Current concepts of progressive muscular dystrophy hold that it is a disease in which, through genetic error, there are intrinsic defects in muscle metabolism, although the nature of these hypothetical defects remains unrecognized. It is the purpose of this report to describe one approach to the problem and to show that there are defects in the metabolism of isolated muscle from patients with progressive muscular dystrophy. In addition, establishment of non-collagen nitrogen as a reference base provided an opportunity to repeat previous observations on the non-heminic iron content of muscle. Earlier work from this laboratory (5, 6) showed that non-heminic iron (that is, iron not included in chromoproteins, myoglobin, and the cytochromes) occurred in muscle in two varieties; one extractable by reducing agents, the second apparently linked with myosin (7). It had been found that the extractable iron in muscle from dystrophic patients was low in terms either of wet weight of muscle or of fat-free dry weight (8-10).Biopsies of muscle were obtained under general anesthesia. The samples were frozen at once in liquid nitrogen and analyses were performed without delay. Biopsies from patients without muscular dystrophy, controls, were obtained from the right transversus abdominis during operation for appendicitis. Biopsies from patients with muscular dystrophy were obtained from the left transversus abdominis. After freezing the muscle, an aqueous extract was prepared in the Potter-Elvebjem homogenizer held at the temperature of melting ice. The extracting medium was 20 volumes of 0.1 N sodium fluoride. Aliquots were analyzed for non-collagen nitrogen (11), for total nitrogen, for aldolase concurrently by the method of Dounce, Beyer, and Barnett (12,13) and by the method of Sibley and Lehninger (4), and for phosphorylase by the Coris' method (3). In addition, in a number of patients the activity of aldolase in serum was also assessed (14). In three patients interpretation of values for non-collagen nitrogen were checked by determination of collagen and elastin (scleroproteins) by the method of Neumann and Logan (15). Extractable iron was determined colorimetrically with orthophenanthroline, using sodium dithionine as a reducing agent (16).For phosphorylase assay the homogenate was centrifuged, the supernatant extract diluted with two volumes of a mixture of cysteine and NaF, pH 6.0, and left for two minutes at 38°. One-tenth ml. was used for the assay. Final concentration of cysteine was 0.05 M, pH 6.0. For aldolase assay the 1/20 extract was diluted with four volumes of water. Two-tenths ml. were used in the presence of 0.5 ml. 0.01 M fructose diphosphate, hydrazine and collidine buffer pH 7.5.
RESULTSIn Table I, collagen nitrogen is expressed as per cent of total nitrogen in the biopsy specimen. There was a definite increase in collagen nitrogen 794
