5.. m} ‘ is. i» :12. :35. QIfixE: 5... .. xiv; .anknraasfl. r. «.39th h~.'ifir\.v\l.‘.‘(1 taagisu‘: :fifixflufln axiailulflunmrr fitéfifis . \L I}: L. gmmu‘. s. 1 12!; 9)}. 5 iii .. L y .13....v‘170» fix... in 2‘s! I... . ‘ 34 , 1:; 12,1 . 2.5:: 2.. 320:»?! , u. Tim“ . v. . 3.3.55? 51.5 1 z; Hunky“ r {:3 egg-119 3&3- .7 7.1.1.... . ‘ . ). . :49: . 1" . a in, . . 1.1.3: 22?! . . uuj’l‘t‘! t«‘1\‘ . , 1:42 23:11. a E .2 .. 2.25.. flak Erik". szin i : v. (.523 .7-21 nkiu . £52. ~i‘::§ 1w 5:»...1 3.3.1: THESIS LIBRARY Michigan State University This is to certify that the thesis entitled GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF URINARY AND PLASMA ORGANIC ACIDS FROM PATIENTS WITH DUCHENNE'S MUSCULAR DYSTROPHY AND AGE MATCHED CONTROLS presented by Thomas J. Carlson has been accepted towards fulfillment of the requirements for M degree in biochemistry Mi /\ Major professor WMP 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution RETURNING MATERIALS: MSU Place in book drop to 5 remove this checkout from “BRAKE your record. FINES will be charged if book is returned after the date . stamped below. ~— GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF URINARY AND PLASMA ORGANIC ACIDS FROM PATIENTS WITH DUCHENNE'S MUSCULAR DYSTROPHY AND AGE MATCHED CONTROLS By Thomas J. Carlson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Master of Science Department of Biochemistry 1982 (n/fir.’ L) ./7/ ABSTRACT GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF URINARY AND PLASMA ORGANIC ACIDS FROM PATIENTS WITH DUCHENNE'S MUSCULAR DYSTROPHY AND AGE MATCHED CONTROLS By Thomas J. Carlson The objective of this study was to compare organic acid levels in urine and plasma from normal and dystrophic patients in addition to those from normal women and carriers of Duchenne's muscular dystrophy (DMD). Specific goals were to a) determine differences in the levels of organic acids in urine and plasma from DMD patients when compared to age matched controls, b) determine differences in the level of organic acids from normal women and carriers, c) correlate differences in organic acid levels in carriers to differences seen in dystrophic patients and d) speculate what enzyme(s) would be responsible for such a change. Nine metabolites in urine are significantly decreased in dystrophic patients; they are glycerate, 4—deoxythreonate, p-hydroxybenzoate, m-hydroxyphenylacetate, m—hydroxyphenylhydracrylate, hydrocaffeate and unknowns labelled X58, UN40 and UN59. Two metabolites phenylacetate and p-hydroxycinnamate were elevated in the urine of dystrophic patients, the latter two metabolites were not detected in normal controls. Carriers exhibited a significant increase in a-hydroxyisobutyrate and an unknown labelled UN4, while a decrease in a compound labelled X27 was observed. Three metabolites namely citrate, 4-deoxythreonate and an unknown, X1 were significantly decreased while an unknown labelled UN3 was elevated in the plasma of dystrophic patients. There were no correlations between the metabolites that were altered in dystrophic patients when compared to metabolites altered in carrier mothers. To Peace 11' Acknowledgements The spot light here falls on Clarence Suelter, one of the happiest professors a person could ever know. I am grateful for his constant support both financially and intellectually. Without it, I could not have completed this work. To my constant companions and friends at lab, Dave, Vickie and Jeff. Also, the jolly bunch next door, Debra, Jerome, Maria and Shelagh who made every day an experience. Believe me. My thanks go to Charlie, Deb, Julia and Elizabeth for the time I spent with you. Finally my thanks to my family who made this possible. iii Table of Contents Page LIST OF TABLES..... ......... ................................. ..... vi LIST OF FIGURES ...... . ................ ............................vii LIST OF ABBREVIATIONS.. .......... . ..... ...........................viii LITERATURE REVIEW ........................ .... ............. ... ...... 1 Muscular dystrophies...... ................ . ..... ..............1 Muscle metabolism ............................... .... ....... ...2 Objective .......... ........ ............. ......................8 MATERIALS AND METHODS ............ . ..... . .......... ..... ......... ..10 Dystrophic urine and plasma.... ............ ..... ..... ........10 Creatinine Determination....... ................ ....... ....... 10 Urine preparation ...................... ......................11 Silanization of glassware..... ................. ..............11 Isolation of organic acids from urine ............ . .......... .11 Oxime formation.... ............... .... ..... .... ..... .........12 Isolation of organic acids from plasma ......... ..............12 DEAE—Sephadex chromatography or urine samples.... ..... .......13 DEAE—Sephadex chromatography of plasma samples........ ....... 14 Lyophilization of urine and plasma samples ............ .. ..... 14 Derivatization of urine and plasma samples..... ...... ........15 Gas chromatography—mass spectrometry analysis..... ........... 15 Data analysis using MSSMET...................................18 iv 8293 RESULTS..... .......... . ......... . ...... ............................20 DISCUSSION. ............ . ............. 22 Urine........ ......... ........................................22 GIyCerate OOOOOOOOOOOOO 00....00....IO.IO.CCUIOOOOOCOOIOCOOOOOOOZS 4-Deoxythreonateoooo-c oooooo 000.900.03.03 ooooooo one... cccccccc 26 m-Hydroxyphenylhydracrylate, (3-hydroxy-3-(3-hydroxypehnyl)-propanoate ................... 26 3-(3,4-Dihydroxyphenyl)—propanoate, (hydrocaffeic acid) ...... .27 SUMMARY AND CONCLUSIONS ............... ..... ........ . ............... 59 APPENDIX A ......................................................... 62 APPENDIX B .......... . .................... .. .................. ......65 REFERENCES .......................................... . ..... . ....... .68 List of Tables Page 1 Organic Acidemias. ............ . ............ ......... ..... ..30 2 Normalized Metabolites in Urine and Plasma Samples from Normal and Dystrophic Patients....... ...... ...........33 3 The Average Relative Concentration of 11 Metabolites in Urine Samples from Normal and Dystrophic Patients Selected from Table 2 Whose Mean Relative Concentration was Approximately Twice that of the Other Gr0up ............ 41 4 The Average Relative Concentration of 4 metabolites in Plasma Samples from Normal and Dystrophic Patients selected from Table 2 whose Mean relative Concentration was Approximately twice that of the Other Group...... ...... 46 5 Normalized Metabolites in Urine Samples from Normal and Carrier Women ......... ... ........ ...... .......... . ..... 47 6 The Average Relative Concentration of 5 metabolites in Urine Samples from Normal and Carrier Women Selected from Table 5 whose mean Relative Concentration was Approximately Twice that of the Other Group ........................ ....... ..... 56 vi List of Figures Ease. Metabolic Pathways in the Metabolism of phenylalanine and p—tyrosine...............................32 Scatter Plots of 4-Deoxythreonate, 3—Hydroxy—3-(3-hydroxyphenyl)-propanoate (m-hydroxyphenylhydracrylate), m-Hydroxyphenylacetate, p-Hydroxybenzoate, Glycerate, and 3-(3,4-Dihydroxyphenyl)-propanoate (hydrocaffeate) found in Urine from Normal (N) and Dystrophic (D) Patients ................................................ ..43 Scatter Plots of Citrate and 4—Deoxythreonate Found in Plasma from Normal and Dystrophic Patients .......................... . ............. . ........ ..55 Scatter Plots for a—Hydroxyisobutyrate Found in Urine from Carriers of DMD and Normal Age Matched Women .......... 58 vii ADP AMP a.m.u. ATP BSTFA DEAE DMD DNA DOPA DOPAC DOPamine GDP GTP p—HBA p—HCA HGH m-HPAA p-HPAA p-HPLA p—HPPA id MSSMET PAA RNA List of Abbreviations Adenosine diphosphate Adenosine monophosphate Atomic mass unit Adenosine triphosphate Bis-trimethysilyltrifluoroacetamide Diethylaminoethyl Duchenne's muscular dystrophy Deoxyribonucleic acid 3-(3,4-Dihydroxyphenyl)alanine 3,4-Dihydroxyphenylacetic acid 2-(3,4-Dihydroxyphenyl)ethylamine Guanosine diphosphate Guanosine triphosphate p—Hydroxybenzoic acid p-Hydroxycinnamic acid Human growth hormone m—Hydroxyphenylacetic acid p—Hydroxyphenylacetic acid p-Hydroxyphenyllactic acid p-Hydroxyphenylpyruvic acid Inner diameter Mass spectral metabolite Phenylacetic acid Ribonucleic acid viii Literature Review Muscular Dystrophies: The muscular dystrophies encompass several inherited diseases characterized by genetic and clinical lines of evidence (96). Patients with limb girdle dystrophy exhibit hypertrophy of the shoulder and pelvic regions initially while hypertrophy of the calves and deltoids occurs later on. It is an autosomal recessive disorder but autosomal dominant inherited patterns have been found affecting the limbs, shoulder and pelvic girdle regions. In facioscapulohumeral muscular dystrophy, onset occurs in early adolescence with the first symptoms involving weakness of the shoulder and facial areas while the pelvic girdle regions are spared. The disease is defined as an autosomal dominant trait. Myotonic dystrophy is also an autosomal dominant disorder. Symptoms are variable with onset occuring between ages from birth and 60 years old affecting the facial, temporal and cranial musculature. The latter being the most affected. The most devastating of the muscular dystrophies is Duchenne's muscular dystrophy (DMD), transmitted as an X—linked recessive mutation affecting mainly boys (1). The genetic defect and localization have not been determined but the disease appears to be the result of a single gene mutation (6). By age 9 to 12 years the severity of the disease confines boys to wheelchairs with death usually following in the second to third decade (1). 2 Four theories concerning the pathogenesis of the muscular dystrophies have been proposed. Proponents of the neurogenic theory argue that a neural disturbance is involved resulting from an abnormality of the motor end plates and decreased motor unit activity (76,77,20). Inadequate blood flow occuring in the microcirculation leading to degeneration of myofibrillar tissue has given credence to the vascular theory (14). An increased permeability of the membrane particularly the sarcolemma constitutes the basic thesis of the membrane theory (133,96,74,97). The fourth hypothesis that involves connective tissue alterations which may lead to ischemia (vascular theory) is discussed by Cazzato 1968 (14). Excellent reviews of muscular dystrophy are given by Rowland, 1976, 1980, (96,98), Sweeny and Brown, 1981, (118), Furukawa and Peter, 1972, (35), and Jerusalem, 1976, (61). The use of animals with inherited myopathies are commonly used as models for human muscular dystrophies. Mice with hereditary muscular dystrophy that follow simple Mendelian inheritance as an autosomal recessive disorder are the most widely studied (102,38,37). Syrian hamsters identified as BIO 1.5, transmit a polymyopathy as an autosomal recessive disorder (49), while dystrophy in chickens is a single gene defect inherited as an autosomal recessive trait (132). Use of these animal and avian models is advantageous since acquiring human muscle for study is limited. It is generally agreed that they are not identical to human muscular dystrOphy. Muscle Metabolism: Because of the importance of proteins in the structure and function of muscle tissue, investigators have concentrated on the 3 metabolism of proteins in these animal models. The pathological changes that occur in muscular dystrophy could be attributed to the mechanism which controls protein turnover. Since muscle metabolism is dependent on the synthesis and the degradation of proteins, the protein concentration in muscle tissue depends upon the balance between synthesis and breakdown. In Duchenne's muscular dystrophy, muscle wasting is not rapid, thus increased protein synthesis occurs along with increased degradation. However, there must be an imbalance between myofibrillar protein degradation and synthesis where synthesis lags behind degradation. Increased myofibrillar protein catabolism in boys with Duchenne's muscular dystrophy was shown (5,78, and 128) using 3-methylhistidine excretion as an index for catabolism of the muscle protein actin and myosin. Only one paper has emerged in which muscle protein synthesis and degradation rates have been measured jn_vivo in patients with DMD (94). The authors used the stable isotope tracer 1—13C-leucine by intravenous feeding and measured incorporation in myofibrillar protein. An increase in protein degradation was not observed. The results indicated supressed protein synthesis. This is opposite to what increased excretion of 3—methyl-histidine suggests; there is an increase in degradation of muscle proteins. Ionasescu et 31. 1971, (54) noted that patients with DMD exhibited an increase in amino acid associated with polyribosomes. However, the increase was indicative of increased collagen synthesis. Increased rates of protein synthesis over degradation are found in dystrophic hamsters (41) while a net loss of protein is seen in dystrophic mice (109,110,89) and chicken (129). The increased protein 4 synthesis over degradation in dystrophic hamster was thought to be due to an increase in ribosomes (41) or an elevated activity of enzymes involved in biosynthesis. An increase in degradation of many myofibrillar proteins was shown by Garber gt al., 1980, (38). Incubation of 129 Red mouse and CS7BL dystrophic mouse gastronemius and soleus muscle in modified media showed increased leucine, alanine and glutamine incorporation in vitro into proteins of the dystrophic line. Release of these three amino acids into the media was also increased (38). These data suggest increased protein synthesis and increased protein degradation in the dystrophic mouse lines. Other investigators have also reported increased rates of incorporation of amino acids into muscle proteins in dystrophic mice (64,109,110). Garber et al., 1980, (37), provided evidence that dystrophic mice exhibited decreased adrenergic and seratonergic responsiveness which provides an explanation why an increase in glutamine and alanine released into the media was observed in dystrophic mice (38); it was reported earlier by Garber 23.21-’ 1976, (36), that catecholamines can inhibit biosynthesis of glutamine and alanine in rats. In other work concerning catecholamines, Gorden and Dowben, 1966, (46) found an increase in urinary excretion of dopamine, adrenaline and noradrenaline, and increased levels of adrenaline and noradrenaline in skeletal and heart muscle from dystrophic mice. In DMD patients, urinary excretion of total amines which include epinephrine, metanephrine and 3—methoxytyramine increase as the disease progresses (21). Epinephrine and metanephrine and their metabolites in urine and plasma from dystrophic patients were also found elevated (112) while normal patterns were seen by Mendell, 1972, (79). Catecholamines play an important role as neurotransmitters and vasoactive agents thus their role in DMD has been suggested since ischemia can be induced. A large body of evidence has shown a marked rise in calcium in the myoplasm of skeletal muscle (103,28,120). The elevated rise in calcium coincides with damage to the myofilaments but the mechanism involved is unknown. Duncan, 1978, (28), suggests that the rise in calcium levels followed by degradation of muscle fibers is a multi-step process resulting in a dissociation of protein subunits in muscle tissue rather than a proteolytic attack. However, raised calcium levels may act to activate proteases both lysozomal and nonlysozomal which are involved in myofibrillar protein turnover (24). Specific activity of a serine protease was elevated in the muscle of dystrophic mice (104) and man (63) while specific activity of cathepsin B was elevated in mice (104), chicken (53) and man (86). Two reports are concerned with a calcium activated protease which removes Z-disks from myofibrils and degrades tropin, tropomyosin and C-protein (24 and 25 respectively) while a calcium activated neutral protease degrades a-actinin and Z-lines (93). Leupeptin, a protease inhibitor of cathepsin B and calcium activated proteases (114), was shown to retard atrophy and slow down protein degradation in dystrophic mice (72) and chickens (114). Abnormalities in purine nucleotide metabolism was suggested due to a decrease in high energy phosphate compounds, reduced levels of ATP and glycogen and an abnormal ATP/ADP ratio in patients with muscular dystrophy (126), while reduced levels of ATP and high concentrations of AMP, GDP and GTP in dystrophic mice were observed (135). In C57 and 129 Red dystrophic mice, dystrophic chickens and one patient with Duchenne's muscular dystrophy exhibit a decreased adenine plus inosine to guanine ratio (108). In contrast to these findings, Lochner and Brink, 1967, (73) find no abnormal alterations in ATP, ADP or high energy creatine phosphate levels in dystrophic syrian hamsters. This finding could be explained on the basis of the type of animal model used, but Farrell and Olson, 1973, (33) showed no alterations in creatine phosphate or ATP concentrations in dystrophic mice or chicken when expressed to noncollagen muscle protein. An increased turnover of adenine nucleotides in patients with Duchenne's was suggested based on the increased excretion in urinary uric acid and adenine nucleotides (7). Because of increased fat and connective tissue infiltration and the implications of a generalized defect in the muscle membrane in dystrophic muscle, researchers have probed into alterations in lipid metabolism. Neutral lipids and triglycerides in muscle from patients with DMD are unchanged, however, sphingomylin is increased (85), while phosphatidylcholine and phosphatidylethanolamine are decreased when compared to healthy individuals (69). In dystrophic mice sphingomylin was decreased in the sciatic nerve and plasma phosphatidylcholine increased (70). Plasma enzyme levels are a diagnostic marker of patients with muscular dystrophy resulting from a generalized membrane defect and leakage from muscle fiber. Creatine kinase (E.C.2.7.3.2) the most useful in diagnosis and rather specific to muscle tissue is increased dramatically and the most consistently in Duchenne's muscular dystrophy while carriers exhibit a marked rise 67% of the time (88). Other factors influencing protein biochemistry in skeletal muscle include insulin, human growth hormone (HGH) and polyamines. In 17 patients with DMD, Depirro gt_al., 1982, (26) noted reduced insulin receptors although binding of insulin to monocytes and glucose metabolism was unaltered. HGH is believed to stimulate RNA polymerase activity (130) thus increase polyamine concentrations. Polyamines help in formation of polysomes and also stimulate DNA-dependent RNA polymerase (66), thus stimulating protein synthesis (65). In limb girdle and myotonic patients muscle levels of spermidine and cadaverine were increased while DMD patients exhibited nonnal levels before HGH treatment (100). Russell and Stern, 1981, (101) observed an increase in urinary polyamines putresine, spermidine and spermine. HGH treatment resulted in an anabolic response in limb—girdle and myotonic patients and a catabolic response in DMD patients (100). All of the patients had normal endogenous HGH (99). Research on DMD for the past 30 years has not uncovered the biochemical abnormality characteristic of the genetic lesion. Stengel-Ruthdowski and Barthelmai, 1973, (111) were unable to find any change in metabolite concentrations in the citric acid cycle or the Embden-Meyerhof pathway in muscle biopsies from patients with DMD. An excellent overview of glycogen, glucose and lactate production and glucose assimilation in patients with DMD is given by Ellis, 1980, (31). Intracellular concentrations of K, Na, Mg, P and S in dystrophic chickens are unaltered (81). Carnitine, which acts as the major transport system of fatty acid acyl groups in fatty acid oxidation into the matrix of the mitochondria (34) could be affected since muscle in dystrophic mice exhibit altered fatty acid metabolism (117). Urinary excretion of carnitine in dystrophic patients was shown to be elevated 8 by Maebashi gt al., 1974, (75) but unaltered by DiMaruo and Rowland, 1976 (27). Objective: The objective of this study was to compare the levels of organic acids in urine and plasma from human patients with DMD to the levels found in age matched controls. Urine samples from females known to be carriers of DMD were also studied. Organic acids are defined as water soluble carboxylic acids with or without hydroxyl or 0x0 groups, amino acid conjugates and short chain fatty acids; amino acids are excluded (122). In the early 1950's and 1960's an amino acid screening program identified some amino acidemias. The main drawback when quantitating amino acids is that defects in enzyme activities could only be found in the first two steps of amino acid metabolism. However, if organic acids, the products of amino acid metabolism, are analyzed, abnormalities in metabolic pathways resulting in marked increase or decrease in an organic acid or acids not only gives information of an enzyme defect(s) later in the pathway but provides elucidation of the normal metabolic pathways in humans. Over 150 metabolic disorders involving a single enzyme defect have been identified (12). Despite extensive studies of enzyme activities and some metabolites in muscle, no one has attempted to complete a "profile” of the organic acids in urine and plasma of normal and dystrophic boys. Organic acid profiles have been carried out on human newborns (8), children with neuroblastoma (40), normal adults (71), diabetic and ketotic patients (82), effects of diet on excretion patterns (15), and other urinary excretion patterns concerned with diseases (59,30). By using gas chromatography-mass spectrometry it has been estimated that 9 20% of the compounds found in biological fluids can be detected, due to the fact that only compounds with a high enough vapor pressure or compounds which can be derivatized to increase their vapor pressure can be analyzed (60). An excellent review on profiling of body fluids states "that if one were able to identify and determine the concentrations of all compounds inside the human body, including both high and low molecular weight substances, one would probably find that almost every known disease would result in characteristic changes in the biochemical composition of the cells and of the body fluids” (60). The power of gas chromatography-mass spectrometry in the elucida- tion of enzymatic defects in metabolism was first shown by Tanaka gt gl,, 1966, (121) through the discovery of isovaleric acidemia. Organic acidemias are characterized by elevated organic acid(s) resulting from an unutilized substrate of a defective enzyme or the product of an enzymatic reaction where the pathway of the unutilized substrate has been diverted (122). The discovery of isovaleric acidemia was followed by the discovery of methymalonic aciduria by Oberholzer gt _l., 1967, (84) and Stokke gt_gl,, 1967, (113), propionic acidemia by Homnes gt gl., 1968, (50) and pyroglutamic aciduria by Jellum gt_gl., 1970, (58). A list of organic acidemias although not complete, and the enzyme(s) affected are given in Table 1. There are over 45 disorders recognized by the accumulation of organic acids (17). Excellent reviews on organic acid disorders and diagnosis are given by Tanaka et al., 1975, (121), Goodman and Markey, 1981, (43), and Chalmers and Lawson, 1982, (17). Materials and Methods Dystrophic urine and plasma: Urine and plasma samples were generously collected by Dr. Ristow, Department of Osteopathic Medicine, M.S.U., E. Lansing, MI and Dr. Nigro, Martin Place Hospital-East, Detroit, MI. An instruction sheet and questionnare of drugs or antibiotics taken were provided for each patient approximately 3 days prior to collection (Appendix A). Patients were asked to fast 12 h before collection of urine and plasma samples and to indicate food taken in the 12 h fast on the questionnare. Blood samples were collected in B-D Vacutainer heparinized tubes, (Becton Dickinson Co., Rutherford, NJ) and centrifuged at the hospital. Care was taken in drawing the blood to prevent hemolysis. Both urine and plasma samples were placed on ice and transported back to the lab where they were placed in 3 dram vials, labelled and stored at -60°C until analyzed. Creatinine Detennination: Saturated picric acid (Aldrich Chemical Co., Milwaukee, MI) was prepared by adding excess picric acid to water and stirred for 1 h. After setting overnight in the dark the solution was then filtered and stored in a colored flask. Alkaline picrate was made fresh daily by combining 7.5 ml of 10% sodium hydroxide to 100 ml of saturated picric acid. Fifty ul of a urine sample was added to 1 ml of alkaline picric acid, vortexed and incubated at room temperature for 10 min. Aliquots (0,20,50,75,IOO,150, and 200 ul) of creatinine standard 10 11 (Aldrich Chemical Co., Milwaukee WI) solution at 1 mg/ml was added to different test tubes containing 1 ml alkaline picrate. After incubation for 10 min, samples were diluted to 5 ml with deionized water and read at 505 nm using a Beckman DU Spectrophotometer (2400) (Fullerton, CA). Summary of creatinine concentrations of all urines examined are given in appendix B. Urine Preparation: Frozen urine samples were thawed in warm water until all of precipitates dissolved. After equilibrating at room temperature, 1 ml of urine was added to a preweighed 1 dram vial and weighed. One ml of water was weighed in the same manner with a clean vial. The difference between the weight of one ml of water and one ml of urine was taken as an estimate of urinary solids. An aliquot of the urine to give 15 mg of urinary solids was diluted to 1 ml and analyzed. Normalizing in this manner avoided the need to dilute samples later in the protocol. Summary of weights and volume of urine analyzed are given in appendix B. §jlanization of glassware: Acid washed glassware were immersed in a 3% solution of dimethyldichlorosilane (Sigma Chemical Co., St. Louis, MO) in hexanes (Mallinckrodt, Paris, KY) for 10 min followed by two rinses with methanol (MCB Manufacturing Chemists, Inc., Cincinnati, OH) and dried at room temperature. Isolation of Organic acids from Urine: Urinary organic acids were analyzed by a modification of a procedure by Thompson and Markey, 1975 (123). An aliquot of urine to give 15 mg of urinary solids was placed in a silanized 20 ml screw top 12 test tube and diluted to 1 ml with deionized water; 10 ul of 1 mg/ml tropic acid (Aldrich Chemical Co., Milwaukee, WI) was also added as an internal standard. Inorganic phosphate was precipitated as its insoluble barium salt by adding 1 ml of 0.1 M Ba(OH)2'8H20 (J.T. Baker Chemical Co., Phillipsburg, NJ) and incubating at room tempera- ture for 10 min. The precipitate was pelleted in a clinical centrifuge at medium speed, the supernatant was saved and the pellet was washed twice with 1 ml of deionized water; all supernatants were pooled. Oxime Formation: After precipitation of inorganic phosphate, oximes of a—ketoacids were formed by adding 190 ul (3.8 mg) of hydroxylamine-hydrochloride (2 g/100 ml pyridine, Fisher Scientific Co., Fair Lawn, NJ) vortexed and incubated at 80°C for 20 min. After cooling to room temperature the pH was adjusted to 6.5 using 6—8 drops of 2 M glacial acetic acid (Mallinckrodt, St. Louis, MO) before applying to a DEAE-Sephadex column. Isolation of Organic Acids from Plasma: The procedure for the isolation of organic acids from plasma is a modification of a procedure by Issachar and Sweeley, 1981 (55). Frozen plasma samples were thawed to room temperature and vortexed. A 0.5 ml aliquot of plasma along with 4 ul oleic acid (Calbiochem—Behring, La Jolla, CA) and 10 ug tropic acid were placed in a 20 ml screw top silanized test tube and diluted to 2 ml with deionized water. The mixture was sonicated for 30 seconds and placed in an ultrafiltration cell (Amicon Corp., Lexington, MS). The plasma was filtered over a diaflo-ultrafilter with a 1000 M.w. cutoff (Amicon, Danvers, MA) at 40 p.s.i. until approximately 100 ul remained. The test tube which 13 contained the plasma solution was rinsed with 2 ml of water and passed through the ultrafiltration cell. This procedure was repeated with an additional 2 ml of water. The ultrafiltrate (6 ml) was collected in a 20 ml screw top test tube (silanized) and inorganic phosphate was precipitated as its insoluble barium salt by adding 1.0 ml of 0.1 M Ba(0H)2-8 H20 and incubated for 10 min at room temperature. The mixture was pelleted and the supernatant collected. The pellet was washed twice with 0.5 ml deionized water and the supernatants pooled. Oximes were prepared by adding 35 ul (0.70 mg) of hydroxylamine- hydrochloride to the combined supernatants, vortexed and heated at 45°C for 45 min. After cooling to room temperature the pH was adjusted to 6.5 with 4-6 drops of 2.0 M glacial acetic acid before applying to a DEAE-Sephadex column. DEAE-Sephadex Chromatography of Urine Samples: The pyridinium acetate form of DEAE—Sephadex-AZS (Pharmacia Fine Chemicals, Piscataway, NJ) was prepared by swelling the resin in 1.5 M pyridinium acetate for 48 h at room temperature, with periodic changes of buffer. A 1.5 M solution of pyridinium acetate was prepared by adding 120.9 ml pyridine (MCB Manufacturing Chemists, Inc., Cincinnati, OH) and 87 ml glacial acetic acid and diluting to 1 l in a volumetric flask with deionized water. The DEAE—Sephadex-AZS was then equilibrated in 0.5 M pyridinium acetate for 24 h at R.T. with frequent changes of buffer. A 0.5 M solution of pyridinium acetate was prepared by adding 40.3 ml pyridine and 29 ml glacial acetic acid and diluting to 1 l in a volumetric flask with deionized water. A pasteur pipet (silanized) with a silanized glass wool plug was filled with 1.5 ml of DEAE-Sephadex and washed with 10 ml of 0.5 M pyridinium acetate before 14 applying the sample. A glass funnel (silanized) was fitted to the pipet with a piece of teflon tubing. The urine sample was loaded on to the column and then washed with 7.0 ml deionized water. Organic acids and other anionic molecules were eluted with 10 ml of 1.5 M pyridinium acetate. The eluent was collected in a 250 ml round bottom flask (silanized) and shell frozen in an acetone and dry ice bath. DEAE-Sephadex Chromatography of Plasma Samples: Plasma samples were applied to a DEAE Sephadex column at 4°C to prevent C02, a natural buffer in blood, from coming out of solution and destroying the column. A pasteur pipet with a glass wool plug was filled with 1.5 ml of resin and equilibrated with 0.5 M pyridinium acetate (4°C). After the sample had entered the column, it was washed with 7.0 ml of deionized water. As soon as the water wash was complete the column was transferred to room temperature and immediately eluted with 10 ml of 1.5 M pyridinium acetate. The move to room temperature allows easier elution of anionic molecules from the column than at 4°C. The eluate is collected in a 250 ml round bottom flask and shell frozen in a dry ice acetone bath. tyophilization of Urine and Plasma Samples: Urine and plasma samples were removed from the lyophilizer as soon as they were free of ice crystals but not yet dry. Flasks were rinsed 3 times with 1.0 ml of methanol and transferred to a 1 dram vial (silanized) with a Teflon liner screw top cap. Samples were evaporated gently under a stream of nitrogen at 37°C with a Meyer N-Evap Analytical Evaporator (Organomation Ass. Inc., Northborough, MA), and placed in an evacuated desiccator over phosphorus pentoxide (J.T. Baker Chemical Co., Phillipsburg, NJ) for 16 h. 15 Derivatization of Urine and Plasma Samples: Dry pyridine was prepared by refluxing 100 ml of pyridine over 5.0 g of barium oxide (MCB Manufacturing Chemists, Inc., Cincinnati, 0H) for 2h and distilled into a dry reagent bottle and stored in a screw top container containing Drierite. Sylon BSTFA (bis-trimethylsilyltri— fluoroacetamide with 1% trimethylchlorosilane, (Supelco, Inc., Bellefonte, PA) was obtained in 1.0 ml ampoules and stored in a container containing Drierite at -20°C. Derivatizing agent was prepared by adding BSTFA to dry pyridine in the ratio of 4:1. Using a 500 ul Hamilton syringe IOO ul of derivatizing agent was added to each urine sample and sealed immediately and incubated at 80°C for 1 h. Plasma samples were derivatized in the same manner except 50 ul of derivatizing agent was used. After heating, the entire sample was cooled to room temperature and transfered with a 500 ul Hamilton syringe to a 100 ul disposable micropipet which was broken in half and flame sealed at one end. The pipet was filled only halfway with sample and flamed sealed by pinching and pulling the end of glass away with forceps. The sealed samples were placed in a 10 ml screw top test tube and stored at 4°C in the dark. Before another sample was transfered the syringe was washed with a 1:1 mixture of methanol/chloroform and rinsed with dry hexane and blown dry with nitrogen. Dry hexane was prepared by refluxing 100 ml of hexane (MCB Manufacturing Chemists, Inc., Cincinnati, OH) over 5.0 g barium oxide and distilling into a dry reagent bottle and placed in a screw top container containing Drierite. Gas Chromatography-Mass Spectrometry Analysis: A 3 M X 2 mm id Pye Unicam gas chromatograph column (Sargent Welch, Skokie, IL) that fit an LKB 2091 gas chromatograph-mass 16 spectrometer (LKB Produktur, Stockholm, Sweden) was washed with soapy water six times by aspiration and rinsed with distilled water and acetone and dried in an oven at 80°C. The column was then silanized and rinsed with methanol and dried at 80°C. The column was then packed with 5% OV-17 on 100/120 Supercoport (Supelco, Inc., Bellefonte, PA). The detector end was fitted with a silanized glass wool and hooked to an aspirator. The injection port end was placed into the packing material. Aspiration continued while the column was gently tapped with a soft piece of plastic (A Sharpie will do) to make sure the column is packed tightly. The column was filled with OV—17 up to 3 cm of the injector port end and plugged with silanized glass wool. The packed column is conditioned at 290°C for at least 24 h in a gas chromatograph with the detector end disconnected and nitrogen flow rate at least 30 ml/min. Before sample analysis the mass spectrometer is calibrated with perflurokerosene. In actual analysis the oven temperature is programmed at 55—265°C at 3°C/min, nitrogen flow rate 30 ml/min, ion source temperature 280°C, electron energy 70 eV, trap current 50 uA and accelerating voltage at 3.5 kV. Using a 10 ul Hamilton syringe a 2-3 ul hexane chaser is drawn up and .2 ul of hydrocarbon mixture-decane (64 ul), undecane (70 ul), dodecane (70 ul), tetradecane (46 ul), hexadecane (51 ul), octodecane (50 mg), eicosane (66 mg), tetracosane (50 mg), and hexacosane (50 mg) dissolved in 10 ml hexane (Applied Science, State College, PA and Aldrich Chemical Co., Milwaukee WI) and 2 - 3 ul of sample is then taken and injected into the gas chromatograph. After the solvent front has passed, data collection is started and the spectrum is scanned from 35-630 a.m.u.'s every 4 seconds. Approximately 1000 scans are taken during each run and stored 17 temporarily on a Cailus disk drive on a pdp 8e computer system (DEC, Schaumburg, IL). Data are then transferred to a pdp 11/44 (DEC, Schaumburg, IL) computer system. All programs for the Pdp 8e were written by Norman Young (134). Pdp 11/44 programs were written by Lee Nestover except the mass spectral metabolite program (MSSMET). MSSMET was rewritten and reorganized with considerable additions made by Lee. All programs were written with the guidance of Dr. C.C. Sweeley and Dr. J.F. Holland. A difficult problem when comparing biological fluids is the choice of method for reporting data. In urine, for example, should it be reported in terms of mg/g of creatinine, mg/24 h of urine, or mg/ kg of body weight? Since the patient's liquid intake affects the volume of the urine and thus the concentration of metabolites, some investigators have relied on the amount of creatinine excreted as a basis for normalization. Creatinine, a by-product of creatine and phosphocreatine metabolism in muscle is related to the muscle mass of an individual (47). However, patients with Duchenne's muscular dystrophy have lower creatine phosphate and creatine in the muscle and reduced muscle mass, which reduces the concentrations of creatinine in the urine. Others have also shown that urinary excretion of creatinine in normal subjects is age dependent and thus doesn't accurately reflect the concentration of other metabolites, such as a-amino nitrogens (2). As was mentioned in the literature review, the excretion of 3—methylhistidine was used as an index for the rate of myosin and actin degradation (5,78, and 128). When expressed per unit of creatinine, an index of muscle mass (47), an increase in this ratio reflected an index in myofibrillar degradation. A problem arises though since l8 3-methylhistidine has been shown to arise from actin in nonmuscle tissues, skin and gastrointestinal muscle (80). Since urinary excretion of creatinine is depressed in patients with DMD, excretion of 3-methyhistidine from nonmuscle tissue would increase the 3-methyhistidine/creatinine ratio (94). Throughout this study an aliquot of urine representing 15 mg of urinary solids was used to normalize the sample. By standardizing each sample in this manner all operations (dilution, etc.) can be kept the same. The data are then expressed in relative terms of each metabolite in the 15 mg sample rather than on a concentration basis. In the case of blood samples, volume can be used as a parameter for normalization since blood volume in mammals does not change extensively. Data analysis using MSSMET: MSSMET is a mass spectral metabolite program developed by Gates gt 21-: (39). Data are generated by entering the volume (ml) of urine used, the concentration of 1 ml of urine in mg and the weight (ug) of internal standard (tropic acid). With this information the program uses the area of the designated ion which differentiates that compound from any other compound in the library and calculates its concentration relative to the area of the designated ion of tropic acid and the 15 mg of urinary solids using the following formula: R=kAcefl1 Ai'Cu'Vu where R is the ratio of ug of metabolite/mg of urinary solids, Ad is the area of the designated ion of a particular compound, A, is the area of the designated ion of the internal standard tropic acid, W1 19 is the weight of tropic acid added, Cu is the concentration expressed as urinary solids/ml of urine and Vu is the volume of urine processed. The constant (k) is a quantitation or response factor which can be determined using a pure reference compound. When a (k) factor is not available, the computer automatically sets the value to 1.0. Since the majority of the compounds in the library do not have a (k) value assigned to them, the concentrations reported here are relative and cannot be reported as ug of compound/mg urinary solids. Relative concentrations of metabolites in plasma samples were generated using the formula described above for urine; the Cu term was entered as 1 mg/ml. Results The number of organic acids and other compounds detected by MSSMET averaged approximately 156 for urine and 70 for plasma samples and 68 unknown metabolites. The mean relative concentrations and standard deviations of metabolites in urine and plasma from normal and dystrophic patients are shown in Table 2. Metabolites in urine and plasma shown in Table 2 were analyzed for statistical differences between the two groups of patients by selecting metabolites whose mean relative concentration was twice that of the other group or greater. The "student” t-test was used to determine the statistical significance for those selected compounds. In urine samples from normal and dystrophic patients, 11 metabolites (Table 3) were selected from Table 2 whose relative mean concentration were approximately twice that of the other group or greater and had a confidence limit of 95% or better. The majority of metabolites shown in Table 3 are depressed in patients with Duchenne's muscular dystrophy. Only phenylacetate, and p-hydroxycinnamate are elevated in dystrophic patients over controls. Some of the metabolites (shown with asterisks in Table 3) are shown in scatter plots in figures 2—4. Compounds X58, UN40, and UN59 were not represented in scatter plots even though the confidence limit was 95% or greater because their identity was uncertain; metabolites phenylacetate, and p-hydroxycinnamate were not included because of the extremely low level present in urine samples from both groups of patients. 20 21 In plasma samples from normal and dystrophic patients, 4 metabolites were selected from Table 2 whose relative mean concentration were approximately twice that of the other group or greater and had a confidence limit of 95% or better (Table 4). Two metabolites which were depressed in dystrophic patients when compared to controls shown in Table 4 with asterisks are represented in scatter plots in figures 5. They were chosen on the basis of their confidence limit of better than 95%. Metabolites labelled X1, UN3 were not included in scatter plots because they were unknown. Metabolites in urine from carrier women of Duchenne's muscular dystrophy compared to urine from normal women of the same age group are shown in Table 5. The same criteria in selecting metabolites from normal and dystrophic patients was used in selecting metabolites in Table 5 that were significantly different between carrier and normal women. Five metabolites whose mean relative concentrations were approximately twice that of the other group or better and exhibited a confidence limit of 95% or above are shown in Table 6. Oleic acid in normal women is elevated 30—fold, while the compound labelled X44 (an aspirin metabolite) is elevated SOO-fold over carrier women. Discussion The profiling of organic acids in biological fluids from human patients by gas chromatography-mass spectrometry offers the investigator a clinical picture of the metabolic state of the individual. Our profiling results show that 11 metabolites (representing approximately 7% of the compounds detected) in urine from normal and dystrophic patients and 4 metabolites in plasma from normal and dystrophic patients were significantly altered (within a 95% confidence limit). The remaining metabolites were not significantly different. The origin and/or significance of each of these metabolite differences will be discussed. Urine One group of metabolites which are significantly different between normal and dystrophic patients, is involved in the metabolism of tyrosine and phenylalanine. Figure 1 shows the metabolic interrelationships of tyrosine and phenylalanine; the metabolite with altered levels are bracketed; compounds which cannot be detected are underlined; the other metabolites do not exhibit altered levels. The major route of p-tyrosine metabolism involves transamination to p—hydroxyphenylpyruvic acid (p-HPPA) by the action of glutamate aminotransferase (62) and oxidation to p-hydroxyphenylacetic acid (p-HPAA) and conversion to p—hydroxybenzoic acid (p—HBA) (67); p-HBA is significantly decreased in dystrophic patients. p-HBA is also formed by dehydrogenation of p-hydroxyphenyllactic acid (p-HPLA) yielding 22 l r 23 p-hydroxycinnamic acid (p-HCA) (elevated in dystrophic patients), which can be directly converted to p-HBA (9) (figure 1). m-Hydroxyphenylacetic acid (m-HPAA), another compound which is significantly decreased in dystrophic patients (Table 3), can be formed from m-tyrosine, a metabolite of phenylalanine. Phenylalanine may either be m-hydroxylated to m-tyrosine (51) or oxidatively decarboxylated to phenylethylamine (62). The latter two compounds are both precursors to m-tyramine biosynthesis (51,23,10) (Figure 1). Other routes for formation of m-HPAA include p—hydroxylation of phenylalanine (10,51) to p-tyrosine followed by m-hydroxylation to 3-(3,4—dihydroxyphenyl) alanine (DOPA). DOPA is then oxidatively decarboxylated to 2-(3,4-dihydroxyphenyl)ethylamine (DOPamine) (51,11,46), or converted to 3,4-dihydroxyphenylacetic acid (DOPAC) (21). DOPamine is readily converted to the m-and p-tyramines (51,10) with formation of m—HPAA by oxidative deamination of m—tyramine or oxidative deamination to DOPAC and p—dehydroxylation of DOPAC yielding m-HPAA (45) (figure 1). The biosynthesis of phenylacetic acid (PAA) another metabolite of phenylalanine metabolism is formed by decarboxylation of phenylalanine to B—phenylethylanine and oxidatively deaminated to PAA (62) (figure 1). Other routes in biosynthesis of PAA include transamination to phenylpyruvate which can be metabolized further by reduction to phenyllactate and undergo oxidative decarboxylation (figure 1). The question at this point is: what is the significance of the reduced levels of m—HPAA and p-HBA and increased levels of p-HCA and PAA excreted by patients with DMD. The levels of all four metabolites could be influenced by enzymatic alterations resulting in aberations in 24 phenylalanine and tyrosine (including catecholanine) metabolism. A decrease in p—HBA and m-HPAA levels could also reflect an increased demand of phenylalanine and tyrosine for synthesis of proteins. An increase in protein synthesis in patients with DMD would require an increased flux of amino acids into proteins, and may decrease the steady state levels of metabolites from other pathways leading from phenylalanine and tyrosine. Factors influencing p-HBA and m-HPAA levels other than endogenous alteration(s) in phenylanine or tyrosine metabolism are diet and metabolism by gut flora (83,107,105). In age matched controls, urinary p-HCA was absent while in 5 patients with DMD extremely low level are excreted (Table 3). Booth gt gl., 1960, (9) has shown that p-HCA excretion in man and rabbit is absent indicating a rapid turnover in formation of p-HBA. The low levels detected is made possible by extreme sensitivity of the mass spectrometer. PAA like p—HCA, is present in dystrophic patients and absent in age matched controls (Table 3). A problem in analysis of free PAA is its extreme votality especially when organic solvents and lyophilization are used. Free PAA exretion contributes approximately 7% of total PAA excreted in normal individuals; the majority is excreted as a glutamate conjugate (57). It is likely that most or all of PAA from dystrophic and normal patients was lost due to evaporation. However, it can be argued that free PAA was excreted in larger quantities in dystrophic patients. A good way to protect free PAA from evaporation is to form a salt with triethylamine (44). The remaining 7 compounds which are statistically different in urine from normal and dystrophic patients will be discussed individually. Three of the compounds labelled X58, UN4O and UN59 are 25 unknown, their function and origin cannot be commented on at this time. Glycerate D-Glycerate is formed by the reduction of 3-hydroxypyruvate which arises during the transamination of serine. In mammals, oxidation of L-galactonate by pig liver L—furonate-hydro-lyase yields 2—keto—3-deoxy—L—galactonate, a substrate for 2-keto-3-deoxy-L—fuconate NAD oxidoreductase that generates L-lactate and glycerate (19). Two types of glycerate acidemias occur in humans called L and D-glyceric acidemia (Table 1). L-Glycerate accumulates in large quantities in the catabolism of serine. A defect in D-glyceric dehydrogenase (glyoxylate reductaSe) (EC 1.1.1.26) is thought to prevent reduction of 3-hydroxypyruvate to D-glycerate. The level of 3-hydroxypyruvate thus increases and is reduced to L-glycerate by the action of lactate dehydrogenase (131). D-Glycerate accumulates in D-glycerate acidemia in serine catabolism but the primary defect is unknown (11,126). It is possible that in serine metabolism after transamination to 3-hydroxypyruvate and reduction to D-glycerate, that glycerate kinase is defective, preventing D—glycerate to proceed onto 3—phosphoglycerate. In this study D- and L—glycerate cannot be separated, rather a peak corresponding to a mixture of D- and L-glycerate is present. In summary, a decreased level of glycerate in patients with DMD (Table 3) may reflect some defect in serine metabolism. However, diet may also affect the levels of urinary glycerate. 26 4-deoxythreonate: Three-2,3-dihydroxybutyrate (4—deoxythreonate) classified as a deoxytetronic acid is an oxidized form of'aydeoxyaldoses (87). It was first characterized in infant and adult urine by gas chromatography—mass spectrometry as a normal excreted metabolite (124,16) and found to be excreted in greater quantities with increasing age (124). The level of this compound in urine and plasma samples from dystrophic patients is significantly decreased. The metabolic basis for such a decrease is unknown. Degradation of carbohydrates in normal metabolism may account for its presence (32). A study of excretion patterns of organic acids under different dietary conditions revealed little fluctuation in the deoxy—sugars except for 2-deoxytetronic acid (15). This suggests that 4—deoxythreonic acid may be fonned endogenously (71). m-Hydroxyphenylhydracrylate, (3-hydroxy—3—(3-hydroxyphenyl)- propanoate: Patients with DMD exhibited an almost 3—fold decrease in urinary excretion of m-Hydroxyphenylhydracrylic acid (m-HPHA) when compared to age matched controls. This difference may be the result of dietary differences between the two groups of patients. m-HPHA is a major constituent of human urine ranging from 2 to 150 mg/day (3). The excretion of m-HPHA, first identified by Armstrong and Shaw (3), was lower in patients with neurological disorders while phenylketonuria patients excreted normal to higher levels when compared to healthy individuals (3). The level of m-HPHA excreted was attributed to the type of diet the patients consumed. The levels of m-HPHA can be decreased dramatically when a defined diet is employed (3) or when glucose is the sole dietary intake (115). The origin of m-HPHA is 27 probably from m-hydroxycinnamic or phenylpropanoic acids occurring in plants (4). §;(3,4-dihydroxyphenyl)-propanoate, (hydrocaffeic acid): The precursor of hydrocaffeic acid is probably caffeic acid. Caffeic acid can arise from injestion of coffee or tea (83). The 3-fold elevation of hydrocaffeic acid in normal patients (Table 3) is probably diet related. Plasma Four metabolites in plasma from normal and dystrophic patients were found to be significantly different (within a 95% confidence limit). The metabolite labeled X1 was decreased in dystrophic patients while the metabolite labeled UN3 was increased almost 3-fold in dystrophic patients (Table 4). The origin and function of these two compounds is unknown. The deoxy-sugar, 4—deoxythreonate previously described in urine samples, is interesting to note, since the relative concentration in plasma is also decreased in patients with DMD. The significant decrease in citrate in plasma from patients with DMD is subject to question. The isolation procedure of organic acids from plasma as well as urine employs Ba(OH)2-8H20 to precipitate phosphate and sulfate as their insoluble barium salts. Barium salts of carboxylic acids are soluble except for a few highly polar organic acids such as citrate, oxalate and tartarate (124) that are partially lost using this procedure (123). Carriers Urine samples from carriers of Duchenne's muscular dystrophy and normal age and sex matched controls were analyzed. The origin and identity of compounds labeled UN4 and X27 (Table 6) which are increased 28 and decreased in carriers respectively are unknown and cannot be commented on at this time. The metabolite a-hydroxyisobutyrate (Table 6 and figure 6) is elevated almost 2-fold in carriers; possibly a metabolite of a—oxidation of branched chain fatty acids (56). a-Hydroxyisobutyrate is increased in urine samples from patients with maple syrup urine disease but disappeared with a defined diet (56). The origin of this compound has not been investigated. In three of the normal women, 2 metabolites were extremely elevated eluting before the hydrocarbon tetracosane. The 2 metabolites X44 and oleic acid that coeluted were elevated 500 and 30-fold over carriers respectively. It was determined that one of the three women had injested aspirin before collection of the urine sample and was assumed that the other two women had also. The designated ion for X44 is m/z 324 while confirming ions are m/z 206 and 193 which predominate in the mass spectra in the three urine samples with elevated levels of X44, while the confirming ions of oleic acid are low in abundance. Other investigators (43,17) have shown that 2-hydroxybenzoate and 2-hydroxyhippuric acid are elevated in urine especially the latter when aspirin is injested. The mass spectra of 2-hydroxyhippuric acid correlates well with the metabolite labelled X44 (48). Oleic acid is present, but is buried in the 2-hydroxyhippuric acid peak. The indicative relative concentration of oleic acid in the three samples is probably inflated due to interference from the molecular ion of 2-hydroxyhippuric acid, m/z 339; this is also the m/z value of the designated ion of oleic acid. As mentioned before in the "Results" section the mass spectral metabolite program, MSSMET, uses the area of i cul ar compound. 30 Table 1 Organic Acidemias Disorder Enzyme affected Reference Leucine Metabolism; Isovaleric Acidemia isovaleryl—CoA dehydrogenase 121 3-methylcrotonylglycinemia 3-methylcrotonyl—CoA carboxylase 29,113 3-hydroxy-3-methylglutaric 3-hydroxy-3-methylglutaryl-CoA 119 acidemia lyase (E.C.4.1.3.4) Isoleucine and Valine Metabolism: 2-methyl—3-hydroxybutyric acidemia B-ketothiolase 22,95 Propionic acidemia propionyl-CoA carboxylase 50 Methylmalonic acid acidemia; Type 1 methylmalonyl-CoA mutase 113,84 (apoenzyme) (E.C.5.4.99.2) 18 Type 2 defects in biosynthesis of cobalamine coenzmne Type 3 Methylmalonyl—CoA racemase Lysine and Tryptophan Metabolism: 2—ketoadipic acidemia 2-ketoadipate reductase 91,116 Glutaric acidemia glutaryl-CoA dehydrogenase 42 (E.C.1.3.9.7) Type 2 multiple acyl-CoA dehydrogenase 92 Pyroglutamic acidemia glutathione synthetase 58 (E.C.6.3.2.3) Tyrosinemias: Type 1 unknown 13 Type 2 tyrosine aminotransferase 106 (E.C.2.6.1.5) and 4-hydroxyphenyl- pyruvic oxidase (E.C.1.13.11.27) Lysine and Tryptophan Metabolism Hyperglycerolemia glycerol kinase 90 Lactic aciduria and lactate dehydrogenase 68 acidosis D-Glyceric acidemia unknown 11 .m—m>m— uwgmp—w awawzxm uo: ow mmpwponmpue Lospo ms» mumcwpcwccs mew umuomuou we poccmo sows: muczoaeoo .umawxomen mew me>mF wpwponwuos uwgmpp< .wcwmogxpuq ucm mcwcmficpxcmsq we Emmponmuwe asp cw mxwzcuma owFonmumz .~ wczmwd 32 IOOo-N IONQ\ ofim%o\\ «£60378 278.37.: A<3>q§cond IOOO I NI© LI 2082;5an IooUon-NI 24.: a N Iooo- Io- NIo <&n_Iu (JQI Q IOOQ: IO IO :0 Io EmI-s o-NWU Io Iooo-w-NI IoooI Io /2/o+n.o& Q o @ Iooo Iooo-IonIo 33 I. 000. 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The average relative concentration of 4 metabolites in plasma samples from normal and dystrophic patients selected from Table 2 whose mean relative concentration was approximately twice that of the other group or greater. The standard deviation and the confidence level determined by the "student" t-test are given for each metabolite. COMPOUND NORMAL (11)b DYSTROPHIC (12)b P VALUEa CITRATE .579 I .713 (6)C .057 I .123 (3) <.025* x1 .576 I .397 (9) .226 I .296 (7) <.05 UN3 .873 I 1.26 (9) 3.53 I 2.90 (12) <.025 4—DEOXYTHREONATE .255 H .137 (10) .002 l+ .105 (6) <.005* a Determined by student t-test. b Number of plasmas examined. C Number of samples containing this metabolite. * Metabolites shown in scatter plots. 47 Income-n ID—‘Qtn Mmm m AAA A MAAA AAA—AA AAA m 6") M Ln Lb AAA AA [.0 o—l L") 4529.39: 2..» PIE—5:8 3353 we $9.52 u .vm~>_mcc mopasmm he Leczsc Pmuo» a .38 3.8: 32:53 22:35 93 3 uni—Eng!” . o.~A A 5.9“ .w. A..m A c.~. u_zoz=.>=m>xomo-e . o.m_ A ..IN ... A... 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Nzo_ouz.o_z_a»a-e.N u.zoz.mh>xowo-m .o.=_oA-o. u.oN2Nm.>=AAz-o Q_ANUZN=N u.o_oNzN.=I-m.N u_m<==I NAZI 0.2.002m u.pm0<.>xwzo.u>u mmx 0.20m::»>xswcuv 0.e~zwm 2:30.290 Avwacvucou. m w—aw» 49 ... CNN. A IAN. ... N... A IAN. .0__8Aa._AA.AAAAI. NAxozo>=-N ... o.m. A can. ... mm.N A ANm. N..NUZN=I.XC=.N2-I MAW w.n. A AN.. .w. mA.A A om.m .Ae_xo A.ALAA=.NA.A.-I. N=.xo u.o.=Nz<.z.Ic.o-N m ow.. A o... ... o..m A N.N. . x=-m oo.o ... Awm. A AoN. .0.L=A_. N.ozxo=..2-I .m. om.m A Ao.A ... mN.m A om.. ovz: .A. ANI. A mm.. .m. nmm. A AA._ U..NU<.>ZNIN>XOIQ.=-O “AW owe. A AA.. oo.o .U.AAA..AIAII-A. u.ozzwza-m-..omo>I-N N mo.. A mam. .m. mo.m A mN._ mmz: .m. m... A No._ .m. AN.. A NAN. .6.EA.=_A. u_o.omz<.zmeoz.z<-N .m. .m.o A N.m. .m. No.m A .N.N . u.og=.-N-u.oszm.xoz=.=-m MN. omm. A Nmm. Mm. .wm. A N... .U_EIA=.ooe>a. u_.>xommN-N N. ON.. A mm.. A. m... A No.. u.oszm.xozN.z-z .m. eo.. A om.m .0. mm.. A mm.m mmzs oo.o ... man. A No.. 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A AAA. AA ANN. A AII. Au_=.Zo=A=A. I.IIZIAZAI.IAZ.I.AZA-A.N MA. AAN. A AAA. MNW Amm. A AAA. ZAALAALA>AEAI. u_AIUAI»ZwZAAAAZAAZAI-A.m N. AN.m A no.m A mo.w A IA.m AAI AN. AA.” A AA.A AA. Ao.A A AA._ N AAAA-UZIAAAIIZAAI. I.INZAIZZ0:_IZ_I-A.A MAW m.NN A w.AA MNW w.AN A A.Nm A.IZZIAZIZZIZZIZAZAIZIIZ-= m m.NA A A._A A mm.N A AA.N A.AMAAI>ZAZAZA¢ZI>Z_a-m.N AA. AA.N A A... AA. AI.N A AA.A IZAAIAIZZAZZIAAAIZZ_I-A.A Am. mN.N A m.A. AN. IA.A A N.AA AAAA__=~>AEAZ. I.AWIAIZZMZAAZIZANZ-A-ZZIAIZZ-A AA. ..IA A AA.A AA. IN.N A IN.A AZIAIAI-A.I-IZOAIAAA. I_oNme>AIZAIZ-A->AIZIZZ-A oo.o A AA.. A AI.A A AANA-NZAZAAIAZIAAAIAA AA. AA.A A AA.A AN. AA.A A Am.o AmZI AA. AA.N A NAA. AA. A.AA A IN.A AmZI AA. AA.A A AI.A AA. NA.A A AA.A AAZI Am. AA.N A AA.N An. AN.N A IA.A ZA._IALAAA.-A. AZINZWIAZAAIZZ_I-A.N Am. AA.A A Am.A AA. AA.. A AAA. ZA._A_=~>. I.INZIIZZIZAWZ-A-ZAIZIZZ-A AI. mow. A NAN. AA. IA.N A NA.A AA__AZAZAAAAA. I.IZAAAAAIZIAZINZHA-A.I Am. A.N. A AA.A Am. AA.A A AA.N UZZA.IIA_ An. AA.A A AA.N ”A. NA.A A IA.A AA_A_A~A. u.o_IIZAZIZ AA. AA.I A AN.A Am II.I A AN.A I.IZAAIIAZAIA ZN. AAA. A AIM. “A NA.N A AAA. I AAAZ-A.ZIAIAAAonzA-m.-m quz<0ucxoxc>zuz mAz: AAAzAAAA-MAAAAAzA-A.-A vAz: quzmumax evx vozZA:LIA quza u_ozAoczAum CA2: 22:0;zoo Atw=:_ucoo. m AAAAA .muwronnwme scam Low cm>wm mgm ,mocouwmcoo use cowpmguzmocou w>wuurwg cams wsb .czogm mgm mucmwuua owsaogumxv vac FwELo: sag» mama—q cw ucsom mumcomgszxowuu¢ vac muaLuwu we mac—q mepmom .m meamvm 55 N .082! I05 D 4-deoxythreonic 2551137 N P < .005 D LOO .50 IO 0571123 D 579: TB N o o o ‘2 3 O 56 Table 6. The average relative concentration of 5 metabolites in urine samples from normal and carrier women selected from Table 5 whose mean relative concentration was approximately twice that of the other group or greater. The standard deviation and the confidence level determined by the "student" t-test are given for each metabolite. COMPOUND NORMAL (5)b CARRIER (7)b P VALUEa a—HYDROXYISOBUTYRATE 24.3 i 14.8 (5)c 45.2 1 13.1 (7) <.05* UN4 10.8 113.8 (3) 31.3 1 6.91 (7) <.01 X27 29.0 i 17.3 (5) 10.1 i 11.5 (5) <.05 X44 (aspirin metabolite) 503 i 571 (3)d .510 i 1.25 (2) <.05 9-0CTADECENOIC (OLEIC) 28.7 1 33.0 (3) .179 i .510 (1) <.05 a Determined by student t-test. b Number of urines examined. C Number of samples containing this metabolite Personal comunication * Metabolite shown in scatter plot. Figures 6. Scatter plots for a-hydroxyisobutyrate found in urine from carriers of DMD and normal age matched women is shown. The mean relative concentration and confidence limit are given. l00.0 50.0 10.0 c N (I- hydroxyisobuiyric P < .05 Summary and Conclusions The main objective of this study was to compare metabolite levels in urine and plasma from normal and dystrophic patients and to screen metabolite levels in urine from carriers and normal women. Nine of the 11 compounds in urine that were found statistically different between normal and dystrophic individuals were decreased in dystrophic patients. However, the levels of none of these metabolites are consistently different between normal and dystrophic patients. Two of the compounds that were depressed, m-hydroxyphenylacetate and p-hydroxbenzoate are metabolites of phenylalanine and tyrosine metabolites. Four other metabolites, glycerate, 4-deoxythreonate, hydrocaffeic acid and m-hydroxyphenyhydracrylate function in other pathways in metabolism. The remaining 3 metabolites are unknown. Two metabolites phenylacetate and p-hydroxycinnmate were elevated in the urine of dystrophic patients, the latter two were not detected in normal controls. Free phenylacetate was probably lost by evaporation, however, it was detected in dystrophic patients. Since these two metabolites were present in very small amounts, additional work is necessary before any significance can be given to the data. Free and conjugated PAA excreted in the urine from normal and dystrophic patients could be measured by converting free PAA to a less volatile triethylamine salt. If free PAA is found to be elevated in dystrophic patients it would then help explain why decreased levels of m-HPAA and p-HBA are seen (see figure 1). Decreased levels of m-HPAA and p-HBA may 59 60 be the result of an increase in phenylalanine and tyrosine incorporation into protein synthesis. Thus the steady state levels of m—HPAA and p-HBA could be affected. If an increased flux of phenylalanine and tyrosine into protein synthesis is correct, one would expect that other metabolite levels in phenylalanine and tyrosine metabolism may also be affected. The levels of precursors to p-HBA formation are detected (these include p—HPLA, p-HPPA and p-HPAA) however, they remain unaltered when compared to normal controls. The activity of the enzyme(s) involved in conversion of p-HPAA to p-HBA may be lower in dystrophic patients. This would probably be a secondary effect of the disease. Precursors to m-HPAA formation include the phenylamines, m-tyrosine, phenylethylamine and m-tyramine. Unfortunately these metabolites cannot be detected using this purification procedure. The levels of these metabolites mentioned above could be measured though by keeping the neutral and cationic fractions from the DEAE-Sephadex column. Other precursors of m—HPAA include the catecholamine metabolites DOPamine and DOPAC. Although DOPamine cannot be detected, DOPAC is and remains unchanged in dystrophic patients. This agrees well with studies done by Dalmaz gt al., (1979), (21) however, they found an increase in urinary levels of DOPamine in patients with DMD. This increase in DOPamine levels may affect the levels of m—HPAA in dystrophic patients (see figure 1). The variables inherent in this type of study are many. Variation in metabolism between individuals, probably the major factor (15), can affect metabolite levels. Differences in physical activity could also have an influence. Metabolism by gut flora does contribute to the level of some metabolites (mainly phenolic acids). Variation in diet 61 between individuals can also influence the levels of metabolites, but the extent is not great (15). Four of the patients with DMD had consumed some type of medication (appendix B). However, the medication did not seem to change any metabolite level or produce any unusual metabolites. Only in the case of injested aspirin in control women did an unusual metabolite occur. The literature contains nothing concerning the origin or function of 4-deoxythreonate. It may or may not be advantageous to pursue how this metabolite functions in metabolism and why a decrease is seen in dystrophic patients. The urinary excretion patterns of 4-deoxythreonate have been studied by other investigators. A decrease in urinary levels occurs when normal patients switch from a low carbohydrate diet to a diet high in carbohydrates (15). This is contradictory to what was stated under ”Discussion”, that the precursor to 4-deoxythreonate might be from carbohydrates degradation. You would expect a switch from low to high carbohydrate diet to increase the amount of 4-deoxythreonate excreted in urine if this is correct. In plasma samples from normal and dystrophic patients, the compound labelled UN3 is elevated significantly (over 3-fold) in dystrophic patients. It would be interesting to determine the identity of this compound and why urinary levels are not altered. One of the primary reasons for measuring urinary metabolite levels in normal and carrier women was to try and correlate any metabolite differences to the same metabolite(s) altered between normal and dystrophic patients. This could then have been used as another diagnostic tool to help aid in detection of carriers of DMD. However, such a correlation was not present. Appendix A Instruction and Questionnare Sheets INSTRUCTIONS Please follow these instructions as carefully as you can. 1. If possible, do not take any non-prescription drugs or alcohol for 72 hours (3 days) prior to the time you collect urine. However, it is important that you do not discontinue any drugs important to your health. Since even simple drugs like aspirin are known to affect the composition of urine, it is easier for us to interpret the results if we know whether you have taken any drugs or alcohol within the past 72 hours. If at all possible, please fill out the attached yellow form. 2. The day before you donate your sample, eat breakfast, lunch and dinner as normal. However, we ask that you refrain from eating or drinking (except water) after 7 p.m. the evening before you collect urine. If for any reason something is consumed after this time, please make a note of the item(s). 3. In the morning before eating breakfast, collect the urine sample as follows: Start to urinate, allow first portion of urine to go directly into the toilet. Then use the container provided to collect the remainder of the urine. Do not worry about having too little urine for us to use, as a very small amount is needed for the analysis. 62 63 4. Put the plastic cap on the container of urine: make certain it is tight. You may rinse the outside of the container with water if you wish. Place urine sample into the plastic bag provided. 5. Place the age, sex, and first name only of the donor on the label. All information will be kept strictly confidential. 64 DIETARY SURVEY Why the need for a fasting morning urine This is because the composition of urine is influenced by what you eat and drink. By not eating after your normal dinner, you give your body time to digest dinner and eliminate products of that meal. Much of what appears in your urine in the morning, then, will be a reflection of your body's natural night-time activities rather than the contents of any snacks or other food or drink consumed after dinner. However, should you forget and eat anyway, note that information. DRUG AND MEDICATION SURVEY In the 72 hours (3 days) preceding the time of urine collection, did you consune any of the following Yes No 1. Vitamins 2. Diet pills 3. Birth control pills 4. Sedatives 5. Aspirin or Anacin or Bufferin 6. Marijuana 7. LSD 8. Heroin 9. Alcohol or alcoholic beverages 10. Other non prescription drugs (specify which) 11. Prescription medication (specify if known) 12. I have consumed one or more items from 6 to 10 above. However, I would prefer not to specify which drugs(s). Age Sex Appendix B Summary of urine samples These data are a summary of creatinine concentrations, weight of urinary solids per 1 ml of urine and volume of urine analyzed. Information obtained from information sheets handed out are also included (appendix A). 65 Normal patients (urine) mg/ml Identification Age Sex creatinine Dgpgs 123128271 9 F 1.04 None 123128272 9 M .810 " 123128273 6 M 1.58 ” 123128274 10 F 2.53 ” 124128273 9 F 1.14 ” 104028274 15 M 1.19 " 105028272 10 F 1.79 " 113028271 6 M — ” 113028272 8 M .900 " 113028273 9 M 1.08 ” 114028271 7 M 1.82 ” 114028271 11 M 1.49 ” 114028273 11 M 2.69 ” 114028274 10 F 1.79 ” 125028271 10 F .670 ” 101058271 12 M 1.84 ” Average and Standard deviation 1.51 :.610 volume of urine solids/ml analyzed urine ml mg .62 24.3 .44 34.5 .26 57.5 .26 57.9 .31 I 47.9 .37 40.4 .28 54.1 .23 66.0 .34 44.0 .55 27.5 .22 69.1 .34 44.0 .31 47.8 .35 42.8 .44 33.9 .26 58.9 47.0 i 13.1 66 Dystrophic patients (urine) volume mg/ml of urine solids/ml Identification Age Sex creatinine Drugs analyzed urine ml mg 106018271 12 M .180 None .61 24.5 106018272 15 M .590 ” .29 51.9 106018274 10 M .520 ” .35 42.7 109018271 10 M .500 ” .39 38.3 109018272 20 M .430 ” .30 50.5 104028276 9 M .530 ” .17 90.5 104028277 11 M .710 ” .22 68.9 105028275 16 M .220 ” .33 45.3 107048271 13 M .470 Tofranil .30 50.2 107048272 10 M .270 Phenobarbital .39 38.1 107048273 9 M .970 None .29 51.7 107048274 8 M .480 Allopurinol .33 45.7 107048275 12 M .520 None .29 51.1 109048271 8 M .460 " .41 36.5 130048271 14 M 1.12 " .20 73.4 130048272 10 M .410 ” .26 56.5 Average and Standard deviation .524 i .245 51.0 i 15.9 Identification 101058275 101058276 101058277 105058271 105058272 Ass 37 29 4O 4O 34 67 Normal Women (urine) mg/ml ereatinine average and standard deviation I06018273 130048273 130048274 130048275 101058272 101058273 101058274 average and standard deviation 34 31 34 36 44 Drugs of urine aspririn none aspirin Carrier Women (urine) 2.17 1.07 1.14 1.46 1.00 1.43 + none H hydrduiril .427 volume analyzed mg .51 .28 .43 .30 .33 .43 .48 .47 .33 .33 .36 .40 ml 54. 54. 35. 50. 45. 48. 34. 31. 31. 45. 45. 41. 37. 38. #4:- m-nP-N 1 solids/ml urine i 8.00 1+ 5.92 10. 11. 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