t among the e n d o c r i n e g l a n d s . A h i e r a r c h y was not yet w i t h i n the realms of a theory; the p i t u i t a r y had yet to be d e c l a r e d the m a s t e r g l a n d . However, r e c i p r o c i t y between g l a n d s as s u g g e s t e d by Timme was e v i d e n t t h r o u g h e x t i r p a t i o n e x p e r i m e n t s and muscular d y s t r o p h y was proposed to be a " p l u r i g l a n d u l a r " d i s e a s e . In such a case, d i f f e r e n t glands were c o n c e i v a b l y r e s p o n s i b l e f o r the d i s o r d e r i n d i f f e r e n t i n d i v i d u a l s but t h e r e was a f i n a l common pathway to the i n t e r a c t i o n of glands which r e s u l t e d i n muscular dyst r o p h y . In 1921 B r o c k and Kay r e p o r t e d t h r e e c a s e s , which a c c o r d i n g to m u l t i p l e symptoms, i n v o l v e d d i s t u r b a n c e s of a p l u r i g l a n d u l a r n a t u r e . M u s c u l a r s t r e n g t h , d y s t r o p h y and myotonia accompanied the endocrine symptoms. E x t r a c t s from the t h y r o i d , p i t u i t a r y , adrenals and t e s t i c l e s had a l l shown t h e i r a b i l i t y t o s t i m u l a t e muscular performance which gave 64 weight to the proposed p l u r i g l a n d u l a r nature o f muscular d y s t r o p h y but the f i n a l e f f e c t of the i n t e r a c t i o n c o u l d a p p a r e n t l y vary with respect to the muscular system. Only i n t h e d y s t r o p h i c c a s e d i d h y p o g l y c e m i a c o n c u r w i t h c r e a t i n u r i a . The i n v e s t i g a t i o n of neuropathy as regards changes i n b l o o d a n d u r i n a r y c o n s t i t u e n t s a s s o c i a t e d w i t h e n d o c r i n o p a t h i e s was i n e v i t a b l e . Gibson and M a r t i n (1922) f o u n d h y p o g l y c e m i a w i t h no c o n c o m i t a n t c r e a t i n u r i a i n myasthenia g r a v i s w h i l e c r e a t i n u r i a was unaccompanied by hypoglycemia i n p r o g r e s s i v e muscular atrophy. Hypoglycemia and c r e a t i n u r i a were the m e t a b o l i c c o n s t a n t s o f a l l cases with pseudohypertrophic muscular dystrophy. Although Janney et a l . (1918) concluded t h a t muscular dystrophy was a p l u r i g l a n d u l a r d i s e a s e , one case e x h i b i t e d symptoms which were i n t e r p r e t e d as strong evidence i n favour of the p i t u i t a r y as the primary c a u s a t i v e g l a n d . The head appeared to be d i s p r o p o r t i o n a t e l y l a r g e , the jaw was w e l l -developed, the growth of the b o d i l y h a i r was c o n s i d e r e d to be abnormally e x c e s s i v e , the f a c i a l muscle was h y p e r t r o p h i e d and t h e tongue g r e a t l y e n l a r g e d . These symptoms were i n c o r r e c t l y c o n s i d e r e d to be those of acromegaly f o r which a tumour of the a n t e r i o r lobe of the p i t u i t a r y was known to be i n v o l v e d . Wright (1925) a l s o s i n g l e d out the p i t u i t a r y as being a e t i o l o g i c a l l y s i g n i f i c a n t i n muscular dystrophy a c c o r d i n g 65 t o r e p u t e d p i t u i t a r y symptoms, i n c l u d i n g an u n d e r - s i z e d s e l l a t u r c i c a , r a r e f a c t i o n of the bony t i s s u e s , a round head and f a c e , s p a c e d t e e t h , heavy c e n t r a l i n c i s o r s , smooth m o t t l e d s k i n , poor n a i l s and s e x u a l under-development i n n i n e t e e n d y s t r o p h i c i n d i v i d u a l s . A weak p u l s e i n t h e nineteen cases was b e l i e v e d to be evidence of a d i s t u r b a n c e i n the p o s t e r i o r lobe of the p i t u i t a r y , s p e c i f i c a l l y , and t h e o v e r a l l symptoms i n v o k e d t r e a t m e n t w i t h p i t u i t a r y e x t r a c t s . The h i s t o l o g i c a l f e a t u r e s of the b i o p s i e s f o r t h e s e c a s e s was c o n s i s t e n t w i t h the d i a g n o s i s ; but i t i s p o s s i b l e t h a t they were i n d i v i d u a l s with myotonic dystrophy i n which the myotonia was not e v i d e n t . Mental d e f i c i e n c y was common among the n i n e t e e n , twelve of whom were males and s e x u a l underdevelopment i s a l s o c ompatible w i t h myotonic d y s t r o p h y . I n a n y e v e n t w h e t h e r s y m p t o m s w e r e m i s i n t e r p r e t e d or c a s e s were m i s d i a g n o s e d , the symptoms a s s o c i a t e d w i t h a p i t u i t a r y d i s t u r b a n c e were many. However, knowledge of the physiology of the p i t u i t a r y was inadequate to e x p l a i n the m u l t i p l i c i t y of syndromes documented by 1925. With the d i s c o v e r y of the p a n c r e a t i c f a c t o r , i n s u l i n , i n 1925, the hormone theory was e s s e n t i a l l y proven. During the f o l l o w i n g f i f t e e n y e a r s the s c i e n c e of e n d o c r i n o l o g y advanced very s w i f t l y . By 1940, the a n t e r i o r p i t u i t a r y had been determined to be the source of t r o p h i c f a c t o r s which r e g u l a t e d other glands and t h e r e f o r e , the a n t e r i o r p i t u i t a r y 6 6 was the master g l a n d . S t e r o i d hormones were a l s o among the g l a n d u l a r f a c t o r s to be d i s c o v e r e d during t h i s time p e r i o d . During the l a t e 1930's the s t e r o i d hormones, g e n e r a l l y , and the male sex hormone, t e s t o s t e r o n e , p a r t i c u l a r l y , was found to have an e f f e c t upon the growth and development of t h e m u s c u l a r s y s t e m . Shank e t a l . (1944) f o u n d t h e c r e a t i n u r i a of c h i l d h o o d to drop f o l l o w i n g i n t r a m u s c u l a r i n j e c t i o n s of t e s t o s t e r o n e for a p e r i o d of ten to f o u r t e e n d ays w h i c h was i n t e r p r e t e d as i n c r e a s e d d e p o s i t i o n o f c r e a t i n e i n the muscle. T h i s o b s e r v a t i o n was made b e f o r e the c r e a t i n u r i a of muscular dystrophy was d i s m i s s e d as a consequence of muscle d e g e n e r a t i o n and the e f f e c t of the hormone upon the most common form of m u scular d y s t r o p h y a f f l i c t i n g males d u r i n g c h i l d h o o d was i n v e s t i g a t e d . The c r e a t i n u r i a of m o d e r a t e l y a d v a n c e d d y s t r o p h y was n o t i n f l u e n c e d by the hormone but symptoms were the same as i n normal c h i l d r e n , that i s , there was a rebounding of c r e a t i n e e x c r e t i o n to a l e v e l t h a t was h i g h e r than the o r i g i n a l . When m e t h y l t e s t o s t e r o n e was a d m i n i s t e r e d o r a l l y t o d y s t r o p h i c c h i l d r e n c r e a t i n e and g u a n i d i n e a c e t i c a c i d l e v e l s i n the u r i n e r o s e , r e t u r n i n g to p r e v i o u s l e v e l s upon c e s s a t i o n o f a d m i n i s t r a t i o n , but the e f f e c t was s i m p l y a t t r i b u t e d t o s t i m u l a t i o n of c r e a t i n e s y n t h e s i s . In a s u b s e q u e n t s t u d y H o a g l a n d et a l . (1945) c o n f i r m e d t h e e f f e c t s of methyl t e s t o s t e r o n e upon c r e a t i n u r i a and noted the l a c k of e f f e c t upon the c r e a t i n i n e output as w e l l . 67 By 1950, the o u t p u t o f 1 7 k e t o - s t e r o i d s was know to i n d i c a t e t e s t o s t e r o n e p r o d u c t i o n i n males. T y l e r and P e r k o f f (1951) and Danowski et a l (1956a) measured a normal output which r e f l e c t e d the normal development o f e x t e r n a l g e n i t a l i a and secondary sex c h a r a c t e r i s t i c s of d y s t r o p h i c i n d i v i d u a l s . The m a l e n e s s o f m u s c u l a r d y s t r o p h y was not r e l a t e d t o s t e r o i d hormone p r o d u c t i o n by the gonads. The adrenal c o r t e x , i t had been l e a r n e d , r e g u l a t e d the blood e l e c t r o l y t e s i n c l u d i n g sodium, potassium, c a l c i u m and phosphate and t h e r e f o r e t h e i r c o n c e n t r a t i o n s were an index o f a d r e n a l c o r t i c a l f u n c t i o n . The a n t e r i o r p i t u i t a r y hormone, a d r e n o c o r t i c o t r o p h i c hormone, was i s o l a t e d and the r e s p o n s e o f t h e b l o o d e l e c t r o l y t e c o n c e n t r a t i o n t o i t s a d m i n i s t r a t i o n was a n o t h e r i n d e x o f a d r e n a l c o r t i c a l f u n c t i o n . T y l e r and P e r k o f f (1951) and Danowski et a l . (1956a) r e p o r t e d the a d r e n a l c o r t e x as b e i n g normal i n muscular dystrophy a c c o r d i n g to such t e s t s . The nerve a c t i v a t e s the muscle t o c o n t r a c t which i n tu r n moves the s k e l e t o n and thus i n the same way that nerve and muscle a r e i n s e p a r a b l e there i s a c l o s e r e l a t i o n s h i p between bone and muscle i n motion. I t i s f o r t h i s reason th a t the osseous changes i n muscular dystrophy brought some c o n t r o v e r s y . The debate had been q u i e t e d u n t i l Timme (1917) p l a c e d the muscular as w e l l as the s k e l e t a l changes at the f o o t , as i t were, of the p i n e a l g l a n d . I t i s very d i f f i c u l t t o s a y whether the c a s e s of Timme were t r u l y a t y p e of 68 muscular dystrophy but if one considers the fact that the composition of the urine was normal as regards creatine and creatinine one might be inclined to think not. Pineal "shadows" were not a consistent feature of those involved in the study of the disease as an endocrinopathy, in fact, they were rare. Girdany and Danowski (1956) reported that there were no pineal calcifications in definitely dystrophic children which refuted the original conclusion of Timme because by this time diagnostic indications had come to include the elevation of serum aldolase 'which was quite striking in muscular dystrophy. In any case, the question as to the source of the-skeletal changes resurfaced: Epstein and Abramson (1941) reported alternations in the X-rays of seven patients diagnosed with the disease to include a reduction in the size of the scapula, larger humeral heads and smaller glenoid fossa, slender humeral shafts. Similar changes were seen in the femur, tibia, fibula, radius and ulna. The pelvis was small with flaring iliac bones and ischial spines in those who had been wheelchair bound for many years and these changes were considered to be the result of immobility. However, there was no correlation between the girth of muscle and bone, the bones of pseudohypertrophied limbs sometimes being slender while normal architecture of bone was seen in atrophied limbs. There was no correlation between the ability to move the skeleton and its structural 69 changes which were said to be unlike those of poliomyelitis. Aside from the specific changes associated with confinement to a wheelchair, the changes were not in the opinion of Epstein and Abramson, explicable on the basis of disuse alone. Girdany and Danowski (1956) disagreed when they found a positive correlation between muscular weakness and the degree of scoliosis, caninization, overtubulation and coaxa valga in dystrophic children. The opinion was that these skeletal abnormalities were consistent with disuse and immobility. Tyler and Perkoff (1951) found narrow shafts, thin cortices and r a r i f i e d heads in the humerus of dystrophic individuals. They were not willing to dismiss the changes as being due to disuse, but the structural changes of the bone were not the same, not as severe as that seen in disease of the parathyroid for which the bone was determined to be the primary target tissue and the function of which became assessable according to blood concentrations of calcium and phosphorous. The serum levels of both were measured as being normal by Tyler and Perkoff and although they were somewhat elevated in those assessed by Danowski et a l . (1956a) the pattern of elevation was not one of any endocrine d i s o r d e r . Despite a complete r e s o l u t i o n concerning the exact nature and source of the skeletal changes in muscular dystrophy, the endocrine system, the parathyroid specifically, was dismissed as the culprit. 70 Hypoglycemia consequent to e x t i r p a t i o n of the t h y r o i d was of course the r e s u l t of a d e c e l e r a t i o n of a l l m e t a b o l i c p r o c e s s e s . The b a s a l m e t a b o l i c r a t e was c o n s i s t e n t l y measured as being subnormal i n d y s t r o p h i c i n d i v i d u a l s but the r e d u c t i o n i n m uscular mass was c o n s i d e r e d to be the reason f o r the slower r a t e and the t h y r o i d was concluded to be f u n c t i o n i n g n o r m a l l y (Shank et a l . , 1944; T y l e r and P e r k o f f , 1951; Danowski et a l . , 1956a). The hyperglycemia o f t h e p a t i e n t s o f Hughes and S h r i v a s t a v a (1927) was r e s i g n e d as being the r e s u l t of a s m a l l e r u t i l i z a t i o n and a b i l i t y o f the muscle t o s t o r e g l u c o s e and t h e r e f o r e a c o n s e q u e n c e o f m u s c u l a r d e g e n e r a t i o n . L i k e w i s e , t h e d e c r e a s e d responses to i n s u l i n r e p orted by Danowski et a l . (1956) were r e c o n c i l e d as b e i n g s e c o n d a r y t o m u s c u l a r d e t e r i o r a t i o n . The theory of U n d e r h i l l and Fine (1911) had proven to be c o r r e c t . That i s , the p a n c r e a t i c f a c t o r , i n s u l i n , kept the blood glucose low and the l i v e r glycogen high while the a d r e n a l f a c t o r which turned out to be a c o r t i c a l s t e r o i d promoted the d e g r a d a t i o n of l i v e r c a r b o h y d r a t e s t o r e s to e l e v a t e t h e b l o o d g l u c o s e . O p p o s i t e e f f e c t s o f t h e p a n c r e a t i c and a d r e n a l f a c t o r s thus s e r v e d to m a i n t a i n a c o n s t a n t l e v e l of blood g l u c o s e . However, i t would appear that r e s t i n g blood g l u c o s e i s not always a r e l i a b l e index of c a r b o h y d r a t e m e t a b o l i s m i n i n d i v i d u a l s beset by muscular d i s e a s e by v i r t u e o f t h e r o l e o f g l u c o s e i n e n e r g y 71 m e t a b o l i s m . The p a t i e n t s of Danowski et a l . (1956) were normal i n terms of blood glucose while a l l those of Shank et a l . and T y l e r and P e r k o f f (1951) had low f a s t i n g b l o o d g l u c o s e . I suspect that the o r i g i n a l hypoglycemic p a t i e n t of McCrudden and S a r g e n t ' s was m i s d i a g n o s e d because the c r e a t i n i n e output was normal i n t h i s c a s e . N e v e r t h e l e s s , the nine s u b j e c t s of Janney et a l . (1918), at l e a s t seven of whom I b e l i e v e were diagnosed c o r r e c t l y , a l l had low f a s t i n g blood g l u c o s e . A low f a s t i n g blood glucose may be r e l a t e d to the degeneration of the muscle i n muscular dystrophy and i t s r e d u c e d c a p a c i t y t o s t o r e g l y c o g e n w h i c h w o u l d c o n t r i b u t e t o f a s t i n g b l o o d g l u c o s e . L i k e w i s e , t h e g l u c o s u r i a reported i n the cases of Janney et a l . who were i n advanced stages may have been caused by the r e d u c t i o n i n muscular mass and a s m a l l e r need f o r g l u c o s e . The responses of the p a t i e n t s of Shank et a l . (1944), T y l e r and P e r k o f f (1951) and Danowski et a l . (1956) to o r a l l y or i n t r a v e n o u s l y a d m i n i s t e r e d glucose which were measured according to blood c o n c e n t r a t i o n s were a l l w i t h i n normal l i m i t s . Carbohydrate metabolism was deemed to be u n d i s t u r b e d . In 1956(c) Danowski et a l . t r e a t e d d y s t r o p h i c c h i l d r e n w i t h a p r e p a r a t i o n of human p i t u i t a r y growth f a c t o r , which caused the resumption of growth i n immature mice from which the p i t u i t a r y had been removed. T h e r e was no b e n e f i t f o l l o w i n g such treatment. Thus, t h i s p a r t i c u l a r f a c t o r of the a n t e r i o r p i t u i t a r y , an excess of which causes acromegaly 72 i n a d u l t h o o d , c o u l d not be r e s p o n s i b l e f o r the d i s o r d e r . S i n c e t h e o t h e r e n d o c r i n e g l a n d s were a s s e s s e d a s f u n c t i o n i n g p r o p e r l y , the p i t u i t a r y was a l s o r e l i e v e d of any r e s p o n s i b i l i t y as the c a u s a t i v e g l a n d . The study o f muscular d y s t r o p h y as an en d o c r i n o p a t h y was l a u n c h e d by w h a t , i n my e s t i m a t i o n , were t h e m i s d i a g n o s e d c a s e s of McCrudden and Sargent i n 1916 and Timme i n 1917 a t a t i m e when both, e n d o c r i n o l o g y and met a b o l i s m were immature s c i e n c e s . In n e i t h e r case was th e r e any mention made of a muscle biopsy but because the muscle p r e s e n t s a d y s t r o p h i c h i s t o l o g y i n some e n d o c r i n e d i s o r d e r s the b i o p s y may not have a l t e r e d the d i a g n o s e s . M u s c u l a r d i s e a s e can be a c o n s e q u e n c e o f p a r a t h y r o i d i n s u f f i c i e n c y which may mimic muscular d y s t r o p h y but the serum l e v e l s o f c a l c i u m and phosphorous must be used t o c o n f i r m the d i a g n o s i s . Postmortem examination w i l l r e v e a l p a t h o l o g y o f the gland which was a p o i n t i n d i s f a v o u r of muscular d y s t r o p h y as an endocrinopathy i n that there were ver y few p o s i t i v e n e c r o p s i c r e p o r t s f o r those diagnosed as d y s t r o p h i c . A d y s t r o p h i c h i s t o l o g y i s a l s o seen i n i n h e r i t e d d i s e a s e o f c a r b o h y d r a t e metabolism. W h i l s t the knowledge and u n d e r s t a n d i n g of the e n d o c r i n e system expanded w i t h methods f o r a s s e s s i n g the f u n c t i o n o f g l a n d s becoming a v a i l a b l e , carbohydrate metabolism was determined to i n v o l v e an o r d e r e d sequence of b i o c h e m i c a l r e a c t i o n s governed by 73 enzymes. Over t i m e , t h e r e f o r e , j u s t as t h e n e r v e was m e t a p h o r i c a l l y s e v e r e d f r o m t h e m u s c l e o f m u s c u l a r dystrophy, endocrinology and metabolism were d i s t a n c e d . As t h e m u s c l e depends upon t h e n e r v e f o r i t s v i a b i l i t y , metabolism i s r e g u l a t e d by the endocrine g l a n d s . However, as t h e m u s c l e has a l i f e o f i t s own, m e t a b o l i s m i s a d i s t i n c t e n t i t y . The s e p a r a t i o n between endocrinology and m e t a b o l i s m was i n d i c a t e d by T y l e r and P e r k o f f (1951) who asked: Is p r o g r e s s i v e muscular dystrophy an endocrine or metabolic d i s o r d e r ? At the end of t h e i r i n v e s t i g a t i o n they found no s u p p o r t f o r a p r i m a r y endocrine d i s t u r b a n c e and c o n c l u d e d t h a t c r e a t i n u r i a was t h e o n l y m e t a b o l i c abnormality i n the d i s e a s e . S h o r t l y t h e r e a f t e r c r e a t i n u r i a was determined to be an epiphenomenon. Muscular Dystrophy as a Neuropathy By t h e end o f t h e n i n e t e e n t h c e n t u r y , postmortem examination had demonstrated th a t t r i v i a l changes i n a s p e c t s o t h e r than the s p i n a l motor neurons o c c u r r e d i n muscular dystr o p h y . Gowers (1902) regarded these t r i f l i n g changes as b e i n g i n s i g n i f i c a n t : they were "thought to suggest t h a t some day i t may be d i s c o v e r e d the muscular c o n d i t i o n depends a f t e r a l l on a morbid s t a t e of the s p i n a l c o r d . The idea i s a r e l i c of the o l d f a n c y , f o r i t was nothing more that t h e r e a r e i n t h e s p i n a l c o r d s p e c i a l t r o p h i c c e n t r e s f o r the muscles apart from the motor c e l l s of the a n t e r i o r cornua. 74 I t i s a d o c t r i n e t h a t s h o u l d have d i e d of i n a n i t i o n l o n g ago, p u r e l y h y p o t h e t i c a l , every d e f i n i t e f a c t i s opposed to. i t . " Before there was knowledge concerning the e x i s t e n c e of p h o s p h o c r e a t i n e and i t s r o l e i n the c h e m i s t r y of muscular c o n t r a c t i o n , muscle tonus was b e l i e v e d to be the e q u i v a l e n t of " p o s t u r a l c o n t r a c t i o n " . That i s , tonus was thought to i n v o l v e the c o n t r a c t i o n of muscle used i n the maintenance of p o s t u r e or f i x a t i o n of the l i m b s , r a t h e r than c o n t r a c t i o n used i n t h e i r m o t i v a t i o n . In 1910, P e k e l h a r i n g and Van Hoogenhuyze6 put f o r t h what some regarded as an i n g e n i o u s theory p e r t a i n i n g to tonus which stemmed from experiments on c a t s i n which c o n n e c t i o n s between the m i d b r a i n and s p i n a l c o r d were s e v e r e d . The r e s u l t a n t c o n d i t i o n , d e c e r e b r a t e r i g i d i t y , was c o n s i d e r e d to be tonus i n e x a g g e r a t i o n . I f the p e r i p h e r a l nerves were cut as w e l l , to make the muscle e n t i r e l y f l a c c i d , s u c h m u s c l e was low i n c r e a t i n e as compared to the muscles i n a r i g i d s t a t e . The lower content of the f l a c c i d muscle was not i n t e r p r e t e d as being due to a l o s s of c r e a t i n e . R a t h e r , the h e i g h t e n e d content o f the t a u t muscle was proposed to be a c h e m i c a l product of the p h y s i o l o g i c a l phenomenon of tonus. Rigor m o r t i s and the prolonged c o n t r a c t u r e of e x c i s e d f r o g muscle induced by the a p p l i c a t i o n of chemicals such as 6C i t e d by Hunter (1928). 75 n i c o t i n e , c a f f e i n e , v e r a t r i n e , calcium c h l o r i d e and sodium thiocyanate i n conjunction with e l e c t r i c a l s t i m u l a t i o n were a l s o thought to approach p h y s i o l o g i c a l tonus. The g r e a t e r c r e a t i n e measured i n r i g o r mortis and prolonged c o n t r a c t u r e as o p p o s e d t o m u s c l e t h a t had j u s t been e l e c t r i c a l l y s t i m u l a t e d to c o n t r a c t supported the theory that c r e a t i n e was a m e t a b o l i c product of tonus. In accordance with the f o r m a t i o n of c r e a t i n e through a process s p e c i a l to muscle tonus, the lower and higher blood c r e a t i n e a s s o c i a t e d with dementia precox or m e l a n c h o l i a and c a t a t o n i c r i g i d i t y , r e s p e c t i v e l y , were a l s o thought to support the r e l a t i o n s h i p between c r e a t i n e and muscle ten s i o n (Looney, 1924). In 1916 R e i s s e r7 found that muscle c r e a t i n e content was u n a f f e c t e d by the a p p l i c a t i o n of c u r a r e t o somatic nerve endings w h i l e a d r e n a l i n and c a f f e i n e i n c r e a s e d content i n p r o p o r t i o n t o t h e i r a b i l i t y to e x c i t e symptoms o f t h e s y m p a t h e t i c nervous system. The e f f e c t s of s y m p a t h e t i c e x c i t a n t s o c c u r r e d even i n c u r a r i z e d animals but not when c e n t r a l - s y m p a t h e t i c output was prevented by nerve s e c t i o n . There seemed to be a c o n n e c t i o n between c r e a t i n e and the sympathetic nervous system which of course was one based on blood flow and the s t i m u l a t i o n of metabolism, but there was e v i d e n c e t h a t the s k e l e t a l , muscle i t s e l f r a t h e r than the b l o o d v e s s e l s s e r v i n g i t was i n n e r v a t e d by s y m p a t h e t i c C i t e d by Hunter, (1928). 76 n e r v e s as w e l l as s o m a t i c . T h e r e f o r e , i f c r e a t i n e was a p r o d u c t o f tonus and i t s p r o d u c t i o n was governed by the sympathetic n e r v e s , then i t f o l l o w e d t h a t the s y m p a t h e t i c nerves were r e s p o n s i b l e f o r tonus which i s p r e c i s e l y what had been s u g g e s t e d upon d i s c o v e r y o f the apparent d u a l i n n e r v a t i o n of the s k e l e t a l muscle. The p u t a t i v e double i n n e r v a t i o n of the s k e l e t a l muscle by somatic and sympathetic nerves was o r i g i n a l l y based on the experiments of Boeke (1909-1911).8 C r a n i a l nerves were severed near the p l a c e s where they emerged from the c e n t r a l nervous system. A f t e r three and a h a l f days which was the time a l l o w e d f o r nerve d e g e n e r a t i o n , the s k e l e t a l muscle i n n e r v a t e d by the corresponding nerves was examined. Using a s i l v e r s t a i n i n g t e c h n i q u e , nonmyelinated nerve f i b r e s were s a i d t o be observed i n the s u p e r i o r o b l i q u e muscle of the eye i n the cat and the s k e l e t a l muscle f i b r e s of the tongue i n the hedgehog. Because s y m p a t h e t i c nerve f i b r e s were nonmyelinated Boeke suggested that the nonmyelinated f i b r e s w h i c h he c l a i m e d t e r m i n a t e d i n s k e l e t a l m uscle f i b r e s belonged to the sympathetic nervous system and were perhaps the anatomical substratum of tonus. F u r t h e r i n v e s t i g a t i o n , i n which both v e n t r a l and d o r s a l r o o t s of the c e r v i c a l or t h o r a c i c p o r t i o n s of the s p i n a l cord were severed i n c a t s , a p p a r e n t l y confirmed the e x i s t e n c e of nonmyelinated f i b r e s C i t e d by Hinsey (1934). 77 t e r m i n a t i n g i n s k e l e t a l m u s c l e . Such f i b r e s were not demonstrable when the thoracico-lumbar sympathetic trunk was removed. I t was t h e r e f o r e concluded that a s k e l e t a l muscle f i b r e r e c e i v e d both somatic and sympathetic i n n e r v a t i o n s , the s y m p a t h e t i c f i b r e b e i n g an " a c c e s s o r y " f i b r e which imparted a t o n i c f u n c t i o n t o the muscle. H u n t e r (1925) d i s a g r e e d w i t h t h e n o t i o n t h a t two d i f f e r e n t nerve f i b r e s were i n n e r v a t i n g the same muscle f i b r e . In no case of examination of the nerves p r o c e e d i n g to the l i m b s of the goat and fowl c o u l d he f i n d a muscle f i b r e which r e c e i v e d both myelinated and nonmyelinated nerve f i b r e s . A l though i t was not e s t a b l i s h e d , he f e l t that i n view of the two types of s t r i a t e d muscle i n mammals i t was c o n c e i v a b l e t h a t a p a r t i c u l a r muscle may be comprised of d i f f e r e n t f i b r e t y p e s . He p r o p o s e d t h a t the mode o f i n n e r v a t i o n of the s k e l e t a l muscle was one i n which f i b r e s w h i c h were r e s p o n s i b l e f o r " p l a s t i c " t o n e r e c e i v e d a n o n m y e l i n a t e d s y m p a t h e t i c e f f e r e n t and the " c o n t r a c t i l e " f i b r e s were a c t i v a t e d by myelinated somatic e f f e r e n t s . The mode of s k e l e t a l muscle i n n e r v a t i o n proposed by Hunter was s u g g e s t e d as p r o v i d i n g a new avenue t h r o u g h w h i c h t h e p a t h o l o g y of muscular dystrophy could be s t u d i e d , which of c o u r s e meant t h a t postmortem e x a m i n a t i o n s h o u l d not be l i m i t e d t o the somatic motor nerves and v e n t r a l horns of the s p i n a l c o r d . Regardless of whether sympathetic nerves were i n n e r v a t i n g the same or separate muscle f i b r e s , though, the 78 s u p p o s e d s y m p a t h e t i c i n n e r v a t i o n o f the s k e l e t a l muscle c o u l d not be ignored as regards the pathogenesis of muscular d y s t r o p h y . B o e k e and H u n t e r d i s a g r e e d upon a n o t h e r p o i n t c o n c e r n i n g t h e c l a i m e d s y m p a t h e t i c i n n e r v a t i o n o f t h e s k e l e t a l muscle. While the l a t t e r b e l i e v e d that the muscle s p i n d l e r e c e i v e d a s y m p a t h e t i c e f f e r e n t , the former was c o n v i n c e d of the s o m a t i c i n n e r v a t i o n of the i n t r a f u s a l m u s c l e f i b r e . Gowers (1896) had s p e c u l a t e d a b o u t the p o s s i b l e r o l e of the muscle s p i n d l e i n the pathogenesis of m u s c u l a r d y s t r o p h y by v i r t u e o f i t s l o c a t i o n i n t h e i n t e r s t i c e s of the e x t r a f u s a l muscle f i b r e s where c o n n e c t i v e t i s s u e m u l t i p l i c a t i o n was o c c u r r i n g . C o n n e c t i v e t i s s u e p r o l i f e r a t i o n seemed to support the e x i s t e n c e of a t r o p h i c d i s t u r b a n c e i n m u s c u l a r d y s t r o p h y . In a d d i t i o n t o the proposed f u n c t i o n of the sympathetic nervous system i n the p r o d u c t i o n of tonus, i t was a l s o suggested that sympathetic n e r v e s might e x e r t a t r o p h i c i n f l u e n c e upon the m u s c l e . Bramwell (1925) suggested that perhaps the muscle death and c o n n e c t i v e t i s s u e abundance i n muscular dystrophy were the consequences of a l a c k or absence of a t r o p h i c i n f l u e n c e s u p p l i e d by t h e s y m p a t h e t i c n e r v e s . He r e g a r d e d the a c c e p t a n c e o f m u s c u l a r d y s t r o p h y as b e i n g a " n e g a t i v e c o n c e p t i o n b a s e d on n e g a t i v e f i n d i n g s i n t h e n e r v o u s s y s t e m " . L i k e H u n t e r he a d v o c a t e d a more e x t e n s i v e examination of the nervous system i n the d i s e a s e . 79 Kure and h i s Japanese coworkers devoted much of t h e i r r e s e a r c h between 1920 and 1931 t o t h e s t u d y o f t h e sympathetic nervous system. They were i n no doubt about the s y m p a t h e t i c i n n e r v a t i o n of the s k e l e t a l muscle and the r e s u l t s of a study i n 1922 apparently supported the r o l e of c r e a t i n e as a p r o d u c t of t o n u s . Both t r o p i c and t o n i c f u n c t i o n s s u g g e s t e d to be governed by the s y m p a t h e t i c i n n e r v a t i o n were i n e v i d e n c e i n m u s c u l a r d y s t r o p h y . C o n n e c t i v e t i s s u e p r o l i f e r a t i o n o c c u r r e d i n m u s c l e s , the muscles i n v o l v e d i n f i x a t i o n of the limbs being the most v u l n e r a b l e . Kure et a l .9 reported a t r o p h i c and d y s t r o p h i c c h a n g e s i n c l u d i n g c o n n e c t i v e t i s s u e p r o l i f e r a t i o n and h y p e r t r o p h i e d f i b r e s , i n the muscle o f an e x p e r i m e n t a l animal from which the sympathetic i n n e r v a t i o n was b e l i e v e d t o be r e m o v e d . S i m i l a r h i s t o l o g i c a l c h a n g e s were s u b s e q u e n t l y observed i n muscle a f t e r the n e r v e s of the p o s t e r i o r r o o t s were s e c t i o n e d . A p o r t i o n of the nerve emanating from the d o r s a l roots were claimed to be composed of e f f e r e n t f i b r e s by t h i s group of workers. Kure et a l . b e l i e v e d they had v e r i f i e d t h i s c l a i m i n 1931 but b e f o r e t h i s t i m e t h e y were c e r t a i n t h a t the p o s t e r i o r r o o t s c o n t r i b u t e d t o the t r o p h i c s t a t e of the m u s c l e . These a l l e g e d e f f e r e n t f i b r e s o f the d o r s a l r o o t were termed " p a r a s y m p a t h e t i c " because they " a i d e d and a b e t t e d " the 9C i t e d by Tower (1931a). 80 t r o p h i c f u n c t i o n o f the s y m p a t h e t i c f i b r e s . U n l i k e the sympathetic f i b r e s , which were c o n f i n e d to the t h o r a c i c o -lumbar p o r t i o n s of the s p i n a l c o r d , the "p a r a s y m p a t h e t i c " f i b r e s were s a i d to be demonstrated i n a l l segments of the s p i n a l c o r d . Postmortem examination went beyond the p e r i p h e r a l motor nerves and s p i n a l c o r d i n muscular d y s t r o p h y . Westphal ( 1 9 2 7 )1 0 r e p o r t e d l e s i o n s i n bot h the s p i n a l c o r d and b r a i n s t e m i n a case d i a g n o s e d as d y s t r o p h i c who s u f f e r e d from symptoms a t t r i b u t a b l e to the b r a i n , i n c l u d i n g a t h e t o t i c and c h o r e i f o r m movements. Changes i n the b a s a l g a n g l i a were documented i n another case of supposed dystr o p h y by Henke and Seeger (1927).1 1 On the b a s i s of these two cases along with an e a r l i e r one i n which degenerative changes were found i n the m i d b r a i n , (Rocas and C r u t c h e t , 1908)1 2 and on the f o u n d a t i o n o f Kure et a l . ' s work, another h y p o t h e s i s was f o r m e d . T h a t m u s c u l a r d y s t r o p h y was t h e r e s u l t o f a d e g e n e r a t i v e l e s i o n i n the m i d b r a i n which was the t r o p h i c c e n t r e f o r the autonomic nervous system was the h y p o t h e s i s . I f t h i s was s o , i t seemed re a s o n a b l e to Hough (1931) tha t the endocrine symptoms r e p o r t e d i n the disease were due to a deranged autonomic i n n e r v a t i o n of the endocrine g l a n d s . The 'Cited by Hough (1931). C i t e d by Hough (1931). C i t e d by Hough (1931). 81 endocrine glands were known to f u n c t i o n with v a r y i n g degrees o f dependence upon t h e i r i n n e r v a t i o n i n c o n t r a s t t o the s k e l e t a l muscle which was c o m p l e t e l y dependent upon the s o m a t i c nerve f o r f u n c t i o n . A t r o p h i c f u n c t i o n f o r the sympathetic or autonomic nerve with r e s p e c t to the s k e l e t a l muscle was seemingly c o n s i s t e n t . A c c o r d i n g to experiments which t e s t e d the a b i l i t y of a d r e n a l i n and p i l o c a r p i n e to produce autonomic symptoms i n e x p e r i m e n t a l a n i m a l s , Kure and O k i n a k a1 3 d e v e l o p e d a t r e a t m e n t f o r m u l a f o r m u s c u l a r d y s t r o p h y . A d r e n a l i n comprised the g r e a t e r p r o p o r t i o n of the mixture which was f i r s t u s e d t o t r e a t d y s t r o p h i c i n d i v i d u a l s i n 1930. S u b c u t a n e o u s i n j e c t i o n s o f a d r e n a l i n plus^ p i l o c a r p i n e improved the s t r e n g t h of d y s t r o p h i c muscle only a few hours f o l l o w i n g t r e a t m e n t and l a s t e d as l o n g as t w e l v e h o u r s , h a v i n g a c u m u l a t i v e e f f e c t w i t h r e p e a t e d i n j e c t i o n s . A s t o u n d i n g r e s u l t s were seen i n a g i r l who, a f t e r f o r t y i n j e c t i o n s w a l k e d f o u r or f i v e k i l o m e t e r s w i t h o u t her c r u t c h e s and a boy, who a p p a r e n t l y resumed h i s work as a t i l l e r o f t h e s o i l f o l l o w i n g f i f t y i n j e c t i o n s . U n f o r t u n a t e l y , symptomatic improvement was p r o p o r t i o n a l to the stage of the d i s e a s e , that i s , improvement was g r e a t e r i n the l e s s advanced c a s e s . Even though there were s t r i k i n g t h e r a p e u t i c r e s u l t s Kure and Okinaka d i d not b e l i e v e that C i t e d by Hough (1931). 82 dystrophy was completely c u r a b l e by t h e i r treatment. Hough (1933) was i n a g r e e m e n t i n t h a t a l l o f h i s p a t i e n t s b e n e f i t e d from a d r e n a l i n p l u s p i l o c a r p i n e but the treatment was not c u r a t i v e . T h e t r o p h i c i n f l u e n c e o f s y m p a t h e t i c a n d " p a r a s y m p a t h e t i c " n e r v e s was d i s p r o v e n almost as soon as treatment with a d r e n a l i n and p i l o c a r p i n e began. When Tower (1931a) removed e i t h e r the s t e l l a t e g a n g l i o n , the d o r s a l r o o t s or both t o g e t h e r i n e x p e r i m e n t a l animals the l i m b s r e t a i n e d t h e i r g i r t h and the muscle showed no g r o s s or m i c r o s c o p i c a l changes suggestive of atrophy or d y s t r o p h y . The t h e o r y o f t h e s y m p a t h e t i c i n n e r v a t i o n o f the s k e l e t a l muscle f i b r e was a l s o d i s p r o v e n by Tower (1931b). W i t h the a p p l i c a t i o n o f t h r e e m o r p h o l o g i c a l s t a i n i n g t e c h n i q u e s e m p l o y i n g s a l t s o f s i l v e r , g o l d c h l o r i d e or m e t h y l e n e b l u e , no e v i d e n c e f o r t h e t e r m i n a t i o n o f s y m p a t h e t i c or "parasympathetic" f i b r e s c o u l d be found i n s k e l e t a l m u s c l e . The o n l y c h a n g e s i n t h e t e r m i n a l i n n e r v a t i o n of the e x t r a f u s a l muscle f i b r e s were those that o c c u r r e d f o l l o w i n g s e c t i o n o f the v e n t r a l r o o t s . Upon d o r s a l root s e c t i o n only the a f f e r e n t f i b r e s of the muscle s p i n d l e were i n absence thereby r e f u t i n g the a s s e r t i o n that the d o r s a l roots i n c l u d e d e f f e r e n t f i b r e s . The a r b o r i z a t i o n of the somatic nerve f i b r e and i t s i n n e r v a t i o n of m u l t i p l e muscle f i b r e s , the motor u n i t , had been d i s c o v e r e d i n 1925 and t h e o n l y n o n m y e l i n a t e d n e r v e f i b r e s t e r m i n a t i n g on 83 s k e l e t a l muscle were determined to be branches of m y e l i n a t e d f i b r e s . Whether the changes i n the nervous system of those diagnosed as d y s t r o p h i c were a c t u a l , t r i v i a l or o t h e r w i s e , the somatic nerves were confirmed to be the only e f f e r e n t i n n e r v a t i o n of the s k e l e t a l muscle and c o u l d t h e r e f o r e be the only source of a t r o p h i c i n f l u e n c e . By the same token, tonus c o u l d only be governed by the somatic nerves. Vitamin E and the F i r s t Animal Model The study of u r i n a r y c o n s t i t u e n t s i n muscular dystrophy e n r i c h e d t h e knowledge o f t h e d i s e a s e and p r o v i d e d a n o n i n v a s i v e means of i n v e s t i g a t i o n . Study employing the muscle i t s e l f was a necessary aspect of s c i e n t i f i c endeavour p e r t a i n i n g to the d i s e a s e as w e l l , but was r e s t r i c t e d by the number and a v a i l a b i l i t y of human s u b j e c t s . An animal model f i n a l l y a r r i v e d and provided more m a t e r i a l f o r s t u d y , a new t r e n d i n r e s e a r c h and another agent f o r treatment of the d i s e a s e . Vitamin E was f i r s t recognized i n vegetable o i l and was shown to be n e c e s s a r y f o r the maintenance of f e r t i l i t y i n a n i m a l s . Goettsch and Pappenheimer (1931) intended to study r e p r o d u c t i o n i n guinea p i g s s u s t a i n e d on a s y n t h e t i c d i e t c a l l e d D i e t 13 which was t r e a t e d w i t h e t h e r i a l f e r r i c c h l o r i d e to render i t d e f i c i e n t i n v i t a m i n E. However, the animals d i e d prematurely and postmortem examination r e v e a l e d n e c r o s i s of the s k e l e t a l muscle unaccompanied by any changes 84 i n the s p i n a l cord of p e r i p h e r a l nerves. The h i s t o l o g i c a l f e a t u r e s of the muscle, which included a r e d u c t i o n i n f i b r e s i z e and number w i t h r e p l a c e m e n t by f a t and c o n n e c t i v e t i s s u e , were s i m i l a r to those of human muscular dystrophy; c o n s e q u e n t l y , the term " N u t r i t i o n a l Muscular D y s t r o p h y " (NMD) was coined to d e s c r i b e the syndrome. Ra b b i t s were a f f e c t e d i n the same way as guinea p i g s but r a t s were i n s u s c e p t i b l e to Diet 13's e f f e c t upon muscle and m o r t a l i t y and c o u l d be used t o v e r i f y D i e t 13's d e f i c i e n c y of v i t a m i n E by the i n f e r t i l i t y i t p r o d u c e d . V i t a m i n E was i n i t i a l l y and l o g i c a l l y assumed to be the a e t i o l o g i c a l agent of NMD, however, the a d d i t i o n of wheat germ o i l which was an abundant source of vi t a m i n E d i d not prevent the development of dystrophy and Diet 11 which was i d e n t i c a l to Diet 13 but without treatment by e t h e r a l f e r r i c c h l o r i d e induced the same moribund c o n d i t i o n . Vitamin E was i n a seemingly paradoxic p o s i t i o n as the a e t i o l o g i c a l f a c t o r of NMD and impetus was g i v e n to study of the e f f e c t s o f d i e t a r y agents upon the neuromuscular system and the a b i l i t y of such agents to cure NMD of Diet 11 or any other dystrophy producing d i e t . Cod l i v e r o i l and l a r d were both c o n s t i t u e n t s of Die t 11 and a s y n t h e t i c d i e t used by Madsen et a l . (1933) which induced l e s i o n s confined to the s k e l e t a l and c a r d i a c muscle of h e r b i v o r o u s animals i n c l u d i n g goats, r a b b i t s and guinea p i g s . On the b a s i s of p a t h o l o g i c a l changes i n the c a r d i a c 85 muscle of goats and r a b b i t s a t t r i b u t a b l e to l a r g e doses of cod l i v e r o i l , Madsen and Maynard i n v e s t i g a t e d cod l i v e r o i l as the p o s s i b l e a e t i o l o g i c a l f a c t o r of animal dystrophy by s u b s t i t u t i n g a v i t a m i n A-D concentrate f o r cod l i v e r o i l as a source of these v i t a m i n s i n the d i e t . The e l i m i n a t i o n of cod l i v e r o i l from the d i e t , a l t h o u g h the a n i m a l s s t i l l became d y s t r o p h i c , p r o l o n g e d t h e i r s u r v i v a l and t h e sugges t i o n was made that some f a c t o r i n the n o n s a p o n i f i a b l e f r a c t i o n of cod l i v e r o i l was perhaps r e s p o n s i b l e f o r the muscular l e s i o n s . Madsen (1936) s u b s e q u e n t l y s t u d i e d the e f f e c t s o f a n i m a l v e r s u s v e g e t a b l e f a t i n the d i e t by t e s t i n g combinations o f cod l i v e r o i l or A-D co n c e n t r a t e s as v i t a m i n s o u r c e s and l a r d o r c o t t o n s e e d o i l as f a t requirements f o r t h e i r a b i l i t y to induce muscular l e s i o n s . The l e s i o n s induced by d i e t s which i n c l u d e d the combination of cod l i v e r o i l and l a r d were the most severe. There was no a p p r e c i a b l e d i f f e r e n c e when c o t t o n s e e d o i l was s u b s t i t u t e d f o r l a r d but c o t t o n s e e d o i l p l u s t h e A-D c o n c e n t r a t e seemed t o p r o t e c t by d e l a y i n g the o n s e t of dys t r o p h y . Lard and v i t a m i n A gave no p r o t e c t i o n but when l a r d was o m i t t e d f r o m t h e d i e t w i t h o u t s u b s t i t u t i o n n u t r i t i o n a l f a i l u r e s t i l l o ccurred and t h e r e f o r e animal f a t was r e j e c t e d as the c a u s a t i v e agent. A f a c t o r or f a c t o r s c o n t a i n e d i n cod l i v e r o i l , the a d d i t i o n of which made a g r e a t e r p r o p o r t i o n o f a n i m a l s on a s t r i c t g r a i n d i e t d y s t r o p h i c ( M o r g u l i s and S p e n c e r , 1 9 3 6 ) , w h i c h were 86 r e s p o n s i b l e f o r t h e c o n d i t i o n r e m a i n e d a p o s s i b i l i t y , though. A l f a l f a and l e t t u c e were d i e t a r y agents which prevented the n u t r i t i o n a l f a i l u r e r e s u l t i n g from a s t r i c t g r a i n d i e t ( M o r g u l i s and S p e n c e r , 1936). A l f a l f a a l s o r e s t o r e d the muscle o f p l a n t e a t i n g animals that were d y s t r o p h i c from G o e t t s c h and Pappenheimer's D i e t 11 and a l t h o u g h l e t t u c e delayed the onset of the dystrophy of t h i s d i e t i t was not c u r a t i v e by i t s e l f . Wheat germ o i l alone d i d not cure but when l e t t u c e , which was a l s o known to c o n t a i n s i g n i f i c a n t amounts of v i t a m i n E, and wheat germ o i l were a d m i n i s t e r e d t o g e t h e r they r e t u r n e d a d y s t r o p h i c and d y i n g a n i m a l t o normal. Dry a l f a l f a or e x t r a c t s of a l f a l f a were i n c a p a b l e of p r e v e n t i n g or c u r i n g a d y s t r o p h i c c o n d i t i o n but d r y a l f a l f a p l u s wheat germ o i l was. I f the a l f a l f a was f r e s h i t gave complete cure as d i d wheat germ i f i t was whole. Although vitamin E was present i n both p r e v e n t i o n s and cures i t appeared not to be the s i n g u l a r f a c t o r . M o r g u l i s et a l . (1938) s u g g e s t e d t h a t two f a c t o r s , a l i p i d s o l u b l e which appeared to be i d e n t i c a l to vi t a m i n E and a water s o l u b l e , were i n v o l v e d i n the c u r e of NMD, which was a p o s s i b l e e x p l a n a t i o n f o r the a b i l i t y of Di e t 11 to induce NMD and the i n a b i l i t y of v i t a m i n E to cure the c o n d i t i o n . In the few o f f s p r i n g produced by r a t s r a i s e d on Di e t 13 the o c c a s i o n a l n e c r o t i c f i b r e was observed i n the s k e l e t a l muscle which suggested the p o t e n t i a l f o r p r o d u c i n g NMD i n 87 th e s e a p p a r e n t l y i n s u s c e p t i b l e a n i m a l s . Rats were i n f a c t t h e f i r s t a n i m a l s i n which a neuromuscular e f f e c t o f a v i t a m i n E d e f i c i e n c y was d e m o n s t r a t e d (Evans and B u r r , 1928). I f r a t s d e p r i v e d of v i t a m i n E were allowed t o mate e a r l y i n l i f e they showed some f e r t i l i t y as d i d those r a i s e d on D i e t 13 and the s u c k l i n g progeny of such r a t s became p a r a l y z e d , a c o n d i t i o n which c o u l d be prevented i f wheat germ o i l was administered during the l a c t a t i o n p e r i o d . The neuromuscular system was not examined i n these c a s e s but l a t e r s t u d y i n i t i a l l y i n d i c t e d the nervous system as the p r i m a r y t a r g e t o f the d i e t a r y d e f i c i e n c y . Postmortem examination of r a t s which became p a r a l y z e d on a v i t a m i n E f r e e d i e t d i s c l o s e d p a t h o l o g i c a l changes w i t h i n the CNS ( E i n a r s o n and R i n g s t e d , 1938; Burr et a l . , 1936). O l c o t t (1938) f i n a l l y succeeded i n p r o d u c i n g a myopathy i n r a t s which i s what E i n a r s o n and Ringsted (1938)1 4 observed i n the e a r l i e s t stages of the neuromuscular c o n d i t i o n i n r a t s which u l t i m a t e l y i n v o l v e d the CNS. I t was thus suggested t h a t the m u s c u l a r s y s t e m was most v u l n e r a b l e to a d e f i c i e n c y o f v i t a m i n E during development, the nervous system being more s u s c e p t i b l e i n a d u l t l i f e . I t i s d i f f i c u l t to understand why r a t s r e c e i v i n g D i e t 13 which were young r a t s were r e s i s t a n t to the e f f e c t upon the neuromuscular system. The f a c t that vitamin E d i d not C i t e d by B i c k n e l l , 1940. 88 cure NMD of D i e t 13 i s a l s o i n e x p l i c a b l e when n a t u r a l and s y n t h e t i c p r e p a r a t i o n s of v i t a m i n E f i n a l l y d i s p l a y e d t h e i r a b i l i t y t o b o t h remedy and p r e v e n t NMD o f a v i t a m i n E d e f i c i e n c y i n r a t s , r a b b i t s , guinea p i g s and dogs ( S h i m i t o r i et a l . , 1939; McKenzie and McCollum, 1939; Anderson et a l . , 1 9 3 9 ) . The t o c o p h e r o l s were the a c t i v e i n g r e d i e n t s o f v i t a m i n E , a l p h a t o c o p h e r o l b e i n g the most b i o l o g i c a l l y a c t i v e , w h i c h by i t s e l f c u r e d NMD, but whether a l p h a t o c o p h e r o l i t s e l f c o n s t i t u t e d v i t a m i n E or was s i m p l y a member o f a c o m p l e x l i k e v i t a m i n B was a p o i n t o f u n c e r t a i n t y . In any case, v i t a m i n E was f i n a l l y i d e n t i f i e d as t h e s i n g u l a r a e t i o l o g i c a l agent o f NMD which became r e p r o d u c i b l e i n r a t s ( P a p p e n h e i m e r , 1939) and o t h e r e x p e r i m e n t a l animals and became the f i r s t animal model of human muscular dystrophy. The s t r o n g resemblance between the h i s t o l o g y of NMD and that of human muscular dystrophy provided a sound b a s i s f o r t r e a t m e n t o f the human d i s o r d e r w i t h v i t a m i n E. A l s o , because the p a t h o l o g i c a l changes i n CNS of r a t s s u b j e c t e d to a v i t a m i n E d e f i c i e n c y by E i n a r s o n and R i n g s t e d (1938) i n v o l v e d t h e lower motor neurons and d e s c e n d i n g motor t r a c t s , a m y o t r o p h i c l a t e r a l s c l e r o s i s (ALS) and s p i n a l m uscular a t r o p h y were i n c l u d e d i n the treatment s t u d i e s . B i c k n e l l (1940) r e p o r t e d t h a t t w e l v e o f e i g h t e e n males diagnosed w i t h pseudohypertrophic muscular dy s t r o p h y , e l e v e n o f whom were under age f i f t e e n , were g r e a t l y i m p r o v e d 89 f o l l o w i n g treatment with whole wheat germ f o r p e r i o d s of two t o f i v e months. A female w i t h amyotonia c o n g e n i t a a l s o improved f o l l o w i n g such treatment f o r t h r e e months and a complete cure appeared to be near accomplishment i n a case of ALS. In B i c k n e l l ' s e s t i m a t i o n v i t a m i n E was "one of the g r e a t advances i n g e n e r a l medicine of the century" and he was c e r t a i n t h a t b o t h m u s c u l a r d y s t r o p h y and ALS were d e f i c i e n c y d i s e a s e s w h i c h were c u r a b l e by d i f f e r e n t c o n s t i t u e n t s of vitamin E a c c o r d i n g to the view that v i t a m i n E was a complex l i k e v i t a m i n B. The v i t a m i n B c o n t a i n e d i n y e a s t t a b l e t s seemed t o e n h a n c e t h e e f f e c t o f v i t a m i n E i n wheat germ o i l a d m i n i s t e r e d by Stone (1940) t o s u f f e r e r s o f m u s c u l a r d y s t r o p h y i n c l u d i n g t h e p s e u d o h y p e r t r o p h i c a n d f a c i o s c a p u l o h u m e r a l forms. Improvement was seen i n a l l by an i n c r e a s e i n muscular s t r e n g t h , a decrease i n f a t i g u e and a change i n the t e x t u r e o f the muscle t o make i t " l e s s rubbery" and these changes were o f t e n seen i n those who had not b e n e f i t e d from amino a c i d therapy. Because v i t a m i n B6 caused f a s i c u l a t i o n s and c o n v u l s i o n s i n r a t s r e c e i v i n g l a r g e doses, Stone t r e a t e d cases of ne u r a l atrophy with v i t a m i n B i n y e a s t t a b l e t s . As v i t a m i n B enhanced the e f f e c t o f v i t a m i n E i n muscular dy s t r o p h y , so v i t a m i n E i n c r e a s e d the p o s i t i v e e f f e c t o f v i t a m i n B i n n e u r o p a t h y . P o s i t i v e r e s u l t s were a l s o seen i n two cases of ps e u d o h y p e r t r o p h i c muscular dystrophy and two p a t i e n t s with amyotonia c o n g e n i t a 90 t o whom wheat germ o i l was a d m i n i s t e r e d by Stone and M a n c h e s t e r ( 1 9 4 1 ) . Because t h e s e c o n d i t i o n s i n v o l v e d c h i l d h o o d and i n f a n c y i t was suggested th a t both were the e x p r e s s i o n of d e f i c i e n c y stemming from a complete lack or i n e f f i c i e n t t r a n s f e r of v i t a m i n E across the p l a c e n t a d u r i n g the p r e n a t a l p e r i o d . A d e f i c i e n c y of any k i n d of v i t a m i n E i n muscular d y s t r o p h y s p e c i f i c a l l y or myopathy g e n e r a l l y , was not s u p p o r t e d by the r e s u l t s of other treatment s t u d i e s w i t h v i t a m i n E and the u s e f u l n e s s of v i t a m i n E i n the treatment of the d i s e a s e was very l i m i t e d . Regardless of the form of v i t a m i n E, whether i t was administered as crude p r e p a r a t i o n o f wheat g e r m , wheat germ o i l or as s y n t h e t i c a l p h a t o c o p h e r o l , and i r r e s p e c t i v e of the route of a d m i n i s t r a t i o n used i n other s t u d i e s , none of the aforementioned c o n d i t i o n s were a m e l i o r a t e d to any s i g n i f i c a n t degree (Shelden et a l . , 1940; F e r r e b e e e t a l . , 1941; S c h w a r z e t a l . , 1942; F i t z g e r a l d and M c A r d l e , 1942; M i n t o t and F r a n k , 1944). F i t z g e r a l d and M c A r d l e (1942) found t h a t v i t a m i n E was e q u a l l y i n e f f i c i e n t i n neuropathy and myopathy, alone or i n c o n j u n c t i o n w i t h v i t a m i n B6 thereby d i s c r e d i t i n g the a l l e g e d complementary a c t i o n s of v i t a m i n s E and B. Minot and Frank (1944) found no s i g n i f i c a n t changes i n e i g h t males with the pseudohypertrophic form ranging i n age from f i v e to nineteen y e a r s , d e m o n s t r a t i n g t h a t t h e e f f e c t of v i t a m i n E was e q u a l l y negative at a l l stages of the d i s e a s e . That v i t a m i n 91 E d i d not amend the d i s o r d e r of metabolism which e x i s t e d i n m u s c u l a r d y s t r o p h y was t h e i n e v i t a b l e c o n c l u s i o n o f treatment s t u d i e s . B i c k n e l l (1940) thought t h a t a d i e t a r y d e f i c i e n c y of v i t a m i n E was p l a u s i b l e because wheat germ was not eaten r e g u l a r l y , l e t t u c e i n salads was not a f a s h i o n a b l e food at the time and breads were made with r e f i n e d f l o u r . D e s p i t e the n e g a t i v e r e p o r t s r e g a r d i n g the t h e r a p e u t i c e f f e c t o f vita m i n E and because a d e f i c i e n c y could be a m a n i f e s t a t i o n of i m p a i r e d a b s o r b t i o n from the g a s t r o i n t e s t i n a l t r a c t , a p o s i t i v e a p p r o a c h was t a k e n i n s e a r c h o f a d e f e c t i n a b s o r b t i o n . Minot and Frank (1944) s t u d i e d the t o c o p h e r o l content of b l o o d serum u s i n g a c o l o u r i m e t r i c method which measured u n o x i d i z e d t o c o p h e r o l . Resting l e v e l s i n c h i l d r e n with p s e u d o h y p e r t r o p h i c muscular dystrophy were comparable to l e v e l s i n a d u l t s with ALS which Weschsler et a l . (1941) reported as being normal. F o l l o w i n g o r a l a d m i n i s t r a t i o n of a l p h a - t o c o p h e r o l there was a subsequent and undelayed r i s e i n serum l e v e l s which i n d i c a t e d t h a t a b s o r b t i o n was not imp a i r e d . Assuming that v i t a m i n E had a p h y s i o l o g i c a l r o l e , a d e f e c t i n u t i l i z a t i o n was s t i l l p o s s i b l e . A l t h o u g h t h e u n c h a n g i n g p a t t e r n s o f c r e a t i n e and c r e a t i n i n e e x c r e t i o n s u p p o r t e d the l a c k o f v i t a m i n E's e f f e c t upon muscular performance ( F i t z g e r a l d and M c A r d l e , 1942), c r e a t i n u r i a had yet to be dismissed as a consequence of muscular d e g e n e r a t i o n . T h e r e f o r e when c r e a t i n u r i a was 92 a l t e r e d even without any concomitant c l i n i c a l improvement i t was s t i l l c o n sidered to be of s i g n i f i c a n c e . M i l h o r a t et a l . (1945) r e p o r t e d t h a t the c r e a t i n u r i a i n cases of muscular dystro p h y and d e r m a t o m y o s i t i s , d i s e a s e i n v o l v i n g s k i n and m u s c l e , d e c r e a s e d a f t e r the t h i r d week of treatment w i t h whole wheat germ. The e f f e c t was no g r e a t e r when the wheat germ was incubated i n human g a s t r i c j u i c e , brewer's y e a s t , e x t r a c t of hog pancreas or e x t r a c t of beef l i v e r . Exposure to a i r or treatm e n t w i t h e t h y l e n e d i c h l o r i d e which would o x i d i z e a component of the wheat germ or remove l i p i d s o l u b l e s u b s t a n c e s , r e s p e c t i v e l y , d i d on the o t h e r hand r e s u l t i n a small e r e f f e c t upon c r e a t i n u r i a . M i l h o r a t and B a r t e l s (1945) found that c r e a t i n u r i a i n h a l f o f t h e i r d y s t r o p h i c p a t i e n t s was reduced when a l p h a t o c o p h e r o l rather than whole wheat germ was f i r s t i ncubated i n the stomach of a normal man. When alpha t o c o p h e r o l was r e f l u x e d w i t h e t h y l e n e d i c h l o r i d e , dibromide, c h l o r o h y d r i n or b r o m o h y d r i n , r e f l u x e d with e t h y l e n e d i c h l o r i d e i n the presence o f a s c o r b i c a c i d or heated on an autoclave i n an atmosphere o f n i t r o g e n , c r e a t i n u r i a d e c r e a s e d i n s i x t y percent o f d y s t r o p h i c p a t i e n t s . That a condensation product of a l p h a t o c o p h e r o l was the b i o l o g i c a l l y a c t i v e substance emerged from these r e s u l t s . According to the absence of an e f f e c t upon c r e a t i n u r i a i t was not an e s t e r of phosphate, a c e t a t e or s u c c i n a t e . 93 I n o s i t o l f u l f i l l e d the c r i t e r i a f o r the substance which r e n d e r e d a l p h a t o c o p h e r o l p h y s i o l o g i c a l l y a c t i v e i n t h a t t o c o p h e r o l - i n o s i t o l was s o l u b l e i n w a t e r , i n o s i t o l was a v a i l a b l e i n the g a s t r o i n t e s t i n a l t r a c t as w e l l as b e i n g p r e s e n t i n wheat germ. Equimolar c o n c e n t r a t i o n s of a l p h a t o c o p h e r o l and benzene h e x a c h l o r i d e i n a l c o h o l c o n t a i n i n g potassium hydroxide were thus r e f l u x e d on a steam bath i n an atmosphere of n i t r o g e n . A n a l y s i s of the product was not c o n c l u s i v e but i t appeared to be a monoether of t o c o p h e r o l -i n o s i t o l and i t was much more e f f e c t i v e i n r e d u c i n g c r e a t i n u r i a than was wheat germ. Uncondensed toco p h e r o l and i n o s i t o l a l s o had d e f i n i t e and p o s i t i v e e f f e c t s upon c r e a t i n u r i a , the e f f e c t being 1/30 to 1/8 as e f f e c t i v e as the condensation p r o d u c t . The gene had been d e f i n e d as t h e s m a l l e s t u n i t o f i n h e r i t a n c e , t h a t one gene produced one enzyme had been h y p o t h e s i z e d and t h e r e was e v i d e n c e t h a t the d i f f e r e n t c l i n i c a l e n t i t i e s of muscular d y s t r o p h y were i n h e r i t e d by d i f f e r e n t d e f e c t i v e genes. Thus, i t was proposed that i f the f o r m a t i o n of t o c o p h e r o l - i n o s i t o l i n v o l v e d a number of r e a c t i o n s c a t a l y z e d by d i f f e r e n t enzymes, the v a r i o u s c l i n i c a l e n t i t i e s c o u l d be the r e s u l t of d i f f e r e n t d e f e c t i v e enzymes of the s y n t h e t i c sequence. I f t h i s were so, then t h e s e v e r e s t f o r m o f m u s c u l a r d y s t r o p h y , t h e p s e u d o -h y p e r t r o p h i c t y p e , was expected to b e n e f i t from condensed t o c o p h e r o l - i n o s i t o l treatment w h i l e the more benign forms 94 would respond to treatment with uncondensed tocopherol and inositol. The hypothesis did not withstand the test of treatment and was immediately discarded. Assuming that vitamin E had a physiological action there was also evidence derived from study employing the model that phosphorylated tocopherol was the active form. The use of the animal model had two aims: to seek the underlying cause of the dystrophy with application to the human disease and to ascertain the physiological role of vitamin E. In contrast to atrophic, denervated muscle for which oxygen consumption was normal (Knowlton and Hines, 19834), oxygen consumption appeared to be greatly elevated in the muscle of NMD (Victor, 1934). It was conceivable in the view of Houchin and Mattil (1942) that vitamin E was related to the oxidative processes of skeletal muscle the knowledge of which was limited to the role of succinate dehyrogenase in the catalysis of the Kreb's cycle reaction: succinate -*• fumarate from which hydrogen atoms were transferred to oxygen via the cytochromes. The oxygen consumption of dystrophic muscle was thus investigated in vivo at varying intervals following oral administration of alpha tocopherol. A normal rate was eventually attained and Houchin and Mattil (1942b) reasoned that if alpha tocopherol was responsible for the decrease, parenteral administration should reduce the time required for the attainment of a normal rate. Because tocopherol-phosphoate was soluble in 95 water i t was used f o r the p a r e n t e r a l a d m i n i s t r a t i o n and the time involved i n the d e c l i n e of oxygen consumption towards a n o r m a l r a t e was i n f a c t r e d u c e d . U n p h o s p h o r y l a t e d tocopherol d i d not lower the elevated oxygen consumption of d y s t r o p h i c muscle i n v i t r o while phosphorylated t o c o p h e r o l l o w e r e d the h i g h s u c c i n a t e dehyrogenase a c t i v i t y which accompanied the excessive oxygen use (Houchin, 1942). It appeared then that alpha t o c o p h e r o l , very p o s s i b l y t h e p h o s p h o r y l a t e d f o r m , was a b i o l o g i c a l o x i d a n t p a r t i c i p a t i n g d i r e c t l y i n or r e g u l a t i n g o x i d a t i o n - r e d u c t i o n r e a c t i o n s . The sugg e s t i o n was made that s i n c e the redox p o t e n t i a l o f a l p h a t o c o p h e r o l was s l i g h t l y l e s s t h a n cytochrome C, alpha tocopherol could perhaps act as a s o r t of "brake" by i n h i b i t i n g the t r a n s f e r of hydrogen atoms to oxygen and without such a braking system o x i d a t i o n would "run r i o t " . Just as i n the human c o n d i t i o n c r e a t i n e content of muscle i n NMD was lowered (Goettsch and Brown, 1938) and t h e r e was thus an i n v e r s e r e l a t i o n s h i p between c r e a t i n e content and oxygen consumption which agreed with the g r e a t e r oxygen consumption by c a r d i a c muscle c o n t a i n i n g much l e s s c r e a t i n e than s k e l e t a l . I f oxygen consumption d i d "run r i o t " as the r e s u l t of a tocopherol d e f i c i e n c y the r e d u c t i o n i n c r e a t i n e was thought to be the sequel which would l e a d to muscle degeneration. The c l a i m of excessive oxygen consumption by d y s t r o p h i c muscle was u l t i m a t e l y b e l i e v e d to be refuted by a d i f f e r e n t 96 c h o i c e o f c o n t r o l a n i m a l . Both oxygen consumption and s u c c i n a t e dehyrogenase a c t i v i t y were demonstrated to be much low e r i n o l d e r a n i m a l s used i n e a r l i e r s t u d i e s t h a n i n younger a n i m a l s . With the employment of younger c o n t r o l animals oxygen consumption i n d y s t r o p h i c muscle was found to be v i r t u a l l y the same as i n c o n t r o l s ( B a s i n s k i and Hummel, 1947; Hummel and B a s i n s k i , 1948). Further study p e r t a i n i n g to the p h y s i o l o g i c a l r o l e of v i t a m i n E was thus deemed to be w i t h o u t m e r i t u n t i l more was known about the m e t a b o l i c p r o c e s s e s u n d e r l y i n g NMD. D u r i n g t h e 1 9 5 0 ' s g r e a t s t r i d e s w e r e made i n b i o c h e m i s t r y w i t h t h e d e l i n e a t i o n o f m e t a b o l i c pathways, i d e n t i f i c a t i o n of enzymes i n v o l v e d and means f o r a s s a y i n g the a c t i v i t y of such enzymes. A p p l i c a t i o n of these advances was made with r e s p e c t to the a v i t a m i n o s i s E animal model, which i n turn were a p p l i e d to the human c o n d i t i o n . One of the a p p l i c a t i o n s o f m e t a b o l i c d i s t u r b a n c e s i n the animal model to human muscular dystrophy fo l l o w e d the d i s c o v e r y of a m i n o a c i d u r i a i n t h e human c o n d i t i o n (Aimes and R i s l e y , 1947). Using paper chromatography, then a new q u a l i t a t i v e method which e x p l o i t s the v a r i a b l e d i s t a n c e s t h a t amino a c i d s move on paper immersed i n a p a r t i c u l a r s o l v e n t , a m u l t i p l i c i t y of s p o t s , rather than the normal two or three were d e l i v e r e d t o paper by the u r i n e of those w i t h the d i s e a s e . In view of the i n c r e a s e i n p r o t e o l y t i c a c t i v i t y of muscle homogenates from the animal model (Weinstock et a l . , 97 1955) the e x c e s s i v e a c t i v i t y of a c a t h e t p t i c enzyme was proposed as the p l a u s i b l e cause of aminoaciduria and muscle degeneration i n the human c o n d i t i o n . By t h a t time c r e a t i n u r i a , the major d r i v i n g f o r c e i n r e s e a r c h r e g a r d i n g muscular d y s t r o p h y , was d i s m i s s e d as b e i n g non-pathognmonic. There was a l s o evidence f o r the demotion, as i t were, of p h o s p h o c r e a t i n e as .the immediate energy source f o r muscular c o n t r a c t i o n which seemed to g i v e a d d e d w e i g h t t o t h e a p p a r e n t s e c o n d a r y n a t u r e o f c r e a t i n u r i a . The h y d r o l y s i s of ATP by the enzyme, ATPase, w h i c h was d e t e r m i n e d t o be an i n t e g r a l p a r t o f t h e c o n t r a c t i l e component, myosin, was c o n s i d e r e d by many to p r o v i d e th e immediate energy f o r m u s c u l a r c o n t r a c t i o n . Knowledge o f t h e mechanism o f m u s c u l a r c o n t r a c t i o n had advanced w i t h the d i s c o v e r y of a c t i n and myosin and t h e i r a b i l i t y to a s s o c i a t e i n the presence of ATP and a d i v a l e n t a c t i o n i n v i t r o . The a p p l i c a t i o n of t h i s knowledge to the a v i t a m i n o s i s E model was, however, q u i t e l i m i t e d because the g e n e t i c a spect of muscular d y s t r o p h y was i n d i s p u t a b l e and t h e r e f o r e the Bar Harbour h e r i t a b l e mouse d y s t r o p h i e s became the p r e f e r r e d a n i m a l model. Use of the V i t a m i n E model ceased by about 1960 and i n v e s t i g a t i o n of the p h y s i o l o g i c a l r o l e of v i t a m i n E was thus i n h i b i t e d . I t s r o l e i n man, i f any, has yet to be f u l l y e l u c i d a t e d . PART I I I DUCHENNE MUSCULAR DYSTROPHY, AN X-LINKED RECESSIVE TRAIT NEUROPATHY? 98 The c l i n i c a l e n t i t i e s of muscular dystrophy were not as E r b b e l i e v e d , t h a t i s , v a r i a n t s o f t h e same d i s e a s e . R a t h e r , t h e y were d e t e r m i n e d t o be d i s t i n c t d i s e a s e s d i c t a t e d by d i f f e r e n t genes. In 1961, John Walton extended the d e f i n i t i o n of muscular dystrophy and c a l l e d "them" as opposed to " i t " , a group of g e n e t i c a l l y determined, primary, p r o g r e s s i v e , d e g e n e r a t i v e m y o p a t h i e s . By t h a t t i m e , c l a s s i f i c a t i o n a c c o r d i n g to c l i n i c a l - g e n e t i c c r i t e r i a was w e l l e s t a b l i s h e d and i t was g e n e r a l l y accepted that Duchenne m u s c u l a r d y s t r o p h y was an X - l i n k e d r e c e s s i v e t r a i t . However, i t was not u n t i l the l a t e 1970's when c y t o l o g y proved that the gene r e s p o n s i b l e f o r the d i s o r d e r was indeed h a r b o u r e d by t h e X chromosome, f o l l o w i n g w h i c h DNA technology was employed as a means of l o c a l i z i n g the gene. I t i s DNA t e c h n o l o g y and r e s e a r c h founded on the knowledge d e r i v e d from molecular a n a l y s i s of both Duchenne and myotonic d y s t r o p h y which has dominated t h i s f i e l d of endeavour f o r the l a s t f i v e years and w i l l u l t i m a t e l y s o l v e the mystery of the pathogenesis of each. With the advances i n the f i e l d of g e n e t i c s during the 1970's t h i s approach to the study of Duchenne muscular dystrophy was i n e v i t a b l e and has d i s c o u r a g e d the resurgence of study p e r t a i n i n g to any neurogenic h y p o t h e s i s . Part I I I has three b a s i c aims: f i r s t l y , to show the way i n which the X - l i n k e d n e s s of the gene r e s p o n s i b l e f o r Duchenne dystrophy was determined. Secondly, to recount the 99 e v e n t s i n v o l v e d i n f o u r s e p a r a t e a r e a s of r e s e a r c h which culminated i n support f o r the neurogenic h y p o t h e s i s . These f o u r a r e a s a r e : mental d e f i c i e n c y i n Duchenne d y s t r o p h y a c c o r d i n g to c l i n i c a l assessment, d i f f e r e n t i a l d i a g n o s i s of myopathy v s . neuropathy, the t r o p h i c i n f l u e n c e s of the nerve upon t h e m u s c l e , and t h e p a t h o l o g y o f i n t r a m u s c u l a r i n n e r v a t i o n . T h i r d l y , t o e n u n c i a t e t h e n e u r o g e n i c h y p o t h e s i s and f o l l o w the steps towards i t s d i s c r e d i b i l i t y . T h e X - l i n k e d n e s s o f D u c h e n n e D y s t r o p h y Mendel's idea of " p a r t i c u l a t e " i n h e r i t a n c e was based on t h e r e s u l t s o b t a i n e d f r o m e x p e r i m e n t s r e g a r d i n g t h e m o r p h o l o g i c a l t r a i t s i n p l a n t s . The p e t a l colour of f l o w e r s was c o n s e r v e d throughout s u c c e s s i v e g e n e r a t i o n s of c r o s s e s between p l a n t s b e a r i n g p u r p l e and white f l o w e r s , t h a t i s , the c o l o u r s were un c h a n g e d , t h e r e was no l i g h t e n i n g or d a r k e n i n g as p r e d i c t e d by t h e t h e o r y o f " b l e n d i n g " i n h e r i t a n c e . M endel i n v e n t e d t h e terms d o m i n a n t and r e c e s s i v e to e x p l a i n the p r o p o r t i o n s of p u r p l e and white p e t a l c o l o u r i n s u c c e s s i v e g e n e r a t i o n s , the t r a i t with the g r e a t e s t numbers i n the f i r s t . g e n e r a t i o n of the c r o s s being the dominant, the l e s s e r numbers belonging to the r e c e s s i v e t r a i t . I t was not u n t i l i t was n o t i c e d that the behaviour of m o r p h o l o g i c a l t r a i t s , as d e s c r i b e d by Mendel p a r a l l e l e d the behaviour of chromosomes during sex c e l l d i v i s i o n , w i t h r e s p e c t to s e g r e g a t i o n and independent assortment, th a t i t 100 was f i n a l l y recognized that the "particles" of inheritance were integral parts of the chromosome. Therefore, the "particles" mixed but remained discreet in the event of union between sperm and ovum. It was the scrutiny of overt traits in the established matings of the proverbial f r u i t f l y , Drosophila, that eventually defined the gene as the smallest particle or unit of inheritance. Another combined effort between cytology and genetics proved the sex-linkedness of a gene. Cytology proved the existence of autosomes and sex chromosomes while the study of eye colour in Drosophila demonstrated that the gene responsible for the t r a i t was harboured by the X chromosome. A sex-linked gene generated a p a r t i c u l a r pattern of inheritance according to pedigree analysis as did an autosomal dominant or autosomal recessive t r a i t . Therefore pedigree analysis of human traits or disorders became the tool of genetic dissection, the means by which the Mendelian or genetic nature of a t r a i t could be inferred. The inevitable pedigree analysis of muscular dystrophy pointed towards the recessive nature of heredity in the pseudohypertrophic type of the disease. Minkowski and Sidler (1927)15 found that a l l those beset by this form of muscular dystrophy in a secluded Swiss vil l a g e , for which Cited by Polachek (1941). 101 g e n e a l o g i c a l records were a v a i l a b l e f o r a three hundred year p e r i o d , were o f f s p r i n g o f p a r e n t s , b o t h o f whom were descendants of e i t h e r of two f a m i l i e s . Barker (1930)1 6 a l s o r e p o r t e d t h a t p s e u d o h y p e r t r o p h i c muscular d y s t r o p h y was no r m a l l y perpetuated as a r e c e s s i v e t r a i t . As more f a m i l y p e d i g r e e s became a v a i l a b l e f o r pseudohypertrophic muscular d y s t r o p h y w h i c h , as Gowers n o t e d , seemed t o o c c u r s p o n t a n e o u s l y i n f a m i l i e s with no p r e v i o u s h i s t o r y of the d i s e a s e , the sex - l i n k e d n e s s of the d i s o r d e r became apparent. M i l h o r a t and Wolff (1943) reported that 26 males i n h e r i t e d t h i s type o f m u s c u l a r d y s t r o p h y i n the same manner as hae m o p h i l i a , by a simple s e x - l i n k e d r e c e s s i v e mode. In one p a r t i c u l a r g e n e a l o g i c t r e e a healthy female had two a f f e c t e d sons who were f a t h e r e d by d i f f e r e n t men, one of the sons h a v i n g a h e a l t h y female s i s t e r . The mother a l s o had a s i s t e r with an a f f e c t e d son thereby r e i n f o r c i n g the apparent t r a n s m i s s i o n of the d i s o r d e r through the normal female l i n e of the f a m i l y . T h e a u t o s o m a l d o m i n a n t t r a n s m i s s i o n o f t h e faci o s c a p u l o h u m e r a l type, s p e c i f i c a l l y , a l s o became evident through p e d i g r e e a n a l y s i s and i n f a c t Hough d i s c o v e r e d by 1936 that the i n h e r i t a n c e of muscular dystrophy had been v i a dominant, r e c e s s i v e and s e x - l i n k e d modes. M i l h o r a t and Wolff (1943) confirmed the i n h e r i t a n c e of muscular dystrophy 'Cited by Hough (1931). 102 by the three d i f f e r e n t modes while n o t i n g that there was a r e l a t i o n s h i p between age of onset and c l i n i c a l c o u r s e , an e a r l i e r onset c o r r e l a t i n g with a f a s t e r pace of p r o g r e s s i o n and t h i s r e l a t i o n s h i p h e l d w i t h i n a g i v e n p e d i g r e e . Before t h e g e n e had b e e n d e f i n e d a s t h e s m a l l e s t u n i t o f i n h e r i t a n c e Gordon (1923) came to the c o n c l u s i o n that e i t h e r t h e p s e u d o h y p e r t r o p h i c o r a t r o p h i c t y p e o f m u s c u l a r d y s t r o p h y , but not bo t h , would occur w i t h i n a giv e n f a m i l y i n which case they were h e r e d i t a r y d i s o r d e r s that bred t r u e . By the time t h a t Beadle and Tatum (1941) proposed the one gene-one enzyme hypothesis there was ample evidence d e r i v e d f r o m p e d i g r e e a n a l y s i s t o s u p p o r t t h e n o t i o n t h a t the i n d i v i d u a l types of muscular dystrophy were i n a c t u a l f a c t d i f f e r e n t Mendelian t r a i t s . D e spite the accumulating evidence f o r the s e p a r a t i o n of the v a r i e t i e s of muscular dystrophy on the b a s i s of g e n e t i c s t h e r e remained some r e s i s t a n c e to the s e p a r a t i o n , some of which was unfounded. The c o n c l u s i o n s of B e l l (1943) who r e s o l v e d to r e t a i n the u n i t a r y nature of muscular dystrophy were on the other hand w e l l - s u p p o r t e d . She found that a l l t h r e e modes o f i n h e r i t a n c e o c c u r r e d i n a l l o f t h e t r a d i t i o n a l c a t e g o r i e s which suggested to her that the same gene was r e s p o n s i b l e f o r the d i f f e r e n t c l i n i c a l e n t i t i e s but a d d i t i o n a l g e n e t i c and environmental f a c t o r s c o u l d r e s u l t i n any o f the t h r e e modes of i n h e r i t a n c e . M i s d i a g n o s i s was u n q u e s t i o n a b l y r e s p o n s i b l e f o r her r e s u l t s because the 103 d i a g n o s i s o f some c a s e s a c c o r d i n g t o t h e t r a d i t i o n a l c a t e g o r i e s w a s i n a p p r o p r i a t e . F o r e x a m p l e , "pseudohypertrophic", rather than c o n f i n i n g i t s a p p l i c a t i o n t o t h e c l i n i c a l syndrome c o r r e s p o n d i n g t o Duchenne's o r i g i n a l d e s c r i p t i o n was a p p l i e d to those with any kind of m u s c u l a r e n l a r g e m e n t . T h i s m i s a p p l i c a t i o n i s w e l l -i l l u s t r a t e d i n a study by M i l l s et a l . (1924) i n which a 29 year o l d female who was " f l e s h y " i n appearance and t h e r e f o r e diagnosed as the pseudohypertrophic type was the e x c e p t i o n to the f i v e other a t r o p h i c members of the f a m i l y . On the grounds t h a t the t y p e s of muscular d y s t r o p h y were not g e n e t i c a l l y d i s t i n c t B e l l p r o p o s e d a new and s i m p l i f i e d c l a s s i f i c a t i o n scheme r e m i n i s c e n t o f E r b ' s , c o n s i s t i n g of t h r e e c a t e g o r i e s : a) pseudohypertrophic; b) a t r o p h i c without f a c i a l involvement; and c) a l l those with f a c i a l i n v o l v e m e n t . T y l e r and Wintrobe (1950) proposed a two category c l a s s i f i c a t i o n system which was again l i k e that of the other scheme of Erb's i n that age of onset was the main f e a t u r e o f one c a t e g o r y d e s i g n a t e d " c h i l d h o o d " , the o t h e r b e i n g c a l l e d " f a c i o s c a p u l o h u m e r a l " . The c l i n i c a l c r i t e r i a of the chi l d h o o d type i n c l u d e d the m a n i f e s t a t i o n of muscular weakness " o f t e n " accompanied by pseudohypertrophy i n the a x i a l or p e l v i c musculature, the "usual" s p a r i n g of the f a c i a l musculature w h i l s t degeneration of muscles below the neck progressed r a p i d l y , and i t "ne a r l y always" a f f e c t e d males. A wave of r e c l a s s i f i c a t i o n had begun wi t h B e l l i n 104 1943 b u t by 1950 t h a t a p a r t i c u l a r t y p e o f m u s c u l a r d y s t r o p h y was g e n e t i c a l l y d i s t i n c t was r e c o g n i z e d by the m a j o r i t y . T y l e r and Stephens (1951) found th a t only 7 of 33 k i n d r e d s d i a g n o s e d as t h e c h i l d h o o d t y p e i n h e r i t e d the d i s e a s e as a s e x - l i n k e d r e c e s s i v e t r a i t and t h e r e f o r e the c h i l d h o o d t y p e d i d not " n e a r l y always" a f f e c t males as o r i g i n a l l y suggested. "Childhood" (although i t was used by some f o r immediate f u t u r e s t u d y ) , was not u n i f y i n g i n terms of g e n e t i c s . I t was a d e s i g n a t i o n , l i k e p s e u d o h y p e r t r o p h i c , which was e a s i l y m i s a p p l i e d and i t coul d not be s u b d i v i d e d i n any way t h a t w o u l d c l e a r l y d e m a r c a t e g e n e t i c a l l y d i s t i n c t i v e c l i n i c a l e n t i t i e s . A f t e r s t u d y i n g a l l known f a m i l i e s s u f f e r i n g from m u s c u l a r d y s t r o p h y i n N o r t h e r n I r e l a n d S t e v e n s o n (1953) found 27 f a m i l i e s fraught with the disease conforming to the c l a s s i c a l d e s c r i p t i o n o f Duchenne, 26 of the 27 f a m i l i e s d e m o n s t r a t i n g a s e x - l i n k e d r e c e s s i v e i n h e r i t a n c e . He p r o p o s e d t o c a l l t h e d i s e a s e "Duchenne t y p e r a p i d l y p r o g r e s s i v e muscular dystrophy of young boys". The name was int e n d e d t o envelop o u t s t a n d i n g f e a t u r e s and to e l i m i n a t e u n c e r t a i n t y i n d i a g n o s i s , but what the r e a s s o c i a t i o n o f D u c h e n n e ' s name d i d was t o r e i d e n t i f y t h e f o r m e r p s e u d o h y p e r t r o p h i c p a r a l y s i s , the o r i g i n a l " h y p e r t r o p h i c p a r a p l e g i a o f i n f a n c y of c e r e b r a l o r i g i n " , making i t once again an e n t i t y unto i t s own. 105 Walton and N a t t r a s s (1954) d i f f e r e d from Stevenson i n the assignment of Duchenne's name i n that "Duchenne type" of t h e i r c l a s s i f i c a t i o n scheme was more i n c l u s i v e . I t was sub d i v i d e d a c c o r d i n g to c l i n i c a l g e n e t i c e n t i t i e s to i n c l u d e t h e s e v e r e form a f f e c t i n g the m a j o r i t y who were males a c c o r d i n g t o the o r i g i n a l d e s c r i p t i o n o f Duchenne and a benign X - l i n k e d form which was i d e n t i c a l to the severe form i n the p a t t e r n of muscular involvement a f f e c t i n g about 10% of the t o t a l . Stevenson b e l i e v e d that the female cases of pseudohypertrophic p a r a l y s i s d e s c r i b e d by Gowers and o t h e r s had been m i s d i a g n o s e d i n view of the u n i s e x u a l nature o f Duchenne1s p a t i e n t s . W a l t o n and N a t t r a s , i n c o n t r a s t , a s s i g n e d Duchenne's name to a r a r e l y o c c u r i n g m u s c u l a r d y s t r o p h y i n f e m a l e s w h i c h was a l s o v i r t u a l l y i n d i s t i n g u i s h a b l e i n the p a t t e r n of muscular involvement and i n h e r i t e d as an autosomal dominant t r a i t . Some c o n f u s i o n , r e g a r d i n g the p r e c i s e d i a g n o s i s s t i l l remained but because t h e s e v e r e X - l i n k e d f o r m was t h e most common, a n y d e s i g n a t i o n o f Duchenne type f o r immediate f u t u r e s t u d y would be m o s t l y c o n f i n e d t o t h o s e f o r w h i c h " r a p i d l y p r o g r e s s i v e m u s c u l a r d y s t r o p h y o f y o u n g b o y s " was a p p l i c a b l e . In 1955, Becker and Keiner c i t e d a new form of muscular dystrophy which a c t u a l l y conformed to the d e s c r i p t i o n of the benign X - l i n k e d Duchenne type of Walton and N a t t r a s s (1954). I t was not u n t i l the e a r l y 1970's that Becker's name was 106 f i r m l y s e c u r e d t o t h i s t y p e of m u s c u l a r d y s t r o p h y but l i n k a g e s t u d i e s d u r i n g the 1960's c l e a r l y d i f f e r e n t i a t e d between the benign and severe X - l i n k e d forms. It was hoped that l i n k a g e s t u d i e s , which i n v o l v e d p e d igree a n a l y s i s of d i f f e r e n t X - l i n k e d t r a i t s , would demonstrate the c l o s e n e s s of two genes which were presumed to be harboured by the X chromosome t h e r e b y p r o v i n g the X - l i n k e d n e s s o f them. U n f o r t u n a t e l y , l i n k a g e between deutan c o l o u r b l i n d n e s s or the gene f o r the Xg b l o o d group and t h a t f o r Duchenne d y s t r o p h y was not demonstrated. Such s t u d i e s d i d on the o t h e r hand prove t h a t the genes f o r Duchenne and Becker d y s t r o p h y were not i d e n t i c a l but whether or not they a r e a l l e l i c has yet to be v e r i f i e d . The f a c i o s c a p u l o h u m e r a l v a r i e t y was c o n f i r m e d to be i n h e r i t e d v i a an autosomal dominant f a c t o r w h i l e a form which was named "limb g i r d l e " was determined to be i n h e r i t e d as an a u t o s o m a l r e c e s s i v e t r a i t ( W a l t o n , 1 9 6 4 ) . The "autosomalness" of these p a r t i c u l a r types needed no f u r t h e r v e r i f i c a t i o n beyond pedigree a n a l y s i s because u n l i k e those a f f l i c t e d by Duchenne dystrophy i n d i v i d u a l s beset by these types were able to reproduce. E a r l i e r pedigree analyses of Duchenne muscular d y s t r o p h y ( P o l a c h e k , 1941; Ray-Brown, 1951) d e m o n s t r a t e d X - l i n k e d n e s s but t h e d i s e a s e was committed to a p u r e l y sex bound r e c e s s i v e f a c t o r with one r e s e r v a t i o n : none of the a f f l i c t e d had produced o f f s p r i n g . A f t e r a m i n o a c i d u r i a had been d i s c o v e r e d u s i n g p a p e r 107 chromatography Bland (1955) t e s t e d the u r i n e of mothers and female s i b l i n g s of boys with Duchenne dystrophy. Although the number was l e s s than i n d y s t r o p h i c boys more than the normal two or three spots were d e l i v e r e d to paper by the u r i n e of these females. I f the gene f o r the d i s e a s e was indeed propagated through the female l i n e as an X - l i n k e d r e c e s s i v e t r a i t , i t was suggested t h a t the o c c u r r e n c e of e x c e s s amino a c i d s of mothers and s i s t e r s who would be c a r r i e r s supported a g e n e t i c anomaly that was h i n t e d at but not f u l l y e x p r e s s e d . Such as s u g g e s t i o n was i n f a c t one w i t h i n s i g h t f o r i t was made l o n g b e f o r e Lyon (1962) h y p o t h e s i z e d t h e random a c t i v a t i o n o f t h e f e m a l e X chromosome which would r e s u l t i n a combination of a c t i v e X chromosomes, some h a r b o u r i n g the d e f e c t i v e gene, i n the m u l t i n u c l e a t e d muscle f i b r e . S u b c l i n i c a l s i g n s of the d i s e a s e i n supposed female c a r r i e r s of the d e l e t e r i o u s gene i n c l u d i n g m u s c u l a r weakness, h i s t o l o g i c a l changes and e l e v a t e d serum enzymes supported both Lyon's hypothesis and the X - l i n k e d n e s s o f the gene. That genes c o u l d mutate spontaneously had explained the occurrence of the d i s o r d e r i n f a m i l i e s with no p r e v i o u s h i s t o r y of the d i s e a s e , but w h i l e the X - l i n k a g e of the gene was very probable i n two b r o t h e r s of a mother w i t h no f a m i l y h i s t o r y , autosomal r e c e s s i v e t r a n s m i s s i o n c o u l d not be r u l e d out c o m p l e t e l y (Emery, 1966). 108 The f e m a l e c a s e s o f D u c h e n n e - 1 i k e d y s t r o p h y w i t h autosomal i n h e r i t a n c e gave cause f o r debate and Moser (1984) r e c o n c i l e d the matter by s u g g e s t i n g that these cases were a c t u a l l y cases of s p i n a l muscular atrophy or another type of l i m b - g i r d l e muscular d y s t r o p h y . I t was, i n f a c t , female cases of Duchenne-like dystrophy that u l t i m a t e l y proved the X - l i n k e d n e s s of the gene f o r the d i s e a s e , but such c a s e s i n v o l v e d s t r u c t u r a l or n u m e r i c a l a b n o r m a l i t i e s of the X chromosome. In 1965 F e r r i e r et a l . p r o v i d e d c y t o g e n i c evidence with a report i n v o l v i n g a 7 1/2 year o l d g i r l , whose c e l l s were sometimes minus a complete X chromosome and other times a p a r t i a l one. She was t i n y i n s t a t u r e , b r o a d - s h o u l d e r e d , narrow-hipped and w i t h i n f a n t i l e female g e n i t a l i a . Her muscles were weakening and her serum c r e a t i n e phosphokinase l e v e l s were h i g h e r than her b r o t h e r ' s who was a l r e a d y c o n f i n e d t o a w h e e l c h a i r w i t h Duchenne d y s t r o p h y . Her s i s t e r was normal and a l l d i a g n o t i c i n d i c a t i o n s p o i n t e d towards Duchenne muscular dystrophy and Turner's syndrome. The nondysjunction of X chromosomes was more l i k e l y to occur when male, rather than female, sex c e l l s were d i v i d i n g and t h e r e f o r e , T u r n e r ' s syndrome was a c o n d i t i o n i n which a p a t e r n a l X chromosome had been l o s t d u r i n g m e o s i s . A maternal X chromosome c o i n c i d i n g with Duchenne dystrophy was h i g h l y c o n v i n c i n g o f t h e X - l i n k e d n e s s o f t h e g e n e r e s p o n s i b l e f o r the d i s o r d e r . 109 In 1977, Gomez e t a l . r e p o r t e d a s i t u a t i o n which i n v o l v e d a p a i r of i d e n t i c a l female t w i n s , one of whom was diagnos e d w i t h Duchenne d y s t r o p h y . Although the a f f e c t e d twin s i s t e r was, i n terms of the X chromosome, q u i t e normal, her mother e x h i b i t e d s u b c l i n i c a l s i g n s of muscular dystrophy a n d h e r b r o t h e r was w h e e l c h a i r bound w i t h Duchenne d y s t r o p h y . I t was t h e r e f o r e assumed that the i n a c t i v a t i o n of the X chromosome had been v e r y s e l e c t i v e r a t h e r than haphazard. C y t o l o g i c study had demonstrated that d u r i n g sex c e l l d i v i s i o n s two p a r t s of nonhomologous chromosomes c o u l d be exchanged to produce what i s c a l l e d a t r a n s l o c a t i o n . I f a t r a n s l o c a t i o n i n v o l v i n g the X chromosome and an autosome o c c u r r e d i n a female the i n a c t i v a t i o n of the X chromosome was d i s c o v e r e d to favour the normal X chromosome. In 1979, Lindenbaum et a l . p r o v i d e d the c y t o g e n i c evidence which gave the f i n a l proof of the X-linkedness of the gene f o r Duchenne d y s t r o p h y and l e d t h e way to m o l e c u l a r a n a l y s i s o f the d i s o r d e r . They d i s c o v e r e d a female with what appeared to be Duchenne dystrophy and although she had no r e l a t i v e s with the d i s o r d e r , the d i a g n o s i s was confirmed a c c o r d i n g t o the muscle b i o p s y , EMG and an e l e v a t e d c r e a t i n e phosphokinase. C y t o l o g i c a n a l y s i s d e m o n s t r a t e d t h a t a t r a n s l o c a t i o n i n v o l v i n g the X chromosome and autosome no. 1 had o c c u r r e d , the breakpoint being at the Xp21 l o c u s . The same.X-autosome t r a n s l o c a t i o n was subsequently found i n other females beset 110 by Duchenne dystrophy which set the stage f o r the i s o l a t i o n of the gene using DNA probes. The C e r e b r a l D e f e c t Mental r e t a r d a t i o n i n Duchenne's d i s e a s e was d i s m i s s e d as b e i n g u n r e l a t e d t o the d e g e n e r a t i o n of the s k e l e t a l muscle d u r i n g the n i n e t e e n t h c e n t u r y . T h e r e a f t e r , such s t a t e m e n t s as "the p a t i e n t was m e n t a l l y d e f i c i e n t " were commonly f e a t u r e d i n the case h i s t o r i e s and c l i n i c a l notes p e r t a i n i n g to t h o s e w i t h what became known as Duchenne dystrophy. With r e s p e c t to p a t h o g e n e s i s , though, they were c o m p l e t e l y i g n o r e d . Mental d e f i c i e n c y was r e d i s c o v e r e d as i t w e re, i n the 1950's and was a g a i n viewed w i t h i n the context of p a t h o g e n e s i s . In S c h e i n f e l d ' s "The New You and H e r e d i t y " (1950) the statement was made t h a t the same gene was r e s p o n s i b l e f o r the m e n t a l d e t e r i o r a t i o n which accompanied the m u s c u l a r d e g e n e r a t i o n o f p s e u d o h y p e r t r o p h i c m u s c u l a r d y s t r o p h y . Subsequent i n v e s t i g a t i o n was embarked upon with the purpose of v a l i d a t i n g or i n v a l i d a t i n g t h i s s t a t e m e n t , and a l s o because there was much i n t e r e s t r e g a r d i n g the p s y c h o l o g i c a l a s p e c t s o f d e b i l i t a t i n g d i s e a s e i n c h i l d r e n . R e p o r t s c o n c e r n i n g subsequent i n v e s t i g a t i o n seemed to r e f u t e the n o t i o n of any mental i n s u f f i c i e n c y i n the Duchenne type of d y s t r o p h y . Walton and N a t t r a s s (1954) found 48 cases of the d i s o r d e r to be m e n t a l l y sound, T r u i t t (1954) r e p o r t e d the I l l normal i n t e l l e c t u a l f u n c t i o n i n g of 72 boys with the d i s e a s e which agreed with the negative a s s o c i a t i o n between muscular and mental i n c a p a c i t y according to F l e i g e l (1955). Morrow and Cohen (1954) i n seeming disagreement found tha t 50% of t h e i r 29 p a t i e n t s , 24 of whom were boys and most of whom were of elementary school age, were not ab r e a s t of other c h i l d r e n i n terms of academic achievement. S i x t y - f i v e p e r c e n t d i s p l a y e d r e a d i n g d i f f i c u l t i e s and a p p r o x i m a t e l y h a l f were l a c k i n g i n a r i t h m e t i c s k i l l s . The average IQ f o r the group was ten p o i n t s below the normal; n o n e t h e l e s s , the a p p a r e n t d e p r e s s i o n o f i n t e l l e c t u a l f u n c t i o n i n g was a t t r i b u t e d to p s y c h o l o g i c a l and emotional f a c t o r s consequent t o p h y s i c a l d i s a b i l i t y . I n t r o v e r s i o n and a p a t h y were c o n s i d e r e d to be the sequel to p h y s i c a l d i s a b i l i t y and were b e l i e v e d to i n t e r f e r e w i t h i n t e l l e c t u a l performance of a c h i l d w i t h i n any give n s e t t i n g . Sherwin and McCully (1961) found t h a t 15 boys w i t h Duchenne muscular d y s t r o p h y were e s s e n t i a l l y normal w i t h i n the sphere of v e r b a l i n t e l l i g e n c e w h i c h o f c o u r s e d i f f e r e d f r o m D u c h e n n e ' s o r i g i n a l d e s c r i p t i o n a hundred years b e f o r e . There was thus some e v i d e n c e t h a t Duchenne muscular d y s t r o p h y d i d not i n v o l v e c o n c o m i t a n t mental i n s u f f i c i e n c y . I f such was the c a s e , p r o g r e s s i v e m e n t a l d e t e r i o r a t i o n c o u l d not accompany p r o g r e s s i v e muscular d e g e n e r a t i o n . In s p i t e of the d e c l a r e d absence of mental r e t a r d a t i o n i n the 48 cases of Walton and N a t t r a s (1954) four were s a i d 112 to be " s l i g h t l y backward". F o l l o w i n g S c h e i n f e l d ' s r a t h e r i s o l a t e d and a l l u s i v e statement there a l s o emerged evidence i n support of the ex i s t e n c e of mental d e f i c i e n c y i n Duchenne muscular d y s t r o p h y . T h i s p o s i t i v e evidence c a r r i e d much more w e i g h t t h a n t h e o p p o s i n g e v i d e n c e i n t h e f i n a l a n a l y s i s . Becker (1953)1 7 found t h a t 12% of a p a r t i c u l a r group o f c h i l d r e n w i t h m u s c u l a r d y s t r o p h y were i n f a c t mentally r e t a r d e d . J u s t as Morrow and Cohen (1954) found a mean subnormal IQ i n t h e i r p a t i e n t s , A l l e n and Rodgin (1960) i n a s t u d y i n v o l v i n g males r a n g i n g i n age from 2 t o 23 y e a r s , d i s c o v e r e d a mean subnormal IQ of 82. However, i n co n t r a s t to Morrow and Cohen, A l l e n and Rodgin decided t h a t w h i l s t emotional and p s y c h o l o g i c a l f a c t o r s c o u l d c o n t r i b u t e to a d e p r e s s i o n of i n t e l l e c t u a l f u n c t i o n i n g , they c o u l d not a c c o u n t f o r t h e d e g r e e o f m e n t a l d e f i c i e n c y nor t h e f r e q u e n c y . F i f t e e n of the 30 p a t i e n t s i n t h i s study were assessed as having a s i g n i f i c a n t l y subnormal IQ. Worden and Vignos (1962) confirmed the e x i s t e n c e of a subnormal i n t e l l e c t i n Duchenne muscular d y s t r o p h y w i t h t h e i r f i n d i n g s i n 38 c h i l d r e n between age 4 and 17. The group gave a mean IQ of 83, 26 of the 38 s c o r i n g an IQ below 90. C h i l d r e n with diabetes m e l l i t u s scored a mean IQ of 107 wh i l e those s u f f e r i n g from amyotonia congenita gave a mean IQ o f 118. T h e r e f o r e , i t a p p e a r e d t h a t e m o t i o n a l and C i t e d by Rosman and Kakulas (1966). I 113 p s y c h o l o g i c a l r e a c t i o n s to disease of p h y s i c a l d i s a b i l i t y i n c h i l d h o o d g e n e r a l l y , were not r e s p o n s i b l e f o r the mental i n s u f f i c i e n c y a s s o c i a t e d with Duchenne muscular d y s t r o p h y . The f a c t that poor academic performance was witnessed i n the v e r y e a r l y stages of the d i s e a s e when d i s a b i l i t y was very s l i g h t and the g r e a t e r i n c i d e n c e of mental impairment i n d y s t r o p h i c c h i l d r e n as compared to the normal p o p u l a t i o n a l s o supported the s p e c i f i c i t y of the r e l a t i o n s h i p between mental r e t a r d a t i o n and Duchenne muscular dystrophy. A l t h o u g h the e x i s t e n c e of the c e r e b r a l d e f e c t was seemingly demonstrated i t s p r o g r e s s i o n was not because IQ d i d not equate with the degree of muscular degeneration and r e s u l t a n t d i s a b i l i t y . For example Worden and Vignos found no p o s i t i v e r e l a t i o n s h i p between IQ and c r e a t i n e / c r e a t i n i n e e x c r e t i o n . A f o l l o w up t e s t on 7 boys 2 1/2 years a f t e r the i n i t i a l assessment a l s o r e v e a l e d no change i n a subnormal m e n t a l s t a t u s , t h e r e b y d e m o n s t r a t i n g t h a t the c e r e b r a l d e f e c t which was seen i n a s i g n i f i c a n t number with Duchenne muscular dystrophy was one of a s t a t i c n a t u r e . When a p o s i t i v e a s s o c i a t i o n between Duchenne muscular dystrophy and mental r e t a r d a t i o n a c c o r d i n g to IQ t e s t i n g was f i r s t r e p o r t e d W i n f i e l d et a l . ( 1 9 5 8 ) , i n s e a r c h o f a f u n c t i o n a l impairment of the b r a i n , employed the EEG as an avenue of i n v e s t i g a t i o n . A b n o r m a l i t i e s i n c l u d i n g 14 and 6 per s e c o n d p o s i t i v e s p i k e s were d i s c o v e r e d i n 9 o f 12 d y s t r o p h i c c h i l d r e n . Wayne & Brown-Mayers (1959) found 114 g e n e r a l a b n o r m a l i t i e s i n b o t h d y s t r o p h i c c h i l d r e n and a d u l t s . The 14 and 6 per second p o s i t i v e s p i k e s were again among t h e a b n o r m a l i t i e s s e e n i n 3 o f 8 w i t h t h e f a c i o s c a p u l o h u m e r a l form and 28 of 62 w i t h the Duchenne type o f d y s t r o p h y by P e r l s t e i n e t a l . ( 1 9 6 0 ) . The d i s e a s e p r o c e s s of both forms was thus suggested to i n v o l v e a " b a s i c m a t u r a t i o n a l n e u r o p h y s i o l o g i c a l " component. The study of B a r w i c k e t a l . ( 1965) i n v o l v e d t h e v a r i o u s c l i n i c a l v a r i e t i e s o f d y s t r o p h y i n c l u d i n g t h e D u c h e n n e , f a c i o s c a p u l o h u m e r a l types and myotonic dystrophy, as w e l l . O n l y 15% o f t h e Duchenne t y p e d i s p l a y e d a b n o r m a l EEG p a t t e r n s as d i d 23% of the f a c i o s c a p u l o h u m e r a l c a s e s . On t h e o t h e r hand 61% o f t h o s e w i t h m y o t o n i c d y s t r o p h y d i s p l a y e d a b e r r a n t EEGs which c o m p l i e d w i t h the w e l l -documented mental d e f i c i e n c y i n t h i s "impure" dyst r o p h y . Mental impairment was e s t a b l i s h e d i n 17 of 29 cases of myotonic dystrophy by Maas and Paterson (1937), four of whom were a d u l t s with the mental e q u i v a l e n t of ten years of age. A c o n g e n i t a l mental d e f i c i e n c y was a l s o seen i n another 11. E i g h t of the 15 p a t i e n t s of Walton and Nattras (1954) with m y o t o n i c d y s t r o p h y were s a i d t o be m e n t a l l y i m p a i r e d . T w e n t y - t w o p e r c e n t o f 319 c a s e s o f t h e d i s o r d e r i n S w i t z e r l a n d s t u d i e d by K l e i n (1957) had a s u b n o r m a l i n t e l l i g e n c e and Dodge et a l . (1965) found t h r e e of nine cases to be mentally d e f e c t i v e . 115 There was a l s o evidence of mental d e c l i n e i n myotonic d y s t r o p h y . Maas and Paterson (1937) spoke of i n t e l l e c t u a l r e t r o g r e s s i o n i n the d i s o r d e r , as d i d Walton and N a t t r a s ( 1 9 5 4 ) , who d e s c r i b e d the d e t e r i o r a t i o n o f i n t e l l e c t and memory i n three i n d i v i d u a l s and dementia i n another t h r e e . V anier (1960) t e s t e d the IQ of one p a r t i c u l a r boy s u f f e r i n g f r o m t h e d i s e a s e a t age s i x and a g a i n a t age t e n and d i s c o v e r e d t h a t IQ had f a l l e n from 85 t o 70 g i v i n g a d e f i n i t e i n d i c a t i o n of the p o t e n t i a l p r o g r e s s i v e nature of the c e r e b r a l a s p e c t . S c h e i n f e l d (1950) a c t u a l l y o f f e r e d no s u p p o r t i n g e v i d e n c e f o r the s t a t e m e n t r e g a r d i n g mental d e t e r i o r a t i o n i n Duchenne muscular dystrophy and perhaps he was a c t u a l l y r e f e r r i n g t o misdiagnosed cases of myotonic d y s t r o p h y . In any c a s e , m e n t a l d e f i c i e n c y i n m y o t o n i c d y s t r o p h y s e r v e d t o r e i n f o r c e the r e a l i t y of the c e r e b r a l d e f e c t i n Duchenne dystrophy. A f t e r the theory which proposed that muscular dystrophy was the r e s u l t of a t r o p h i c d i s t u r b a n c e o r i g i n a t i n g i n the supposed autonomic i n n e r v a t i o n of the s k e l e t a l muscle was a b a n d o n e d , p o s t m o r t e m e x a m i n a t i o n r e t u r n e d t o t h e confinement of the p e r i p h e r a l motor nerves and s p i n a l cord f o r the main purpose of c o n f i r m i n g the myopathic nature of the d i a g n o s i s . In seeming a n t i c i p a t i o n o f S c h e i n f e l d ' s s t a t e m e n t and b e c a u s e o f t h e d o c u m e n t a t i o n o f m e n t a l impairment i n m y o t o n i c d y s t r o p h y , Black and R a v i n (1947) took examination t o the higher c e n t r e s of myotonic dystrophy 116 where they d i s c o v e r e d no g r o s s or h i s t o l o g i c a l c h a n g e s . L i k e w i s e , Becker (1953)1 8 observed no changes i n the b r a i n of " t r u e " d y strophy, the i n v e s t i g a t i o n of which was spurred by the d i s c o v e r y of mental r e t a r d a t i o n i n h i s s u b j e c t s . With the i n c r e a s i n g e v i d e n c e f o r , and t h e r e f o r e g r e a t e r e m p h a s i s upon m e n t a l d e f i c i e n c y i n b o t h m y o t o n i c and Duchenne d y s t r o p h y , Rosman and K a k u l a s (1966) f o c u s e d a t t e n t i o n upon the b r a i n s of both d i s o r d e r s at autopsy. In three myotonic and three Duchenne type cases a s t a t i c mental d e f e c t corresponded to c e r e b r a l a b n o r m a l i t i e s i n c l u d i n g a r e d u c e d b r a i n w e i g h t , abnormal a r c h i t e c t u r e o f n e u r o n a l arrangement o f the c o r t e x and e c t o p i c neurons, which were e x p l a i n e d by the r e s u l t o f improper n e u r o n a l m i g r a t i o n d u r i n g d evelopment. The maldevelopment o f the c e r e b r a l hemispheres was proposed to be due to the g e n e t i c anomaly of e i t h e r d i s e a s e which a f f e c t e d the b r a i n at a c r i t i c a l and v u l n e r a b l e time during development. The r e s u l t s of a subsequent study by Rosman and Rebeiz (1967) s u p p o r t e d t h e p r o p o s a l f o r m y o t o n i c d y s t r o p h y . H i s t o l o g i c a l changes i n the muscle of t h r e e p a t i e n t s , i n which a nonprogressive i n t e l l e c t u a l impairment was evidenced by a " c e r e b r a l d y s g e n e s i s " as p r e v i o u s l y d e s c r i b e d , were much more severe than i n a p a t i e n t with no i n t e l l e c t u a l and v e r y m i n o r c e r e b r a l a b n o r m a l i t i e s . T he m u s c u l a r , C i t e d by Rosman and Kakulas (1966). 117 i n t e l l e c t u a l and c e r e b r a l aspects of myotonic dystrophy a l l seemed t o be r e l a t e d . T h e y were c o n s i d e r e d t o be c o n s e q u e n c e s of the d e f e c t i v e gene r e s p o n s i b l e f o r the d i s o r d e r which had been determined to be i n h e r i t e d as an autosomal dominant t r a i t . A s i m i l a r s i t u a t i o n was reported f o r Duchenne dystrophy by Rosman (1970), who found a c e r e b r a l d y s g e n e s i s i n one i n d i v i d u a l with a subnormal i n t e l l i g e n c e . There was a l s o a c o r r e l a t i o n between i n t e l l e c t u a l c a p a c i t y and d e g e n e r a t i v e changes i n the muscle i n t e n o t h e r s , s i x o f whom were i n t e l l e c t u a l l y impaired and who were at d i f f e r e n t stages of the d i s e a s e . However, Dubowitz and Crome (1969) found no evidence of a c e r e b r a l maldevelopment i n Duchenne d y s t r o p h y . Although minor h i s t o l o g i c a l a b n o r m a l i t i e s were d i s c o v e r e d i n the b r a i n of one who had been mentally r e t a r d e d , they were dismissed as being u n r e l a t e d to the muscular changes. By the time Zellweger and Hanson (1967) had confirmed the presence and n o n p r o g r e s s i o n of the c e r e b r a l d e f e c t i n Duchenne m u s c u l a r d y s t r o p h y a c c o r d i n g t o IQ t e s t i n g , a l t h o u g h t h e EEG had n o t d e m o n s t r a t e d any f u n c t i o n a l i m p a i r m e n t , i t a p p e a r e d t h a t t h e c e r e b r a l d e f e c t was indigenous to the b r a i n . There was no c o r r e l a t i o n between serum enzymes, which were thought to be l i b e r a t e d from d e g e n e r a t i n g m u s c l e , and IQ (Worden and V i g n o s , 1 9 6 2 ) , making i t u n l i k e l y that the c e r e b r a l aspect was a secondary e f f e c t . Dubowitz (1965) found that a g r e a t e r p r o p o r t i o n of 118 c h i l d r e n who became d y s t r o p h i c and who were subnormal i n i n t e l l i g e n c e , were delayed i n r e a c h i n g m a t u r a t i o n a l motor m i l e s t o n e s , i n c l u d i n g s i t t i n g and s t a n d i n g without support and w a l k i n g , t h a n were t h o s e w i t h an a v e r a g e or above average i n t e l l i g e n c e . It appeared th a t the c e r e b r a l d e f e c t was primary to the b r a i n but the q u e s t i o n of why the d e f e c t was not p r e s e n t or p e r c e p t i b l e i n a l l c a s e s of Duchenne dystrophy was more d i f f i c u l t to answer. The cases with a p o s i t i v e f a m i l y h i s t o r y of the d i s e a s e were more f r e q u e n t l y mentally r e t a r d e d (Dubowitz, 1965) and Cohen et a l . (1968) r e p o r t e d 94.9% c o n c o r d a n c e between a f f e c t e d male s i b l i n g s , that i s , i f one d y s t r o p h i c brother was m e n t a l l y d e f e c t i v e the other was as w e l l . P r o s s e r et a l . ( 1 9 6 9 ) , i n agreement with the f i n d i n g s of Worden and V i g n o s ( 1962), found that i n g e n e r a l the IQ of boys wi t h muscular dystrophy was lower than t h e i r u n a f f e c t e d s i b l i n g s but the d i s p a r i t y between a f f e c t e d and u n a f f e c t e d s i b l i n g s v a r i e d from f a m i l y t o f a m i l y , the d i f f e r e n c e i n magnitude b e i n g g r e a t e s t i n f a m i l i e s of g r e a t e r i n t e l l i g e n c e . A subnormal i n t e l l i g e n c e i n female c a r r i e r s seemed p l a u s i b l e i n view of the o t h e r s u b c l i n i c a l s i g n s , but the d a t a of P r o s s e r e t a l . ( 1 9 6 9 ) d i d n o t c o n f o r m t o s u c h as p o s s i b i l i t y . There was no d i f f e r e n c e between the IQ's of heterozygous females and other u n a f f e c t e d f a m i l y members nor was t h e r e any d i f f e r e n c e between IQ's of s i b l i n g s of c a r r i e r s and n o n c a r r i e r s . 119 Cohen et a l . (1968) proposed four g e n e t i c t h e o r i e s to account f o r the inconstancy of the c e r e b r a l d e f e c t , three of which assumed the e x i s t e n c e of two separate genes: a) one gene was r e s p o n s i b l e f o r muscular d y s t r o p h y w i t h normal i n t e l l i g e n c e and a n o t h e r f o r m u s c u l a r d y s t r o p h y w i t h subnormal i n t e l l i g e n c e ; b) two genes which were l i n k e d d i c t a t e d the muscular and c e r e b r a l d e f e c t s , s e p a r a t e l y ; c) two genes which were not l i n k e d but i n t e r a c t e d governed the muscular and c e r e b r a l aspects s e p a r a t e l y . The f o u r t h theory proposed that a s i n g l e gene was r e s p o n s i b l e f o r muscular and mental d e f e c t s but that there was l i m i t e d e x p r e s s i o n of the gene w i t h i n t h e CNS w h i c h was i n f l u e n c e d by f a m i l i a l p r e d i s p o s i t i o n . The knowledge of gene e x p r e s s i o n was u n f o r t u n a t e l y q u i t e l i m i t e d at that time. More i m p o r t a n t l y , a b i o c h e m i c a l a b n o r m a l i t y c o n s e q u e n t t o t h e d e f e c t i v e p r o t e i n governed by the i m p e r f e c t gene a c c o r d i n g to the s i n g l e gene h y p o t h e s i s was not a v a i l a b l e . T h e r e f o r e no neurochemical abnormality c o u l d be i n v e s t i g a t e d . By 1970 the frequent obtuseness of i n t e l l e c t i n t h i s d i s e a s e o f t h e v o l u n t a r y m u s c l e , noted by Duchenne the c e n t u r y b e f o r e , was c o n f i r m e d . Very l i t t l e more c o u l d be added r e g a r d i n g cause and e f f e c t c o n c e r n i n g the c e r e b r a l d e f e c t i n e i t h e r Duchenne or myotonic dystr o p h y . Even s o , j u s t as a p p a r e n t m e n t a l d e f i c i e n c y was t h e b a s i s o f Duchenne's a s s e r t i o n t h a t t h e d i s e a s e was "of c e r e b r a l 120 o r i g i n " , t h e c e r e b r a l d e f e c t s i n Duchenne and m y o t o n i c dystrophy l e n t support to the neurogenic h y p o t h e s i s . T h e D i f f e r e n t i a l D i a g n o s i s o f M y o p a t h y v s . N e u r o p a t h y Erb d i v i d e d neuromuscular d i s e a s e i n t o myopathy and neuropathy a c c o r d i n g t o the h i s t o l o g i c a l f e a t u r e s of the mus c l e . Group a t r o p h y , a h i s t o l o g i c a l f e a t u r e which i s considered to be t y p i c a l of neuropathy, i n cases of Duchenne dystrophy, was c i t e d by McComas et a l . i n 1971c as a p o i n t very much i n favour of the neurogenic h y p o t h e s i s of muscular dystrophy. However, i t was fe a t u r e s t y p i c a l of myopathy i n the b i o p s i e s of those w i t h p u r p o r t e d n e u r o p a t h i e s which b l u r r e d t h e l i n e , as i t w e r e , d i v i d i n g myopathy and neuropathy, making the myogenic nature of muscular dystrophy somewhat q u e s t i o n a b l e . D u r i n g the " m e t a b o l i c phase" of mus c u l a r d y s t r o p h y , r e s e a r c h employing human muscle was devoted p r i m a r i l y t o b i o c h e m i c a l a n a l y s i s , by v i r t u e o f advances w i t h i n the sphere of energy metabolism and the c h e m i s t r y of muscular c o n t r a c t i o n . When no sharp d i s t i n c t i o n between myopathy and neuropathy was made i n terms of chemical composition at the end of the 1930's (Reinhold and K i n g s l e y , 1938), and i n view o f t h e a p p a r e n t l y u n d i s t u r b e d m e c h a n i s m o f m u s c u l a r c o n t r a c t i o n i n muscular dystrophy, r e s e a r c h returned to the q u a l i t a t i v e examination of muscle using the l i g h t microscope ( H a s s i n , 1943). 121 T h r o u g h o u t t h e m e t a b o l i c p h a s e the p a t h o l o g i c a l r e a c t i o n s o f m u s c l e c o n t i n u e d t o be t h e p u r p o s e o f e x p e r i m e n t a l i n q u i r y which employed a n i m a l s u b j e c t s . Regeneration of muscle was found to occur i n p a t h o l o g i c a l s t a t e s other than that which r e s u l t e d from d e n e r v a t i o n . As a r e s u l t of a renewed i n t e r e s t i n the pathology of muscular dystrophy, muscular disease i n g e n e r a l , the l i s t of c r i t e r i a of f e a t u r e s c o n s i d e r e d to be d i a g n o s t i c of myopathy was expanded. By 1960, the l i s t s t i l l i n c l u d e d random v a r i a t i o n of f i b r e s i z e , connective t i s s u e p r o l i f e r a t i o n , degenerative changes, f i b r e s p l i t t i n g and c e n t r a l n u c l e i . Phagocytosis and r e g e n e r a t i v e changes, namely, b a s o p h i l i a , v e s i c u l a r n u c l e i and p r o m i n e n t n u c l e o l i , had been a d d e d . The d i s c o v e r y of the motor u n i t had brought understanding to the contiguous p o s i t i o n s of normal and a t r o p h i e d f i b r e s and the random v a r i a t i o n o f f i b r e s i z e i n m u s c u l a r d y s t r o p h y r e i n f o r c e d the myogenic nature of the group. During the 50's c o l l a t e r a l s p r o u t i n g by nerves was f i r s t observed and thus e x p l a i n e d the c l u s t e r s of a t r o p h i c f i b r e s of group atrophy seen the b i o p s i e s of neuropathy. Group atrophy was e v i d e n c e of r e i n n e r v a t i o n of f i b r e s which had l o s t t h e i r n e r v e , as i t were, by c o l l a t e r a l s p r o u t s o f s u r v i v i n g healthy nerves. Although the muscle biopsy had not p r o v i d e d a g r e a t d e a l of i n s i g h t i n t o the pathogenesis of muscular d y s t r o p h y w h i c h i n terms o f h i s t o l o g y was s t i l l t h e 122 e q u i v a l e n t of myopathy, the h i s t o l o g y of the muscle was inescapably of d i a g n o s t i c v a l u e . Duchenne's " l o c a l i s e d e l e c t r i s a t i o n " was supplanted by t h e e l e c t r o m y o g r a m (EMG) which r a t h e r than t e s t i n g the c o n t r a c t i l e r e s p o n s e o f m u s c l e t o the a p p l i c a t i o n o f e l e c t r i c c u r r e n t , recorded the e l e c t r i c a l a c t i v i t y of muscle p r o d u c e d by i t s c o n t r a c t i o n . A l a r g e number of a c t i o n p o t e n t i a l s w e r e o b s e r v e d t o be common a n d e v e n c h a r a c t e r i s t i c of muscular dystrophy. However, the number o f s p i k e s was not n e c e s s a r i l y a s e n s i t i v e i n d e x o f d y s t r o p h i c muscle; r a t h e r , the f e a t u r e s of the i n d i v i d u a l s p i k e s were d i s c o v e r e d to be a l t e r e d ( K u g e l b e r g , 1947, 1949). The amplitude was found very o f t e n to be decreased and, most important, the d u r a t i o n was shortened, i n d i c a t i n g a l o s s of f i b r e numbers comprising the motor u n i t . There was thus a g r e a t e r synchrony between i n d i v i d u a l f i b r e s of the u n i t with i t s c o n t r a c t i o n . The presence of p o l y p h a s i c p o t e n t i a l s which were a l s o smaller and b r i e f e r than normal and were i n t e r p r e t e d as the r e s u l t o f the " a n a t o m i c a l d i s i n t e g r a t i o n of the motor u n i t " were c o m p a t i b l e w i t h random a t t a c k upon i n d i v i d u a l m u s c l e f i b r e s . T h e s e parameters, p o t e n t i a l s of short d u r a t i o n and low amplitude, which were sometimes p o l y p h a s i c , a l s o a p p l i e d to myotonic dystrophy and myasthenia g r a v i s . They q u i c k l y became the s t andard f e a t u r e s of the EMG which were used as i n d i c a t o r s of myopathy. 123 F i b r i l l a t i o n p o t e n t i a l s and i n c r e a s e d i n s e r t i o n a l a c t i v i t y were found to be c h a r a c t e r i s t i c of neuropathy. In c o n t r a s t to muscular dystrophy, s p e c i f i c a l l y , i n which t h e r e was a d i s p r o p o r t i o n a t e d e c r e a s e i n t h e s t r e n g t h o f c o n t r a c t i o n corresponding to a p a r t i c u l a r number of s p i k e s , the number of p o t e n t i a l s was p r o p o r t i o n a l to s t r e n g t h i n n e u r o p a t h y i n w h i c h whole motor u n i t s were a f f e c t e d . " G i a n t " p o t e n t i a l s w i t h long d u r a t i o n s and high amplitudes were i n t e r p r e t e d as being evidence of r e i n n e r v a t i o n of motor u n i t s which had l o s t t h e i r nerve, as i t were, by c o l l a t e r a l s o f h e a l t h y nerve f i b r e s i n c h r o n i c d e n e r v a t i n g d i s e a s e . " G i a n t " p o t e n t i a l s were t h e r e f o r e the e l e c t r o p h y s i o l o g i c a l c o r r e l a t e of group atrophy seen i n b i o p s i e s . The EMG, i n a d d i t i o n t o the muscle b i o p s y , proved to be of v a l u e i n e s t a b l i s h i n g a d i a g n o s i s e s p e c i a l l y when the c l i n i c a l syndrome was i n q u e s t i o n . The muscle biopsy and the EMG v e r i f i e d the consensus as t o t h e n a t u r e o f C h a r c o t - M a r i e - T o o t h d i s e a s e (CMT) u l t i m a t e l y reached t h r o u g h postmortem e x a m i n a t i o n . The study of Brodal et a l . (1953) supported the neurogenesis of CMT as d i d t h a t of Adams et a l . ( 1953) who s t a t e d w i t h r e s p e c t t o the m u s c l e b i o p s y s p e c i f i c a l l y t h a t : " T h i s p a t h o l o g i c a l p i c t u r e i s undoubtedly that of a c h r o n i c n e u r a l muscular atrophy and not a d y s t r o p h y . " However, n e a r l y a decade l a t e r the impression g i v e n by 12 of 17 b i o p s i e d cases i n an i n v e s t i g a t i o n by Haase and Shy (1960) was one of 124 m y o p a t h y . S p e c i f i c m y o p a t h i c c h a n g e s were much more frequent i n CMT, f o r which p a t h o l o g i c a l changes were seen i n the d i s t a l aspect of the p e r i p h e r a l n e r v e , as compared to f o u r t e e n cases of amyotrophic l a t e r a l s c l e r o s i s (ALS). Only one of the 14 cases o f ALS showed d e g e n e r a t i v e changes as compared to 59% of those with CMT. Regeneration of muscle was common i n CMT a s i n d i c a t e d by p h a g o c y t o s i s , i n f l a m m a t i o n , b a s o p h i l i a and p r o m i n e n t n u c l e o l i . Such f e a t u r e s were c o m p a r a t i v e l y i n f r e q u e n t i n ALS as were c o n n e c t i v e t i s s u e p r o l i f e r a t i o n and c e n t r a l n u c l e i . Group l e s i o n s were a l s o seen i n 100% of those with ALS while only 70% w i t h CMT d i s p l a y e d s u c h e v i d e n c e o f n e u r o p a t h y . Myopathic f e a t u r e s were a l s o r e p o r t e d i n a case of CMT by T y r e r and S u t h e r l a n d (1961) and a l t h o u g h t h e c a s e s o f M y r i a n t h o p o l o u s (1964) gave EMG's t y p i c a l of neuropathy, b i o p s i e s had "myopathic components". L i k e the muscular d y s t r o p h i e s CMT was determined to be h e r i t a b l e , an autosomal dominant p a t t e r n with i n c o m p l e t e p e n e t r a n c e b e i n g r e p o r t e d by L u c a s and F o r s t e r ( 1 9 6 2 ) . Lucas and F o r s t e r took note of r e p o r t s concerning c l i n i c a l s y n d r o m e s w h i c h were i n t r a n s i t i o n b e t w e e n m u s c u l a r dystrophy and Charcot-Marie-Tooth. They a l s o c o n s i d e r e d the e x i s t e n c e of C h a r c o t - M a r i e - T o o t h and F r e i d r i c h ' s a t a x i a , which was a l s o known to be h e r i t a b l e , w i t h i n one f a m i l y . With these f a c t s and with the knowledge of other s t u d i e s and the r e s u l t s r egarding b i o p s i e s from t h e i r own they put f o r t h 125 a t h e o r y . I f any one of a t h e o r e t i c a l group of enzymes belonging to a p a r t i c u l a r aspect of metabolism o p e r a t i n g i n the neuromuscular system was d e f e c t i v e , a combined myopathy-neuropathy was proposed to be the r e s u l t . The brunt of the d i s e a s e p r o c e s s would depend upon the p a r t i c u l a r enzyme i n v o l v e d and the d y s t r o p h i c b i o p s i e s of CMT o b s e r v e d by Lucas and F o r s t e r and others were t h e r e f o r e thought to be e x p l i c a b l e . Within the context of t h i s theory, F r e i d r i c h ' s a t a x i a , C h a r c o t - M a r i e - T o o t h and muscular d y s t r o p h y were r e p r e s e n t a t i v e of s p e c i f i c p o s i t i o n s on a continuum of n e u r o m u s c u l a r d i s e a s e , CMT b e i n g the " m i d d l e man" o f myopathy and neuropathy. The f a m i l i a l aspect of a c o n d i t i o n i n 12 i n d i v i d u a l s i n which i n t a c t s e n s i t i v i t y accompanied muscular weakness and w a s t i n g r e i n f o r c e d the o r i g i n a l d i a g n o s i s of m u s c u l a r d y s t r o p h y . A f t e r f u r t h e r e x amination and c o l l a b o r a t i o n using the a n c i l l a r y procedures of the muscle biopsy and the EMG, Kugelberg and Welander (1956) a l t e r e d t h e i r d i a g n o s i s and d e f i n e d a new syndrome which they c a l l e d " H e r e d o f a m i l i a l j u v e n i l e m u s c u l a r a t r o p h y " , w h i c h o t h e r s c a l l e d "pseudomyopathic muscular atrophy", but which would a c q u i r e the t i t l e of "Kugelberg-Welander s p i n a l muscular a t r o p h y " . The d i s c o v e r y of t h i s new syndrome and the q u e s t i o n as to i t s nature i s reminiscent of the myogenic versus neurogenic debate of the a d u l t form of p r o g r e s s i v e muscular a t r o p h y d u r i n g the n i n e t e e n t h c e n t u r y . The Kugelberg-Welander 126 s y n d r o m e r e m i n d e d t h o s e i n v o l v e d i n t h e f i e l d o f n e u r o m u s c u l a r d i s e a s e of the a b i l i t y of n e u r o p a t h y t o c l i n i c a l l y simulate myopathy. T s u k a g o s h i et a l . (1965) r e p o r t e d f i v e c a s e s which c l i n i c a l l y r e s e m b l e d l i m b - g i r d l e m u s c u l a r d y s t r o p h y . Although they were of adult onset, they were con s i d e r e d to be cases of Kugelberg-Welander s p i n a l muscular atrophy. The EMG and b i o p s i e s of these f i v e i n d i v i d u a l s and those of f i f t e e n others with the disease described by Gardner-Medwin e t a l . ( 1 9 6 7 ) , showed f e a t u r e s t y p i c a l o f n e u r o p a t h y . Myopathic f e a t u r e s were a l s o seen among b i o p s i e s . C e n t r a l n u c l e i , n e c r o s i s and s t r u c t u r a l changes were seen i n the c a s e s of Tsukagoshi et a l . w h i l e v a c u o l a r d e g e n e r a t i o n , h y a l i n i z a t i o n , phagocytosis and the o c c a s i o n a l h y p e r t r o p h i e d f i b r e were among the changes i n b i o p s i e d cases of Gardner-Medwin et a l . In view of these myopathic f e a t u r e s i t was i n i t i a l l y suggested that Kugelberg-Welander s p i n a l muscular atrophy was a combined myopathic-neurogenic atrophy as had been suggested f o r Charcot-Marie-Tooth. Gardner-Medwin et a l . u l t i m a t e l y d i s m i s s e d t h i s c h a r g e i n l i g h t o f the myopathic h i s t o l o g i c a l f e a t u r e s that had been observed i n d e n e r v a t e d muscle of e x p e r i m e n t a l animals (Adams et a l . , 1962). In 1949, S i b l e y and Lehninger found the serum l e v e l s of the cytoplasmic enzyme, a l d o l a s e , to be g r o s s l y e l e v a t e d i n d y s t r o p h i c c h i l d r e n . E l e v a t e d serum a l d o l a s e , a l o n g w i t h 127 the elevation of other cytoplasmic enzymes involved in ATP production, which were subsequently discovered, became diagnostic of muscular dystrophy. In 1959 Ebashi et a l . developed an assay for creatine phosphokinase because of its importance in muscle metabolism. The ability to measure serum levels of creatine phosphokinase proved very fruitful. The degree of elevation of this enzyme was greatest of a l l cytoplasmic enzymes. As a result, the elevation of creatine phosphokinase was considered to be the most sensitive index of primary muscular disease. However, elevations in purported neuropathies were not consistent with the specificity for myopathy. Tsukagoshi et a l . (1965) found elevations in Charcot-Marie-Tooth disease. Some of those with Kugelberg-Welander spinal muscular atrophy in the study of Gardner-Medwin et a l . (1967) had elevated levels. Dawson (1966) determined the rate of cytoplasmic enzyme efflux in both dystrophic and denervated chicken muscle. The enzymes included creatine phosphokinase, lactate dehydrogenase, malic dehydrogenase, aldolase, aspartate transaminase, and phosphoglucomutase. The rate of efflux of a l l was accelerated and comparable in the dystrophic and denervated muscle. Neuropathy and myopathy were thus not as distinct in terms of enzyme leakage as originally believed. Although myopathic changes in muscle could result in a relatively short period of time following denervation in experimental animals (Adams et a l . , 1962) the human 128 s i t u a t i o n was more d i f f i c u l t to a s s e s s . Drachman et a l . (1967) took i t upon t h e m s e l v e s t o p r o v i d e t h e f i r s t s y s t e m a t i c s t u d y i n o r d e r to answer the q u e s t i o n as t o whether myopathic f e a t u r e s i n human muscle c o u l d be the r e s u l t of c h r o n i c d e n e r v a t i o n . Nine i n d i v i d u a l s who had been s t r i c k e n by p o l i o m y e l i t i s 10 to 54 years p r i o r to the s t u d y were s e l e c t e d as e x a m p l e s o f " p u r e , p a r t i a l , l o n g s t a n d i n g d e n e r v a t i o n " . Myopathic f e a t u r e s , i n c l u d i n g degenerative and regenerative changes, c e n t r a l n u c l e i being the most common myopathic f e a t u r e , were i n abundance i n seven b i o p s i e s which gave the same combined m y o p a t h i c -n e u r o p a t h i c p i c t u r e s that were seen i n Charcot-Marie-Tooth and Kugelberg-Welander SMA. It was t h e r e f o r e concluded that myopathic changes c o u l d be i n c i d e n t a l to d e n e r v a t i o n i n human c h r o n i c d e n e r v a t i n g d i s e a s e . However, a random v a r i a t i o n i n f i b r e s i z e was the only h i s t o l o g i c a l f e a t u r e s e e n i n t h r e e b i o p s i e s . T h i s came as a s u r p r i s e and u l t i m a t e l y l e d Drachman et a l . to suggest that i n view of the myopathic f e a t u r e s i n b i o p s i e s of d e c l a r e d neuropathic d i s e a s e , that perhaps i t was time to reopen the q u e s t i o n as t o t h e myogenic v e r s u s n e u r o g e n i c o r i g i n o f m u s c u l a r d i s o r d e r s i n c l u d i n g muscular dystrophy. Just as myopathic f e a t u r e s i n the b i o p s i e s of p o l i o m y e l i t i s had s e r v e d t o confirm Erb " i n h i s s c r u p l e s " when he proposed the o r i g i n a l t r o p h i c t h e o r y of muscular d y s t r o p h y , such h i s t o l o g i c a l 129 f e a t u r e s i n p o l i o m y e l i t i s supported Drachman et a l . ' s n o t i o n t h a t c e r t a i n myopathies were neuropathies i n d i s g u i s e . T h e P a t h o l o g y o f t h e I n t r a m u s c u l a r M o t o r I n n e r v a t i o n When Hunter (1925) proposed the i n v e s t i g a t i o n o f the a l l e g e d sympathetic i n n e r v a t i o n as regards the p a t h o g e n e s i s of muscular d y s t r o p h y , he suggested the same f o r myasthenia g r a v i s and myotonic d y s t r o p h y . Both d i s e a s e s were a l s o i n c l u d e d w i t h m u s c u l a r d y s t r o p h y i n t h e s t u d y o f t h e mechanism of c o n t r a c t i o n i n muscular d i s e a s e ( N e v i n , 1934). In g e n e r a l , d u r i n g t h e " m e t a b o l i c p h a s e " o f m u s c u l a r d y s t r o p h y , r e s e a r c h p e r t a i n i n g t o m y a s t h e n i a g r a v i s and myotonic dystrophy p a r a l l e l e d that of muscular d y s t r o p h y . W i t h t h e u s e o f t h e same b a s i c s i l v e r s t a i n i n g t e c h n i q u e Tower (1931) used t o s t u d y the i n t r a m u s c u l a r i n n e r v a t i o n , a redundant i n n e r v a t i o n c o u l d be demonstrated i n myopathy w h i l e the presence of empty Schwann tubes was i n d i c a t i v e of neuropathy (Bowden and Gutmann, 1945). The i n t r a v i t a l s t a i n i n g w i t h methylene b l u e , which e n t a i l e d subcutaneous i n j e c t i o n of the dye bef o r e the muscle t i s s u e was e x t r a c t e d ( C o e r s , 1952) e v e n t u a l l y p r o v i d e d much g r e a t e r d e t a i l to the i n t r a m u s c u l a r i n n e r v a t i o n . Changes i n the s i z e and s t r u c t u r e o f e n d p l a t e s and nerve f i b r e s c o u l d be v i s u a l i z e d and the number of muscle f i b r e s i n n e r v a t e d by an axon or the t e r m i n a l i n n e r v a t i o n r a t i o c o u l d be e s t i m a t e d . An i n c r e a s e i n t e r m i n a l i n n e r v a t i o n r a t i o s as a consequence 130 o f c o l l a t e r a l , t e r m i n a l or u l t r a t e r m i n a l a r b o r i z a t i o n o f n e r v e f i b r e s was d i s c o v e r e d t o be c h a r a c t e r i s t i c o f neuropathy. The m u s c l e o f m y a s t h e n i a g r a v i s d i d not p r e s e n t a d y s t r o p h i c h i s t o l o g y , and i n i t i a l l y i t was regarded as being r e l a t i v e l y f r e e o f p a t h o l o g y u n t i l i t was more c l o s e l y i n v e s t i g a t e d . I t was not the pathology o f the muscle per s e , t h o u g h , but the p a t h o l o g y of the i n t r a m u s c u l a r motor i n n e r v a t i o n w h i c h p r o v i d e d the most i m p o r t a n t e v i d e n c e re g a r d i n g p a t h o g e n e s i s . Coers and Woolf (1954)1 9 were a b l e t o d i s c e r n q u a l i t a t i v e changes which gave e v i d e n c e o f a neu r a l d e f e c t . E n d p l a t e s were elongated and c h o l i n e s t e r a s e p r e p a r a t i o n d e m o n s t r a t e d a b n o r m a l i t i e s i n the s u b n e u r a l a p p a r a t u s . C o e r s and Desmedt ( 1 9 5 9 )2 0 c o n f i r m e d t h e e x i s t e n c e o f the " d y s p l a s t i c " a r b o r i s a t i o n o f the n e r v e t e r m i n a l which was s p e c i f i c f o r the d i s e a s e . A " d y s t r o p h i c " a r b o r i s a t i o n was a l s o d e s c r i b e d and as t h i s f e a t u r e was c o n s i d e r e d t o be a s e c o n d a r y r e a c t i o n t o m u s c u l a r d e g e n e r a t i o n i n d y s t r o p h y , i t was regarded as secondary i n m y a s t h e n i a g r a v i s . E l e c t r o n m i c r o s c o p y v e r i f i e d t h e p r e s e n c e o f changes i n the subneural a p p a r a t u s (Zacks et a l . , 1962). C i t e d by Walton (1981). 'Cited by Walton (1981). 131 C o e r s and Woolf ( 1959) used the same methylene b l u e s t a i n i n g technique used to examine the t e r m i n a l i n n e r v a t i o n of m y a s t h e n i a g r a v i s , i n the s t u d y of the i n t r a m u s c u l a r nerve f i b r e s i n myotonic dyst r o p h y . A b n o r m a l i t i e s i n c l u d e d p r o f u s e a r b o r i s a t i o n of the nerve t e r m i n a l , l a r g e r t h a n normal e n d p l a t e s and r a m i f i c a t i o n of nerve t e r m i n a l s around i n d i v i d u a l muscle f i b r e s . These changes were con s i d e r e d to be the r e s u l t of hypertrophy and degenerative changes i n the m u s c l e . MacDermot (1961) confirmed the e x i s t e n c e of these a b n o r m a l i t i e s but o b s e r v e d o t h e r a b n o r m a l i t i e s , as w e l l . Axons of the d i s t a l neuron were abnormal, sometimes g i v i n g the appearance of d e g e n e r a t i o n . In view of these a d d i t i o n a l c h a n g e s , MacDermot pr o p o s e d t h a t a d e f e c t i n the d i s t a l n e u r o n was i n p a r t r e s p o n s i b l e f o r the p a t h o g e n e s i s o f m y o t o n i c d y s t r o p h y , i n c o n j u n c t i o n w i t h t h e d e f e c t indigenous to the muscle f i b r e . A p a r t i c u l a r s t r a i n of the Bar Harbour h e r i t a b l e mouse d y s t r o p h i e s , s t r a i n 129, was c o n f i r m e d t o be a myopathy a c c o r d i n g t o the absence of a n e u r a l l e s i o n . S t r u c t u r a l changes i n the subneural apparatus and t e r m i n a l a r b o r i s a t i o n were at f i r s t regarded as being secondary m a n i f e s t a t i o n s of a p r i m a r y d i s e a s e p r o c e s s . However, t h e p o s s i b i l i t y remained t h a t the m o r p h o l o g i c a l changes were primary. In order to v e r i f y the nature of the changes i n mouse dystrophy Conrad e t a l . (1964) i n v e s t i g a t e d the m i n i a t u r e e n d p l a t e p o t e n t i a l s o f the d y s t r o p h i c m u s c l e as a p h y s i o l o g i c a l 132 i n d i c a t o r o f the s t a t e o f the p r e s y n a p t i c t e r m i n a l and s u b n e u r a l a p p a r a t u s . They d i s c o v e r e d q u i t e u n e x p e c t e d l y that the frequency of m i n i a t u r e endplate p o t e n t i a l s was much r e d u c e d , as was the i n c r e a s e i n f r e q u e n c y i n response to h i g h s o d i u m c o n c e n t r a t i o n s . Such changes were seen i n m y a s t h e n i a g r a v i s and d e n e r v a t i o n w h i c h weakened t h e m y o p a t h i c i t y of t h i s p a r t i c u l a r animal model. The r e s t i n g membrane p o t e n t i a l s of t h e d y s t r o p h i c muscle were a l s o measured as being comparable to that of denervated muscle, which was lower than n o r m a l . McComas and Mrozek (1967) r e p o r t e d t h a t the muscle f i b r e s of t h i s s t r a i n from which e l e c t r i c a l a c t i v i t y could be recorded by the i n s e r t i o n of an e l e c t r o d e , v e r y commonly d i d n o t r e s p o n d when t h e i n n e r v a t i n g n e r v e was s t i m u l a t e d . S u ch f i b r e s were c o n s i d e r e d to be " f u n c t i o n a l l y " d enervated. T h i s e f f e c t i v e d e n e r v a t i o n c o u l d a l s o be r e l a t e d t o t h e m o r p h o l o g i c a l changes i n the p r e s y n a p t i c t e r m i n a l and subneural apparatus w h i c h w e r e t h e n deemed t o be o f i m p o r t a n c e i n t h e pathogenesis of the c o n d i t i o n . The pathology of the i n t r a m u s c u l a r motor i n n e r v a t i o n i n the animal model, and myotonic dystrophy made the secondary n a t u r e o f t e r m i n a l a r b o r i s a t i o n o f t h e n e r v e f i b r e i n m u s c u l a r d y s t r o p h y somewhat s u s p e c t . The s u s p i c i o n was s t r e n g t h e n e d by the e v i d e n c e f o r a p r e s y n a p t i c d e f e c t i n m y a s t h e n i a g r a v i s . In a d d i t i o n t o the p a t h o l o g y of the i n t r a m u s c u l a r motor i n n e r v a t i o n and t h e d e c r e a s e i n 133 m i n i a t u r e e n d p l a t e p o t e n t i a l f r e q u e n c y , the amplitude of m i n i a t u r e endplate p o t e n t i a l was reduced. The r e d u c t i o n was i n t e r p r e t e d t o be t h e r e s u l t o f s m a l l e r q u a n t a o f a c e t y l c h o l i n e released by the p r e s y n a p t i c t e r m i n a l . Trophic Influences of Nerve Upon Muscle A f t e r the somatic nerves were confirmed to be the only e f f e r e n t i n n e r v a t i o n of the s k e l e t a l muscle i n 1931, Tower (1935) v e r i f i e d the t r o p h i c power of the animating nerve by m o n i t o r i n g the a t r o p h y and d e g e n e r a t i o n of muscle which succeeded d e n e r v a t i o n . She a l s o demonstrated i n 1937 that i t was not simply the s t r u c t u r a l i n t e g r i t y of the nerve which was n e c e s s a r y to m a i n t a i n a w e l l - n o u r i s h e d s t a t e of the muscle, but the a c t i v i t y of the nerve as w e l l . In order to render the s p i n a l cord completely q u i e s c e n t and t h e r e f o r e d e p r i v e the motor neurons of the r e c e i p t of any impulses, the lumbar and s a c r a l d o r s a l r o o t s were s e c t i o n e d and the s p i n a l c o r d t r a n s e c t e d above and below. T h i s p r o c e d u r e r e s u l t e d i n a r e d u c t i o n i n the volume of the m u s c u l a r system. M i c r o s c o p i c a l l y , c o n n e c t i v e t i s s u e p r o l i f e r a t i o n accompanied the atrophy of the i n d i v i d u a l f i b r e s . Meanwhile, Dale and Feldberg (1934) had announced the r o l e of a c e t y l c h o l i n e as the chemical communicator between nerve and muscle. A c e t y l c h o l i n e was detected i n the medium p e r f u s i n g the l e g of an a n a e s t h e t i z e d a n i m a l f o l l o w i n g s t i m u l a t i o n of the s c i a t i c nerve. A f t e r the muscle had been 134 a c t i v a t e d to c o n t r a c t a c e t y l c h o l i n e was no longer d e t e c t a b l e and was assumed t o be degraded by l o c a l s o u r c e s of the enzyme, c h o l i n e s t e r a s e . The a n t i c h o l i n e s t e r a s e d r u g , physostigmine, produced a p o s i t i v e e f f e c t upon the muscular f a t i g u a b i l i t y o f myasthenia g r a v i s i n 1934 (Walker). t h i s was the f i r s t pharmacological evidence which i m p l i c a t e d the neuromuscular j u n c t i o n i n the pathogenesis of the d i s e a s e . T h e r e a f t e r a n d w i t h c o n f i r m a t i o n o f t h e r o l e o f a c e t y l c h o l i n e i n n e u r o m u s c u l a r t r a n s m i s s i o n , a n a c e t y l c h o l i n e d e f i c i e n c y became a p l a u s i b l e c a u s a t i v e f a c t o r r e s p o n s i b l e f o r the muscular c o n d i t i o n . The s y n t h e s i s of a c e t y l c h o l i n e was found to be v e r y n o t i c e a b l y promoted by vi t a m i n E i n 1945 (Torda and W o l f f ) , a time when the p h y s i o l o g i c a l r o l e of vi t a m i n E was s t i l l b e i n g p u r s u e d . B e f o r e t h e a v i t a m i n o s i s E model was c o m p l e t e l y s u p p l a n t e d by the Bar Harbour h e r i t a b l e mouse d y s t r o p h i e s Hove and C o p e l a n d (1954) r e a s o n e d t h a t i f v i t a m i n E was the p h y s i o l o g i c a l a c t i v a t o r of a c e t y l c h o l i n e s y n t h e s i s a v i t a m i n E d e f i c i e n c y c o u l d r e s u l t i n an a c e t y l c h o l i n e d e f i c i e n c y . A d e a r t h of a c e t y l c h o l i n e was a p o s s i b l e u n d e r l y i n g cause of n u t r i t i o n a l muscular dystrophy. In order t o t e s t t h i s h y p o t h e s i s , r a t s were r a i s e d on a d i e t r e p l e t e w i t h v i t a m i n E but d e p l e t e d i n c h o l i n e , the p r e c u r s o r of a c e t y l c h o l i n e . L e s i o n s which were c o n f i n e d to the s k e l e t a l muscle were the consequence of t h i s d i e t . The changes i n c r e a t i n e / c r e a t i n i n e e x c r e t i o n , u n l i k e those of 135 the a v i t a m i n o s i s E model which were rather a b r u p t , were more g r a d u a l , and t h e r e f o r e more l i k e the human c o n d i t i o n . An i n t e r r u p t e d nervous i n f l u e n c e stemming from an a c e t y l c h o l i n e d e f i c i e n c y , which was p o s s i b l y r e l a t e d t o a d e f e c t i n s y n t h e s i s or u t i l i z a t i o n of a c e t y l c h o l i n e , was suggested as a new a p p r o a c h to the p a t h o g e n e s i s of the human muscular d y s t r o p h i e s . Such a s u g g e s t i o n i m p l i e d that a c e t y l c h o l i n e was a t r o p h i c f a c t o r but t h e r e was yet no s t r o n g evidence f o r a r o l e other than i n the a c t i v a t i o n of the muscle. The r o l e o f a c e t y l c h o l i n e i n t h e p a t h o g e n e s i s of m y a s t h e n i a g r a v i s must have been the f o u n d a t i o n of the p r o p o s a l . During the 1930's the s u p e r s e n s i t i v i t y of muscle t o the a p p l i c a t i o n o f a c e t y l c h o l i n e was d i s c o v e r e d t o be a consequence of denervation but the mechanism r e s p o n s i b l e f o r t h i s h y p e r s e n s i t i v i t y was g i v e n no a t t e n t i o n . In 1959, Axelson and T h e s l e f f took advantage of the new experimental t e c h n i q u e of i o n t o p h o r e t i c a p p l i c a t i o n and the measurement of e l e c t r i c a l changes p r o d u c e d by such a p p l i c a t i o n to a s m a l l a r e a o f t i s s u e by m i c r o e l e c t r o d e s . A c c o r d i n g t o e l e c t r i c a l a c t i v i t y i n d i c a t i v e o f d e p o l a r i z a t i o n , i n r e s p o n s e to the a p p l i c a t i o n of a c e t y l c h o l i n e , d e n e r v a t e d muscle g r a d u a l l y became s e n s i t i v e to a c e t y l c h o l i n e over i t s - e n t i r e l e n g t h . Diamond and M i l e d i (1959) found f e t a l r a t m u s c l e t o be u n i f o r m l y s e n s i t i v e t o the a p p l i c a t i o n of a c e t y l c h o l i n e . Following b i r t h there was a narrowing of the r e c e p t o r s e n s i t i v e area to c o i n c i d e with the d i s t r i c t of the 136 e n d p l a t e and t h e r e f o r e , a r e l a t i o n s h i p between d e n e r v a t i o n and development was i n e v i d e n c e . As w e l l as r e f e r r i n g to the n u t r i t i o n of t i s s u e s , " t r o p h i c " a l s o r e f e r s to t h e i r growth and development. T h e s l e f f (1960) produced the same u n i f o r m s e n s i t i v i t y to a c e t y l c h o l i n e by the a p p l i c a t i o n of bo t u l i n u m t o x i n , which was known to i n h i b i t the r e l e a s e of a c e t y l c h o l i n e from the p r e s y n a p t i c t e r m i n a l . I t appeared t h a t a c e t y l c h o l i n e was r e s p o n s i b l e f o r the changes i n i t s re c e p t o r zone with d e n e r v a t i o n and the r o l e of a c e t y l c h o l i n e as a t r o p h i c f a c t o r was c o n c e i v a b l e . In order to study the p o t e n t i a l e f f e c t of a c e t y l c h o l i n e , d u r i n g growth and development, chick embryos were employed. U n l i k e o t h e r s t a n d a r d e x p e r i m e n t a l a n i m a l s , they do not r e q u i r e a muscle-nerve connection f o r r e s p i r a t i o n , and would t h e r e f o r e s u r v i v e s u r g i c a l or p h a r m a c o l o g i c a l d e n e r v a t i o n p r o c e d u r e s . The e x p e r i m e n t s o f Ahmed (1964), Drachman (1964) and Drachman (1967) employed botulinum t o x i n , or post s y n a p t i c i n h i b i t o r s o f a c e t y l c h o l i n e . A l l r e s u l t e d i n gross and m i c r o s c o p i c a l f e a t u r e s of the s k e l e t a l muscle at b i r t h which were s i m i l a r t o those o f d e n e r v a t i o n . Not o n l y was a c e t y l c h o l i n e r e l e a s e d from the ne r v e t e r m i n a l w i t h the a r r i v a l o f an i m p u l s e , r e l e a s e o c c u r r e d spontaneously when n e r v e i m p u l s e t r a n s m i s s i o n was i n t e r r u p t e d . T h i s n o n q u a n t a l , spontaneous r e l e a s e was a s s o c i a t e d w i t h the m i n i a t u r e e n d p l a t e p o t e n t i a l s which F a t t and Katz measured fro m t h e e n d p l a t e o f r e s t i n g a m p h i b i a n muscle i n 1952. 137 Drachman f e l t t h a t the spontaneous nonquantal r e l e a s e of a c e t y l c h o l i n e , was the t r o p h i c f a c t o r of the muscle. Ahmed d i d not h e s i t a t e to suggest that "these o b s e r v a t i o n s may be o f i n t e r e s t t o t h e n e u r o m u s c u l a r p a t h o l o g i s t a n d e m b r y o l o g i s t i n t h e a s s e s s m e n t o f d y s t r o p h i e s and maldevelopment of the s k e l e t a l muscle." Animal muscle was o r i g i n a l l y d i v i d e d a n a t o m i c a l l y i n t o r e d and w h i t e , and p h y s i o l o g i c a l l y i n t o slow and f a s t t w i t c h , r e s p e c t i v e l y . With the advent of h i s t o c h e m i s t r y d u r i n g the 1950's, which made q u a l i t a t i v e a n a l y s i s of enzyme a c t i v i t y w i t h i n t h e c e l l p o s s i b l e , a n o t h e r means o f c l a s s i f i c a t i o n was d e v i s e d . White muscle r e a c t e d s t r o n g l y f o r m y o s i n A T P a s e , p h o s p h o r y l a s e and o t h e r g l y c o l y t i c enzymes. However, the r e a c t i o n of i n d i v i d u a l f i b r e s of the w h i t e , f a s t t w i t c h f l e x o r d i g i t o r u m longus v a r i e d to g i v e a checkerboard appearance of f i b r e s r e a c t i n g s t r o n g l y , weakly a n d i n a n i n t e r m e d i a t e f a s h i o n . T h e s t r o n g l y r e a c t i v e f i b r e s which were p r i m a r i l y a n a e r o b i c , depending upon glycogen s t o r e s for energy metabolism, gained the name Type I I . The f i b r e s of the slow t w i t c h s o l e u s r e a c t e d w e a k l y and u n i f o r m l y f o r p h o s p h o r y l a s e or A T P a s e but h i s t o c h e m i s t r y r e v e a l e d h i g h a c t i v i t y of the N A D - l i n k e d dehydrogenase. Type I f i b r e s were designated as those f o r which metabolism was p r i m a r i l y a e r o b i c . I t was d i s c o v e r e d that at b i r t h a l l mammalian muscles were r e l a t i v e l y slow i n t h e i r c o n t r a c t i o n - r e l a x a t i o n times 138 and w i t h development the p o t e n t i a l l y f a s t became f a s t e r w h i l e the slow became somewhat f a s t e r b e f o r e s l o w i n g t o t h e i r l o w e s t s p e e d which was m a i n t a i n e d t h r o u g h l i f e . B u l l e r e t a l . (1960a) found t h a t when the s p i n a l cord was t r a n s e c t e d and the a f f e r e n t impulses from the muscle were i n t e r r u p t e d , the slow t w i t c h muscles f a i l e d to develop t h e i r c h a r a c t e r i s t i c speeds. B u l l e r e t a l . (1960b), denervated t h e s o l e u s a n d f l e x o r d i g i t o r u m l o n g u s m u s c l e o f e x p e r i m e n t a l a n i m a l s and r e i n n e r v a t e d each w i t h the nerve o r i g i n a l l y s u p p l y i n g the o t h e r . T h i s " c r o s s - i n n e r v a t i o n " r e s u l t e d i n a s l o w i n g of the t w i t c h time f o r the former l y f a s t f l e x o r d i g i t o r u m longus (FDL) and a s h o r t e n i n g of the c o n t r a c t i o n - r e l a x a t i o n time of the s o l e u s . In the same way that muscle was uniform i n t w i t c h time at b i r t h , the h i s t o c h e m i s t r y of animal muscle was found to be the same from one muscle t o the n e x t . The immature s o l e u s was s i m i l a r t o t h e a d u l t m u s c l e i n t e r m s o f h i s t o c h e m i s t r y w hile the FDL was uniform at b i r t h , becoming "mixed" i n f i b r e type with development. Enzyme a c t i v i t y s p e c i f i c f o r e i t h e r Type I or Type I I f i b r e s was l o s t f o l l o w i n g d e n e r v a t i o n but c r o s s i n n e r v a t i o n e x p e r i m e n t s p r o v e d t h a t i t was i n f a c t the nerve which possessed the power to determine the metabolic p r o p e r t i e s of the muscle. C r o s s - i n n e r v a t i o n experiments r e s u l t e d i n a r e v e r s a l o f enzyme p r o f i l e s c o r r e s p o n d i n g t o t w i t c h t i m e s . That i s , with a r e d u c t i o n i n t w i t c h time t h e r e was an i n c r e a s e i n the 139 a c t i v i t i e s o f ATPase and other g l y c o l y t i c enzymes while an i n c r e a s e i n o x i d a t i v e enzyme a c t i v i t y a c c o m p a n i e d a l e n g t h e n i n g of t w i t c h time (Dubowitz and Romanul, 1967; Romanul and Van der Meulen, 1966; Dubowitz, 1967). These changes o c c u r r e d i n b o t h newborn and a d u l t a n i m a l s and t h e r e f o r e i t a p p e a r e d t h a t t h e same m e c h a n i s m was r e s p o n s i b l e f o r both the d i f f e r e n t i a t i o n and maintenance of m e t a b o l i c p a r a m e t e r s i n t h e s k e l e t a l m u s c l e . Dubowitz (1967) c o n c l u d e d t h a t " I f the c e n t r a l nervous system has such a p r o f o u n d c o n t r o l of the s k e l e t a l muscle, i t r a i s e s the q u e s t i o n as to whether s o - c a l l e d "primary" d i s e a s e s of m u s c l e , s u c h as m u s c u l a r d y s t r o p h y , a r e not p e r h a p s secondary to a derangement of these c o n t r o l l i n g f a c t o r s i n the nervous system." The Neurogenic Hypothesis The d i v i d i n g l i n e between myopathy and neuropathy i n terms o f h i s t o l o g y , i n t r a m u s c u l a r i n n e r v a t i o n and serum enzymes had become i n d i s t i n c t by 1970 and with the knowledge c o n c e r n i n g t h e p o w e r f u l i n f l u e n c e of the nerve upon the metabolic processes of the muscle, the focus had s h i f t e d to the i n n e r v a t i n g nerve. McComas et a l . (1971a) developed an e l e c t r o p h y s i o l o g i c a l t e c h n i q u e which was i n t e n d e d f o r the study of the d e n e r v a t i n g p r o c e s s , but would i n e v i t a b l y be a p p l i e d to purported myopathy. The method of McComas et a l . was designed f o r the e s t i m a t i o n of the number of motor u n i t s 140 i n a p a r t i c u l a r muscle o f an i n d i v i d u a l . The numbers d e r i v e d from normal i n d i v i d u a l s were a c t u a l l y c o m p a t i b l e w i t h those o b t a i n e d from f i b r e counts of the i n n e r v a t i n g nerve d e r i v e d from autopsy, thereby g i v i n g some c r e d i b i l i t y to the method. The method i n v o l v e d the graded s t i m u l a t i o n of the e x t e n s o r d i g i t o r u m b r e v i s muscle of the f o o t , f o r w hich o n l y one e n d p l a t e zone c o u l d be f o u n d . With the i n c r e a s i n g s t r e n g t h of s t i m u l a t i o n of the i n n e r v a t i n g nerve, an i n c r e m e n t a l and summating response a c c o r d i n g to a c t i o n p o t e n t i a l s was o b s e r v e d . Assuming t h a t each i n c r e m e n t corresponded to one motor u n i t the number of motor u n i t s was c a l c u l a t e d by d i v i d i n g the maximum amplitude produced with maximum c o n t r a c t i o n by t h e a v e r a g e a m p l i t u d e o f t h e incremental responses. When the technique of McComas et a l . was used to assess the f u n c t i o n a l i n n e r v a t i o n of myotonic dystrophy (McComas et a l . , 1971b), the incremental responses were w i t h i n a normal range i n d i c a t i n g the normal s i z e of the motor u n i t s . The maximum a m p l i t u d e on t h e o t h e r hand was r e d u c e d which r e s u l t e d i n the c a l c u l a t i o n of a subnormal number of motor u n i t s . The same s i t u a t i o n a p p l i e d to Duchenne d y s t r o p h y (McComas et a l . , 1971c). U n l i k e myotonic dystrophy, which demonstrated a p r o g r e s s i v e r e d u c t i o n i n motor u n i t numbers w h i l e r e t a i n i n g s i z e w i t h the advancement of the d i s e a s e , t h e r e was no c o r r e l a t i o n between the number of motor u n i t s 141 and the age of the c h i l d . The number of motor u n i t s was i n v a r i a b l y subnormal, though. A r e d u c t i o n i n the number of f u n c t i o n a l motor u n i t s was a l s o m e a s u r e d i n m y a s t h e n i a g r a v i s , l i m b - g i r d l e and f a c i o s c a p u l o h u m e r a l muscular dystrophy ( S i c a and McComas, 1971) but p o t e n t i a l s with l a r g e r than normal amplitudes were generated by maximal c o n t r a c t i o n i n the l a t t e r two. These a b n o r m a l l y l a r g e a m p l i t u d e s were i n t e r p r e t e d as t h e s t r o n g e s t e v i d e n c e i n f a v o u r of the t h e o r y which emerged from t h i s study of f u n c t i o n i n g motor u n i t s . The neurogenic hypothesis of muscular dystrophy was embodied by the theory of the " s i c k motor u n i t " . The theory was founded on the t r o p h i c i n f l u e n c e of the nerve upon the muscle and attempted to u n i f y neuromuscular d i s e a s e on t h i s b a s i s . A c c o r d i n g to t h e t h e o r y ( o f t h e s i c k motor u n i t ) a l l n e u r o m u s c u l a r d i s e a s e s , i n c l u d i n g the m u s c u l a r d y s t r o p h i e s , were the r e s u l t o f a d e f e c t i v e o r a b s e n t t r o p h i c i n f l u e n c e . T h e r e f o r e , they i n v a r i a b l y i n v o l v e d the whole motor u n i t , not the i n d i v i d u a l muscle f i b r e . The g r e a t e r t h a n n o r m a l a m p l i t u d e s f o r m a x i m a l c o n t r a c t i o n i n limb g i r d l e and facioscapulohumeral muscular d y s t r o p h y w e r e b e l i e v e d t o be t h e r e s u l t o f t h e r e i n n e r v a t i o n of motor u n i t s by c o l l a t e r a l s s p r o u t e d by h e a l t h y n e r v e s . In t h e c a s e s of Duchenne and m y o t o n i c dystrophy the " s i c k n e s s " of the nerve was such that i t was l e s s capable of s p r o u t i n g c o l l a t e r a l s f o r r e i n n e r v a t i o n and 142 the i n a b i l i t y of nerves to sprout c o l l a t e r a l s was supposed t o be r e s p o n s i b l e f o r the m y o p a t h i c h i s t o l o g i e s o f the muscular d y s t r o p h i e s g e n e r a l l y . The ever presence of group a t r o p h y i n the b i o p s i e s o f so c a l l e d n e u r o p a t h i e s was e v i d e n c e of an ongoing d e n e r v a t i o n - r e i n n e r v a t i o n p r o c e s s , which was sometimes incomplete or i n e f f i c i e n t , g i v i n g r i s e t o t y p i c a l myopathic f e a t u r e s . The myopathic b i o p s i e s of d i s e a s e s such as p o l i o m y e l i t i s were according to the theory o f t h e s i c k m o t o r u n i t , c o m p a t i b l e w i t h D r a c h m a n ' s c o n c l u s i o n s f o l l o w i n g h i s study of t h i s c h r o n i c d e n e r v a t i n g d i s e a s e . That i s , they were the consequence of the l a c k of or the un s u s t a i n e d r e i n n e r v a t i o n of motor u n i t s by h e a l t h y n e r v e s . The t h e o r y a l s o p r e d i c t e d the death of a t l e a s t some motor neurons i n muscular d y s t r o p h y , the r e a l i t y of w h i c h was s u p p o r t e d by t h e p e r s o n a l c o m m u n i c a t i o n o f Tomlinson to McComas. Tomlinson reported that t h e r e was a l o s s o f s p i n a l motor neurons i n t h r e e cases o f Duchenne muscular dystrophy. The neurogenic hypothesis of muscular dystrophy aroused d e b a t e a n d c o n t r o v e r s y a n d a t f i r s t r e c e i v e d some e x p e r i m e n t a l s u p p o r t . D y s t r o p h i c m u s c l e w h i c h was t r a n s p l a n t e d t o normal mice r e g e n e r a t e d and i n d o i n g so a t t a i n e d normal p h y s i o l o g i c a l p r o p e r t i e s i n c l u d i n g the a b i l i t y to c o n t r a c t ( S a l a f s k y , 1971). G a l l u p and Dubowitz (1973) c u l t u r e d combinations of f e t a l neural t i s s u e coupled w i t h a d u l t muscle d e r i v e d from normal and d y s t r o p h i c mice. 143 Normal and d y s t r o p h i c muscle regenerated i n the presence of normal n e u r a l t i s s u e w h i l e muscle coupled w i t h d y s t r o p h i c embryonic s p i n a l c o r d was m o r p h o l o g i c a l l y abnormal. Only one of ten c r o s s - i n n e r v a t i o n s i n d y s t r o p h i c mice produced c o n v e r s i o n of muscle i n terms of metabolic and c o n t r a c t i l e p r o p e r t i e s (Law and Atwood 1972). The t r o p h i c i n c a p a c i t y of the nerve i n the animal model was seemingly demonstrated. With r e s p e c t to the human c o n d i t i o n when muscle was e x t r a c t e d through biopsy and grown i n c u l t u r e c o n t a i n i n g the necessary n u t r i t i v e requirements i t showed no m o r p h o l o g i c a l a b n o r m a l i t i e s , seemingly demonstrating that the muscle was equipped with a l l the n e c e s s i t i e s f o r growth and s u r v i v a l i f g i v e n the p r o p e r n u t r i t i o n ( B i s h o p et a l . 1 9 7 1 ) . The t e c h n i q u e of McComas e t a l . was ex t e n d e d t o M c A r d l e ' s d i s e a s e which was known to i n v o l v e a d e f i c i e n c y i n the g l y c o g e n d e g r a d i n g e n z y m e , p h o s p h o r y l a s e , a n d t o t h y r o t o x i c o s i s which a f f e c t s the metabolic rate of a l l c e l l s (Upton et a l . , 1973). Again, a r e d u c t i o n i n the number of motor u n i t s with the r e t e n t i o n of s i z e was measured i n both and "neuropathy" was assigned which i n view of the profound e f f e c t of the nerve upon the metabolism of muscle had some m e r i t . As r e g a r d s the c e r e b r a l d e f e c t s of Duchenne and my o t o n i c d y s t r o p h y , a l t h o u g h i t was r e c o g n i z e d t h a t the genes f o r each c o u l d be expressed i n both b r a i n and muscle, the r o l e of the n e u r a l element was accentuated by v i r t u e of i t s powerful i n f l u e n c e upon muscle metabolism. 144 A l l too soon, though, the tenets of muscular dystrophy as t h e r e s u l t o f a n e u r o g e n e s i s were c h a l l e n g e d and t h e r e f o r e , j u s t as the neurogenic t h e o r i e s o f the past had r e c e i v e d o n l y ephemeral support the e v i d e n c e d a g a i n s t the t h e o r y o f McComas e t a l . a c c r u e d q u i c k l y . H a r r i s and M a r s h a l l (1973) found v e r y few d y s t r o p h i c f i b r e s t o be " f u n c t i o n a l l y " denervated as proposed by McComas and Mrozek (1967). In f a c t , a few of the muscle f i b r e s i n normal mice d i d n o t g e n e r a t e a c t i o n p o t e n t i a l s i n r e s p o n s e t o s t i m u l a t i o n o f the i n n e r v a t i n g n e r v e . F u r t h e r e v i d e n c e a g a i n s t the " f u n c t i o n a l " d e n e r v a t i o n of d y s t r o p h i c muscle was p r o v i d e d by the l a c k o f t e t r o d o t o x i n r e s i s t a n t a c t i o n p o t e n t i a l s or e x t r a j u n c t i o n a l a c e t y l c h o l i n e a c t i v i t y . When the d y s t r o p h i c muscle was denervated t e t r o d o t o x i n r e s i s t a n t a c t i o n p o t e n t i a l s appeared on or about the same time as they d i d i n denervated normal muscle g i v i n g a d d i t i o n a l weight to the f u n c t i o n a l i n n e r v a t i o n o f mouse d y s t r o p h i c m u s c l e . T h e r e was a l s o no l o s s o f s p i n a l motor neurons i n t h i s s t r a i n (Papapetropoulos and B r a d l e y , 1972), and t h e r e f o r e d e n e r v a t i o n , f u n c t i o n a l or a c t u a l , was not a f e a t u r e of the animal model. The changes i n the t e r m i n a l i n n e r v a t i o n were, as o r i g i n a l l y c o n s i d e r e d to be, secondary m a n i f e s t a t i o n s of muscular d e g e n e r a t i o n . Using e s s e n t i a l l y the same c u l t u r e technique as G a l l u p and Dubowitz (1973) Paul and Powell (1974) ob t a i n e d o p p o s i t e r e s u l t s f o r n e r v e - m u s c l e c u l t u r e s . N o r m a l m u s c l e 145 regenerated r e g a r d l e s s o f the source of neural t i s s u e . The presence of a normal embryonic s p i n a l c o r d d i d not prevent the a b o r t i v e r e g e n e r a t i o n which t h e d y s t r o p h i c m u s c l e e x p r e s s e d when c u l t u r e d a p a r t o f n e u r a l t i s s u e . The u l t i m a t e proof of the t r o p h i c c a p a b i l i t y of the nerve i n the d y s t r o p h i c mouse model was provided by Law et a l . i n 1976. Cross u n i o n of nerves from d y s t r o p h i c mice to normal mice demonstrated that the d y s t r o p h i c nerve possessed the normal t r o p h i c c a p a b i l i t i e s . The f o r m e r l y slow t w i t c h s o l e u s i n n o r m a l m i c e a t t a i n e d c o n t r a c t i l e and h i s t o c h e m i c a l p r o p e r t i e s t y p i c a l o f muscles n o r m a l l y i n n e r v a t e d by the t i b i a l nerve. The t r o p h i c power of the d y s t r o p h i c nerve was e q u i v a l e n t to that of the normal nerve according to c r o s s -i n n e r v a t i o n of nerves w i t h i n normal mice. In 1973, Coers et a l . r e p o r t e d an i n c r e a s e d t e r m i n a l , i n n e r v a t i o n r a t i o i n lower motor neuron di s e a s e i n c l u d i n g a myotrophic l a t e r a l s c l e r o s i s , s p i n a l muscular atrophy and p o l i o m y e l i t i s . T h i s i n c r e a s e d t e r m i n a l i n n e r v a t i o n r a t i o as oppos e d t o a redundant i n n e r v a t i o n , r e f l e c t e d t e r m i n a l a r b o r i s a t i o n of the neuron i n these d i s o r d e r s . The r a t i o was normal i n m y a s t h e n i a g r a v i s . A l t h o u g h t h e r e was an i n c r e a s e i n ' s o m e c a s e s o f l i m b - g i r d l e a n d f a c i o s c a p u l o h u m e r a l muscular d y s t r o p h y , those f o r Duchenne d y s t r o p h y were normal, r e f l e c t i n g the l a c k of q u a l i t a t i v e changes i n the i n t r a m u s c u l a r i n n e r v a t i o n . Post s y n a p t i c f o l d s , which were a l s o seen i n m y a s t h e n i a g r a v i s , were 146 observed i n Duchenne dystrophy using the e l e c t r o n microscope (Jerusalem et a l . , 1974). However, the lack of p r e s y n a p t i c changes and the f a c t t h a t a h e a l t h y nerve terminated on a n e c r o t i c muscle f i b r e were thought to be evidence a g a i n s t a s i c k motor neuron. The o r i g i n a l r e p o r t of Tomlinson regarding the l o s s of s p i n a l motor neurons i n c a s e s of Duchenne d y s t r o p h y was c o n t r a d i c t e d by Tomlinson et a l . i n 1974. There were no s i g n i f i c a n t changes or r e d u c t i o n i n the number of s p i n a l motor neurons s u p p l y i n g the lower limbs i n s i x cases of the d i s e a s e . Another i n d i v i d u a l i n c l u d e d i n t h i s s t u d y was o r i g i n a l l y diagnosed as Duchenne dystrophy. S h o r t l y b e f o r e d e a t h t h e d i a g n o s i s was a l t e r e d t o t h a t o f K u g e l b e r g -Welander s p i n a l m u s c u l a r d y s t r o p h y a c c o r d i n g to an EMG t y p i c a l of neuropathy. T h i s a l t e r e d d i a g n o s i s was confirmed a t a u t o p s y by t h e l o s s o f s p i n a l m o t o r n e u r o n s . M i s d i a g n o s i s was p e r h a p s r e s p o n s i b l e f o r t h e r e s u l t s r e g a r d i n g the three cases c i t e d by McComas i n support of the neurogenic h y p o t h e s i s . In any event, Duchenne dystrophy was confirmed to be a d i s e a s e with no apparent pathology of the s p i n a l c o r d . Both q u a l i t a t i v e and q u a n t i t a t i v e changes were observed i n myotonic d y s t r o p h y by Coers et a l . (1973). I t was the o n l y dystrophy f o r which a r e d u c t i o n i n the number of motor u n i t s was estimated u s i n g an e l e c t r o p h y s i o l o g i c a l technique which i n v o l v e d computer a n a l y s i s of the p o t e n t i a l s evoked 147 from the extensor d i g i t o r u m b r e v i s ( B a l l a n t y n e and Hansen, 1974). The extensor d i g i t o r u m b r e v i s muscle was employed f o r e l e c t r o p h y s i o l o g i c a l s t u d y , i n p a r t by v i r t u e o f convenience; the s k i n o v e r l y i n g the muscle i s t h i n and the b e l l y o f the muscle i s f l a t which w i l l minimize the amount of e l e c t r i c a l i n t e r f e r e n c e from n e i g h b o u r i n g m u s c l e s . McComas e t a l . v e r i f i e d t h e d i a g n o s i s o f s u b j e c t s p a r t i c i p a t i n g i n t h e i r i n v e s t i g a t i o n s w i t h the use of the EMG. P o t e n t i a l s c o n s i d e r e d to be t y p i c a l of myopathy were demonstrated i n proximal muscles which were v i s i b l y a f f e c t e d by the d i s e a s e p r o c e s s . By studying the e l e c t r o p h y s i o l o g y of a d i s t a l m u s c l e l i k e t he e x t e n s o r d i g i t o r u m i t was b e l i e v e d t h a t g r e a t e r i n s i g h t c o u l d be gained c o n c e r n i n g p a t h o g e n e s i s , because the d i s e a s e p r o c e s s was not f u l l y f l e d g e d i n such muscles. In order to prove that the r e s u l t s o b t a i n e d by t h e i r method were not l i m i t e d to one muscle McComas et a l . (1974) e x t e n d e d t h e e s t i m a t i o n o f f u n c t i o n i n g motor u n i t s i n muscular dystrophy to the soleus of the foot and the thenar and hypothenar muscles of the hand. A r e d u c t i o n i n the number of motor u n i t s was i n f a c t c a l c u l a t e d . However, r e g a r d l e s s of the muscle employed, the p r e c i s e procedure of McComas et a l . was d i s c r e d i t e d by the computer a n a l y s i s method o f B a l l a n t y n e and Hansen (1974) and by t h a t o f P a n a y i o t o p o u l o s e t a l . ( 1 9 7 4 ) . S c a r p a l e z o s a n d P a n a y i o t o p o u l o s (1973) a s s e r t e d t h a t d i f f e r e n c e s i n the 148 l a t e n c i e s of p o t e n t i a l s a r i s i n g from the muscle f i b r e s of t h e e x t e n s o r d i g i t o r u m b r e v i s c o u l d r e s u l t i n m i s s e d p o t e n t i a l s , which might be f u r t h e r obscured by the n o i s e l e v e l o f the r e c o r d i n g s y s t e m . P a n a y i o t o p o u l o s et a l . (1974) used e s s e n t i a l l y the same technique, but photographed r e s p o n s e s to graded s t i m u l a t i o n and measured the evoked p o t e n t i a l s a f t e r a m p l i f i c a t i o n i n a m i c r o f i l m r e a d e r p r i n t e r . They found t h a t the number of motor u n i t s was normal i n nine cases of Duchenne dystrophy while t h e i r s i z e was g r e a t l y r e d u c e d . A c c o r d i n g to the same p r o c e d u r e , P a n a y i o t o p o u l o s and S c a r p e l e z o s (1974) r e p o r t e d t h e r e d u c t i o n i n the s i z e with the r e t e n t i o n of the numbers of motor u n i t s i n l i m b - g i r d l e d y s t r o p h y . There was a l o s s of u n i t s i n cases of s p i n a l muscular atrophy, those that were s u r v i v i n g b eing normal i n s i z e . The g i a n t p o t e n t i a l s i n l i m b - g i r d l e dystrophy f i r s t observed by McComas et a l . were a t t r i b u t e d to the h y p e r t r o p h y o f the e x t e n s o r d i g i t o r u m b r e v i s i n t h i s form of muscular dystrophy, the presence of which was suggested to be a p o s s i b l e p o i n t of d i a g n o s t i c d i s t i n c t i o n between t h i s t y p e o f d y s t r o p h y and s p i n a l muscular atrophy. Doubt had been cast upon the method of McComas et a l . , and the neurogenic h y p o t h e s i s o f muscular d y s t r o p h y . The a b s e n c e o f a p p a r e n t p a t h o l o g y t o the n e r v o u s s y s t e m a c c o r d i n g to postmortem examination and the lack of severe changes i n the i n t r a m u s c u l a r i n n e r v a t i o n a l s o weakened the 149 hypothesis. When Bishop et a l . (1971) studied the regenerative capacity of dystrophic muscle they used the explant tissue technique in which fragments of muscle were immersed in tissue culture medium. Askanas et a l . (1975) demonstrated the ability of muscle derived from Duchenne dystrophy to regenerate in cultures established according to the explant technique. Emery (1977) also reported the ability of muscle extracted from male fetuses at risk for Duchenne dystrophy to grow and differentiate in such cultures. Unfortunately, for those who firmly believed in the neurogenic hypothesis, the behavior of cells within a culture established by the disaggregation technique pointed towards a defect which was indigenous to the muscle fibre in Duchenne dystrophy. Muscle tissue was first reduced to a suspension of single cells by the use of proteolytic enzymes in the disaggregation technique. With this method Thompson et a l . (1977) observed the abnormal clustering of cells obtained from Duchenne dystrophy and interpreted this observation as an indication of the myogenic nature of the defect in the disease. The possibility remained that a trophic disturbance existed as evidenced by histological changes in the muscle of a male fetus at risk for the disease (Emery 1977). Regardless of the potential reality of a trophic influence gone awry in the disease, though, there was insufficient knowledge pertaining to the trophic 150 i n f l u e n c e and no other means of a s s e s s i n g a d i s r u p t e d or d e f e c t i v e i n f l u e n c e . In the meantime, the s t u d y of B l a c k e t a l . (1974) redrew the l i n e , as i t were, between myopathy and neuropathy a c c o r d i n g the muscle b i o p s y , the EMG and serum enzyme. A t o t a l o f 105 i n d i v i d u a l s were d i v i d e d i n t o t h r e e groups: those who had been c l i n i c a l l y d i a g n o s e d , those diagnosed a c c o r d i n g t o b i o p s i e s and EMG's and t h o s e f o r whom a d i a g n o s i s c o u l d not be e s t a b l i s h e d . P o t e n t i a l s with short d u r a t i o n and low amplitude and those which were p o l y p h a s i c were s t i l l used as the c r i t e r i a f o r a myopathic EMG, but c e r t a i n h i s t o l o g i c a l f e a t u r e s , which were con s i d e r e d to be t y p i c a l of myopathy i n the p a s t , were excluded and regarded as b e i n g n o n d i a g n o s t i c . I t was d e g e n e r a t i v e a n d r e g e n e r a t i v e changes which were used i n the assignment of myopathy to b i o p s i e s , while random v a r i a t i o n i n f i b r e s i z e , connective t i s s u e p r o l i f e r a t i o n , f i b r e s p l i t t i n g and c e n t r a l n u c l e a t i o n were excluded from the l i s t of c r i t e r i a . Ninety percent of the 105 cases were concordant f o r b i o p s i e s and EMG's, that i s , e i t h e r both were n e u r o p a t h i c or both were myopathic and the l e v e l of serum c r e a t i n e phosphokinase was a l s o i n agreement. I t was thus c o n c l u d e d t h a t "a t r u e d i v i s i o n between myopathy and neuropathy e x i s t s " . The r e a s o n f o r the re-emergence of the n e u r o g e n i c h y p o t h e s i s of muscular dystrophy i s perhaps summarized by the words of Gowers w r i t t e n i n 1902: "The v i t a l i t y of the 151 muscle presents a strange d u p l i c i t y . The n u t r i t i o n of the m u s c l e depends on t h a t of the n e r v e s through which i t s f u n c t i o n i s c a l l e d f o r t h . " By the 1970's the way i n which the nerve a c t i v a t e s the muscle was w e l l known and w i t h the a r r i v a l of immunocytochemical t e c h n i q u e s , r a t h e r than an a c e t y l c h o l i n e d e f i c i e n c y , a p o s t s y n a p t i c a c e t y l c h o l i n e r e c e p t o r d e f i c i e n c y was d e t e r m i n e d to be the u n d e r l y i n g cause of myasthenia g r a v i s . Further support f o r the r o l e of a c e t y l c h o l i n e as a t r o p h i c f a c t o r was provided (Cohner 1974, 1 9 8 0 ) . However, as p r e v i o u s l y s t a t e d , t h e k n o w l e d g e p e r t a i n i n g to the way i n which the nerve e x e r t s i t s t r o p h i c i n f l u e n c e upon the s k e l e t a l muscle was inadequate to s u s t a i n r e s e a r c h p e r t a i n i n g to a t r o p h i c theory f o r the pathogenesis of muscular d y s t r o p h y . The v e s t i g e s of the neurogenic hypothesis were e v i d e n t i n the p r o p o s a l of Vrbova (1985). She suggested t h a t the p r o t e i n i n v o l v e d i n Duchenne dystrophy was s i t u a t e d i n the muscle and was necessary f o r the growth and development of the muscle. Without the proper f u n c t i o n of t h i s p r o t e i n , the muscle co u l d not withstand the demands pl a c e d upon i t by the nerve during the m a t u r a t i o n a l p e r i o d and the nerve would there b y impose pathology upon the muscle. I t would appear t h a t j u s t as the focus had s h i f t e d to the post s y n a p t i c s i d e f o r myasthenia g r a v i s , so had that f o r muscular d y s t r o p h y . The pendulum had swung back, as i t were, to the myogenic 152 view on the b a s i s of the absence of a neural l e s i o n and a d y s t r o p h i c h i s t o l o g y . The C a r d i a c Defect I f Duchenne muscular d y s t r o p h y was the r e s u l t of a neurogenic phenomenon one would expect to see the e f f e c t s of some s i c k motor u n i t s i n female c a r r i e r s . That i s , b i o p s i e s should r e v e a l changes i n complete u n i t s d i s p e r s e d .within the m u s c l e a c c o r d i n g t o t h e random i n a c t i v a t i o n o f the X c hromosome i n t h e u n i n u c l e a t e d m o t o r n e u r o n . The h i s t o l o g i c a l changes i n the b i o p s i e s of heterozygous females a r e , on the c o n t r a r y , q u i t e d i f f u s e . Among the c l i n i c a l m a n i f e s t a t i o n s of female c a r r i e r s are e l e c t r o c a r d i o g r a p h i c a b n o r m a l i t i e s which o c c u r i n about 10% and i n f a c t such a b n o r m a l i t i e s may occur i n muscular dystrophy i n g e n e r a l . M y o c a r d i a l involvement i n muscular dystrophy became evident d u r i n g the n i n e t e e n t h century through postmortem examination w h i c h d i s c l o s e d h i s t o l o g i c a l f e a t u r e s i n the myocardium r e s e m b l i n g t h o s e o f t h e s k e l e t a l m u s c l e . T h e e l e c t r o c a r d i o g r a m (ECG) e v e n t u a l l y r e v e a l e d a b n o r m a l i t i e s which were c o n s i s t e n t with the involvement of the heart i n t h e d y s t r o p h i c p r o c e s s ; h o w e v e r , i t was n o t u n t i l r e c l a s s i f i c a t i o n began a c c o r d i n g t o c l i n i c a l and g e n e t i c c r i t e r i a t h a t the d i s t i n c t e l e c t r o c a r d i o g r a m f o r Duchenne muscular dystrophy was r e c o g n i z e d . Abnormally deep Q waves s i m i l a r to those seen i n m y o c a r d i a l i n f a r c t i o n and t a l l R 153 waves in the right precordial leads were discovered to be the most consistent and specific features for the X-linked recessive Duchenne dystrophy, being infrequent in the other forms including the form designated Duchenne type with autosomal recessive inheritance (Emery, 1972; Perloff et a l . , 1966; P e r l o f f et a l . , 1967; Slucka, 1967). The c h a r a c t e r i s t i c ECG p r o f i l e which occurs in the greater proportion of Duchenne muscular dystrophy patients can therefore be of diagnostic significance. Tachycardia is also very common in Duchenne muscular dystrophy and the fact that i t was demonstrated to persist during sleep, a time when the heart rate normally f a l l s , is a point in favour of its nonnervous origin (Kraus, 1932; Boas and Lowenburg, 1931). It was recognized in the 1930's that the accelerated heart rate was a consequence of the combination of the weakness of the heart muscle and a reduced venous return due to the degeneration of the muscle. It was later v e r i f i e d that while stroke volume may be reduced the cardiac output may be increased by virtue of the increased number of contractions per unit of time. Chenard et a l . (1988), used left ventricular ejection time (LVET) and pre-ejection period (PEP) as indices of left ventricular performance in 177 males with Duchenne dystrophy ranging in age from 6 to 21 years. Their findings were a prolonged PEP and an abbreviated LVET. While there was no correlation between age and heart rate, which normally decreases with 154 age, the PEP/LVET r a t i o increased with age beyond age t e n , thereby confirming the weakness of the myocardium. The p r o g r e s s i v e weakening of the myocardium and the ECG p a t t e r n appear to be e x p l i c a b l e by the h i s t o l o g i c a l changes w h i c h b e g i n i n t h e p o s t e r o b a s a l segment o f t h e l e f t v e n t r i c l e and w h i c h may s p r e a d to i n v o l v e t h e l e f t v e n t r i c u l a r f r e e w a l l t o e v e n t u a l l y become d i f f u s e t r a n s m u r a l f i b r o s i s ( S a n y a l e t a l . , 1 9 7 8 ) . C a r d i a c a b n o r m a l i t i e s i n Duchenne dystrophy are i n evidence during i t s course but p a t i e n t s r a r e l y show any s i g n s of c a r d i a c d i s e a s e . This masking of the c a r d i a c defect i s a p p a r e n t l y due to the p r o g r e s s i v e d e t e r i o r a t i o n o f t h e s k e l e t a l muscular system. Although i d e n t i c a l features are not seen i n the ECG' s of female c a r r i e r s , a bnormalities have been r e p o r t e d . The a l g e b r a i c sum of R and S wave amplitudes were s i g n i f i c a n t l y greater i n some c a r r i e r s of the l e t h a l gene as compared to n o r m a l s or women w i t h l i m b g i r d l e m u s c u l a r d y s t r o p h y ( E m e r g y , 1969 ; Lane et a l . , 1980 ) . In v i e w o f t h e a b n o r m a l i t i e s i n female c a r r i e r s and the rather p e c u l i a r and unique f e a t u r e s of the ECG which, although not a b s o l u t e l y c o n s t a n t , occur i n the majority and occur i n s i b l i n g s with the d i s o r d e r , i t would appear that the d y s t r o p h i c process of the heart muscle i s g e n e t i c a l l y determined. Given that the same gene d i c t a t e s both c a r d i a c and s k e l e t a l d e g e n e r a t i v e changes and knowing that the i n n e r v a t i o n of the heart i s not 155 necessary for f u n c t i o n or t r o p h i c s t a t e of the myocardium by v i r t u e of the f a c t t h a t t r a n s p l a n t a t i o n i s s u c c e s s f u l i r r e s p e c t i v e of i n n e r v a t i o n , the c a r d i a c d e f e c t i n Duchenne d y s t r o p h y i s s t r o n g e v i d e n c e a g a i n s t any n e u r o g e n i c hypothesis for the pathogenesis of the d i s o r d e r . The Membrane Defect In 1975 M o k r i and E n g e l f i r s t b r ought l i g h t t o a membrane abnormality i n the very e a r l y stages of the d i s e a s e as d i s c l o s e d by the e l e c t r o n microscope. Recent work has demonstrated that the p r o t e i n involved i n Duchenne dystrophy i s e i t h e r embedded i n the plasma membrane or i s l o c a t e d on the i n t r a c e l l u l a r s i d e of and a s s o c i a t e d with the plasma membrane. Wharten and colleagues (CBC r a d i o , 1988), whose e x p e r i m e n t a l r e s u l t s f a v o u r the former l o c a t i o n of the p r o t e i n , have proposed that the p r o t e i n normally p r o v i d e s a s t r u c t u r a l d u r a b i l i t y to the c e l l membrane. They a r e convinced that the disease i s due to a complete lack of or the reduced e x p r e s s i o n of the p r o t e i n which r e n d e r s the muscle f i b r e very v u l n e r a b l e i n terms of mechanical s t r a i n . At b i r t h we see a seemingly p e r f e c t and healthy i n f a n t whose muscle i s immature, i t s a c t i o n l i m i t e d . I t i s only a f t e r he has d e v e l o p e d t o a t t a i n an u p r i g h t p o s t u r e i n f u l f i l l m e n t of the b i p e d a l n e s s of man that the d i s e a s e i s c l i n i c a l l y m anifest and t h e r e a f t e r progresses very s w i f t l y towards.a hopeless and f a t a l outcome. It appears t h a t , as 156 Vrbova suggested, the muscle cannot withstand the demands placed upon i t by the animating nerve and i n t h i s sense the disease could be considered to be a neuropathy; however, i t i s not t h e a c t i v a t i o n o f t h e m u s c l e i t s e l f but t h e c o n t r a c t i o n produced by a c t i v a t i o n which seems to i n i t i a t e the disease p r o c e s s . Therefore, i t i s the use of the muscle which appears to be the p r i m a r y cause of the e v e n t u a l d e g e n e r a t i o n . The f a c t that the heart i s a l s o s u b j e c t to m e chanical s t r a i n s u p p o r t s the r o l e of mechanics as the i n i t i a t i n g event. Metabolic Consequences of a Membrane Defect In v i t r o s t u d y has shown t h a t c r e a t i n e e n t e r s the muscle c e l l v i a an energy dependent process and, the h a l f time of c r e a t i n e i s l o n g , being 30 - 35 days, i n d i c a t i n g that most of that which i s normally taken up by the s k e l e t a l muscle f i b r e i s r e t a i n e d u n t i l i t i s l o s t through c o n v e r s i o n t o c r e a t i n i n e ( F i t c h and S h i e l d s , 1966). S h o r t e r than normal h a l f times f o r the decrease i n u r i n a r y c r e a t i n i n e b e a r i n g a C14 l a b e l d e r i v e d from a d m i n i s t e r e d c r e a t i n i n e c o u l d be e v i d e n c e o f e n h a n c e d e n t r y i n t h e m u s c u l a r d y s t r o p h i e s , but t h i s would not account f o r the l o s s of c r e a t i n e and because the r e d u c t i o n i n l a b e l l e d u r i n a r y c r e a t i n e p a r a l l e l e d that of c r e a t i n i n e , F i t c h et a l . (1968) concluded that i n t r a c e l l u l a r trapping was impaired. 157 S i x t y percent of muscle c r e a t i n e i s phosphorylated and because the unphosphorylated form e n t e r s and a r e l a t i v e l y s m a l l amount escapes a c c o r d i n g to i n v i t r o e xperimentation one might t h i n k that p h o s p h o r y l a t i o n and t h e r e f o r e the ATP producing processes are i n v o l v e d i n i t s r e t e n t i o n . However, F i t c h and S h i e l d s (1966) f o u n d t h a t w h i l e a n a e r o b i o s i s i n h i b i t e d t h e e n t r y o f c r e a t i n e , i t s l o s s was l i t t l e a f f e c t e d and t h e y t h e r e f o r e suggested t h a t once c r e a t i n e e n t e r s the c e l l a r e s t r i c t i v e membrane impedes i t s e f f l u x . A c c o r d i n g to such a mechanism, i f the integument of the plasma membrane i s l o s t through d e g e n e r a t i v e p r o c e s s e s i n the muscular d y s t r o p h i e s i t c o u l d account f o r enhanced e n t r y and i m p a i r e d i n t r a c e l l u l a r t r a p p i n g . The f a c t t h a t h a l f times f o r the decrease i n l a b e l l e d c r e a t i n i n e o r i g i n a t i n g i n a d m i n i s t e r e d c r e a t i n e a r e s h o r t e r i n d i f f e r e n t t y p e s o f muscular d y s t r o p h y ( F i t c h et a l . , 1968) and t h e y become shorter with the advancement of Duchenne muscular dystrophy s p e c i f i c a l l y ( F i t c h and S i n g o n , 1964) are c o n s i s t e n t w i t h c r e a t i n u r i a as an e v e n t which i s secondary t o muscular d e g e n e r a t i o n . On t h e o t h e r h a n d , th e h a l f t i m e s f o r Duchenne muscular dystrophy are s i g n i f i c a n t l y s h o r t e r than f o r other v a r i e t i e s of muscular dystrophy and the chemical composition of the muscle i n Duchenne dystrophy seems to be s p e c i f i c f o r the d i s e a s e . V i g n o s a n d Warner (1963) f o u n d t h a t w h i l e t o t a l c r e a t i n e was reduced i n p a t i e n t s with neurogenic a t r o p h y , an 158 a d u l t f o r m o f d y s t r o p h y and Duchenne d y s t r o p h y , t h e p h o s p h o c r e a t i n e c o n t e n t s w e r e d i f f e r e n t i n e a c h . P h o s p h o c r e a t i n e content was normal i n a d u l t d y s t r o p h y and reduced i n both Duchenne d y s t r o p h y and neurogenic atrophy but the r e d u c t i o n i n the former was q u i t e s t r i k i n g , the l a t t e r b e i n g r e l a t i v e l y m oderate. S i m i l a r l y , the t o t a l phosphate and ATP were normal i n the a d u l t dystrophy while t h e i r r e d u c t i o n s were g r e a t e s t i n Duchenne dystr o p h y . The ADP c o n c e n t r a t i o n was normal i n a l l t h r e e g r o u p s , a l l i n d i v i d u a l s b e i n g ambulatory and t h e r e f o r e i n the e a r l i e r s t a g e s o f d i s e a s e , but the r a t i o of ATP/ADP was 1.57 i n Duchenne dystrophy as compared to 2.5 i n the a d u l t dystrophy and n o r m a l s w h i c h ra n g e d i n age from 3 t o 50 y e a r s . Assuming t h a t the s u b j e c t s p a r t i c i p a t i n g i n the study o f R e i n h o l d and K i n g s l e y (1938) were c a s e s o f Duchenne d y s t r o p h y , which i s h i g h l y l i k e l y , t h e i r r e s u l t s r e g a r d i n g the c h e m i c a l composition of the muscle are compatible with t h o s e o f V i g n o s and Warner. I f the membrane n o r m a l l y r e s t r i c t s c r e a t i n e e f f l u x , a primary l e s i o n of the membrane, rather than i n t e r r u p t i o n which i s i n c i d e n t a l to degeneration i s s t r o n g l y supported. T h i s may e x p l a i n why the s t i m u l a t i o n o f c r e a t i n e s y n t h e s i s by g l y c i n e a d m i n i s t r a t i o n had a p o s i t i v e e f f e c t upon p a t i e n t s i n the e a r l i e r stages of the d i s e a s e , by p a r t i a l l y and t e m p o r a r i l y amending a dearth of phosphocreatine. 159 M a l i g n a n t h y p e r t h e r m i a i s a p o t e n t i a l l y f a t a l c o n d i t i o n , o f t e n a f f e c t i n g young m a l e s . The m e t a b o l i c processes of the muscle i n t h i s c o n d i t i o n are u n c o n t r o l l a b l y a c c e l e r a t e d , u s u a l l y by the a d m i n i s t r a t i o n of a n e s t h e t i c s , to the p o i n t at which heat p r o d u c t i o n exceeds heat l o s s . I n c r e a s e d s e r u m l e v e l s o f p o t a s s i u m , and c r e a t i n e phosphokinase i n d i c a t e changes i n membrane p e r m e a b i l i t y as does hypocalcaemia. The f a c t that there are reported cases of Duchenne d y s t r o p h y who have s u f f e r e d from m a l i g n a n t h y p e r t h e r m i a i n r e s p o n s e t o t h e a d m i n i s t r a t i o n o f a n e s t h e t i c s (Smith et a l . , 1985; Wang et a l . , 1986; Sethna et a l . , 1986) supports the exis t e n c e of an a l t e r e d membrane p e r m e a b i l i t y due to a primary l e s i o n . I t i s i n p a r t the i n f l u x of c a l c i u m which s t i m u l a t e s the metabolic processes of the muscle and the increase of muscle calcium i n Duchenne dystrophy could be a r e f l e c t i o n of increased access of t h i s i o n t o the muscle f i b r e as a r e s u l t of an i n t e r r u p t e d membrane. E l e v a t i o n of serum CPK and o t h e r c y t o p l a s m i c enzymes c o u l d then be due to a c o m b i n a t i o n o f e s c a p e , i n c r e a s e d t u r n o v e r as a r e s u l t of escape and i n c r e a s e d turnover as a r e s u l t of calcium i n f l u x . Increases i n serum c r e a t i n e phosphokinase i n het e r o z y g o u s females f o l l o w i n g e x e r c i s e (Moser, 1984) are a l s o c o n s i s t e n t with a d i s r u p t i o n i n the plasma membrane. S y m p t o m a t i c a m e l i o r a t i o n by a d r e n a l i n ( p l u s p i l o c a r p i n e ) c o u l d be e x p l a i n e d by i t s s t i m u l a t i o n of the 160 ATP producing p r o c e s s e s , thereby t e m p o r a r i l y a v e r t i n g the impending n u t r i t i o n a l f a i l u r e of the muscle f i b r e . Although an i n c r e a s e i n c a l c i u m may i n i t i a l l y s e r v e to s t i m u l a t e m e t a b o l i s m , t h e i n t e g r i t y o f t h e m u s c l e f i b r e w o uld u l t i m a t e l y be threatened by an i n t e r r u p t e d sarcolemma and t h i s c o u l d v e r y w e l l be t h e r e a s o n f o r t h e s e g m e n t a l n e c r o s i s which i s a prominent h i s t o l o g i c a l f e a t u r e . C e l l c u l t u r e s t u d y has d e m o n s t r a t e d t h a t c r e a t i n e may be a s p e c i f i c a c t i v a t o r of p r o t e i n s y n t h e s i s i n the muscle f i b r e ( Ingwall et a l . 1972, Ingwall et a l . 1974, Ingwall 1976). I f t h e membrane i s i n t e r r u p t e d d u r i n g t h e c o u r s e o f r e g e n e r a t i o n , a d e a r t h of the "food" of muscle, as F o l i n c a l l e d i t , c o u l d be the major r e a s o n f o r the a b o r t i v e r e g e n e r a t i o n e v i d e n c e d by b a s o p h i l i c f i b r e s which never r e a t t a i n a normal s i z e . The m e t a b o l i c consequences of a membrane d e f e c t c o u l d be r e s p o n s i b l e f o r t h e t r u l y " d i f f i c u l t n u t r i t i o n " which e x i s t s i n Duchenne dystrophy. The Question of the C e r e b r a l Defect The phenomenon of i n t e l l i g e n c e i s indeed complex and i s comprised of many components. There has been disagreement a b o u t whether i n t e l l i g e n c e i s g e n e r a l l y i m p a i r e d i n a p e r c e n t a g e of those w i t h Duchenne d y s t r o p h y , or whether v e r b a l i n t e l l i g e n c e per se i s s e l e c t i v e l y d e p r e s s e d . For example, i n c o n t r a s t to Prosser et a l . (1969), who concluded t h a t t h e r e was a uniform d e p r e s s i o n of i n t e l l i g e n c e i n a 161 p e r c e n t a g e o f t h e i r s u b j e c t s , Marsh and Munsat (1974) i n t e r p r e t e d r e s u l t s c o n c e r n i n g the c l i n i c a l assessment o f t h e i r p a t i e n t s t o f a v o u r a s e l e c t i v e , n o n p r o g r e s s i v e impairment o f v e r b a l i n t e l l i g e n c e . I f the l a t t e r i s the a c t u a l s i t u a t i o n , the ques t i o n s t i l l remains: i s the d e f e c t indigenous to the b r a i n ? The s p e e c h o f t h o s e who s u f f e r f r o m t h e a l l e g e d c e r e b r a l d e f e c t i s , as Duchenne f i r s t d e s c r i b e d , slow and m i s a r t i c u l a t e d . In view of the f a c t t h a t the tongue may e n l a r g e , i t i s q u i t e p o s s i b l e t h a t t h e m u s c l e s o f v e r b a l i z a t i o n may be a f f e c t e d i n such a way as to make the c o o r d i n a t i o n of muscles d i f f i c u l t to g i v e a s l o w e r , s l u r r e d speech p a t t e r n . The d i f f e r e n c e between the r e d u c t i o n of and the complete l a c k of expre s s i o n of the p r o t e i n may e x p l a i n the i n c o n s t a n c y of the reputed c e r e b r a l d e f e c t , the l a t t e r b e i n g r e s p o n s i b l e f o r i t s p r e s e n c e . The m e t a b o l i c consequences of a t o t a l lack of the p r o t e i n c o u l d manifest themselves e a r l i e r i n the d i s e a s e and may e x p l a i n why those with an a p p a r e n t l y subnormal I n t e l l i g e n c e a re r e t a r d e d i n reaching m a t u r a t i o n a l milestones (Dubowitz 1965). A g r e a t e r s e v e r i t y o f h i s t o l o g i c a l changes i n a s s o c i a t i o n w i t h an apparent i n t e l l e c t u a l d e f i c i e n c y (Rosman 1970) c o u l d a l s o be the r e s u l t of a complete as opposed to an incomplete lack of a membrane embedded p r o t e i n which i m p a r t s a s t r u c t u r a l d u r a b i l i t y to the muscle f i b r e . The t o t a l absence of such a p r o t e i n i n f i b r e s of muscles p a r t i c i p a t i n g i n the p r o d u c t i o n 162 of speech may be such that an impairment of so c a l l e d v e r b a l i n t e l l i g e n c e remains s t a t i o n a r y . I f t h e c e r e b r a l d e f e c t i s , on t h e o t h e r h a n d , indigenous to the b r a i n , the p r o t e i n must have a r o l e other t h a n o r i n a d d i t i o n t o t h a t o f p r o v i d i n g s t r u c t u r a l d u r a b i l i t y . I propose i n t h i s case that the p r o t e i n has a dual f u n c t i o n : that of imparting s t r u c t u r a l d u r a b i l i t y and that of impeding the e f f l u x of c r e a t i n e . There must be a mechanism t h a t a c c o u n t s f o r t h e d i f f e r e n c e s i n c r e a t i n e c o n c e n t r a t i o n i n the s k e l e t a l muscle, heart and b r a i n . I f a d i f f e r e n c e i n d e n s i t i e s of the membrane-embedded p r o t e i n n o r m a l l y a c c o u n t e d f o r the c o n c e n t r a t i o n d i f f e r e n c e s , a complete absence of the p r o t e i n i n the b r a i n which i s not s u b j e c t t o m e c h a n i c a l s t r a i n m i g h t a c c o u n t f o r a n o n progressive, subnormal i n t e l l i g e n c e . The k i n e t i c s of the isozyme f o r c r e a t i n e phosphokinase i n the b r a i n r e f l e c t the s m a l l e r need f o r p h o s p h o c r e a t i n e , without which a s t a t i c c e r e b r a l d e f e c t might accompany the muscular changes. In t h i s c a s e , D u c h enne d y s t r o p h y would not be a " p u r e " myopathy, but by v i r t u e of the inconstancy of the c e r e b r a l d e f e c t i t c o u l d n e i t h e r be a " t r u e " neuropathy. Again, a t o t a l l a c k or r e d u c e d e x p r e s s i o n o f the p r o t e i n c o u l d account f o r the inconstancy of the c e r e b r a l d e f e c t . 1 6 3 B i b l i o g r a p h y Adams, R.D., Denny-Brown, D., Pearson, CM., Diseases of Mu s c l e , 1st e d i t i o n , London, 1953, 2nd ed. Kimpton, London, 1962. Ahmed, Y.Y., Anat. R e c , 155, 133-138, 1964. A l l e n D.E., Johnson, A.G., Wolff, A.L., J . Anat. 105, 1969. A l l e n , J . E . and Rodgin, D.W., Amer. J . D i s . 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