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Studies of Erythrocyte Membrane Alterations in Duchenne Muscular Dystrophy Bridges, Michael Anthony 1976

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STUDIES OF ERYTHROCYTE MEMBRANE ALTERATIONS IN DUCHENNE MUSCULAR DYSTROPHY by MICHAEL ANTHONY BRIDGES B . S c , U n i v e r s i t y of New Mexico, 1969 A THESIS SUBMITTED IN PARTIAL FUItflUJIENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF PHARMACOLOGY We accept t h i s t h e s i s as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA November, 1976 @ Michael Anthony Bridges, 1976 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Michael Anthony Bridges Department of Pharmacology  The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date November 25, 1976 - i -ABSTRACT LXichenne Muscular Dystrophy (DMD) appears to be a generalized plasma membrane disorder involving many body tissues of affected i n d i v i d u a l s , includ-ing erythrocytes. Investigation of the myopathic aspects of t h i s disease has suffered from d i f f i c u l t i e s i n distinguishing between the immediate effects of the primary l e s i o n i n muscle and the sequelae of muscle fib e r necrosis. How-ever, since there are indications that erythrocytes may also be abnormal i n DMD, i t may be possible to characterize t h i s primary lesion i n these c e l l s . Furthermore, examination of erythrocytes of DMD patients and their female " c a r r i e r " r e l a t i v e s may reveal convenient biochemical markers of the disease which may aid both i n early patient diagnosis and i n c a r r i e r detection. The present investigations comprise a screening study i n which a variety of chemical and biochemical techniques were employed i n order to com-pare the structural and functional characteristics of DMD patient and c a r r i e r erythrocytes with those of normal control erythrocytes. A number of red c e l l abnormalities were found to be present i n erythrocytes from patients with DMD or from their female c a r r i e r r e l a t i v e s : alterations i n erythrocyte membrane phospholipid contents, i n membrane-bound enzymatic a c t i v i t i e s associated with active sodium and potassium transport, as well as i n those believed to be related to active calcium extrusion, and i n the osmotic f r a g i l i t y character-i s t i c s of i n t a c t red c e l l s . Although these findings are s t i l l tentative, they provide evidence supporting the generalized membrane defect hypothesis of DMD, as well as suggest promising avenues for further investigation of the molecular basis of DMD pathogenesis u t i l i z i n g red c e l l s . Recognized mechanisms of c e l l u l a r injury are discussed i n the attempt to reconcile the experimental findings of these studies with those of other investigators, and p a r a l l e l s are drawn between the alterations observed i n DMD erythrocytes and those exhibited by erythrocytes i n various other disorders and i n experi-mental models. - i i i -TABLE OF CONTENTS Page INTRODUCTION General Considerations 1 The Myogenic Hypothesis 2 The Neurogenic Hypothesis 6 The Vascular/Vasoactive Amine Hypothesis 8 The Membrane Defect Hypothesis 10 The Red Blood C e l l i n the Investigation of Duchenne Muscular Dystrophy 13 MATERIALS AND EXPERIMENTAL METHODS Materials 17 Preparation of Erythrocyte Membranes 18 Chemical Characterization of Erythro-cyte Membranes 19 Kinetic Analyses of erythrocyte Membrane Enzymatic A c t i v i t i e s 1. Adenosin-5'-triphophatases 20 2. p-Nitrophenyl Phosphatases 22 3. Acetylcholinesterase 23 Other Techniques Used to Characterize Erythrocyte Membranes 1. Thin Layer Chromatography 24 2. Gel Electrophoresis 25 Assessment of the Osmotic F r a g i l i t y of Intact Erythrocytes Subjected to Hypotonic Stress 25 S t a t i s t i c a l Analysis 26 - i v -Page RESULTS Compositional Analyses of Erythrocyte Membranes 29 Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids 39 Kinetic Analyses of Erythrocyte Membrane Enzymatic A c t i v i t i e s 1. Basal and Total p-Nitrophenyl Phosphatase 50 2. Adenosine-5 1-Triphosphatases 56 3. Acetylcholinesterase 63 Gel Electrophoresis 77 Erythrocyte F r a g i l i t y 82 DISCUSSION 88 BIBLIOGRAPHY 114 APPENDIX 123 - v -LIST OF TABLES No. T i t l e Page I Erythrocyte Membrane S i a l i c Acid Content 31 I I Erythrocyte Membrane Cholesterol Content 32 I I I Erythrocyte Membrane Phospholipid Content 33 IV (A) Erythrocyte Membrane Surface DTNB Titer 35 IV (B) Total Erythrocyte Membrane DTNB Titer 36 V (A) TNBS Titer of Intact Erythrocyte Membranes 37 V (B) TNBS Titer of Detergent-disrupted Erythro-cyte Membranes 38 VI (A) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Phosphatidyl Serin R f 41 VI (B) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Sphingomyelin 42 VI (C) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Phosphatidyl Choline R f 43 VI (D) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Phosphatidyl Ethanolamine R f 44 VII (A) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Phosphatidyl Serine 45 VII (B) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Sphingomyelin 46 VII (C) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Phosphatidyl Choline 47 T i t l e Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Phosphatidyl Ethano-1amine 2+ A c t i v i t y of Erythrocyte Membrane Mg -dependent (Basal) p-Nitrophenyl Phosphatase 2+ A c t i v i t y of Erythrocyte Membrane Mg -dependent, K -stimulated p-Nitrophenyl Phosphatase K +-stimulated Component of Erythrocyte Membrane p-Nitrophenyl Phosphatase: V x K +-stimulated Component of Erythrocyte Membrane p-Nitrophenyl Phosphatase: K q for K 2+ A c t i v i t y of Erythrocyte Membrane Mg -dependent (Basal) Adenosine Triphosphatase 2+ A c t i v i t y of Erythrocyte Membrane Mg -dependent, Na , K -stimulated Adenosine Tr iphosphatase 2+ High A f f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphos-phatase: V „ c max 2+ High A f f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphosphatase K c for C a 2 + 2+ Low A f f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphosphatase V max 2+ Low A f f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphosphatase K f o r C a 2 + .5 - v i i -No. T i t l e Page X (A) Substrate Kinetics of Erythrocyte Membrane Acetylcholinesterase: 75 X (B) Substrate Kinetics of Erythrocyte Membrane Acetylcholinesterase: K ^ for Acetylthio-choline * 76 XI (A) Fluoride I n h i b i t i o n of Erythrocyte Membrane Acetylcholinesterase: H i l l Coefficient 80 XI (B) Fluoride I n h i b i t i o n of Erythrocyte Membrane Acetylcholinesterase: K 5 for F 81 XII Polyacrylamide Gel Electrophoretic Analysis of Erythrocyte Membrane Proteins: Relative Peak Heights Derived from Densitometry Scan 83 - V l l l -LIST OF FIGURES Concentration Dependence of +K -stimulation of Erythrocyte Membrane Mg -dependent p-Nitrophenyl Phosphatase i n Normal and DMD Erythrocytes. Inset Eadie Plot Represent-ation of the Same Data. Concentration Dependence of the Stimulation of Erythrocyte Membrane Mg2+-dependent ATPase by Calcium. Eadie Plot Analysis of the Data i n Figure 2. Concentration Dependence of Acetylthiocholine Hydrolysis by Erythrocyte Membrane Acetylchol-inesterase. Inset Eadie Plot Analysis of the Same Data. Fluoride I n h i b i t i o n of Erythrocyte Membrane Acetylcholinesterase i n Normal and DMD Erythro-cytes. Inset H i l l P lot Analysis of the Same Data. Osmotic F r a g i l i t y of Intact Erythrocytes. Top Panel: DMD and Age-matched Normals. Bottom Panel: Normal Females and Definite Female Carriers. - i x -ACKNCWLEDGFJffiNTS I wish to express my deepest gratitude to Dr. David V. Godin for hi s many contributions to t h i s work and to my graduate education, not the least of which were i n s p i r a t i o n , knowledge and encouragement. I also g r a t e f u l l y acknowledge the invaluable assistance of Dr. P. J . M. MacLeod, who provided the c l i n i c a l materials examined i n these studies and made valuable suggestions during the preparation of t h i s manu-s c r i p t . I would also l i k e to thank Dr. Malcolm Greig, Department of Computer Science, University of B r i t i s h Columbia, and Mr. Guy Costanzo, Research O f f i c e r , City of Vancouver Health Department, for their a s s i s -tance with the s t a t i s t i c a l analyses, Ms. Therese Wan Ng for her expert technical assistance and for her help i n the preparation of the diagrams, and Mrs. Carol Reynish and her c l e r i c a l s t a f f at Thompson & McConnell, Barr i s t e r s and S o l i c i t o r s , White Rock, for their s k i l l f u l execution of t h i s manuscript. F i n a l l y , I wish to acknowledge the kind f i n a n c i a l support of the Medical Research Council of Canada. - X -FOR JACQUELINE HELEN McCONKEY-BRIDGES - 1 -INTRODUCTION General Considerations Attempts to conceptually define the term "muscular dystrophy" have been frustrated by the lack of precise information regarding the homogeneity of the various c l i n i c a l myopathies placed i n t h i s group and their e t i o l o g i -c a l bases. Despite t h i s lack of basic knowledge regarding the nature of the muscular dystrophies i n man, operational d e f i n i t i o n s have been u t i l i z e d i n order to permit the d i f f e r e n t i a l diagnosis of individual cases and to d i s t i n -guish these from the non-dystrophic myopathies. These operational d e f i n i t i o n s take into consideration factors such as the mode of transmission of the d i s -order, i t s age of onset, rate of progression and the l i k e [1]. This a b i l i t y to distinguish between the various c l i n i c a l myopathies i s c r u c i a l to both the investigator, who seeks information on the basic disorder, and the c l i n i c i a n who must properly manage the myopathic patient. Unfortunately, the dystro-phic myopathies are as yet incurable, and the c l i n i c i a n must content himself with mere p a l l i a t i o n of these syndromes [1]. Duchenne muscular dystrophy (DMD), the focus of the present inves-t i g a t i o n , i s a progressive, c r i p p l i n g disease of young males characterized by recessive sex-linked inheritance and an early onset of symptoms; these symp-toms include proximal muscle weakness and atrophy, hypertrophy of the calves i n most cases, muscular contractures, myocardial involvement and a high i n c i -dence of mental retardation, with death usually resulting from respiratory or cardiac f a i l u r e by the second or t h i r d decade [1]. In B r i t i s h Columbia, the Health Surveillance Registry estimates the province-wide frequency of DMD as one case per 6000 l i v e male b i r t h s [2]. One t h i r d of DMD cases are believed to be new mutants; one t h i r d have a previous family history of the disease, and one t h i r d are born to unknowing and often mutant c a r r i e r s [3]. - 2 -A great deal of research has been directed towards characterization of the primary le s i o n underlying DMD. I t should be noted here that much con-fusion i n the l i t e r a t u r e has been generated by researchers who apparently as-sume that the eti o l o g i e s of a l l muscular dystrophies are the same and who therefore seek to characterize the basic defect i n DMD by extrapolation from experimental data derived from the study of other human myopathies and dystro-phic animal models. As w i l l become more evident, there i s no firm evidence that these various muscular dystrophies are pathophysiologically i d e n t i c a l to DMD [4]. Investigators have advanced four d i f f e r e n t hypotheses regarding the possible etiology of DMD: (a) the myogenic hypothesis suggests that the primary defect i n muscular dystrophy l i e s within the muscle f i b e r ; (b) the neurogenic hypothesis seeks to implicate abnormal neurotropism of defective motor neurons i n myopathy; (c) the vascular hypothesis maintains that the primary le s i o n resides i n the microcirculation, leading to sk e l e t a l muscle necrosis; an extension of t h i s hypothesis implicates faulty vasoactive amine handling i n the production of DMD; and (d) the membrane defect hypothesis suggests that muscular dystrophy i s an inherited molecular disorder of c e l l u -l a r membranes with widespread tissue involvement. The experimental evidence upon which these various hypotheses rest warrants some discussion. The Myogenic Hypothesis Although many structural and functional disturbances have been i d e n t i f i e d i n muscle, the chief problem remains: how does one distinguish the primary le s i o n from secondary and t e r t i a r y effects a r i s i n g from muscle fi b e r necrosis, lipogenesis and connective tissue i n f i l t r a t i o n ? Some of the more compelling findings i n studies involving muscle are presented here. - 3 -Hughes has analysed l i p i d extracts of skeletal muscle minces by t h i n layer chromatographic methods [5] and found no a l t e r a t i o n i n t o t a l muscle l i p i d s of DMD patients. However, the content of individual l i p i d fractions was greatly altered and bore a marked s i m i l a r i t y to the pattern observed i n human f e t a l muscle: the cholesterol and sphinogomyelin l i p i d fractions were increased and the phosphatidyl choline f r a c t i o n was de-creased r e l a t i v e to normal. Hughes therefore suggested that DMD might represent a maturation f a i l u r e of skeletal muscle. Similar results had previously been obtained for genetically dystrophic mice [6]. Studies i n myopathic chickens [7] and hamsters [8], however, do not reveal the same pattern of l i p i d f r a c t i o n a l t e r a t i o n s . Dhalla et a l . have quantified the adenosine triphosphatase (ATPase) a c t i v i t i e s of sarcolemmal preparations obtained from patients with DMD and dystrophic hamsters [ 9 ] . In both DMD patients and dystrophic ham-2+ s t e r s , the basal and Ca -dependent ATPase a c t i v i t i e s of muscle sarco-lemma were observed to be increased; the Na +, K+-dependent enzyme a c t i v i t y was found to be depressed i n the human myopathy but elevated i n the hamster. Skeletal muscle sarcolemmal preparations from dystrophic mouse have revealed a pattern almost i d e n t i c a l to that observed i n DMD when assayed for the three ATPase enzyme a c t i v i t i e s [10, 1 1 ] . Dhalla et a l . have suggested that the sarcolemmal abnormalities seen i n DMD might r e s u l t from an a l t e r a t i o n i n the chemical composition of the muscle c e l l membrane [ 9 ] . Such an a l t e r a t i o n might profoundly a f f e c t the functional i n t e g r i t y of the muscle c e l l and be at the root of the dystrophic process. Skeletal muscle sarcoplasmic reticulum (SR) has been the object of extensive investigation i n the search for the primary lesion i n DMD i n view 2+ of the importance of t h i s organelle i n governing Ca a v a i l a b i l i t y i n the - 4 -process of muscle contraction. Takagai et a l . have i d e n t i f i e d compositional abnormalities i n protein and l i p i d fractions of SR vesicles i n DMD [12], the l i p i d a lterations resembling those observed by Hughes i n whole muscle ex-tracts [6]. Functional abnormalities were also found i n SR by these and other 2+ mvestigators: decreased Ca -uptake by SR vesicles was noted i n i n v i t r o 2+ studies [12, 13], as was decreased Ca -dependent ATPase a c t i v i t y [13]. Similar functional alterations have also been shown to occur i n myopathic hamsters [14] and dystrophic chickens [15] , but not i n human Myotonic muscular dystrophy [13]. Takagai et a l . have advised caution i n the interpretation of these findings, because SR vesicles are frequently contaminated with micro-somal material from adipose and connective tissues which have i n f i l t r a t e d muscle as part of the dystrophic process [12]. The presence of these contam-inants i n SR ve s i c l e preparations might well a l t e r the compositional and functional parameters evaluated to a considerable degree. Investigators have also turned their attention to the study of protein synthesis i n DMD muscle. Monckton and Nihei have demonstrated a three-fold increase i n protein synthesis i n heavy polyribosome fractions of DMD muscle [16]. Ionasescu et a l . have confirmed these observations and have shown that heavy polyribosomes from DMD muscle produce four to f i v e times as much collagen as normal heavy polyribosomes [17]. Whether these findings are indicativ e of the primary defect underlying DMD (e.g., a defective genetic mechanism for terminating collagen synthesis [4, 16]), or whether the ob-served alterations merely represent a non-specific response to inju r y , i s impossible to judge without further investigation. Studies of the co n t r a c t i l e apparatus of DMD muscle have been equally inconclusive. Gel electrophoretic analysis of con t r a c t i l e proteins reveal normal protein p r o f i l e s i n DMD material [18], but troponin a c t i v i t y (expressed - 5 -2+ as try p s i i i - s e n s i t i v e Ca -binding capacity of natural actomyosin) was found to be depressed by at l e a s t 50% of normal i n muscle obtained from biopsy of DMD patients [19]. This could r e f l e c t a basic defect i n excitation-contraction coupling or could be a consequence of the presence of excessive proteolytic enzyme a c t i v i t y , often observed i n dystrophic muscle [20, 21]. Actomyosin ATPase a c t i v i t y from dystrophic human muscle has also been shown to be de-pressed r e l a t i v e to normal muscle a c t i v i t y [22]. Strickland and E l l i s have found an interesting abnormality i n DMD muscle which also extends to other body tissues. These investigators had previously demonstrated an abnormally high conversion of glucose to fructose i n dystrophic muscle, with a corresponding drop i n glucose-6-phosphate pro-duction [23]. This shunting of substrate away from the energy-producing gl y -c o l y t i c pathway was shown to r e s u l t from the presence of a modified hexokinase isoenzyme I I i n the DMD muscle f i b e r [24]. Strickland and E l l i s demonstrated t h i s enzyme a l t e r a t i o n by gel electrophoretic methods i n sk e l e t a l muscle, l i v e r and brain tissues obtained post mortem from DMD patients. Such a defect i n hexokinase isoenzyme I I could arise from an inheritable l e s i o n of protein synthesis and could produce widespread damage i n many body tissues by depriv-ing them of e f f i c i e n t g l y c o l y t i c machinery for ATP production and by subject-ing these tissues to noxious l e v e l s of fructose and s o r b i t o l , implicated by some i n the production of diabetic neuropathy [25]. The foregoing discussion serves to i l l u s t r a t e a number of important points: (a) a large number of disturbances of structure and function have been observed i n Duchenne dystrophic muscle; (b) there are great d i f f i c u l t i e s associated with the search for the primary l e s i o n i n t h i s disorder, since t h i s pathogenic factor could e a s i l y be obscured by secondary and t e r t i a r y a l terations a r i s i n g from the dystrophic process; (c) the use of animal models - 6 -of human muscular dystrophy i n the search for the DMD pathogenic lesion i s fraught with p e r i l , because i n the absence of prior knowledge of t h i s basic defect, there can be no v a l i d c r i t e r i o n for deciding which animal model best approximates the human disorder; and (d) some abnormalities observed to occur i n dystrophic muscle can also be demonstrated i n certain other non-muscular tissues [23]. This l a s t point suggests that the primary lesion of DMD may not necessarily reside exclusively i n the muscle c e l l ; i n f a c t , some investigators believe that the le s i o n may not reside i n muscle at a l l . The Neurogenic Hypothesis The neurogenic hypothesis originated with studies on murine muscular dystrophy. Conrad and Glaser examined the f a t i g u a b i l i t y of neuromuscular transmission i n normal and dystrophic mice and found evidence of a slower rate of fatigue i n dystrophic murine muscle which could not be explained by i n -creased muscle responsiveness [26]. To account for t h i s phenomenon, Conrad and Glaser postulated the existence of an a l t e r a t i o n i n neuromuscular transmission independent of any primary muscle dysfunction. This suggestion of abnormal neural a c t i v i t y i n murine dystrophy pro-voked great interest i n myopathic animal models. Transplantation studies i n dystrophic mice [27, 28] and hamsters [29] led to the suggestion that some extramuscular factor governing sk e l e t a l muscle regeneration i s abnormal i n the dystrophic host. In an attempt to i d e n t i f y the abnormal extramuscular factor, Gallup and Dubowitz grew various combinations of normal and abnormal murine nerve and muscle together i n tissue culture [30]. These investigators found: (a) murine dystrophic muscle behaved normally with respect to myotube form-ation and con t r a c t i l e a c t i v i t y when regeneration occurs i n the presence of normal spinal cord c e l l s , but (b) regeneration of both normal and dystrophic - 7 -muscle was severely affected when coupled with spinal cord cultured from mice with muscular dystrophy. Since these tissue culture preparations were free from humoral, vascular and higher central nervous system influences, Gallup and Dubowitz conjectured that neurons i n the spinal cord are by themselves capable of producing myopathic manifestations i n murine muscle. Similar experiments conducted by Hamburgh et a l . f a i l e d to reproduce Gallup and Dubowitz's r e s u l t s [31]. However, other investigators have shown alterations i n the normal functioning of neurons i n murine dystrophy. For example, there i s evidence of abnormal axoplasmic flow of l i p i d s [32] and proteins [33] i n s c i a t i c nerve of animal dystrophy models, as well as a report of defective central and peripheral cholinergic neurons occurring i n these animals [34]. Although unequivocal evidence to support the assertion that neuro-genic influences underlie the production of murine dystrophy i s s t i l l lacking, the r e s u l t s obtained from animal dystrophy studies have stimulated much i n -terest i n the possible neural etiology of progressive muscular dystrophy i n man. The results of some of these investigations of DMD are discussed next. McComas, Sica and Currie were the f i r s t to make the case for a neurogenic etiology of DMD [35]. They observed a 75% reduction i n the number of functioning motor units i n the extensor digitorum brevis muscle of Duchenne dystrophy patients, while recording normal action potential amplitudes for any given motor unit i n these affected children. This suggested that the process of denervation occurring i n DMD i s highly s e l e c t i v e , destroying individual motor units i n their e n t irety. On the basis of these and subsequent experi-ments, McComas et a l . postulated that DMD involves a chronic dysfunction of motor neurons, leading to their physiological f a i l u r e [36]. Other researchers have been less fortunate i n their attempts to demonstrate t h i s selective loss of motor units i n DMD. Their reports reveal no s i g n i f i c a n t difference i n the - 8 -number of motor units between children with DMD and age-matched normal con-t r o l s [ 3 7 , 38, 3 9 ] . The neurogenic hypothesis of DMD pathogenesis i s a t t r a c t i v e , especially since the existence of neurotrophic influences on ske l e t a l muscle i s well established [40], but the present evidence i s not s u f f i c i e n t l y com-p e l l i n g to convince one that t h i s i s the whole story. In t h i s form of muscular dystrophy, pathological alterations extend beyond nerve and skeletal muscle. Perhaps the e t i o l o g i c a l factors responsible for t h i s myopathy arise elsewhere i n the body. The next section discusses other possible l o c i for the Duchenne defect. The Vascular/Vasoactive Amine Hypotheses According to Engel, the e a r l i e s t h i s t o l o g i c a l changes seen i n s k e l -e t a l muscle of patients with p r e - c l i n i c a l and early c l i n i c a l DMD are small f o c i of grouped muscle f i b e r s undergoing necrosis or regeneration — a l l f i b e r s of the group being at about the same stage [41]. Engel believes these focal abnormalities to be so cha r a c t e r i s t i c of DMD that they may be considered to be diagnostic of t h i s disorder [42]. Most hypotheses of DMD pathogenesis cannot adequately explain why abnormal muscle f i b e r s occur i n clusters surrounded by fi b e r s of h i s t o l o g i c -a l l y normal appearance. The vascular hypothesis offers a solution to t h i s problem. I t suggests the p o s s i b i l i t y that the blood supply to these small groups of affected f i b e r s i s compromised, since the size of a given focus could correspond to the area serviced by a terminal a r t e r i o l e [42]. Skeletal muscle lesions similar to those reputed to occur i n DMD have been produced i n rabbits by occlusion of the animal's muscle microvasculature with small doses of i n t r a - a r t e r i a l l y injected dextran p a r t i c l e s [42]. - 9 -Recent reports have disputed Engel's characterization of DMD myo-pathy and the relevance of dextran-embolus model. For example, O'Brien et a l . have suggested that a random d i s t r i b u t i o n of necrotic fi b e r s may be more representative of Duchenne muscular lesions [43]. Furthermore, morphometric studies of Duchenne muscle microvasculature have f a i l e d to demonstrate any abnormality except the r e p l i c a t i o n of the basement membrane of c a p i l l a r i e s , an a l t e r a t i o n found to occur i n a number of systemic disorders [44]. These data cast some doubt upon the v a l i d i t y of the vascular occlusion hypothesis. Investigators, unable to demonstrate gross morphological changes or occlusion of microcirculation i n DMD, have sought evidence for functional ischemia i n the pathogenesis of t h i s disorder, reasoning that i f elevated l e v e l s of c i r c u l a t i n g vasoactive amines (e.g., catecholamines and indole-amines) could be demonstrated, then the vascular hypothesis might yet solve the mystery of DMD etiology. Murphy, Mendell and Engel detected an abnor-mality of vasoactive amine handling associated with p l a t e l e t s obtained from Duchenne dystrophy patients. These authors observed a marked reduction i n the i n i t i a l rate of serotonin (5-HT) uptake by DMD p l a t e l e t s , as well as a lowering of p l a t e l e t 5-HT content [45]. Murphy et a l . have suggested that an analogous abnormality of vasoactive amine uptake may also occur i n the auto-nomic nerve terminals of the intramuscular vasculature, since s i m i l a r i t i e s have been described for amine transport mechanisms i n p l a t e l e t s and i n nerve endings i n brain and i n the periphery [46]. Because neuronal re-uptake i s the major mechanism for termination of the action of certain neurotransmitters i n the synaptic c l e f t [47], a defect i n t h i s re-uptake apparatus might lead to a prolongation of the effects of these vasoactive amines, with resultant damage being produced either by a d i r e c t toxic action upon muscle fib e r s or by ren-dering them ischemic through intense vasoconstriction [45]. The c r e d i b i l i t y - 10 -of t h i s modification of the vascular hypothesis w i l l depend upon the demon-st r a t i o n of such a neuronal uptake defect i n patients with DMD. I t i s clear from the foregoing sections that there are many tissues and organelles i n which DMD patients show deviations from the normal s i t u a t i o n : compositional and enzymatic abnormalities have been described for p r a c t i c a l l y every organelle of the s k e l e t a l muscle c e l l ; defects i n g l y c o l y s i s of s k e l e t a l muscle, l i v e r and brain have been reported; mental retardation of affected children and functional loss of motor neurons have also been observed; alterations i n the c a p i l l a r y basal lamina and abnormal vasoactive amine hand-l i n g by p l a t e l e t s and muscle have been described. Such a d i v e r s i f i e d pattern of alterations c l e a r l y makes i d e n t i f i c a t i o n of the primary lesion i n DMD a d i f f i c u l t problem. But t h i s complex pattern may also be supplying an impor-tant clue: i t might be suggesting a more basic, generalized defect than has been supposed underlies the production of t h i s disorder. The following sec-t i o n continues to elaborate upon the d i f f u s e pathology encountered i n DMD and presents evidence for a generalized c e l l u l a r membrane defect i n the patho-genesis of t h i s syndrome. The Membrane Defect Hypothesis I t has long been recognized that myopathy i n DMD i s not r e s t r i c t e d to s k e l e t a l muscle. Cardiac muscle involvement i s usually associated with t h i s syndrome [1]. In a recent retrospective study of autopsy findings covering a thirteen-year period i n Denmark, Leth and Wulff showed h i s t o l o g i c a l evidence of cardiomyopathy i n 80% of DMD patients investigated [48]. Goto, using gel electrophoretic a n a l y t i c a l methods, was able to demonstrate abnor-mally high l e v e l s of the cardiac isoenzyme of skeletal muscle creatine phos-phokinase (CPK) i n the serum of DMD patients [49]. This may suggest that the - 11 -same pathological processes, which produce "leaky" skeletal muscle plasma membranes and the loss of i n t r a c e l l u l a r enzymes (e.g., CPK), may also be operative i n cardiac muscle. Disturbances of cardiac rhythm, especially persistant tachycardia, are common i n DMD [1]. Moreover, a number of investigators have claimed that the very c h a r a c t e r i s t i c electrographic pattern observed i n t h i s disorder i s of diagnostic value i n distinguishing between DMD and other juvenile forms of progressive muscular dystrophy i n man [50]. Sudden death from cardiac f a i l u r e frequently occurs i n DMD [1]. In the Danish study previously cited [48], 41% of the Duchenne patients who died between 1960-1973 succumbed from cardiac complications of their disease. These reports c l e a r l y document that muscle lesions i n DMD are not r e s t r i c t e d to s k e l e t a l muscle but also extend to cardiac muscle. Recently, evidence has accumulated that erythrocytes from DMD patients exhibit compositional, structural and functional abnormalities. Kunze et a l . have demonstrated a s i g n i f i c a n t increase i n the sphingomyelin content, as well as alterations i n the f a t t y acid composition of phospha-t i d y l ethanolamine and sphingomyelin, of DMD red c e l l membranes [51]. Matheson and Howland have reported that saline-washed erythrocytes from patients with DMD appear d r a s t i c a l l y deformed when viewed by scanning electron microscopy [52]. /Although these s t r u c t u r a l l y modified erythrocytes, termed echinocytes, were observed i n both normal and dystrophic children, the propor-t i o n of these distorted c e l l s to normally appearing c e l l s i n whole blood was very d i f f e r e n t for the two groups. In normal children the percentage of echinocytes ranged from 3-7%, but DMD children showed a range of 20-98% echinocytes i n whole blood. Other investigators have confirmed Matheson and Howland's findings, but a considerable degree of overlap e x i s t s between the - 12 -normal and affected groups [53]. Furthermore, an increased incidence of echinocytes i n whole blood has also been shown to occur i n a number of other c l i n i c a l conditions, including some myopathies [52, 54]. Thus, although the morphological a l t e r a t i o n described here i s not s p e c i f i c to DMD, i t further supports the idea that the pathological process at work i n t h i s disease i s widespread, involving even red blood cells,although i n a stereo-typed way. Abnormalities i n K + fluxes have been reported for Duchenne ery-throcytes. In a recent study, Howland has shown that red blood c e l l s from DMD patients e x h i b i t an abnormally high permeability to potassium; he has also ob-served t h i s a l t e r a t i o n i n brain and l i v e r mitochondria of genetically dystro-phic mice [55]. But Howland could find no evidence to suggest that i n t r a -c e l l u l a r K + l e v e l s are depleted i n DMD erythrocytes. This very high passive K + permeability may be at l e a s t i n part compensated by the reported increase i n active K + transport into red c e l l s i n DMD [56]. Recently, Roses and Appel have demonstrated abnormal a c t i v i t y of erythrocyte membrane protein kinase i n both DMD and Myotonic muscular dys-trophy [57]. Endogeneous protein kinase i s an enzyme system capable of cata-ly z i n g the phosphorylation of certain membrane protein components. Roses and Appel found that i n DMD red c e l l membranes, the phosphorylation of a compon-ent of spectrin (gel electrophoretic band II) i s increased, while i n the myotonic erythrocyte membrane, phosphorylation of component "a" of band I I I i s decreased [58]. I t has been suggested that the state of phosphorylation of membrane components may be important i n determining the structural and func-t i o n a l c h a r a c t e r i s t i c s of the erythrocyte membrane. Consistent with t h i s hypothesis i s the report by Kury and McConnell that the state of phosphory-- 13 -l a t i o n of the erythrocyte membrane, which i s modified i n the presence of adrenaline or prostaglandins, influences the configurational state of membrane l i p i d s , as monitored using the spin-label techniques [59]. The foregoing discussion of the many and varied tissue alterations reported i n DMD including those described i n red c e l l s leads one to suggest that the primary l e s i o n underlying t h i s disorder may well be a generalized one involving c e l l u l a r plasma membranes. This proposed membrane defect could i n -volve alterations i n the s t r u c t u r a l interrelationships between plasma membrane protein and l i p i d components, thereby giving r i s e to myriad secondary changes i n c e l l u l a r functional parameters, including alterations i n the properties of subcellular organelles. Thus the membrane defect hypothesis of DMD could unify the previously discussed c e l l u l a r abnormalities into a coherent whole. However, these experimental findings are s t i l l inconclusive: the pathogenic le s i o n i n DMD has yet to be unequivocally i d e n t i f i e d , and the secondary and t e r t i a r y a l terations which accompany t h i s defect have yet to be rationalized r e l a t i v e to t h i s primary l e s i o n . The Red Blood C e l l i n the Investigation of Duchenne Muscular Dystrophy The use of erythrocytes i n the study of DMD pathogenesis has much to recommend i t : (a) the pathological process underlying DMD markedly i n -fluences red c e l l membrane structure and function; (b) erythrocyte membranes share many features i n common with more complex plasma membrane systems, such as sarcolemma; these features include compositional s i m i l a r i t i e s [60], a oua-+ 2+ bain-sensitive active Na -transport system [61], and an active Ca -extru-sion mechanism [62]; furthermore, there i s good evidence that erythrocytes possess an actomyosin-like system (spectrin associated with red c e l l actin) - 14 -which i s located at their inner membrane surfaces and may be responsible for regulation of red c e l l shape [63, 64]; (c) erythrocytes are not subject to the same degenerative c e l l u l a r processes which may obscure the pathogenic l e s i o n i n ske l e t a l muscle; f i n a l l y , (d) red c e l l s from normal and affected subjects may be obtained by a minimally invasive procedure (venipuncture), and their membranes may be e a s i l y isolated i n a very pure state and i n high y i e l d by routine methods (see Materials and Methods). In short, the red blood c e l l membrane obtained from patients affected with DMD offers a nearly ideal model membrane system for investigative purposes. In addition to supplying information p o t e n t i a l l y useful i n the i d e n t i f i c a t i o n of the molecular basis of DMD, the red c e l l may also be of value i n the early diagnosis of t h i s disorder. At present, the establishment of a d e f i n i t i v e diagnosis of DMD i n the affected c h i l d i s an arduous and time-consuming a f f a i r for the patient and his family. There i s often great d i f f i c u l t y i n distinguishing between DMD, other muscular dystrophies and cer-t a i n non-dystrophic disorders which produce proximal muscle weakness [1]. The diagnosis invariably involves long-term observation of the progression of the patient's symptoms, the performance of numerous c l i n i c a l diagnostic tests for exclusion of the non-dystrophic myopathies, s e r i a l determination of serum le v e l s of skeletal muscle enzymes (e.g., CPK), biopsy of affected muscu-lature and electromyography [1]. This tends to work great hardships upon the patient and his family and poses problems i n advising the parents about the r i s k of DMD i n future pregnancies. Therefore, i t would be of great value to develop a rapid, convenient and minimally invasive c l i n i c a l t e s t , which would unambiguously i d e n t i f y DMD i n the c h i l d presenting with t h i s disorder. I f a biochemical DMD marker could be found i n the erythrocytes of affected - 15 -children and i f t h i s marker were unique to t h i s disease, then such a diagnos-t i c t o o l would be a r e a l i t y . This hypothetical red c e l l DMD marker might even prove to be de-tectable years before the onset of the c l i n i c a l signs of dystrophy, perhaps even as early as the neonatal or even the f e t a l stages. Diagnosis of DMD i n utero by amniocentesis [65, 66] would allow the p o s s i b i l i t y of aborting affected fetuses, while neonatal screening could permit early i n s t i t u t i o n of therapeutic measures i n the p r e - c l i n i c a l stage, i f research into DMD patho-genesis were ultimately to reveal ways to cure or ameliorate the dystrophy. Analysis of erythrocytes may also provide a means of unequivocally determining c a r r i e r status i n female re l a t i v e s of Duchenne muscular dystrophy patients. A variety of i n d i r e c t indices of c a r r i e r status have been used [65]: (a) the physical examination — true c a r r i e r s often have a s l i g h t degree of myopathy which occasionally can be detected as muscular hypertrophy or weak-ness [1], but physical examination rarely reveals these abnormalities; (b) sk e l e t a l muscle biopsy — i n some c a r r i e r s , h i s t o l o g i c a l examination of muscle biopsy material w i l l detect abnormalities, but the d i f f i c u l t y i n obtaining a large enough sample for a d e f i n i t i v e judgment on c a r r i e r status l i m i t s the value of t h i s procedure; (c) quantitative electromyography — EMG detects ab-normalities i n something less than 50% of d e f i n i t e or probable c a r r i e r s ; since the technique i s laborious and often non-definitive, i t s popularity as a de-tection method i s small; (d) determination of serum levels of muscle enzymes — the most successful method to date for detection of c a r r i e r status i s e s t i -mation of serum creatine phosphokinase (CPK) l e v e l s ; CPK determinations are easy to perform, but t h i s test only detects 60-70% of d e f i n i t e c a r r i e r s . C l e a r l y , more convenient and unequivocal means for detecting c a r r i e r status i n DMD are needed. - 16 -When a woman with an affected son seeks genetic counselling, the pro b a b i l i t y of future affected offspring can only be discerned i f the source of her son's disease can be i d e n t i f i e d as either f a m i l i a l or sporadic. I t should be noted that sporadic cases of DMD are thought to occur with a high frequency. If the mother does not show myopathy, an estimation of t h i s p r o b a b i l i t y must be made by examination of the family history. This w i l l usually allow the assignment of the woman to one of three categories d e s c r i -bed by Walton [1]: "Definite c a r r i e r s " are those mothers of an affected son who have also an affected brother, maternal uncle, s i s t e r ' s son or other male r e l a t i v e i n the female l i n e of inheritance; also mothers of affected sons by di f f e r e n t , non-consanguineous fathers. "Probable c a r r i e r s " are the mothers of two or more affected sons, who have no other affected r e l a t i v e s . "Possible c a r r i e r s " are the mothers of isolated cases and the s i s t e r s and other female r e l a t i v e s of affected males. The accepted theory of a high incidence of sporadic DMD has recently been questioned by Roses and Appel [67]. They investigated 21 mothers of DMD patients (3 d e f i n i t e c a r r i e r s , 4 probable c a r r i e r s and 14 possible c a r r i e r s ) , using three d i f f e r e n t methods of evaluating the c a r r i e r status i n these women: (a) detailed physical examination for detection of muscle weakness, (b) electron microscopic examination of erythrocyte morphology to estimate the incidence of echinocytes, and (c) measurement of the extent of endogeneous phosphorylation of erythrocyte membrane spectrin. They demonstrated detect-able muscle weakness i n 19 of the 21 mothers. A l l 21 showed an abnormally high incidence of red c e l l s with shape d i s t o r t i o n s . The mean values for spectrin phosphorylation were i d e n t i c a l for the three c a r r i e r types and were s t a t i s t i c a l l y higher than the mean value for female control subjects. These - 17 -results suggest that many mothers of children showing apparently sporadic DMD are actually c a r r i e r s of the defective gene. Unfortunately, the alterations i n red c e l l morphology and spectrin phosphorylation observed by Roses and Appel do not constitute unambiguous markers of DMD c a r r i e r status, since individual values for the women i n the three c a r r i e r classes often f e l l well within the normal range [53, 67]. Therefore, the quest for r e l i a b l e indices of c a r r i e r status continues. The studies of red c e l l s described i n t h i s thesis are a continu-ation of the search for the pathogenic le s i o n underlying DMD and for unambigu-ous markers of the affected and c a r r i e r states of t h i s disorder. The work re-ported here comprises a screening study; erythrocytes obtained from patients with DMD and their female r e l a t i v e s were analysed by a wide variety of bio-chemical techniques to evaluate red c e l l s tructural and functional character-i s t i c s . S t a t i s t i c a l comparison of these parameters to those obtained for age and sex-matched normal controls has revealed promising areas for future i n -depth investigation. MATERIALS AND EXPERIMENTAL METHODS Materials The following reagents were obtained from Sigma Chemical Company: T r i s (hydroxymethyl) aminomethane (Trizma Base, reagent grade), imidazole (grade I I I ) , disodium adenosine-5 1-triphosphate, p-nitrophenyl phosphate (104 phosphatase substrate), acetylthiocholine hydrochloride, Triton X-100, sodium dodecyl sulfate (SDS), 5,5'-dithiobis-(2-nitrobenzoic acid), p i c r y l sulfonic acid (trinitrobenzenesulfonic a c i d ) , N-acetyl neuraminic acid (type I I I from egg), cholesterol standard, phosphorus standard solution (20 micro-- 18 -grams inorganic P/ml as KH^CPO^)), p-nitrophenol standard solution (10 micromoles/ml). Bovine serum albumin (fraction V) was supplied by Armour Pharmaceutical Company. Ninhydrin ("Baker TLC Reagent") was purchased from Baker Chemical Company. Cyanogum-41, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine (TMED) were obtained from E-C Apparatus Corporation. Bromophenol blue (indicator pH 3.0-4.7) and Coomassie b r i l l i a n t blue s t a i n were supplied by Eastman-Kodak Company. A l l other chemicals were of a n a l y t i c a l reagent q u a l i t y . The water used i n a l l experiments was prepared by double d i s t i l l a t i o n using a Corning glass s t i l l . Preparation of Erythrocyte Membranes Blood for investigation was obtained from patients with muscular dystrophy, their families and age/sex-matched normal controls. Blood samples of approximately 20 ml were drawn from each subject into anticoagulant [1 ml 3.8% (w/v) sodium c i t r a t e per 9 ml blood] and kept at or below 4° C during the preparation of hemoglobin-free erythrocyte membranes [68]. The buffy coat f r a c t i o n was removed during two i n i t i a l isotonic saline washes, and the erythrocyte p e l l e t s were exposed to stepwise osmotic l y s i s i n 0.08, 0.06, 0.04, 0.02 and 0.009 M NaCl, the p e l l e t s being recovered by centrifuga-t i o n following each l y t i c step. After a f i n a l wash with 10 mM T r i s buffer (pH 7.4), erythrocyte membranes were quick-frozen using dry i c e and acetone and stored at -20°C. Chemical and enzymatic analyses of membranes followed a r i g i d time schedule such that a given determination was always performed after the same int e r v a l following preparation. This was of pa r t i c u l a r impor-tance i n the analysis of membrane enzymatic parameters, some of which vary with the period of storage. - 19 -Chemical Characterization of Erythrocyte Membranes Protein content of membrane preparations was determined by the method of Lowry et a l . [69], using bovine serum albumin as standard. Protein contents of membrane suspensions averaged between 3 and 4 mg protein per ml. Phospholipid was estimated by B a r t l e t t ' s modification of the Fiske-SubbaRow assay for inorganic phosphorus [70]. Membrane cholesterol content was deter-mined by the method of Zak et a l . [71]. S i a l i c acid was estimated, following hydrolysis of 0.2 ml of mem-brane suspension i n 0.1 N B^SO^at 80°C for 30 minutes, by the method of Warren [72]. Amino group t i t e r s of intact and detergent-disrupted erythrocyte membranes were measured using trinitrobenzenesulfonic acid as described by Godin and Ng [73]. Membranes (0.2 ml) were incubated at 37 + 0.5°C i n the presence of 1.0 ml 20 mM T r i s buffer (pH 8.0) i n a t o t a l volume of 2.9 ml. In experiments on detergent-disrupted membranes, 0.2 ml of 3% (v/v) Triton X-100 detergent was added to the incubation mixture. The reaction was i n i t i a t e d by addition of 0.1 ml 10 mM trinitrobenzenesulfonic acid (TNBS) solution (pH 8.0) and terminated after 1 hour by addition of 1:1 (by volume) mixture of 1 M HCl and 10% (w/v) sodium dodecyl sulfate. The extent of trinitrophenylation was expressed as the absorbance at 335 nm per mg membrane protein. The surface sulfyhydryl group t i t e r of erythrocyte membranes was estimated by incubating membranes at 37 + 0.5°C i n the presence of 1.0 ml 0.15 M imidazole buffer (pH 7.4) i n a t o t a l volume of 2.9 ml. Determination of t o t a l sulfhydryl group t i t e r required disruption of membranes by addition of sodium dodecyl sulfate at a f i n a l concentration of 1% (w/v) to the reaction - 20 -mixture. Reactions were i n i t i a t e d with 0.1 ml 3 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). Free sulfhydryl group l a b e l l i n g with DTNB i s complete within 30 minutes and involves d i s u l f i d e bond formation between a free sulfhydryl group of the membrane and one 5-thio-2-nitrobenzoic acid (TNB) group DTNB, with simultaneous release of the other molecule of TNB into the medium. The absorbance of the liberated TNB at 412 nm gives a measure of the number of membrane sulfhydryl groups modified. Membrane sulfhydryl t i t e r s are expressed i n units of nanomoles sulfhydryl per mg protein (TNB Molar Extinction Coefficient i s 1.36 x 10 4) [74]. Kinetic Analyses of Erythrocyte Membrane Enzymatic A c t i v i t i e s 1. Adenosine-5'-triphosphatase Erythrocyte membrane adenosine-5'triphosphatase (ATPase) a c t i v i t i e s were assessed under two d i f f e r e n t sets of chemical conditions. 2+ a) Basal (Mg -dependent) and Total (Mg 2 +, Na +, K+-dependent) ATPase The reaction mixture for the determination of t o t a l Na +, K+-ATPase, which i s the sum of the Mg2+-ATPase and Na +, K+-ATPase, consisted of the following components at the f i n a l (mM) concentrations shown i n parentheses: Tris-HCl buffer, pH 7.4 (55 mM); MgCl (3 mM); disodium adenosine-5'-triphos-phate (ATP) (3 mM); NaCl (80 mM); KC1 2(20 mM); ethylene glycolbis-(B-amino-ethyl ether)-N,N'-tetra-acetic acid (EGTA) (0.1 mM); 0.6-0.8 mg membrane 2+ protein, a l l i n a f i n a l volume of 3 ml. The Mg -ATPase component of t o t a l ATPase a c t i v i t y was determined as above except that Na + and K + were omitted from the reaction mixture. In t h i s analysis as well as that described below (section b), corrections were made for non-enzymatic hydrolysis of the ATP substrate. - 21 -b) Mg 2 +, Ca 2 +-dependent ATPase 2+ The reaction mixture for the k i n e t i c analysis of Ca -stimulated, Mg2+-dependent ATPase contained: Tris-HCl buffer, pH 7.4 (55 mM); MgCl 2 (6.4 mM); disodium ATP (2 mM); EGTA (0.1 mM); CaCl 2 (0.067, 0.080, 0.093, 0.100, 0.117, 0.133, 0.167, 0.200, 0.233 and 0.300 mM, corresponding to f i n a l free C a 2 + concentrations of 0.166, 0.326, 1.03, 2.69, 18.0, 29.7, 60.0, 89.4, 119.0 and 179.0 micromolar, respectively)*; 0.3-0.4 mg membrane protein, a l l 2+ i n a f i n a l volume of 3 ml. The basal (Mg -stimulated) ATPase a c t i v i t y rep-2+ resented that a c t i v i t y obtained by omitting Ca from the reaction mixture. ATPase reactions were i n i t i a t e d with membrane protein, incubated for one hour at 37 + 0.5°C and terminated by addition of 1 ml ice cold 20% (w/v) tri c h l o r o a c e t i c acid. Mixtures were centrifuged (30,000 x g, 10 minutes) to remove membrane material, and a 3.0 ml aliquot of supernatant was assayed for inorganic phosphate by the method of Fiske and SubbaRow [ 7 5 ] . Specific a c t i v -i t i e s were expressed as micromoles of inorganic phosphate liberated per hour per mg membrane protein. 2+ As described previously [ 7 6 ] , the kin e t i c s of Ca stimulation of 2+ ATPase are complex. The existence of two d i s t i n c t classes of Ca -stimulated ATPase a c t i v i t i e s was deduced from the biphasic character of Eadie plots pro-duced by p l o t t i n g the reaction rate of Ca 2 +-ATPase ( t o t a l Mg 2 +, Ca 2 +-ATPase a c t i v i t y less basal Mg -ATPase a c t i v i t y ) as a function of rate/Ca concen-t r a t i o n . The Ca concentration producing half maximal stimulation (K r) . 5 and the maximal reaction v e l o c i t y (V x ) were evaluated for each a c t i v i t y ; *The concentrations of free calcium i n the EGTA-Ca2+ buffer system were determined by means of a computer program provided by Dr. B. Roufogalis, Faculty of Pharmaceutical Sciences, The University of B r i t i s h Columbia. - 22 -these kinetic parameters were determined from the slope and Y-intercept, re-spectively, of the best f i t t i n g lines describing linear segments of Eadie plots (See Results). Lines of best f i t were computed using a Compucorp 140 "Statistician" calculator. 2. Basal (Mg^ -dependent) and 2+ + Total (Mg , K -dependent) p-Nitrophenyl Phosphatase The reaction mixture for the kinetic analysis of K +-stimulated p-nitrophenyl phosphatase (NPPase) consisted of the following components at the fin a l (mM) concentrations shown in parentheses: Imidazole-HCl buffer, pH 7.4 (50 mM); MgCl 2 (3 mM); p-nitrophenyl phosphate (3 mM); KC1 (2, 4, 6, 8, 10, 20, 30 mM); 0.6-0.8 mg membrane protein, a l l in a fi n a l volume of 3 ml. The 2+ basal (Mg -stimulated) component of total NPPase activity was determined as above except that KC1 was omitted from the reaction mixture. NPPase reactions were initiated with membrane protein, incubated for one hour at 37 + 0.5°C and terminated by addition of 1 ml ice cold 20% (w/v) trichloroacetic acid. Mixtures were centrifuged (30,000 X g, 10 minutes) to remove membrane material, and a 3.0 ml aliquot of supernatant was rendered alkaline with 1 ml 1.5 M Tris solution. The amount of p-nitrophenol in the basified supernatant was quantified spectophotometrically by measuring the absorbance of the solution at 412 nm. Specific activity of membrane NPPase was expressed as micromoles p-nitrophenol liberated per hour per mg membrane protein. In a l l cases, correction was made for the non-enzymatic hydrolysis of substrate. + Kinetic parameters describing the K -stimulated component of NPPase activity were evaluated using Eadie plot analysis. The K + concen-tration producing half maximal stimulation (K ) and the maximal reaction .5 - 23 -velocity (V__v) were determined from the slope and Y-intercept, respectively, of the linear Eadie plot representation of the data. 3. Acetylcholinesterase Erythrocyte acetylcholinesterase (AChE) activity was determined in reaction mixtures containing the following components in fi n a l concentrations shown in parentheses: Tris-HCl buffer, pH 8.0 (90 mM); 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), pH 8.0 (0.33 mM); acetylthiocholine (0.05, 0.10, 0.20, 0.50 and 1.00 mM); 30-40 micrograms membrane protein, a l l in a final volume of 3.0 ml. The reaction was performed in spectrophotometric cuvettes at room temperature and initiated by addition of substrate. The increase in optical density at 412 nm due to the reaction of liberated thiocholine with DTNB was linear for at least two minutes, and re-action rates were calculated from the average absorbance increase per minute during the f i r s t two minutes of the reaction. AChE activity i s expressed as micromoles thiocholine liberated per minute per mg membrane protein with correction being made for the non-enzymatic breakdown of acetylthiocholine. The kinetic parameters K ^ and V__v for the reaction were determined from . D max the slope and Y-intercept of the best f i t line computed for AChE Eadie plot data. Because NaF inhibition of erythrocyte membrane AChE activity has been reported to be a useful probe of membrane f l u i d i t y [77], the kinetics of inhibition of AChE by NaF were assessed in the same fashion as described above for the substrate kinetic experiments except that enzymatic act i v i t i e s were determined in the presence of increasing concentrations of NaF (0.5-2.5 mM) at a saturating concentration (1 mM) of acetylthiocholine. Kinetic data were analysed using H i l l plots [Log-^Vo/Vj-l) plotted as a function of Log^n NaF - 24 -concentration, where v Q i s the reaction velocity in the absence of inhibitor and v^ i s the velocity at any given concentration of inhibitor]. The kinetic parameters, "n" (which has been reported to be sensitive to the state of f l u i d i t y of the erythrocyte membrane [77]) and K ^ for inhibition, were deter-mined from the slope and the negative antilog-^ of the X-intercept, respec-tively, of the line computed to best f i t the plotted H i l l data. Other Techniques Used to Characterize Erythrocyte Membranes 1. Thin Layer Chromatography Erythrocyte membrane pellets, obtained by centrifugation and con-taining 1.5-2.0 mg protein were extracted twice with 2 ml of a 2:1 (by volume) chloroform-methanol mixture. The extract was washed three times with 1 ml 0.75% (w/v) NaCl solution; the chloroform phases were pooled, and the aqueous phases discarded. Next, the pooled chloroform phases were evaporated to dry-ness and quantitatively spotted on an activated (30 minutes at 110°C) s i l i c a gel F-254 plate (0.25 mm thickness, Brinkmann). The plate was run in a solvent mixture containing chloroform, methanol and ammonia (14:6:1, by volume). Phos-pholipid spots were identified by their values, which were highly reproduc-ible in this solvent system. Visualization of resolved phospholipid spots was accomplished by treatment of the plate with ninhydrin reagent (for phosphatidyl ethanolamine and phosphatidyl serine) and iodine vapour (for phosphatidyl choline and sphingomyelin). The various phospholipid classes were quantified by extraction from the s i l i c a gel (with 1 ml methanol, three times), evapora-tion of the extract to dryness and analysis of the residue for inorganic phosphorus as described previously [70]. - 25 -2. Gel Electrophoresis Erythrocyte membranes were s o l u b i l i z e d and subjected to gel electro-phoretic analysis by the method described by Fairbanks et al.[78] as outlined below. One hundred m i c r o l i t e r aliquots of membrane suspensions (containing 3 to 4 mg protein per ml) were combined with 50 m i c r o l i t e r s of a solution con-taining the following components: Tris-HCl buffer, pH 8.0 (30 mM); sodium dodecyl sulfate (SDS) (3%, w/v); sucrose (10%, w/v); disodium ethylenediamine-tetraacetic acid (EDTA) (3 mM); bromophenol blue (20 micrograms/ml), used as tracking dye; and 2-mercaptoethanol (0.3%, v/v). This mixture was then incu-bated for 20 minutes at 37°C to reduce membrane sulfhydryl groups and achieve membrane s o l u b i l i z a t i o n . Next, aliquots of t h i s mixture, containing 50 micro-grams of protein material (to insure optimal resolution), were applied to a 5% polyacrylamide gel containing 1% SDS. Electrophoresis was performed i n a c i r c u l a t i n g Tris-HCl buffer, pH 8.0 (50 mM), containing 0.1% SDS, with an E-C Corporation gel electrophoresis apparatus (150 v o l t setting). Resolution of membrane protein components was allowed to proceed u n t i l the tracking dye had migrated 10 cm or more from the o r i g i n . The gel was extensively washed with a mixture of methanol, water and g l a c i a l acetic acid (5:5:1, by volume) to remove residual SDS and stained with Coomassie b r i l l i a n t blue dye. Assessment of the Osmotic F r a g i l i t y of Intact Erythrocytes Subjected to Hypotonic Stress  Two m i l l i l i t e r s of whole blood were drawn into c i t r a t e anticoagulant (as described i n "Preparation of Erythrocyte Membranes") and washed twice with cold isotonic s a l i n e . Half a m i l l i l i t e r of these packed erythrocytes were washed with a cold 0.15 M NaCl solution made up i n Tris-HCl buffer, pH 7.0 (15 mM). The erythrocyte p e l l e t , obtained by centrifugation, was resuspended i n 8 ml of t h i s cold NaCl-Tris solution. A 0.2 ml aliquot of these resuspended erythrocytes was then added to 3.8 ml of each of several s a l t solutions [0.080, 0.075, 0.070, 0.065, 0.060, 0.050 and 0.040 M NaCl, made up i n Tris-HCl buffer, pH 7.0 (15 mM)]. Following incubation for 15 minutes at room temperature, samples were centrifuged at 40,000 X g for one minute, and the absorbance at a of the supernatant was read at 540 nm. The percent hemolysis of erythrocytes at a given NaCl concentration was expressed r e l a t i v e to t o t a l hemolysis i n d i s t i l l e d water. S t a t i s t i c a l Analysis Erythrocytes obtained from c l i n i c a l and control subjects were exhaustively analysed by the biochemical methods outlined in the preceeding sections. Data on a variety of red c e l l membrane parameters were generated in the hope of finding in the c l i n i c a l subjects areas of significant departure from normality which might warrant future in-depth investigation. The time required to completely process c l i n i c a l samples was such as to limit somewhat the total number of blood specimens that could be handled. Further, the relatively low incidence of DMD in the general population, compounded with the physical d i f f i c u l t i e s involved in drawing blood from affected subjects, placed some degree of constraint on the acquisition of c l i n i c a l material for analysis. In addition, parental reluctance limited the number of sex/age-matched control blood samples which could be obtained. For these reasons, sample size became an important consideration in the selection of the most appropriate s t a t i s t i c a l method for group comparison of c l i n i c a l and control subjects. The "independent samples t-test" i s often the s t a t i s t i c a l method of - 27 -choice when independent samples are drawn from two different populations and compared on the basis of a given single criterion to determine whether or not the populations d i f f e r . However, selection of this classical parametric method entails a number of assumptions regarding the nature of the populations from which the samples are drawn; in particular, the populations are assumed to be normally distributed and to exhibit homogeneous variances [79]. When the "t-test" i s applied to sampled populations which seriously violate the assumptions underlying this classical parametric method, no great confidence may be placed in the s t a t i s t i c a l result [80]. Procedures, which have been developed to test the applicability of parametric methods to given sampled populations, depend upon sample sizes large enough to permit the ade-quate characterization of these populations. When the number of observations is relatively small, as in the present study, group comparison may be safely made using methods described as "non-parametric" and "distribution-free" [81]. These methods entail far fewer assumptions regarding the nature of the sampled populations and therefore are ideal for comparison of groups of small sample size. These tests are called "distribution-free" because they never assume that the population distribution of variate magnitudes is precisely defined, as do the classical parametric tests; generally, their only assumption is that the sampled population is continuously distributed [80]. Since these tests concern themselves with known sample-linked characteristics (e.g., the rank relationships within a set of pooled observations), and not with estimated population-linked parameters (e.g., variance), they are described as "non-parametric" methods [80]. The Wilcoxon rank sum test is a distribution-free, non-parametric analogue of the independent samples "t-test" [79], and therefore is considered - 28 -to be one of the most useful of the non-parametric methods. According to Armitage, when distributions are normal and variances are equal, the Wilcoxon rank sum test has an asymptotic relative efficiency of 96% of that of the "t-test", and i f the distribution are not normal, the efficiency of the rank test is never less than 86% and may be in f i n i t e l y high [81]. Thus, l i t t l e is sacrificed when performing this test upon data which f u l f i l l s the more s t r i n -gent requirements of the classical parametric methods. An added advantage of the Wilcoxon method i s that calculations are made with ease and rapidity. A sample calculation i s offered elsewhere (see Appendix) to illustrate the use of the Wilcoxon rank sum method of s t a t i s t i c a l analysis. According to Bradley, the median and the interquartile range (i.e. the interval containing the median and the middle-most half of the experimen-ta l observations) are better indices of data location and dispersion than the mean and the standard deviation when the sampled population is non-normal, and these population indicators may even be employed to advantage when normality is assured [80]. Since the precise nature of the underlying populations sampled in this study remains obscure for the reasons previously stated, the mean and the median have both been used to characterize the experimental data for purposes of comparison (see Results). Unfortunately, the interquartile range is not well defined when dealing with samples of relatively small size. Therefore, in place of the interquartile range, the range of a l l sample obser-vations has been chosen, along with the standard deviation, to describe the dispersion of experimental data (see Results). - 29 -RESULTS A variety of chemical and biochemical techniques were employed in order to compare structural and functional characteristics of erythrocytes obtained from DMD patients or their female relatives with those of erythro-cytes obtained from normal controls. With regard to the selection of appro-priate controls, i t should be noted that a l l c l i n i c a l and control blood specimens examined were procured from either female adults, ranging in age from mid-adolescence to middle-age, or children primarily in their pre-adolescent years. The female relatives of DMD patients under study were categorized by the method of Walton [1] as definite, probable or possible carriers of DMD. In this study, definite and probable carriers were con-sidered as a single group due to the limited av a i l a b i l i t y of these subjects. The possible carriers, on the other hand, were more pl e n t i f u l , and the deci-sion was taken to subdivide these subjects on the basis of their relationship to the DMD patients. Thus for purposes of group comparison, three groups of c l i n i c a l subjects have been compared with normal adult females: (a) definite and probable DMD carriers, (b) possible carriers who are mothers of DMD patients and (c) possible carriers who are sisters of DMD patients (see Tables I-XII). Compositional Analyses of Erythrocyte Membranes Abnormal plasma membrane s i a l i c acid levels have been reported to occur in denervated rat skeletal muscle [82] and in erythrocytes obtained from patients with muscular dystrophy associated with severe hemolytic anemia [83]. In this latter case, Balduini et a l . were unable to detect the presence - 30 -of any s i a l i c acid in the erythrocyte membrane material sampled from two middle-aged brothers with this syndrome. These reports suggested that determination of erythrocyte membrane s i a l i c acid content might be of value in the study of DMD red c e l l s . Table I presents the results of this investi-gation. The s i a l i c acid contents of erythrocyte membranes of DMD patients and their female relatives did not significantly differ from those of appro-priate normal controls. It has been reported in various animal models of muscular dystrophy that abnormally high levels of cholesterol are present in skeletal muscle plasma membranes [8, 84], The data presented in Table II suggests that a similar alteration does not occur in the erythrocyte membranes of patients affected with DMD or of their carrier relatives. Phospholipids have also been reported to be subject to considerable modification in animal muscular dystrophy. In myopathic hamster cardiac muscle, total c e l l phospholipids are decreased [8], while the opposite i s seen in skeletal muscle of dystrophic chickens [7]. When erythrocyte mem-branes of DMD patients and carrier relatives were analysed for gross phospho-l i p i d content, no s t a t i s t i c a l l y significant departure from normality was observed (see Table III). The decision to examine the sulfhydryl content of DMD erythrocyte membranes was prompted by the observations of Chou et a l . who demonstrated that treatment of chickens suffering from hereditary muscular dystrophy with penicillamine p a r t i a l l y alleviated the symptoms of the avian disease and appeared to protect muscle c e l l membranes, as evidenced by a decrease in plasma CPK activity [85]. Since pencillamine i s a thiol compound with 31 TABLE I Erythrocyte Membrane S i a l i c Acid Content (units: nanomoles/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" is small) Significance at 0.05 level Age-matched Normal Children 6 98 95 9 4 88-110 -Children with DMD 10 113 107 18 6 93-144 N.S. Normal Female Adults 7 105 101 14 5 85-120 -Definite and Probable DMD Carriers 6 106 107 14 6 80-122 N.S. Possible Carriers: Mothers of DMD Patients 5 113 111 10 5 103-130 N.S. Possible Carriers: Sisters of DMD 7 108 105 16 6 85-134 N.S. Patients - 32 -TABLE I I Erythrocyte Membrane Cholesterol Content (units: micrograms/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 193 195 22 9 168-221 -Children with DMD 9 216 216 26 9 178-275 N.S. Normal Female Adults 8 234 228 48 17 170-328 -Definite and Probable DMD Carriers 6 247 241 30 12 217-289 N.S. Possible Carriers: Mothers of DMD Patients 4 228 229 13 6 213-244 N.S. Possible Carriers: Sisters of DMD Patients 8 206 207 16 6 186-225 N.S. -33 -TABLE III Erythrocyte Membrane Phospholipid Content (units: milligrams phospholipid/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" is small) Significance at 0.05 level Age-matched Normal Children 6 .528 .526 .031 .013 .484-.574 -Children with DMD 6 .523 .484 .087 .036 .448-.639 N.S. Normal Female Adults 8 .596 .599 .068 .024 .478-.721 -Definite and Probable DMD Carriers 5 .531 .530 .074 .033 .439-.623 N.S. Possibl Carriers: Mothers of DMD Patients 3 .618 .615 - -.585 .615 .654 N.S. Possible Carriers: Sisters of DMD 8 .577 .566 .071 .025 .489-.685 N.S. Patients - 34 -reducing properties, these authors suggested that sulfhydryl groups of the muscle plasma membrane might play some important role in avian muscular dystrophy. By analogy, elevated plasma CPK levels observed in human DMD may at least in part be determined by a defect in muscle plasma membrane structure involving membrane sulfhydryl groups — a defect which i f s u f f i -ciently generalized might be detectable in erythrocyte membranes. When intact and detergent-disrupted erythrocyte membranes are reacted with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), the quantity and distribution of membrane sulfhydryls can be estimated. In intact membranes, only surface sulfhydryl groups are accessible to the reagent, while in detergent-disrupted membranes a l l membrane sulfhydryls react, allowing an estimation of latent or buried sulfhydryl groups in the membrane. The data displayed in Tables IV (A and B) reveal no significant differences between c l i n i c a l and control groups with respect to these parameters. Trinitrobenzene sulfonic acid (TNBS) i s a group-specific reagent which introduces a chromophoric trinitrophenyl label onto primary amino groups. Since both protein and phospholipid components of membranes contain primary amino groups, the rate of labelling of intact and detergent-disrupted erythrocytes by TNBS provides a convenient means of assessing the structural integrity of these two important membrane components [73]. As in the DTNB studies, comparison of intact and detergent-disrupted membranes allows one to examine the properties of both surface and latent residues. If gross structural abnormalities occur in DMD erythrocyte membranes, these may alter the characteristics of TNBS incorporation into surface or latent primary amino residues. Examination of Tables V (A and B) w i l l show that in DMD -35 -TABLE IV (A) Erythrocyte Membrane Surface DTNB Titer (units: nanomoles sulfhydryl groups/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 4 28 30 5 2 21-32 -Children with DMD 7 25 28 7 3 14-32 N.S. Normal Female Adults 7 27 25 6 2 22-38 -Definite and Probable DMD Carriers 5 25 24 9 4 13-36 N.S. Possible Carriers Mothers of DMD Patients 3 23 25 - - 20, 25, 26 N.S. Possible Carriers: Sisters 7 24 24 3 1 19-29 N.S. of DMD Patients -36 -TABLE TV (B) Total Erythrocyte Membrane DTNB Titer (units: nanomoles sulfhydryl groups/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where V i s small) Significance at 0.05 l e v e l Age-matched Normal Children 4 53 55 5 3 46-57 -Children with DMD 8 51 54 15 5 28-69 N.S. Normal Female . Adults 7 54 51 9 3 42-69 -Definite and Probable DMD Carriers 5 46 43 7 3 40-57 N.S. Possible Carriers Mothers of DMD Patients 3 50 48 - - 48, 48, 55 N.S. Possible Carriers: Sisters of DMD Patients 7 51 49 8 3 43-70 N.S. -37 -TABLE V (A) TNBS Ti t e r of Intact Erythrocyte Membrane (units: absorbance at 335 nm/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 4 .125 .126 .004 .002 .119-.128 -Children with DMD 7 .128 .128 .019 .007 .104-.161 N.S. Normal Female Adults 7 .126 .124 .029 .011 .096-.187 -Definite and Probable DMD Carriers 4 .143 .146 .019 .009 .119-.162 N.S. Possible Carriers Mothers of DMD Patients 3 .137 .132 - -.129 .132 .149 N.S. Possible Carriers: Sisters of DMD Patients 7 .135 .133 .023 .009 .113-.183 N.S. -38 -TABLE V (B) TNBS Tit e r of Detergent-disrupted Erythrocyte Membranes (units: absorbance at 335 nm/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 4 .728 .734 .039 .020 .675-.767 -Children with DMD 7 .779 .746 .077 .029 .698-.894 N.S. Normal Female Adults 8 .813 .785 .115 .041 .693-1.042 -Definite and Probable DMD Carriers 4 .777 .805 .139 .069 .586-.910 N.S. Possible Carriers Mothers of DMD Patients 3 .783 .779 .017 .010 .768 .779 .802 N.S. Possible Carriers: Sisters of DMD 7 .786 .767 .062 .023 .700-.861 N.S. Patients patients and their female relatives, these parameters do not diffe r s i g n i f i -cantly from normal. Thin Layer Chromatographic Analysis of Erythrocyte Membrane  Phospholipids There is a great deal of evidence that some muscular dystrophies are associated with alterations in membrane phospholipid components. For example, increases in skeletal muscle membrane sphingomyelin fractions have been reported in avian muscular dystrophy [84], vitamin E-deficiency myopathy in calf [86], and human DMD [5]; furthermore, this same alteration has been described by Kunze et a l . for DMD erythrocyte membranes [51]. Phosphatidyl choline i s another membrane phospholipid reported to deviate from normal in muscular dystrophy. Depression of skeletal muscle membrane phosphatidyl cho-line levels occurs in DMD [5], and certain animal dystrophy models [7, 84]. Kunze et a l . have also observed highly significant changes in the fatty acid patterns of phosphatidyl ethanolamine and sphingomyelin in DMD erythrocytes [51]. Alterations, such as decreases in li n o l e i c (18:2) fatty acid components of sphingomyelin with concomitant increases in stearic (18:0) components, might modify phospholipid polarity greatly, thereby affecting fractional migration parameters (Rf's) of phospholipids on thin layer chromato-graphic (TLC) analysis. Membrane phospholipid alterations have also been shown to be associ-ated with erythrocyte morphological abnormalities. Thus, erythrocytes from patients with beta-Thalassemia Major possess well characterized morphological alterations associated with an increase in membrane phosphatidyl choline [87]. Since morphological abnormalities have also been reported to occur in erythro-cytes of DMD patients and possibly in female carriers of the dystrophic t r a i t - 40 -as well, i t was f e l t that detailed analysis of erythrocyte membrane phospho-li p i d s in DMD patients and their female relatives might provide valuable i n -formation on possible membrane alterations in these individuals. Tables VT and VTI present the TLC data for c l i n i c a l and control groups. Tables VI (A-D) summarize the relative mobility (R^) findings for the various phospholipid fractions (phosphatidyl serine, sphingomyelin, phospha-tidyl choline and phosphatidyl ethanolamine), while Tables VII (A-D) describe a quantitative analysis of these erythrocyte membrane phospholipid fractions, expressed as a percentage of total phospholipid. No significant differences could be found between the mobilities of the erythrocyte membrane phospholipids of DMD patients and those of normal children [Tables VI (A-D)]. Similarly, DMD sisters in the possible carrier category show the same TLC migration pat-terns as age and sex-matched normal controls. Unfortunately, insufficient data are available on two other groups: definite/probable carriers and pos-sible carriers who are mothers of affected children; without a larger number of observations in these data-poor groups, one i s unable to assess whether or not the erythrocyte membrane phospholipids of these subjects exhibit normal fractional migration patterns. However, as may be noted in Tables VI (A-D), both data-poor c l i n i c a l groups show at least one R^  observation f a l l i n g within the normal range. On the basis of inspection alone, one might argue that sig-nificant differences between such subjects and their control counterparts are unlikely. The rationales behind this "over-lap argument" and some of i t s shortcomings w i l l be considered in the Discussion section. Despite the need for further assessment of fractional migration parameters for data-poor carrier groups, the data presented in Tables VI (A-D) -41 -TABLE VI (A) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Phosphatidyl Serine (units: f r a c t i o n a l migration r e l a t i v e to solvent front) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 .138 .136 .008 .003 .132-.152 -Children with DMD 6 .125 .118 .025 .010 .102-.161 N.S. Normal Female Adults 6 .151 .145 .021 .009 .134-.189 -Definite and Probable DMD Carriers 2 - - - - .143 .159 Possible Carriers Mothers of DMD Patients 2 - - - - .111 .145 ? Possible Carriers: Sisters of DMD 8 .135 .124 .056 .020 .071-.240 N.S. Patients - 42 -TABLE VI (B) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Sphingomyelin (units: f r a c t i o n a l migration r e l a t i v e to solvent front) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 .219 .220 .013 .005 .200-.240 -Children with DMD 6 .182 .174 .029 .012 .148-.226 N.S. Normal Female Adults 6 .221 .211 .037 .014 .187-.278 -Definite and Probable DMD Carriers 2 - - - - .221 .229 ? Possible Carriers Mothers of DMD Patients 2 - - - - .175 .230 ? Possible Carriers: Sisters of DMD 8 .200 .200 .069 .025 .106-.304 N.S. Patients -43 -TABLE VI (C) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Phosphatidyl Choline (units: f r a c t i o n a l migration r e l a t i v e to solvent front) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 .353 .346 .020 .008 .333-.386 -Children with DMD 6 .324 .319 .043 .018 .273-.391 N.S. Normal Female Adults 6 .362 .351 .046 .019 .310-.438 -Definite and Probable DMD Carriers 2 - - - - .368 .378 ? Possible Carriers Mothers of DMD Patients 2 - - - - .310 .339 ? Possible Carriers: Sisters of DMD 8 .340 .316 .087 .031 .224-.444 N.S. Patients - 44 -TABLE VI (D) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Phosphatidyl Ethanolamine R f (units: f r a c t i o n a l migration r e l a t i v e to solvent front) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 .494 .488 .023 .010 .467-.526 -Children with DMD 6 .457 .451 .049 .020 .398-.528 N.S. Normal Female Adults 6 .484 .473 .042 .017 .439-.556 -Definite and Probable DMD Carriers 2 - - - - .493 .521 Possible Carriers Mothers of DMD Patients 2 - - - - .444 .461 •p Possible Carriers: Sisters of DMD 8 .478 .450 .077 .027 .382-.585 N.S. Patients -45 -TABLE VII (A) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Phosphatidyl Serine (units: percent t o t a l phospholipid fraction) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 20.3 21.0 2.2 0.9 16.2-22.1 -Children with DMD 6 21.3 20.5 3.2 1.3 18.8-27.5 N.S. Normal Female Adults 6 14.9 15.1 4.0 1.6 8.9-20.1 -Definite and Probable DMD Carriers 2 - - - - 13.9 18.7 ? Possible Carriers Mothers of DMD Patients 2 - - - - 17.3 26.0 ? Possible Carriers: S i s t e r s of DMD 8 22.4 21.5 3.4 1.2 18.3-29.0 Signif i c a n t (p<0.01) Patients -46 -TABLE VII (B) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Sphingomyelin (units: percent t o t a l phospholipid fraction) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 14.1 10.7 7.6 3.1 7.9-26.0 -Children with DMD 6 11.9 11.7 2.1 0.8 9.4-14.5 N.S. Normal Female Adults 6 10.1 6.2 7.5 3.1 4.0-22.5 -Definite and Probable DMD Carriers 2 - - - - 7.4 10.8 Possible Carriers Mothers of DMD Patients 2 - - - - 7.1 19.0 ? Possible Carriers: Sisters of DMD 8 12.7 13.5 5.9 2.1 5.0-21.0 N.S. Patients - 47 -TABLE VII (C) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Phosphatidyl Choline (units: percent t o t a l phospholipid fraction) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 6 28.4 28.5 2.0 0.8 26.0-30.8 -Children with DMD 6 27.0 27.3 1.4 0.6 25.1-28.3 N.S. Normal Female Adults 6 29.1 28.4 2.6 1.1 25.7-32.7 -Definite and Probable DMD Carriers 2 - - - - 31.5 34.1 Possible Carriers Mothers of DMD Patients 2 - - - - 27.7 32.5 ? Possible Carriers: Sisters of DMD 8 26.5 26.3 3.1 1.1 22.1-31.5 N.S. Patients - 48 -TABLE VII (D) Thin Layer Chromatographic Analysis of Erythrocyte Membrane Phospholipids: Relative Quantity of Phosphatidyl Ethanolamine (units: percent total phospholipid fraction) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 6 37.2 38.0 4.8 1.9 30.6-43.2 -Children with DMD 6 38.9 37.7 3.5 1.4 36.1-45.6 N.S. Normal Female Adults 6 44.1 47.6 7.7 3.2 33.6-51.7 -Definite and Probable DMD Carriers 2 - - - - 37.9 38.7 ? Possible Carriers Mothers of DMD Patients 2 - - - - 34.5 36.0 ? Possible Carriers: Sisters of DMD 8 37.0 36.9 4.3 1.5 30.6-43.8 N.S. Patients - 49 -seems hardly supportive of the findings reported by Kunze et a l . However, the tabulated data presented here does not reflect the whole story. Upon TLC analysis of erythrocyte membrane material obtained from six DMD patients, three of the affected children's specimens revealed two phospholipid spots migrating on the TLC plate with nearly the same mobility characteristics as normal sphingomyelin. These two spots were in fact supposed to be composi-tionally-modified sphingomyelin, although the truth of this assertion must await further investigation. Attempts to correlate the appearance of the "twin sphingomyelin-like spots" with the source of these children's dystrophy were unsuccessful: two of the three DMD cases were diagnosed as sporadic, one hereditary. This sphingomyelin anomaly has also been observed in two other sub-jects: one the sister of a young adult male with Becker's muscular dystrophy (a more benign form of sex-linked, pseudo-hypertrophic muscular dystrophy than DMD), the other the autosomal recessive (carrier) mother of a child with limb-girdle muscular dystrophy. It i s interesting to note that this woman's af-fected child did not show this particular phospholipid pattern, nor did an unrelated male limb-girdle carrier. These results indicate that TLC analysis of erythrocyte membranes, derived from patients with different types of muscu-lar dystrophies and their carrier relatives, may prove to be a f r u i t f u l re-search tool. The TLC data concerning the relative quantities of the four main phospholipid species have proven to be of interest and suggest continued u t i l -ization of this technique in DMD investigation. Tables VII (A-D) indicate that while no significant quantitative differences seem to occur in the various - 50 -phospholipid fractions of DMD and control children, possible c a r r i e r s who are s i s t e r s of DMD patients c l e a r l y d i f f e r from normality (p<0.01) with re-spect to the phosphatidyl serine content of their red c e l l membranes. Other phospholipid species appear to be present i n normal quantities i n t h i s group's erythrocytes. Again the small number of experimental observations precludes d e f i n i t i v e statements regarding the p r o b a b i l i t y that definite/probable c a r r i e r s and possible c a r r i e r mothers are normal with respect to their erythrocyte mem-brane phospholipid contents. Examination of Table VII (B) reveals a point of some interest: the sphingomyelin content of erythrocyte membranes of c l i n i c a l and control subjects (adults as well as children) shows a high degree of v a r i -a b i l i t y . K inetic Analyses of Erythrocyte Membrane Enzymatic A c t i v i t i e s 1. Basal and Total p-Nitrophenyl Phosphatase 2+ + There i s very good evidence that the Mg -dependent, K -stimulated p-nitrophenyl phosphatase (NPPase) a c t i v i t y observed i n some membranes r e f l e c t s + 2+ the K -stimulated dephosphorylation step i n the hydrolysis of ATP by Mg -de-pendent, Na +, K +-stimulated adenosine-5'-triphosphatase (ATPase), an enzyme intimately associated with the regulation of i n t r a c e l l u l a r sodium and potas-2+ 2+ + sium [88, 89]. Analysis of basal (Mg -dependent) and t o t a l (Mg , K -dependent) NPPase a c t i v i t i e s provides a means of focussing on the ^ - s e n s i -t i v i t y of the membrane sodium-potassium pump's enzymatic machinery without the complication of the p r i o r Na+-dependent phosphorylation step. Brody and Brody have reported NPPase abnormalities i n certain muscu-l a r disorders: these investigators have observed elevated levels of basal NPPase (but not total NPPase or Na +, K+-ATPase) activity in sarcolemmal prepar-ations of denervated rat limb [90] and i n the microsomal fractions of skeletal muscle obtained from rats rendered myotonic by treatment with (2,4-Dichloro-phenoxy) acetic acid (2,4-D) and similar monocarboxylic acids [91]. Recent studies of DMD carriers conducted by Thomson et a l . [92] have provided evidence of reduced total body levels of intracellular potassium in these subjects. Such findings prompted the investigation of NPPase function in erythrocyte mem-branes of DMD patients and carriers. Reference to Tables VIII (A and B) w i l l show that only DMD children differ significantly from normal controls with respect to basal and total NPPase activity levels. The finding that there i s a s t a t i s t i c a l l y significant difference (at the 0.05 level) between total NPPase activities of DMD and con-t r o l red c e l l membranes requires comment. Total NPPase activity represents the sum of basal NPPase and a K +-stimulated component. This K +-stimulated component, when evaluated for each subject by subtracting the basal level from the total level of enzymatic activity, exhibited no significant d i f f e r -ence (at the 0.05 level) between c l i n i c a l and control groups. However, these groups are found to diff e r at the 0.10 level of s t a t i s t i c a l significance. Figure 1 illustrates a typical example of the kinetic analysis of K +-stimulation of erythrocyte membrane NPPase. Here the pooled data for DMD patients and age-matched controls are displayed together in both Michaelis-Menten-type plots and Eadie plots (see insert). The kinetic parameters for K +-stimulation of this enzyme, presented in Tables VIII (C and D), represent the means of individual values obtained by Eadie analyses as previously de-scribed (see Materials and Methods). _ 52 _ TABLE VIII (A) A c t i v i t y of Erythrocyte Membrane Mg -dependent (Basal) p-Nitrophenyl Phosphatase (units: micromoles p-nitrophenol/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 5 .120 .112 .019 .008 .103-.140 -Children with DMD 10 .092 .096 .020 .006 .055-.118 Sig n i f i c a n t Normal Female Adults 9 .098 .095 .026 .009 .057-.139 -Definite and Probable DMD Carriers 6 .106 .104 .034 .014 .069-.164 N.S. Possible Carriers Mothers of DMD Patients 5 .087 .090 .018 .008 .068-.104 N.S. Possible Carriers: Sisters of DMD 8 .092 .088 .020 .007 .059-.120 N.S. Patients -53 -TABLE VIII (B) A c t i v i t y of Erythrocyte Membrane Mg -dependent, K -stimulated p-Nitrophenyl Phosphatase (units: micromoles p-nitrophenol/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 5 .211 .202 .028 .013 .181-.248 -Children with DMD 10 .164 .169 .039 .012 .089-.214 Sign i f i c a n t Normal Female Adults 9 .166 .156 .043 .014 .100-.234 -Definite and Probable DMD Carriers 6 .170 .165 .034 .014 .136-.226 N.S. Possible Carriers Mothers of DMD Patients 5 .172 .173 .045 .020 .113-.217 N.S. Possible Carriers: S i s t e r s of DMD 8 .160 .151 .041 .015 .102-.220 N.S. Patients - 54 -FIGURE 1: Concentration Dependence of K +-stimulation of Erythrocyte Membrane Mg2+-dependent p-Nitrophenyl Phosphatase in Normal and DMD Erythrocytes. Inset Eadie Plot Representation of the Same Data. - 55 -\ z iu .IO • s • a a > B \ » • >v \ » • • CONTROL • D M D •OB k J .4 V B i e x i o " 3 V NITROPHEIMQ [K+] V NITROPHEIMQ , • 8 • a B ^ ^ ^ " ^ • • • (0 Ui J • .• a • o e In B IO BO a o KCI ( mM ) • - 56 -Table VIII (C) reveals that no significant differences are detect-able between the c l i n i c a l and control preparations with respect to the maximal velocity of K+-NPPase activity. The suspicion that the definite/probable carrier group i s heterogeneous, composed of two sub-populations differing with respect to this parameter, i s raised by the marked difference between the two tabulated observations (0.109 and 0.195 micromoles p-nitrophenol/hour/milligram membrane protein). Further experimentation w i l l be required to assess this possibility. /Although not shown in Table VIII (C), the V for K+-NPPase of 3 max erythrocyte membrane material was found to be elevated in three limb-girdle subjects: one affected child and two carrier adults (one male and one female, both of whom were unrelated). Therefore, kinetic analysis of the ^-stimula-tion of red c e l l NPPase may prove to be a useful tool in the characterization of erythrocytes in limb-girdle muscular dystrophy. Of special interest are the kinetic data summarized in Table VIII (D) concerning the K for K + of erythrocyte membrane K+-NPPase. While tendering the now-familiar reservations regarding data-poor groups, one also observes one carrier group which deviates very significantly from normality with respect to K c for K+: possible carriers who are sisters of DMD patients. The proba-b i l i t y i s less than 0.01 that this particular c l i n i c a l population and the con-t r o l population are identical with respect to the K 5 parameter and that the observed differences in group sample means a rise merely by chance sampling from population extremes. 2. Adenosine-5'-Triphosphatases (ATPases) There is much evidence in the literature that in muscular dystrophy -57 -TABLE VIII (C) K +-stimulated Component of Erythrocyte Membrane p-Nitrophenyl Phosphatase: V m a x (units: micromoles p-nitrophenol/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 5 .096 .103 .026 .012 .054-.122 -Children with DMD 6 .091 .086 .020 .008 .076-.128 N.S. Normal Female Adults 6 .096 .098 .033 .013 .054-.134 -Definite and Probable DMD Carriers 2 - - - - .109 .195 Possible Carriers Mothers of DMD Patients 2 - - - - .051 .144 ? Possible Carriers: S i s t e r s of DMD 8 .083 .079 .027 .009 .046-.133 N.S. Patients - 58 -TABLE VIII (D) K -stimulated Component of Erythrocyte Membrane p-Nitrophenyl Phosphatase: K _ for K (units: millimolar) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 5 4.51 4.52 0.69 0.31 3.59-5.47 -Children with DMD 6 5.90 6.36 1.67 0.68 3.53-7.50 N.S. Normal Female Adults 6 5.09 5.13 1.39 0.57 3.61-7.03 -Definite and Probable DMD Carriers 2 - - - - 5.88 6.74 Possible Carriers Mothers of DMD Patients 2 - - - - 4.33 4.64 ? Possible Carriers: Sisters of DMD 8 3.40 3.49 0.50 0.18 2.46-4.18 Significant (p<0.01) Patients - 59 -alterations occur i n plasma membrane Na +, K+-ATPase from a variety of tissues. Abnormal a c t i v i t i e s of t h i s enzyme have been reported i n skeletal muscle sar-colemma of dystrophic mice [11], hamsters [14, 93] and chickens [94], as well as i n DMD muscle biopsy material [9]. The a c t i v i t y of t h i s enzyme i s also altered i n hepatocyte membrane preparations from dystrophic avian l i v e r [94]. That erythrocyte membrane le v e l s of Na +, K+-ATPase a c t i v i t y may be aberrant i n DMD i s suggested by Sha'afi et a l . , who determined that red c e l l s obtained from affected children and female c a r r i e r s show increased active i n f l u x of potassium [56]. Tables IX (A and B) summarize respectively the c l i n i c a l and control 2+ 2+ + + data for the basal (Mg -dependent) and t o t a l (Mg -dependent, Na , K -stimulated) ATPase a c t i v i t i e s found i n erythrocyte membranes. Examination of these two tables reveals no s i g n i f i c a n t differences between DMD patients and normal children with respect to these enzyme a c t i v i t i e s . However, the possi-ble c a r r i e r s who are DMD s i s t e r s do show a s i g n i f i c a n t decrease i n erythrocyte t o t a l Na +, K+-ATPase a c t i v i t y . This finding and the single high value for the definite/probable DMD c a r r i e r group i n Table IX (B) suggest that red c e l l mem-brane ATPase function should receive more detailed study i n the future. With larger sample s i z e s , t h i s parameter may even prove to be s i g n i f i c a n t l y d i f f e r -ent from normal i n DMD patient material. 2+ The important role of Ca -stimulated ATPase a c t i v i t y i n s k e l e t a l muscle function i s well known. Therefore, recent reports of abnormal a c t i v i -t i e s of t h i s enzyme i n s k e l e t a l muscle organelles i n muscular dystrophy are worthy of note: for example, t h i s enzyme a c t i v i t y i s said to be elevated i n sk e l e t a l muscle sarcolemma procured from myopathic hamster [14], dystrophic -60 -TABLE IX (A) A c t i v i t y of Erythrocyte Membrane Mg -dependent (Basal) Adenosine Triphosphatase (units: micromoles inorganic phosphate/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 5 .170 .178 .028 .013 .125-.196 -Children with DMD 3 .182 .172 - -.165 .172 .210 N.S. Normal Female Adults 6 .197 .199 .035 .014 .137-.242 -Definite and Probable DMD Carriers 2 - - - - .210 .232 Possible Carriers Mothers of DMD Patients 1 - - - - .172 ? Possible Carriers: S i s t e r s of DMD Patients 6 .159 .162 .030 .012 .121-.191 N.S. _61 _ TABLE IX (B) 2+ A c t i v i t y of Erythrocyte Membrane Mg -dependent, Na +, K +-stimulated Adenosine Triphosphatase (units: micromoles inorganic phosphate/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 l e v e l Age-matched Normal Children 5 .415 .417 .067 .030 .340-.504 -Children with DMD 3 .343 .323 - -.307 .323 .399 N.S. Normal Female Adults 6 .436 .435 .075 .030 .331-.559 -Definite and Probable DMD Carriers 1 - - - - .621 Possible Carriers Mothers Of DMD Patients 2 - - - - .393 .424 ? Possible Carriers: S i s t e r s of DMD Patients 5 .279 .240 .090 .040 .224-.439 Sign i f i c a n t - 62 -mouse [10] and DMD patient [9] biopsy specimens, while the reverse alteration has been reported for avian muscular dystrophy [94]. Sarcoplasmic reticular 2+ (SR) ac t i v i t i e s of Ca -stimulated ATPase have been reported to be depressed in dystrophic hamster [14], and SR calcium-uptake has been observed to be substantially lower in skeletal muscle of dystrophic chickens than in that of normal controls [15]. Furthermore, the density of 90 angstrom membrane particles in freeze-fractured SR, believed to be ATPase protein, i s s i g n i f i -cantly reduced in avian dystrophy [15]. 2+ Red blood c e l l s also have Ca -ATPase activity present in their plasma membranes [95]. In fact, a number of reports have documented the existence of not one, but two such a c t i v i t i e s , each having different kinetic characteristics and perhaps different locations and/or functional roles in the erythrocyte membrane. These act i v i t i e s are often referred to as "high"-and "low"-affinity Ca -ATPases because of their differing requirements for 2+ calcium [76, 96, 97, 98, 99]. Ca -ATPase activity in the red c e l l membrane i s believed to be associated with an active calcium-extruding mechanism, ener-gized by ATP and serving to maintain low concentrations of calcium in the intact c e l l [98, 100]. There i s some controversy, however, as to the relative roles of the "high"- and "low"-affinity components of the enzyme in active Ca -transport [76, 101]. Experiments with erythrocytes using a divalent cation ionophore to raise intracellular levels of calcium have shown that such increases are associated with morphological alterations in the c e l l s [102, 103]. A defect in calcium extrusion, with resulting accumulation of intracellular calcium, might explain the morphological changes reported in erythrocytes of patients with DMD [52, 53, 54]. The properties of erythrocyte membrane Ca -ATPases were therefore examined in both DMD patients and their female relatives. The data in Figure 2 illustrate the biphasic nature of erythrocyte membrane Ca -ATPase activity in both DMD patients and controls and i s seen even more clearly when data are expressed in the form of Eadie plots (see 2+ Figure 3), from which the kinetic parameters (K _ for Ca and V__„) could • D max be calculated. The results for a l l groups examined are presented in Tables IX (C-F). It i s apparent from Table IX (C and D) that the three DMD patients on-examined with respect to their erythrocyte membrane high-affinity Ca -ATPase activity showed significantly lower maximal rates of ATP hydrolysis than con-trols, but apparently normal K j- values for calcium. This was not the case with the DMD female relatives examined, although the single subject in the possible carriers who are mothers of DMD patients group exhibited an abnorm-al l y low V but a high K K parameter. These results are far from conclu-sive, but they strongly suggest that this enzymatic activity warrants more 2+ intensive investigation in DMD. Although the low-affinity Ca -ATPase activity [Tables IX (E and F)] does not appear to depart significantly from normal, the data from possible carriers, both mothers and sisters of patients, does indicate the need for further study of this ATPase. 3. Acetylcholinesterase (AChE) Investigations into the synthesis and metabolism of acetylcholine (ACh) in tissues of muscular dystrophy animal models have revealed this to be another area of abnormal enzymatic function. For example, Trabucchi et a l . - 64 -FIGURE 2: Concentration Dependence of the Stimulation of Erythrocyte Membrane Mg2+-dependent ATPase by Calcium. - 65 -- 66 -FIGURE 3: Eadie Plot Analysis of the Data in Figure 2. V ( J J MOLES Pi/hr/mg PROTEIN ) p ra b _68 _ TABLE IX (C) 2+ High A f f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphosphatase: ^ m a x (units: micromoles inorganic phosphate/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Significance Individual at 0.05 l e v e l Values (where "n" i s small) Age-matched Normal Children 5 1.05 1.06 0.17 0.08 0.81-1.28 -Children with DMD 3 0.73 0.76 - -0.65 0.76 0.79 Sign i f i c a n t Normal Female Adults 6 0.92 0.89 0.24 0.10 0.58-1.25 -Definite and Probable DMD Carriers 2 - - - - 0.77 1.21 •p Possible Carriers Mothers 1 0.46 ? Of DMD Patients Possible Carriers: Sisters 6 0.80 0.82 0.22 0.09 0.48- N.S. of DMD 1.01 Patients - 69 -TABLE IX (D) 2+ High A f f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphosphatase: K for Ca . 5 2+ (Units: micromolar free Ca ) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Si g n i f i c a n c e at 0.05 l e v e l Age-matched Normal Children 5 .328 .364 .077 .035 .207-.393 -Children with DMD 3 .261 .230 - -.205 .230 .350 N.S. Normal Female Adults 6 .222 .212 .145 .059 .032-.412 -D e f i n i t e and Probable DMD C a r r i e r s 2 - - - - .207 .211 ? Possible C a r r i e r s Mothers of DMD Patients 1 - - - - .574 •? Possible C a r r i e r s : S i s t e r s of DMD 6 .307 .283 .140 .057 .133-.537 N.S. Patients TABLE IX (E) 2+ Low Af f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphosphatase: V x (units: micromoles inorganic phosphate/ hour/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 5 1.35 1.29 0.19 0.09 1.12-1.63 -Children with DMD 3 1.15 - - -0.96 1.14 1.36 N.S. Normal Female Adults 6 1.15 1.15 0.25 0.10 0.80-1.48 -Definite and Probable DMD Carriers 2 - - - - 1.09 1.53 Possible Carriers Mothers of DMD Patients 1 - - - - 0.68 Possible Carriers: Sisters of DMD 6 0.78 0.70 0.38 0.16 0.36-1.24 N.S. (significant at p «=0.10) Patients -71 _ TABLE IX (F) 2+ Low Af f i n i t y Ca -stimulated Component of Erythrocyte Membrane Adenosine Triphosphatase: K ^ for C a 2 + 2+ (units: micromolar free Ca ) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 5 6.74 5.98 2.08 0.93 5.11-10.31 -Children with DMD 3 7.21 8.23 - -4.77 8.23 8.62 N.S. Normal Female Adults 6 5.35 3.99 3.61 1.47 2.66-11.91 -Definite and Probable DMD Carriers 2 - - - - 4.80 7.52 ? Possible Carriers Mothers Of DMD Patients 1 - - - - 12.10 •? Possible Carriers: Sisters of DMD 6 5.41 5.16 1.33 0.54 3.63-7.33 N.S. Patients - 72 -have found some evidence for a possible defect in the synthesis of ACh in central and peripheral cholinergic neurons of muscular dystrophic mice [34]. Other investigators have detected alterations in ACh handling in muscular dystrophy: AChE has been found to exhibit embryonic characteristics in avian myopathy [104]; the activity of AChE has been shown to be depressed in sar-colemmal preparations from dystrophic mouse skeletal muscle [10], and a three-fold increase has been reported to occur in the K ^ for substrate for red c e l l AChE in murine dystrophy [105]. When the kinetics of erythrocyte AChE were investigated in DMD patients and carriers u t i l i z i n g Eadie analysis (see Materials and Methods and Figure 4), no significant departures from normality were observed in either K K for acetylthiocholine (ATCh) or V „ . These results are summarized in Tables X (A and B). Butterfield et a l . have conducted spin label studies of erythrocytes obtained from patients suffering from myotonic muscular dystrophy; their find-ings suggest that the red c e l l membrane matrix in this disorder i s less polar and more fl u i d than the matrix of normal erythrocytes [106, 107]. Such pola-r i t y and f l u i d i t y alterations in myotonic red ce l l s may reflect either gross differences in the membrane composition or more subtle abnormalities in the molecular arrangement of membrane l i p i d and protein constituents. Butterfield et a l . have interpreted their erythrocyte spin label data as evidence that myotonic muscular dystrophy i s a generalized membrane disorder, affecting even red blood c e l l s [106, 107]. Similar arguments might also be advanced in the case of DMD, i f erythrocyte membranes could be shown to display such matrix modifications. - 73 -FIGURE 4: Concentration Dependence of Acetylthiocholine Hydrolysis by Erythrocyte Membrane Acetylcholinesterase. Inset Eadie Plot Analysis of the Same Data. - 74 --75 -TABLE X (A) Substrate Kinetics of Erythrocyte Membrane Acetylcholinesterase: V x (units: micromoles thiocholine liberated/ minute/ milligram membrane protein) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 5 1.34 1.33 0.12 0.05 1.22-1.53 -Children with DMD 10 1.44 1.43 0.22 0.07 1.04-1.82 N.S. Normal Female Adults 9 1.48 1.49 0.20 0.07 1.16-1.81 -Definite and Probable DMD Carriers 6 1.56 1.56 0.13 0.05 1.39-1.74 N.S. Possible Carriers Mothers of DMD Patients 5 1.42 1.32 0.36 0.16 1.05-1.88 N.S. Possible Carriers: Sisters of DMD 8 1.24 1.28 0.29 0.10 0.80-1.72 N.S. Patients -76 -TABLE X (B) Substrate Kinetics of Erythrocyte Membrane Acetylcholinesterase: K 5 for Acetylthiocholine (units: micromolar) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 5 93 93 7 3 84-104 -Children with DMD 10 97 93 10 3 88-112 N.S. Normal Female Adults 9 99 98 13 4 84-129 -Definite and Probable DMD Carriers 6 101 100 12 5 82-116 N.S. Possible Carriers Mothers of DMD Patients 5 105 106 22 10 85-139 N.S. Possible Carriers: Sisters of DMD Patients 8 96 99 11 4 82-111 N.S. - 77 -Fluidity studies were therefore undertaken on DMD c l i n i c a l blood specimens using the approach described by Bloj et a l . [77]. These investiga-tions demonstrated, using rats fed with different fat-supplemented diets, that the degree of NaF-induced allosteric inhibition of erythrocyte AChE (as assessed by the magnitude of the slope of the H i l l plot) i s indicative of the state of membrane f l u i d i t y . Bloj et a l . have found strong correlation between the estimates of membrane f l u i d i t y made by this biochemical method and those made by physical techniques, such as electron spin resonance spectroscopy [77]. Figure 5 shows the effect of increasing concentrations of NaF on the maximal rate of hydrolysis of ATCh by DMD and control erythrocyte membranes, with an inset of the same data in the form of H i l l plots. The K 5 for F~ inhibition of AChE and the H i l l coefficient (the slope of the H i l l plot) are summarized for the various c l i n i c a l and control groups in Tables XI (A and B). It w i l l be noted that no significant differences have been observed in any of the groups studied. Gel Electrophoresis Margareth et a l . were able to demonstrate that the gel electrophore-t i c pattern of microsomal membrane proteins obtained from rat skeletal muscle underwent marked alterations following muscle denervation [108]. Boegman's work on dystrophic mouse has revealed appreciable modification of the protein fraction of skeletal muscle sarcolemma; the affected murine proteins were found to differ from control proteins in quantity and gel migration rate [10]. However, when the gel electrophoretic patterns of erythrocyte membrane pro-teins obtained from DMD patients and carriers were studied by Roses et a l . , - 78 -FIGURE 5: Fluoride Inhibition of Erythrocyte Membrane Acetylcholinesterase in Normal and DMD Erythrocytes. Inset H i l l Plot Analysis of the Same Data. - 79 -IMaF ( m M ) - 8 0 -TABLE XI (A) Flouride Inhibition of Erythrocyte Membrane Acetylcholinesterase: H i l l Coefficient Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 5 0.94 0.90 0.12 0.05 0.84-1.14 -Children with DMD 6 0.97 0.97 0.09 0.04 0.86-1.07 N.S. Normal Female Adults 6 0.96 0.91 0.20 0.08 0.76-1.36 -Definite and Probable DMD Carriers 2 - - - - 0.86 1.16 Possible Carriers Mothers Of DMD Patients 2 - - - - 0.92 1.09 ? Possible Carriers: Sisters of DMD 8 0.95 0.96 0.22 0.08 0.60-1.28 N.S. Patients -81 -TABLE XI (B) Flouride Inhibition of Erythrocyte Membrane Acetylcholinesterase: K ,- for F (units: millimolar) Group n Mean Median SD SE Range or Individual Values (where "n" i s small) Significance at 0.05 level Age-matched Normal Children 5 4.07 4.02 0.93 0.41 2.98-5.38 -Children with DMD 6 4.16 4.20 0.43 0.18 3.67-4.73 N.S. Normal Female Adults 6 4.54 4.50 0.64 0.30 3.82-5.43 -Definite and Probable DMD Carriers 2 - - - - 3.98 4.05 Possible Carriers Mothers of DMD Patients 2 - - - - 3.37 4.22 •? Possible Carriers: Sisters of DMD Patients 8 4.06 4.19 1.01 0.36 2.45-5.48 N.S. -82 -no reproducible departures from normality were observed [57]. Despite this negative report by Roses et a l . , gel electrophoretic analysis of red c e l l membrane proteins was performed upon a small but repre-sentative sample of c l i n i c a l and control blood specimens (see Table XII). The erythrocyte membranes of the six subjects tested were analysed as previously described (see Materials and Methods); the stained gel was optically scanned with a densitometer. No gross differences in the protein fractional migration patterns could be discerned for these six subjects (not shown in Table XII). Table XII provides a comparison of the relative protein contents of several major bands observed when the erythrocyte membrane material of the six subjects under consideration was analysed by gel electrophoresis. The protein content of gel bands I, II, III, V and VI was estimated for each subject by measuring the densitometer scan peak height for each band and expressing this as a percentage of the total height of a l l five protein bands. Examination of these tabulated data w i l l reveal that no gross differences were observed in the quantities of these protein sub-fractions for the c l i n i c a l and control specimens. More subtle differences, i f such exist, would require extensive investigation to ascertain the limits of normality and the v a r i a b i l i t y of c l i n i c a l material. Erythrocyte Fr a g i l i t y Hereditary spherocytosis and hereditary stomatocytosis are hemato-logical disorders both of which are characterized by increased f r a g i l i t y of erythrocytes to hypotonic stress [109, 110]. Furthermore, Kuiper and Llvne have shown that in hereditary spherocytosis there is a marked decrease in -83 -TABLE XII Polyacrylamide Gel Electrophoretic Analysis of Erythrocyte Membrane Proteins: Relative Peak Heights Derived from Densitometry Scan (Each peak height is expressed as the percentage of the total for each individual. Band designation is as described by Fairbanks et al.[78]) Subject Band I Band II Band III Band V Band VI Age-matched Normal 20 30 30 10 10 Child Child with Hereditary 21 32 29 9 10 DMD Child with sporadic 24 29 28 9 10 DMD Normal Female 21 35 23 9 11 Adult Possible Carrier: Mother 23 32 24 9 11 of DMD Patient Possible Carrier: Sister 18 29 31 10 12 of DMD Patient - 84 -the percentage of long chain fatty acid components of red c e l l membrane phos-pholipids [109]. Mentzer et a l . have observed an abnormally high resting membrane permeability to potassium and sodium in hereditary stomatocytosis [111]. Similar changes with respect to potassium fluxes and l i p i d composition have been reported for DMD erythrocytes [51, 55]. Also DMD, like hereditary spherocytosis and stomatocytosis, i s a disorder associated with erythrocyte morphological alterations [52, 53, 54]. Given these findings and despite an apparently cursory study by Miale et a l . which produced only negative findings [112], an assessment of erythrocyte osmotic f r a g i l i t y was deemed appropriate. Such a study was, in fact, considered essential, because hitherto a l l of the experimental methods utilized in these investigations involved erythrocyte membranes (processed to eliminate v i r t u a l l y a l l erythrocyte cytoplasmic compon-ents) rather than intact erythrocytes. It i s known that the properties of isolated leaky erythrocyte membranes [113, 114, 115, 116] or even resealed membrane preparations [117] may di f f e r quite substantially from those of the membrane in the intact c e l l . In this regard, Tanaka and Ohnishi using spin label techniques showed that the asymmetric f l u i d i t y pattern manifested by the membrane of intact red ce l l s i s lost upon hemolysis [118]. Thus the exclu-sive use of erythrocyte membrane material in the study of DMD might result in the failure to detect the more subtle molecular features which characterize this disorder. Figure 6 illustrates the pooled results of the osmotic f r a g i l i t y study of c l i n i c a l and control erythrocytes. The top panel clearly reveals that the red ce l l s of the three DMD patients tested exhibit a greater sus-c e p t i b i l i t y to hemolysis when exposed to hypotonic stress than i s seen in - 85 -FIGURE 6: Osmotic Fra g i l i t y of Intact Erythrocytes. Top Panel: DMD and Age-matched Normals. Bottom Panel: Normal Females and Definite Female Carriers of DMD. - 86 -* CONTROL. • DMD • CONTROL • CARRIERS SO 40 60 80 NaCl (mM) - 87 -the erythrocytes of the four control children tested. Standard error bars are included for the NaCl range of 0.05-0.07 M to illustrate that no overlap exists between these two groups over this range. The lower panel of Figure 6 reveals no significant differences in the osmotic f r a g i l i t y of red cells of definite carriers of DMD (n = 2) and normal female adult controls (n = 3). Standard error bars for these two data pools exhibit considerable overlap and have been omitted from the diagram for purposes of c l a r i t y . - 88 -DISCUSSION The foregoing experimental results comprise a screening study wherein erythrocytes obtained from patients with DMD and their female rela-tives were analysed by a wide variety of analytical techniques to evaluate red c e l l structural and functional characteristics. S t a t i s t i c a l methods were employed in the comparison of erythrocyte parameters in both c l i n i c a l and con-t r o l blood samples. These comparisons have revealed promising areas for future in-depth investigation. It i s hoped that further studies of DMD erythrocytes w i l l contribute to the elucidation of the pathogenic lesion underlying this disorder and w i l l provide unambiguous red c e l l markers of the affected and carrier states in DMD. As indicated in the Results section, a number of red c e l l abnormali-ties were found to be present in erythrocytes from patients with DMD or from their female carrier relatives: (a) the content of phosphatidyl serine in the erythrocyte membrane l i p i d fraction was found to be elevated by 50%; (b) evidence for the poss i b i l i t y of two distinct sphingomyelin populations in erythrocyte membrane li p i d s was suggested by their different fractional migration characteristics upon thin layer chromatographic analysis; (c) mem-2+ brane basal (Mg -dependent) p-NPPase activity was found to be significantly lowered; (d) the apparent dissociation constant (K 5) for potassium stimula-tion of p-NPPase was also observed to be depressed; (e) the activity of 2+ + + erythrocyte membrane Mg -dependent, Na , K -stimulated ATPase was likewise lowered; (f) a decrease was also seen in the maximal velocity of ATP hydroly-2+ 2+ sis by the high-affinity component of erythrocyte Mg -dependent, Ca -stimulated ATPase; (g) the possiblity of similar lowering of the V m a x of the - 89 -Caz_r-ATPase low-affinity component was also noted (p<0.10); and f i n a l l y , (h) an increase in the f r a g i l i t y of intact erythrocytes to osmotic stress was observed. No apparent abnormalities were evident in the erythrocyte mem-brane contents of s i a l i c acid, cholesterol or total phospholipid. Cl i n i c a l red c e l l membranes likewise showed normal primary amino and sulfhydryl group surface and latent t i t e r s . The TLC fractional mobilities and relative quanti-ties of most membrane phospholipid subtractions were normal in DMD patients and their female relatives, as were the kinetic characteristics of erythrocyte membrane AChE and i t s inhibition by NaF. Finally, in a small but representa-tive number of c l i n i c a l blood samples tested, no gross differences were found in the relative quantities and fractional migration properties of red c e l l membrane proteins subjected to gel electrophoretic analysis. Clearly, erythro-cyte membrane phospholipids, the enzyme acti v i t i e s associated with active sodium and potassium transport, as well as those believed to be related to active calcium extrusion, and the osmotic integrity of intact red c e l l s appear to be promising areas for further in-depth investigation of DMD. The results of several lines of investigation [51, 52, 53, 54, 55, 56, 57, 58, 67], as well as those summarized above, suggest that erythrocytes in DMD are abnormal in several respects. These findings, as well as those for other non-muscular DMD tissues (see Introduction), strongly argue in favour of the hypothesis that the defect in DMD i s a generalized one involv-ing a number of membrane systems. It may well be that the essential patho-logical characteristics of this disease are demonstrable in red blood c e l l membranes, and unequivocal markers of DMD and the carrier state may likewise be revealed by erythrocyte research. - 90 -Study of red c e l l s may also provide answers regarding the homo-geneity of DMD and ca r r i e r groups. There have been certain indications that DMD i s not a single pathological e n t i t y , but rather a c o l l e c t i o n of diseases of similar symptomatology, but d i f f e r e n t etiology: For example, there are reports that apparent sporadic and hereditary DMD cases may be distinguished by the extent of palmitate oxidation occuring i n their s k e l e t a l muscle mito-chondria [119] or by the l e v e l s of diphenoloxydase a c t i v i t y found i n their p l a t e l e t s [120]. However, the work of Roses and Appel [67], discussed e a r l i e r (see Introduction), argues against the occurrence of biochemically d i s t i n c t subclasses of DMD c a r r i e r s , since most subjects c l a s s i f i e d as "possible c a r r i e r s " [1] revealed their abnormal status i n exactly the same fashion as did known and probable c a r r i e r s , the erythrocytes of subjects from a l l three groups possessing apparently i d e n t i c a l morphological and functional alterations [67]. The suggestion by Roses and Appel that many possible c a r r i e r s are i n fact true c a r r i e r s of the DMD gene(s), a l b e i t " s i l e n t c a r r i e r s " [65], i s further strengthened by the findings of the present investigation. The possible c a r r i e r s who are s i s t e r s of DMD patients exhibited certain composi-ti o n a l [see Table VII (A)] and functional [see Tables VIII (D) and IX (B)] abnormalities. However, these p a r t i c u l a r alterations were not observed i n the DMD patients or i n other c a r r i e r groups. An e n t i r e l y d i f f e r e n t , though probably related, group of changes were to be found i n DMD erythrocyte mem-branes [see the "twin sphingomyelin-like" TLC spot discussion i n Results, also Tables VIII (A) and IX (C) and Figure 6]. These part i c u l a r alterations likewise were not found i n any of the DMD " c a r r i e r " groups. I t should be noted that any female r e l a t i v e of a c h i l d a f f l i c t e d by DMD may, on the bases - 91 -described i n the Introduction, be designated a " c a r r i e r " ; thus one would expect to f i n d a certain proportion of the possible c a r r i e r group to be perf e c t l y normal. The above observations may well argue for the existence of pathologically d i s t i n c t sub-varieties of the affected and c a r r i e r states i n DMD. However, the data on these altered parameters, p a r t i c u l a r l y i n the definite/probable c a r r i e r s and possible c a r r i e r mothers of DMD patients, are as yet i n s u f f i c i e n t to adequately assess the homogeneity of t h i s disease i n affected children and their female r e l a t i v e s . I t was previously stated (see Results) that the present data could be considered s u f f i c i e n t to rule out the p o s s i b i l i t y that the erythrocytes of definite/probable c a r r i e r s and possible c a r r i e r mothers are abnormal with res-pect to the properties examined here. In nearly every case these two data-poor groups show at least one observation which f a l l s within the normal range of values [see Tables VI (A-D), VII (A-D), VIII (C and D), IX (A-F) and XI (A and B)]. The only exceptions to t h i s rule are seen i n certain single 2+ observations of Ca -ATPases [see Tables IX (B-F)]. These two c a r r i e r groups may well be normal with respect to parameters where data overlap the range of normal values. This "overlap argument" i s only v a l i d for small sample sizes and may be stated as follows: Assume for the moment that the population from which a given data-poor group of individuals i s taken does d i f f e r sub-s t a n t i a l l y from normal with respect to a given parameter; then the p r o b a b i l i t y i s exceedingly small (p«0.05) that random sampling of t h i s abnormal popula-t i o n would by chance alone produce a sample of size n = 1 or n = 2 where one or both observations f a l l within the range of normal values as estimated by a r e l a t i v e l y small sampling of the normal population, n = 6. Thus, i f such an - 92 -overlap of the normal range does occur, one could argue that i t i s highly l i k e l y that the data-poor group i s normal with respect to the parameter under consideration. Underlying t h i s argument are two assumptions: the sampling groups are assumed to r e f l e c t homogeneous populations, and untoward experi-mental errors are assumed to have negligible influence on parameter evaluation. In Table VIII (C), the two divergent values of K+-NPPase V for the max definite/probable c a r r i e r group could suggest that perhaps one or both of these conditions are not f u l f i l l e d i n every case. Clearly, further study of these parameters i s indicated i f the normality of data-poor ca r r i e r groups i s to be assessed. I f abnormalities are found, then the homogeneity of the various c a r r i e r groups and of the DMD patient subclasses (e.g., sporadic and hereditary) may be evaluated. Having i d e n t i f i e d several possible alterations i n erythrocytes of DMD patients and c a r r i e r s , one must now consider how these abnormalities might be related to the o v e r a l l disease process. Since the present findings require extensive v e r i f i c a t i o n and elucidation, any such discussion must involve a good deal of speculation. Therefore in the discussion which follows, certain assumptions have been made for the sake of s i m p l i c i t y . I t i s assumed that (a) a l l cases of DMD r e f l e c t the same underlying pathology ( i . e . , DMD i s a homogeneous disorder that i s not composed of biochemically d i s t i n c t sub-v a r i e t i e s ) and (b) the abnormalities exhibited by known and s i l e n t c a r r i e r s of DMD represent an attenuated form of the pathology underlying the disorder of a f f l i c t e d children. This l a t t e r assumption i s made, because female c a r r i e r s , apparently even s i l e n t ones [67], often exhibit some degree of demonstrable myopathy, although rar e l y are they a f f l i c t e d to the degree - 93 -observed in DMD patients [1]. The discussion which follows attempts to rationalize the various experimental data from this work and from the investigations of others in terms of recognized mechanisms of cellular injury. Numerous parallels are drawn between the alterations observed in DMD red cells and those exhibited by erythrocytes in various other dis-orders and experimental models. Many of the changes described for erythrocytes in DMD (e.g., alterations in membrane ionic permeability and cellular ionic homeostasis [55, 56, 121], transformation of a significant proportion of c e l l s into echinocytes [52, 53, 54, 67], modification of membrane phospholipid subtrac-tions [see sphingomyelin commentary in Results, also Table VII (A)] [51], enhanced osmotic f r a g i l i t y [see Figure 6] and reduction of membrane deform-a b i l i t y [121]) are similar to those observed when red cells are exposed to oxidant injury with subsequent peroxidation of plasma membrane l i p i d s . Oxidant injury may occur in erythrocyte aging and may trigger removal of the red c e l l from the circulation and i t s destruction in the spleen [122]. Aged erythro-cytes exhibit a diminution in the in vivo lipoperoxidation protective system (i.e., glutathione, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase), resulting in peroxidative injury to membrane li p i d s [122], as well as abnormally high levels of potassium conductance, decreased intracellular potassium, increased intracellular sodium, decreased membrane deformability, enhanced osmotic f r a g i l i t y and hemolysis [122, 123, 124]. Lipoperoxidation i s known to be the result of free radical attack on unsatu-rated bonds found within the fatty acid residues of membrane l i p i d s ; once - 94 -begun the process is autocatalytic [124]. Oxidant injury resulting from the deficiency of natural antioxidant vitamin E is believed to underlie the pro-duction of a number of nutritional muscular dystrophies in animals [125]. It is of interest that the echinocyte morphology observed in human DMD erythro-cytes [52, 53, 54, 67] is also to be found in red cells of rats rendered dystrophic by dietary deficiency of vitamin E [126]. Furthermore, the ele-vated potassium conductances seen in human DMD skeletal muscle and red cells [127, 55] also occur in brain and liver mitochondria of vitamin E-deficient rats [55]. That oxidant injury and lipoperoxidation may underlie or at least contribute to many of the abnormalities observed in DMD erythrocytes is suggested by the findings of Kunze et al . , who detected alterations in the fatty acid patterns of phosphatidyl ethanolamine and sphingomyelin [51]. These investigators found that the content of saturated fatty acid residues in these phospholipid subtractions were increased at the expense of unsatur-ated residues. It is known that moderate oxidant injury triggers a pre-lytic redistribution of membrane phospholipid fatty acid residues, the rate of acyla-tion of both saturated and unsaturated fatty acids into these phospholipids being accelerated; however, since lipoperoxidative attack on unsaturated residues is ongoing, these fatty acids continue to be rapidly lost, allowing the accumulation of saturated residues [124]. This increase in saturated fatty acid content in membrane lipids may be responsible for the reduction in deformability observed in erythrocytes subjected to lipoperoxidation. Furthermore, the lysophosphatides produced during membrane lipid oxidant injury may well be involved in enhancing osmotic fragility and precipitating hemolysis in red cells, since even low concentrations of these detergent-like molecules (2 x 10 M) are known to be capable of lysing erythrocytes [128]. Conversion from the discocytic to the echinocytic morphology has also been observed when normal red ce l l s are incubated with lysophosphatides in vitro [52] . In addition to producing l i p i d damage, oxidant injury i s also capable of destroying the sulfhydryl residues of erythrocyte membrane proteins [124]. It is well known that the integrity of these membrane sulfhydryl groups i s required for the maintenance of normal membrane ionic permeability [129]; therefore, the destruction of these residues could have grave consequences on erythrocyte homeostasis. Experiments employing treatment of erythrocytes with sulfydryl-modifying agents have revealed that c e l l s so treated exhibit mem-branes leaky to sodium and potassium [129], decreased deformability and enhanc-ed osmotic f r a g i l i t y [130]. Contrary to the findings of Qmaye and Tappel which suggest that lipoperoxidation injury i s associated with genetic muscular dystrophy in some species of chickens and mice [131], the DTNB labelling studies of erythro-cytes of DMD patients and their female "carrier" relatives failed to show any evidence of loss of membrane sulfhydryl groups [see Tables IV (A and B)]. However, i t might be suggested that the twin "sphingomyelin-like" spots observ-ed upon TLC analysis of some c l i n i c a l blood samples may reflect the occurrence of lipoperoxidative injury in the membranes of these c e l l s , the second spot arising from newly acylated phospholipids rich in saturated fatty acid residues. If lipoperoxidation is occurring in DMD erythrocytes, i t seems unlikely that i t represents the mechanism of primary importance in the pathogenesis of this disease, since these twin "sphingomyelin-like" spots were only observed in half - 96 -of a l l patients analysed. Furthermore, phosphatidyl ethanolamine, which is particularly abundant in poly-unsaturated fatty acids, should have exhibited detectable thin layer chromatographic alterations as a result of re-acylation with saturated fatty acid residues [124], but in fact, no such alterations were seen in any of the clinical samples. These facts might suggest that a more specific mechanism of lipid modification than lipoperoxidation is opera-ting in DMD erythrocytes. Should analysis prove these twin "sphingomyelin-like" TLC spots to be sphingolipids, then one might suspect the occurrence of some abnormality in sphingomyelin synthesis or renewal or perhaps the activation of lipases which selectively attack this phospholipid subtraction. A recent report that short and long-chain triglyceride lipase activities are markedly elevated in skeletal muscle of hereditarily dystrophic mice [132] may have a bearing on this point. Even i f lipoperoxidation does not represent a major cause of lipid modification in DMD erythrocytes, a low level of oxidative stress may be occurring in these cells, compensated for by stimulation of the in vivo l i p -operoxidation protection system [131]. Low levels of oxidant injury might not be discernible as a reduction in membrane sulfhydryl residues [124]; therefore, identification of the occurrence of this subtle, albeit injurious process within DMD red cells might not be possible using techniques which quantify membrane sulfhydryl groups (e.g., the DTNB labelling technique used in the present investigation). However, analysis of erythrocyte contents of glutathione and the various enzymatic components of the in vivo lipoperoxida-tion protective system could provide a sensitive index of membrane peroxida-tive damage. - 97 -If low level peroxidative damage i s occurring within DMD erythro-cytes, i t conceivably could continue to the enhancement of osmotic f r a g i l i t y observed in these c e l l s (see Figure 6). If this i s so, and despite the fact that the erythrocytes of DMD carriers failed to exhibit any abnormality in their response to osmotic stress (see Figure 6), examination of the f r a g i l i t y characteristics of DMD carrier red ce l l s in the presence of thyroxine, a hormone reported to potentiate the hemolytic effects of peroxides on erythro-cytes [122], might unmask the presence of an osmotic defect similar to that seen in the ce l l s of DMD patients and might afford a convenient marker of the carrier state. Another disorder of erythrocytes which produces many alterations similar to those observed in DMD red c e l l s i s hereditary spherocytosis (HS). Consideration of this hematological disorder may shed some light upon the involvement of erythrocytes in DMD pathology. HS i s a genetically transmitted disorder in which erythrocytes exhibit alterations of cellular morphology and membrane l i p i d composition, as well as showing reduced deformability, enhanced osmotic f r a g i l i t y and abnormalities of transmembrane ionic fluxes [109, 110, 129, 133]. The passive permeability of sodium ions in HS has been found to be twice normal [134], but without concomitant membrane leakiness to potassium ions [130]. This high sodium influx i s compensated by an elevated level of active sodium extrusion, energized by abnormally high rates of glycolysis in erythrocytes [135]. DMD erythrocytes, on the other hand, show a different pattern of ionic fluxes; these cells have a predilection to leak potassium (instead of sodium), while maintaining apparently normal intracellu-lar levels of potassium [55, 122]. Normal potassium homeostasis in the face - 98 -of t h i s f i v e - f o l d increase i n conductance [55] may be maintained by a compensa-tory increase i n active potassium i n f l u x , since a s i g n i f i c a n t elevation i n active potassium pumping has been reported to occur i n the erythrocytes of DMD patients and c a r r i e r s ; however, t h i s a l t e r a t i o n i s apparently associated with normal l e v e l s of sodium extrusion [56]. This might suggest a departure from the normal 3:2 sodium-potassium exchange r a t i o i n the sodium-potassium pump of these c e l l s . A stoichiometric a l t e r a t i o n i n the operation of the sodium-potassium exchange pump has been observed by Hull and Roses to occur i n eryth-rocytes obtained from patients suffering from myotonic muscular dystrophy [136]. These investigators have evaluated the exchange r a t i o i n myotonic erythrocytes, finding two sodium ions exchanged for an equal number of potassium ions, and they suggest that t h i s modification may arise from a reduction i n the a f f i n i t y of one of the three binding s i t e s for sodium at the l e v e l of the membrane cation pump [136]. Perhaps the increased active potassium uptake, but normal sodium extrusion, reported i n DMD red c e l l s [56] r e f l e c t s an a l t e r a t i o n , not i n the a f f i n i t y of the pump for sodium as seen i n HS, but rather i n i t s a f f i n i t y for potassium. The membrane sodium-potassium exchange pump i n DMD erythrocytes may have undergone some modification which increases i t s a f f i n i t y for potassium without affecting i t s a f f i n i t y for sodium. Tentative evidence for such a modification might be seen i n the increased a f f i n i t y (decreased K ,.) of membrane p-NPPase for potassium ion observed i n certain c l i n i c a l sub-• 3 j e c t s [see Table V I I I (D)]. I f further investigation bears out t h i s observa-tion i n DMD erythrocytes, then stoichiometric studies of active sodium-potassium exchange i n these c e l l s would be warranted. - 99 -Apparently ionic homeostasis is not precisely maintained in DMD erythrocytes, since a small but significant increase in intracellular sodium is reported to occur in these cells [122]• Secondary to this increase in intracellular sodium, the red cell may imbibe some water, and this may explain the tendency of DMD erythrocytes to lyse in hypo-osmolar media before normal erythrocytes (see Figure 6). The decreased activity of membrane Na +, K +-ATPase noted in the present studies for some clinical subjects [see Table IX (B)] may produce this increase in intracellular sodium levels, but this explanation is not entirely compatible with an alteration in the mechanism of cation pumping just postulated to explain the normal potassium levels found within DMD red cells. Perhaps the observed decreases in enzymatic activities associated with the sodium-potassium pumping mechanism [see Tables VIII (A) and IX (B)] are indicative of the operation of complex homeostatic compensa-tory mechanisms triggered by cation imbalances within the intact erythrocyte. The situation is further complicated by uncertainty as to the extent to which the functional characteristics of the active sodium-potassium transporting mechanism in intact erythrocytes are reflected by the properties of the Na +, K+-stimulated ATPase activity in isolated membranes. One cannot eliminate the possibility, for example, that a decrease in Na +, K+-ATPase activity in isolated membranes may be a reflection of the decreased stability of the enzyme in a pathologically altered membrane under the conditions of membrane isolation. It is interesting, however, that a depression of Na +, K+-ATPase activity has been observed in sarcolemmal membrane preparations derived from DMD skeletal muscle [9] . There is evidence that ionic homeostatic mechanisms are perturbed in DMD skeletal muscle, since abnormally high levels of potassium - 100 -efflux have been reported to occur in these tissues [127]. Furthermore, whole body studies of DMD carriers have revealed a reduction in intracellular potas-sium contents and an elevation in cellular water [92]. Such imbalances might give rise to sublethal injuries in erythrocytes and other non-excitable tissues, but these same defects in ionic homeostasis would be devastating to excitable tissues, perhaps producing many of the myriad pathological changes of DMD muscle and nerve discussed earlier (see Introduction) and ultimately culminating in necrosis [55]. The importance of ionic flux imbalances in the process of muscle c e l l death i s emphasized by the report that treatment of DMD patients with lithium gluconate, purported to be a non-competitive inhibitor of potas-sium translocation in a variety of biological membranes [137], decreases serum CPK activity [138], one of the indices of muscle deterioration in the early stages of this disease [1]. Lithium alone or in combination with other drugs may prove useful in ameliorating the rapid progress of DMD [139]. While alterations in sodium-potassium homeostasis may be invoked to explain deleterious changes which occur in excitable tissues in DMD, as well as some of the functional disturbances observed in DMD and HS erythrocytes (e.g., increased osmotic f r a g i l i t y ) , other abnormalities in the red c e l l s of patients with DMD and HS (e.g., decreased deformability, echinocytic morphology, etc.) are not so readily explained on this basis. However, there i s a good deal of evidence that alterations in cellular membrane li p i d s may represent a more fundamental change in the production of abnormal erythrocytes in DMD and HS, and may underlie skeletal muscle myopathy in DMD. Such a basic modification of membrane integrity could well have profound effects upon the functioning - 101 -of membrane enzymes by al t e r i n g membrane l i p i d - p r o t e i n interactions. For example, Kuiper and Livne have correlated their observation of a reduction i n the quantity of long chain f a t t y acid conjugates of membrane phospholipids i n HS erythrocytes with other features of t h i s hematological disorder (e.g., reduction i n c e l l surface area, increased sodium conductance, enhanced osmotic f r a g i l i t y , etc.) [109]. Despite the difference i n ionic permeability charact-e r i s t i c s between DMD and HS erythrocytes (discussed i n the preceeding para-graphs) , basic modifications i n the phospholipid subfractions of erythrocyte membranes are also observed i n DMD [51] [see Table VII (A) and sphingomyelin commentary i n Results]. These modifications of red c e l l membrane l i p i d s i n DMD may have profound effects on cation permeability, as well as on membrane morphology, f l u i d i t y , deformability and osmotic f r a g i l i t y [124, 128]. Possible mechanisms whereby these changes may occur w i l l be discussed more f u l l y i n the pages which follow. Emphasizing the potential importance of l i p i d modifica-tion i n the underlying pathology i n DMD tissues are the reports of Hughes and Kunze et a l . [5, 51]. These investigators have found s i g n i f i c a n t alterations of l i p i d fractions i n DMD skeletal muscle similar to those seen i n DMD erythro-cytes. However, these findings are somewhat ambiguous since s k e l e t a l muscle necrosis i s usually associated with lipogenesis. For these reasons, i t appears that a thorough biochemical characterization of erythrocyte l i p i d s obtained from DMD patients and their female r e l a t i v e s would be warranted. In addition to u t i l i z i n g TLC analysis of membrane l i p i d s to detect gross abnor-mal i t i e s i n the various phospholipid subfractions, hydrolysis of these sub-fractions followed by gas-liquid chromatography would allow accurate i d e n t i -f i c a t i o n of phospholipid f a t t y acid conjugates i n c l i n i c a l blood specimens. - 102 -Once the composition of erythrocyte membrane l i p i d components is established for DMD patients and carriers, u t i l i z a t i o n of techniques which probe the lipid-protein interactions within intact red c e l l s might prove valuable. Perhaps the sulfhydryl and primary amino group labelling techniques, as well as the biochemical method of assessing membrane f l u i d i t y advanced by Bloj et a l . [77] (see NaF inhibition of AChE in Materials and Methods and Results), may prove more useful in the study of intact erythrocytes than these methods proved to be in the study of isolated red c e l l membranes. The fact that these techniques failed to detect any abnormalities in the fine structure of isolated DMD erythrocyte membranes [see Tables IV (A and B), V (A and B) and XI (A and B)] in no way eliminates the possibility that such abnormalities do occur in the membranes of intact c e l l s . As has been pre-viously discussed (see Results), when isolated erythrocyte membranes ("ghosts") have been rigorously compared to the membranes of intact cells u t i l i z i n g various probes of membrane integrity, red c e l l ghosts were found to deviate considerably from the native structure exhibited by the membranes of intact erythrocytes [113, 114, 115, 116, 117, 118] . Preliminary studies from this laboratory indicate that the kinetic characteristics of substrate hydrolysis by erythrocyte membrane AChE and i t s inhibition by NaF differ markedly between normal intact eythrocytes and ghosts prepared from normal c e l l s (data not pre-sented in Results). While such findings do not negate the value of u t i l i z i n g red c e l l ghosts in the study of DMD, they do suggest caution in the interpre-tation of data obtained from isolated membranes and emphasize the value of using intact erythrocytes wherever possible in these investigations. - 103 -It i s not clear to what extent alterations in membrane lipid-protein interactions in DMD erythrocytes influence calcium homeostasis in these c e l l s , but the results of the present investigations and the work of others indicate that cellular calcium handling may be impaired in DMD tissues. For example, the maximal velocity of the high-affinity component of erythrocyte membrane 2+ Ca -ATPase, believed by Schatzmann and his colleagues [98, 101] to represent the red ce l l ' s calcium extrusion mechanism, is significantly depressed in DMD subjects [see Table IX (C)]. A similar alteration may also be present in the low-affinity component of this enzyme [see Table IX (E)], which Schatzmann et a l . have tentatively identified as a slightly denatured form of the high-2+ a f f i n i t y Ca -ATPase produced during the preparation of red c e l l ghosts [98, 101]. Thus the calcium pump may be defective in DMD erythrocytes. A 2+ similar disturbance in Ca -ATPase activity with impairment of active calcium uptake has been reported to occur in sarcoplasmic reticular vesicles derived from DMD skeletal muscle [12]. It i s not clear whether these apparent abnormalities in the calcium-pumping mechanism identified in DMD erythrocytes and skeletal muscle organelles arise from membrane l i p i d alterations with subsequent modification of membrane lipid-protein interactions, or whether they may represent some basic, inherit-able change in a protein component of this cationic transport mechanism. It is clear, however, that such a change in calcium homeostatic machinery could be potentially deleterious to the cellular integrity of skeletal muscle [140]. Furthermore, this defect could produce many of the abnormalities in cellular morphology and function described for DMD erythrocytes. For example, the - 104 -intracellular accumulation of calcium ions in the DMD red c e l l , secondary to defective calcium extrusion, may predispose the c e l l to leak potassium at an abnormally high rate; the a b i l i t y of calcium to modify potassium permeability in erythrocytes has been well documented by investigators who studied potassium efflux from metabolically depleted red ce l l s incubated in calcium Ringers solu-tion [141, 142]. Similarly, elevation of intracellular calcium concentrations in erythrocytes by introduction of the cation via the antibiotic ionophore A23187 produces a marked increase in potassium efflux, as well as demonstrable reduction in red c e l l deformability [143]. Furthermore, metabolic energy deple-tion and elevation of intracellular calcium ion concentration with A23187 in red c e l l s has been shown to ini t i a t e the transformation from the normal dis-cocytic morphology to the spiculated echinocytic form; this change i s apparently related to the accumulation of 1,2-diacylglycerol within the inner leaflet of the erythrocyte membrane, producing outward evaginations of the membrane ("spicules"); 1,2-diacylglycerol is believed to result from the calcium-stimulated breakdown of membrane phosphatidyl choline by endogeneous phospholi-pase C [102, 103, 145, 146]. Allan et a l . suggest that the mechanism just described may explain the loss of membrane components and the enhanced osmotic f r a g i l i t y observed to occur in aging erythrocytes, as well as in HS red ce l l s [146]. As described in detail earlier, many interesting parallels exist bet-ween the alterations seen in HS erythrocytes and those noted for DMD erythro-cytes. It is hardly surprising therefore that Feig and Guidotti have observed 2+ a deficiency of Ca -ATPase activity in HS red c e l l s , which these investi-gators believe may reflect a basic alteration in the calcium extrusion mechanism - 105 -[147]. It is conceivable that these parallel alterations in DMD and HS red cel l s may arise from a similar defect in calcium homeostasis. The close correlation between the data reported in this thesis [see Tables IX (C and E)], the other published observations of erythrocyte abnormalities in DMD, and the known sequelae of high levels of intracellular calcium established by ionophore studies suggests that potentially valuable insights into DMD pathogenesis may be obtained by a rigorous study of calcium extrusion in these erythrocytes. Another possible locus for the primary defect in DMD erythrocytes might be the microfibrillar elements, which form an anastomosing network attached to the membrane's inner surface [64]. The function of this network has not been established, but some investigators believe that this m i c r o f i b r i l -lar system in red c e l l s represents an actomyosin-like contractile mechanism analogous to that found in the skeletal muscle c e l l [65]. If this i s the case, then characterization of this system in DMD erythrocytes should receive high pr i o r i t y in future investigations, especially since there i s evidence that the contractile elements of DMD skeletal muscle may be defective. Furukawa and Peter have observed decreased actomyosin-ATPase activity and impaired superprecipitation of myosin B derived from skeletal muscle of patients with DMD [22]. The speculation that an actomyosin-like system exists in erythrocytes was initiated in 1962 by Ohnishi who demonstrated with viscometry that skeletal muscle myosin interacts with a protein in the water-soluble extracts of acetone powder preparations of red c e l l ghosts; Ohnishi also observed that this inter-action i s inhibited by addition of ATP to the extract [148]. This protein has subsequently been identified as actin by i t s molecular weight, net charge, - 106 -a b i l i t y to polymerize into filaments with the double h e l i c a l morphology and i t s decoration with heavy meromyosin; i n s i t u erythrocyte a c t i n i s associated with a high molecular weight p r o t e i n l o o s e l y bound to the inner membrane sur-face, c a l l e d " s p e c t r i n " (gel electrophoretic bands I and II [78]), forming the f i b r i l l a r network previously described [64]. The question of whether or not 2+ t h i s s p e c t r i n - a c t i n system possesses a Mg -dependent ATPase a c t i v i t y , l i k e s k e l e t a l muscle actomyosin-ATPase, i s g r e a t l y disputed, some even contending 2+ that s p e c t r i n may be related to one component of the membrane's Ca -ATPase a c t i v i t y [63, 64, 121, 149, 150, 151, 152]. A number of l i n e s of evidence suggest that the s p e c t r i n - a c t i n net-work, together with attached glycophorin, a membrane-traversing prote i n , con s t i t u t e s the cytoskeleton of erythrocytes and i s the c h i e f determinant of erythrocyte shape and deformability [110] — two parameters known to be a l t e r e d i n DMD red c e l l s . For example, when erythrocytes are heated, a discocyte-spherocyte t r a n s i t i o n i s observed to occur at the precise temperature at which a membrane protein possessing ATPase a c t i v i t y denatures [153]; Jacob has strongly suggested that t h i s p rotein i s s p e c t r i n [110]. A s i m i l a r morphologi-c a l a l t e r a t i o n i s observed when erythrocytes are subjected to treatment with v i n b l a s t i n e , a drug which i n t e r a c t s with m i c r o f i b r i l l a r proteins and i s known to denature erythrocyte s p e c t r i n with some s p e c i f i c i t y [154, 155]. F i n a l l y , Jacob e t a l . have found i n d i c a t i o n s that s p e c t r i n polymerization may be defec-t i v e i n HS erythrocytes, c e l l s known to e x h i b i t echinocytic morphology [154]. That the anastomosing s p e c t r i n - a c t i n network attached to the inner surface of erythrocyte membranes may also be capable of influencing red c e l l c a t i o n i c permeability i s suggested by the work of Lubin e t a l . [156]. These i n v e s t i g a -tors found that when erythrocytes obtained from patients s u f f e r i n g from s i c k l e - 107 -c e l l disease are treated with dimethyl adipimidate, a protein cross-linking drug with special a f f i n i t y for glycophorin, the excessively high potassium conductance usually observed in these c e l l s is reduced to normal levels. Lubin and his colleagues further showed that this same drug i s capable of normalizing cation permeability, morphology, and osmotic f r a g i l i t y in erythro-cytes from patients with hereditary stomatocytosis [157]. The above evidence suggests that erythrocyte cationic permeability, morphology, deformability and osmotic f r a g i l i t y may a l l be influenced by the membrane's spectrin-actin micro-f i b r i l l a r network. A defect in this system might underlie the similar altera-tions observed in red c e l l s in HS [154, 155], hereditary stomatocytosis [156, 157] and DMD. Whether this defect represents the primary lesion in these dis-orders or is merely one of the many alterations in membrane structure and function, possibly resulting directly from changes in membrane lipid-protein interactions, remains to be determined. In terms of ongoing investigation of DMD erythrocytes, isolation and biochemical characterization of DMD red c e l l spectrin and actin components might be highly informative. It would also be of interest to determine i f in vitro treatment of DMD erythrocytes with dimethyl adipimidate results in a normalization of membrane functional altera-tions associated with DMD. Finally, the work of Strickland and E l l i s [24] w i l l be briefly con-sidered with a view to suggesting how a generalized metabolic defect in gly-colysis might be deleterious to skeletal muscle ce l l s and erythrocytes, should such a defect prove to be even more primary than the abnormalities observed in lip i d s and some proteins in affected tissues. These investigators demonstrated the occurrence of a compositionally and functionally abnormal isoenzyme II of - 108 -hexokinase in DMD skeletal muscle, resulting in a high conversion of glucose to fructose with a concomitant drop in glucose-6-phosphate production [23, 24]. Strickland and E l l i s have also found evidence of the same enzymatic defect in DMD l i v e r , peripheral nerve and brain, but not in DMD adipose and connective tissue [24] . Such a metabolic abnormality might be expected to produce fatty change in skeletal muscle fibers via the conversion of the large quantities of fructose into triglycerides. Furthermore, this defect might be expected to produce impaired fatty acid synthesis in liver by blocking NADPH generation by the hexose monophosphate pathway [160]. Since mature erythrocytes are incapable of de novo synthesis of fatty acids, but are continuously renewing their l i p i d components via various l i p i d exchange mechanisms, many of which draw upon circulating l i p i d stores in the plasma [128], and since the liver i s a prime contributor to plasma l i p i d stores [160], an alteration in li v e r fatty acid synthesis in DMD would be expected to produce alterations in l i p i d consti-tuents of erythrocytes. Furthermore, i f this glycolytic defect should extend to the erythrocyte i t s e l f in DMD, and since the energy needs of mature red ce l l s are supplied primarily by anaerobic glycolysis and from products of the hexose monophosphate pathway [160], one would expect to observe ATP depletion in the DMD erythrocyte; this in turn could seriously affect the ATP-requiring calcium and sodium-potassium transport pumps and pump-linked transport of non-ionic substances, as well as decreasing the supply of energy required for the various membrane l i p i d renewal pathways. Therefore, a generalized metabolic defect such as the one observed by Strickland and E l l i s might produce sublethal or lethal injuries to erythrocytes, as well as to other body tissues in this disease. It i s interesting to note in this regard that erythrocyte pyruvic - 109 -kinase deficiency, a hemolytic anemia a r i s i n g from a congenitally defective enzyme of the red c e l l ' s g l y c o l y t i c pathway and thereby resulting i n a d e f i -ciency of erythrocyte ATP stores, produces a picture i n erythrocytes strongly resembling that seen i n DMD ( i . e . , echinocytic morphology, an accelerated rate of potassium e f f l u x , compensatory Na +, K+-ATPase pumping, decreased deform-a b i l i t y and osmotic f r a g i l i t y [129, 161]). The foregoing discussion suggests that a thorough study of g l y c o l y t i c metabolism of erythrocytes, l i v e r and other tissues might be of real value i n the investigation of the molecular basis of DMD pathogenesis. In addition to discussing a variety of interesting methodological approaches not already employed i n the present investigation, consideration has been given i n the preceeding pages to suggesting which of the experimen-t a l techniques already employed may prove useful i n further characterization of DMD erythrocytes. Therefore, a b r i e f examination of possible refinements of the present techniques which might improve their research value would be appropriate here. A report by Cohen et a l . [123] on the biochemical characterization of density -separated normal human erythrocytes demonstrated that the age of the various red c e l l populations comprising whole blood has a profound ef f e c t upon the magnitude of erythrocyte compositional and functional parameters. These investigators found that increasing red c e l l age correlates with eleva-t i o n of hemoglobin and i n t r a c e l l u l a r sodium, but with reduction of i n t r a -c e l l u l a r potassium, as well as with reduction i n membrane protein, s i a l i c a cid, phospholipid, cholesterol, and AChE a c t i v i t y [123]. Perhaps the negative results observed i n the present studies of membrane s i a l i c acid, - 110 -cholesterol, phospholipid and AChE activity in DMD erythrocytes arise from a "swamping out" process, whereby significant alterations in erythrocytes of a given age are obscured by the presence of c e l l s of different ages which dis-play normal parameters or alterations in the opposite direction. Segregation of erythrocytes according to c e l l age by use of density-separation techniques may allow greater sensitivity in the characterization of abnormalities in DMD red c e l l s . Another important modification of the present methodology employed in these DMD studies might be to u t i l i z e intact erythrocytes instead of iso-lated red c e l l membrances in the analysis of the membrane f l u i d i t y charac-t e r i s t i c s of c l i n i c a l and control blood c e l l s . Tanaka and Ohnishi have demon-strated that the f l u i d i t y patterns exhibited by the various regions of the erythrocyte membrane, based upon the asymmetrical distribution of membrane phospholipid components, are lost when red c e l l s undergo hemolysis [118]. Since the preparation of isolated plasma membranes of c l i n i c a l and control erythrocytes involves step-wise hemolysis, possible alterations in membrane f l u i d i t y characteristics of DMD erythrocytes may be obscured by these pre-parative operations. Since Bloj et a l . [77] have suggested that the H i l l coefficient for the inhibition by NaF of membrane-bound AChE is a useful probe of membrane f l u i d i t y , and since AChE i s an enzyme located on the outer surface of the erythrocyte membrane, analysis of the membrane f l u i d i t y charac-t e r i s t i c s of intact erythrocytes from DMD and normal subjects should be possible. Preliminary studies on intact red c e l l s conducted in this labora-tory have demonstrated that the kinetic characteristics of substrate hydroly-sis by membrane AChE and i t s inhibition by NaF di f f e r markedly in these c e l l s - I l l -from those observed for isolated erythrocyte membrane preparations. The results of other preliminary studies, i f confirmed by further investigation, may cast some doubt upon the sensitivity of this biochemical technique, pur-ported to be a reliable means of assessing membrane f l u i d i t y characteristics. When normal erythrocyte membranes are treated with agents known to severely modify membrane protein and l i p i d components (e.g., trypsin and phospholipase A), these membranes exhibited H i l l parameters in NaF-AChE studies similar to those obtained for untreated control membranes. Bloj et a l . conducted their experiments on isolated membranes prepared from erythrocytes of rats fed various fat-modified diets [77] and not on human erythrocyte membranes. Therefore, a thorough assessment of the usefulness of this technique in the characterization of f l u i d i t y properties of human control erythrocytes should be conducted prior to undertaking a full-scale study of membrane f l u i d i t y in DMD erythrocytes. Another potentially valuable modification of the present methodology employed in the investigation of DMD erythrocytes would entail analysis of the osmotic f r a g i l i t y characteristics of DMD patient and carrier red ce l l s in the presence of membrane stabilizers of destabilizers. For example, i t might be of considerable c l i n i c a l interest i f non-steroidal anti-inflammatory drugs possessing membrane-stabilizing properties, e.g. indomethacin [162], could be shown to correct the abnormal osmotic f r a g i l i t y of DMD erythrocytes. That indomethacin may be of therapeutic value in DMD i s suggested by a recent report by Bulien and Hughes where treatment of dystrophic hamsters with this drug was shown to reduce CPK levels in the serum of these animals, as well as normalize skeletal muscle levels of this enzyme [163] . These findings suggest - 112 -the p o s s i b i l i t y that the defect i n s k e l e t a l muscle membrane i n t e g r i t y i n muscu-l a r dystrophy, resulting in the release of cytoplasmic enzymes, may have some bearing on the f r a g i l i t y of erythrocyte membranes i n DMD. Also, the a b i l i t y of drugs to correct the defect i n the hypotonic s t a b i l i t y of erythrocytes i n v i t r o may prove to be a useful screening procedure for the evaluation of drugs to be tested for possible b e n e f i c i a l effects i n vivo. With regard to DMD c a r r i e r erythrocytes, no abnormalities i n gross osmotic s t a b i l i t y were detected i n these c e l l s (see Figure 6). However, i t may be possible to unmask more subtle alterations i n the hypotonic s t a b i l i t y of c a r r i e r erythrocytes by examining their s t a b i l i z a t i o n and d e s t a b i l i z a t i o n properties i n the presence of membrane-active drugs (e.g., propranolol), since i t has recently been shown that the effects of these drugs upon the osmotic s t a b i l i t y of erythrocytes i s c r i t i c a l l y dependent upon l i p i d - p r o t e i n interac-tions within the membrane [164]. Comparison of the osmotic behavior of DMD c a r r i e r and normal erythrocytes following treatment with membrane-active drugs may reveal subtle differences i n the fine structures of the membranes of these c e l l s and may even provide a convenient means of identifying the c a r r i e r state i n the female r e l a t i v e s of DMD patients. In summary, the findings of the present investigation, a l b e i t tenta-t i v e , and those of other research workers suggest that DMD i s a generalized disorder a f f e c t i n g many body tissues i n affected children, even erythrocytes. The c e l l u l a r locus of the defect i s s t i l l uncertain, although a strong case may be made for l o c a l i z a t i o n of the l e s i o n i n the plasma membranes of affected c e l l s . The abnormalities i n red c e l l membranes discussed previously lend strong c r e d i b i l i t y to t h i s hypothesis that DMD i s a generalized plasma membrane defect, - 113 -although abnormalities have also been noted in organelle membranes of certain other tissues. A variety of alterations of red c e l l composition and function have been discussed with a view to discerning which alteration might reflect the primary lesion in this disorder and thus gives rise to the other abnormali-ties observed. Llpoperoxidative mechanisms of membrane injury, alterations in ionic homeostasis, abnormalities in erythrocyte microfibrillar components and modifications of membrane l i p i d components with subsequent alteration of l i p i d -protein interactions v i t a l to the maintenance of cellular integrity have a l l been discussed as possible candidates in the search for the primary DMD lesion. Although at present, there is no conclusive means of choosing between these p o s s i b i l i t i e s , the author favors the hypothesis that DMD arises from a funda-mental alteration in membrane l i p i d s , producing various perturbations of mem-brane structure and function which are ultimately lethal in affected c e l l s . The possibility that this alteration in membrane li p i d s might be secondary to a genetic defect in cellular glycolytic machinery has been discussed and pro-posals made as to the mechanics of erythrocyte involvement in such a disorder. 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Diem), Geigy Pharmaceuticals, Montreal, pp. 124-127, (1962) - 123 -APPENDIX This section illustrates the use of the Wilcoxon rank sum method of s t a t i s t i c a l analysis. This method may be employed in the comparison of groups with a minimum sample size of four observations. In the present example, mem-brane cholesterol contents of erythrocytes drawn from Duchenne dystrophic and normal subjects are compared. The experimental observations (expressed as micrograms cholesterol per milligram membrane protein) are as follows: Controls (nj = 6) DMDs (n 2 = 9) 168.1 275.0 167.8 195.3 186.7 177.7 209.1 209.3 203.9 217.4 221.3 219.3 215.9 216.4 220.4 The Wilcoxon rank sum test proceeds in the following manner (79): both samples are combined, ordering the observations from low to high, then a rank is assigned to each observation. For tied observations (i.e., those having the same numerical value), one assigns the corresponding average rank to each t i e . (Observations from the group with the smaller sample size are tagged with an asterisk to permit their identification in the pooled data array.) - 124 -Ordered Array Rank 167.8* 1 168.1* 2 177.7 3 186.7* 4 195.3 5 203.9* 6 209.1* 7 209.3 8 215.9 9 216.4 10 217.4 11 219.3 12 220.4 13 221.3* 14 275.0 15 The test consists of summing the ranks of the group having the smaller sample size. For comparison of samples of equal size, one may sum the ranks of either group. Rank Sum = 1 + 2 + 4 + 6 + 7 + 14 =34 The rank sum is next compared with the tabulated limits, which have been compiled for the desired level of s t a t i s t i c a l significance. In the present example where n-^  = 6 and n 2 = 9, the significance limits for the Wilcoxon test at 2<x= 0.05 are 31-65 [165]. If the rank sum attains these levels or exceeds them in the outward direction, the null hypothesis (i.e., that the population distributions of cholesterol content values in each group are equal) must be rejected. However, in the present example, the rank sum f a l l s within the significance limits range, and therefore, the null hypothesis is accepted: i.e., at the 5% level of s t a t i s t i c a l significance, no discern-ible difference in erythrocyte membrane cholesterol content has been found between samples drawn from Duchenne dystrophic and normal control populations. 

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