This dissertation has been 65—13,267 microfilmed exactly as received PENICK, Ronald Jack, 1933- STUDIES ON GANGLIOSIDES FROM HUMAN AND CALF BRAIN. The Ohio State University, Ph.D., 1965 Chemistry, biological University Microfilms, Inc., Ann Arbor, Michigan STUDIES ON GANGLIOSIDES FROM HUMAN AND CALF BRAIN Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Ronald Jack Penick, B.A., M.Sc. t t X-XXXX The Ohio State University 1965 Approved by Advisor Department of Physiological Chemistry ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. Robert H. McCluer for his suggestion of the problem area and for his invaluable guidance and encouragement throughout the course of this investigation. Sincere thanks to Mrs. E. Coram, Mrs. E. Hock, Mr. E. Prominski, and Mr. W. Hayhow for their technical assistance in various stages of this study. Deepest appreciation is also extended to my wife and family for their forbearance and understanding during this period of inten­ sive study. This graduate program has been made possible through the Depart­ ment of the Air Force, Air University, Air Force Institute of Tech­ nology, Wright-Patterson Air Force Base, Ohio. CONTENTS Page ACKNOWLEDGMENT i i TABLES i i i FIGURES iv PLATES v ABBREVIATIONS _ vi STATEMENT OF PROBLEM 1 HISTORICAL 3 Introduction 3 Thin Layer Chromatography of Gangliosides 5 Ganglioside Structures 8 Gas-Liquid Chromatography of Carbohydrates 12 EXPERIMENTAL 14 Instrumentation 14 M aterials and Standards 15 P reparation of Mixed G angliosides 17 Isolation of Individual Gangliosides 18 Qualitative Analytical Methods 19 Thin Layer Chromatographic Procedures 19 N-acetylneuraminic acid 21 Hexosamine 21 Hexose 22 F a tty acids 22 i i i Page Quantitative Analytical Methods 23 N-acetylneuraminic acid 23 Hexosamine 23 Hexose 24 Glucose and G alactose 24 Sphingosine 27 RESULTS AM> DISCUSSION 28 Glucose and Galactose Assay by Gas-Liquid Chromatography 28 Ganglioside Analyses 48 Neuraminidase Data 54 Fatty Acid Data 57 Thin Layer Chromatographic Properties of Gangliosides 58 SUMMARY 66 BIBLIOGRAPHY 68 AUTOBIOGRAPHY 72 iv TABLES Table Page 1 Relative Retention Times of Some Carbohydrate TMS D eriv ativ es 30 2 GLC Precision Data 37 3 K3 Data 40 4 K4 Data 41 5 fCL Data 43 6 Summary of Hexose Analyses 47 7 G anglioside Component Analyses Data 50 8 Molar Ratios of Ganglioside Components 51 v FIGURES Figure Page 1 Structure and Nomenclature Summary of Brain Gangliosides 11 2 Retention Time vs. Flow Rate 33 3 Theoretical Plates vs. Flow Rate 34 4 Theoretical Plates vs. Temperature 35 5 Gas-Liquid Chromatogram of Hexose Assay 46 v i PLATES P late Page I TLC Patterns of Mixed Gangliosides 59 II TLC Patterns of Individual Gangliosides 61 III Ganglioside TLC Mobilities 62 v i i ABBREVIATIONS GLC Gas-liquid chromatography TLC Thin layer chromatography HMDSZ Hexamenthyl-disilazane TMCS Trimethyl-chlorosilane TMS Trimethyl-silyl GalNac N-acetyl galactosamine NANA N-acetyl neuraminic acid Glu Glucose Gal Galactose Mann Mannitol STATEMENT OF THE PROBLEM Ganglioside is a terra coined by Klenk (l) in 1935 to descri-be a glycolipid preparation which he obtained from the brain of a Tay- Sachs* disease victim. This preparation was shown by Klenk and later by Blix (2) to yield a purple color with the orcinol reaction. This color was demonstrated to be caused by sialic acid, an acidic car­ bohydrate. Klenk (l) reported the presence of sphingosine, stearic acid, glucose, galactose, galactosamine and sialic acid in his gan­ glioside preparation. During the next few years, there were few reports concerning ganglioside. In 1958, Bogoch (3) reported a molecular weight of 250,000 for his ganglioside preparation as determined in an aqueous media by ultracentrifugation techniques and concluded that the mat­ erial was polymeric in nature. Two years later, Klenk and Geilin (4) substituted N,N* -dimeth- ylformamide for water and determined the molecular weight of their ganglioside preparation. They calculated values between 1000 and 2000. At about this same time, investigators began to report that the ganglioside preparations were heterogeneous. Attempts to iso­ late and characterize individual gangliosides resulted in disagreement concerning the molar ratios of the ganglioside components. Judging the purity of the ganglioside preparations was a problem which each investigator had to consider and by 1961 the most common solution was to demonstrate homogeneity with a thin layer chromato­ graphic assay. The value of this homogeneity test was complicated, however, by the fact that the various investigators were using dif­ ferent solvent systems for the assay. Thus, correlation of ganglio­ side data from one laboratory with that from other laboratories was d i f f i c u l t . Attempts to characterize the individual gangliosides by component molar ratios indicated a requirement for reliable glucose and galac­ tose assays at the sub-milligram level. Therefore, an investigation was undertaken to develop analytical procedures to meet these require­ ments. This study resulted in the development of a new gas-liquid chromatographic method which was suitable for the simultaneous quan­ titative analysis of glucose and galactose from sub-milligram amounts of ganglioside. When thisGLC assay was used to characterize a preparation which appeared to be an unreported ganglioside, the data suggested that it was a mixture even though the TLC assay of the preparation indicated that it was homogeneous. Subsequently, it appeared from a study of the most recent literature that no single TLC solvent system was adequate to distinguish the numerous ganglioside species which were reported. Therefore, it was decided to investigate the TLC properties of all the ganglioside preparations available in this laboratory in four commonly used solvent systems. When possible, these data would be compared to data reported in the literature. It was anticipated that these studies would result in a more complete understanding of the chemical nature of the individual gang­ lio s id e s . h isto rica l Introduction In 1935 Klenk (l) first reported the isolation of a glycosphing- olipid which contained a sialic acid residue and coined the term ganglioside. Many workers have since conducted studies concerning the chemical and biochemical properties of ganglioside preparations. During the last two years, Svennerholm (5), Brady et al. (6), and Carter et al. (7) have published reviews which discuss advances made in isolation and characterization of this family of complex glyco- lip id s . Svennerholm (5) discussed the evidence which indicates that the major gangliosides are four in number and vary only with respect to the number and/or the position of the sialic acid residues. One of these gangliosides is a monosialo molecule which is resistant to the hydrolytic action of neuraminidase from V. cholerae or Cl. perfrinqes. The other three major gangliosides can be con­ verted to th is molecule by th e action of th is enzyme according to th e following scheme* ^^^ydisialo I t r i s i a l o — —< r Z —> monosialo ~'~>disialo II The monosialo ganglioside is considered to have a basic structure which is common to all four major gangliosides and may be described as follows* N-acylsphingosine-(l*-i)Glu-(4«-l)Gal-(4«-l)GalNac-(3<-l)Gal 3 r 2 NANA Klenk and Gielen (8) observed a hexosamine-free-disialo-tri- hexoseganglioside from human b rain in which g alacto se was th e only hexose found. Within the last year, Kuhn and Wiegandt (9) reported the isola­ tion of several less complex gangliosides from human brain. These molecules were reported to occur in the order of one to five percent of the total ganglioside preparation. Tettamanti et al. (10) offered evidence for a disialo tetrahex- ose ganglioside, a tetrasialo-trihexose ganglioside, and a second monosialo-trihexose ganglioside, which they isolated from pig brain. Svennerholm (11) recently reported the ganglioside profile from foetal, child and adult brain. This profile was based on the thin layer chromatographic pattern of the mixed gangliosides. From these data he noted that the complexity of the gangliosides increases with age. He also concluded that the ganglioside content increases with age up to about five years, levels off, and then decreases slowly after about forty years. Many investigators have reported changes in the ganglioside profile as affected by various pathological states. This area was extensively reviewed through early 1963 by Johnson (12). This author also discussed the matabolic studies of gangliosides. Other workers have recently reported metabolic studies. O'Brien (13) reported an abnormal ganglioside distribution in metachromatic leukodystrophy patients. Zeman and Alpert (14) studied the gang­ lioside content of brains affected by juvenile amaurotic idiocy. Philippart and Menkes (15) studied patients with Gaucher's disease and proposed a metobolic block involving the catabolism of ganglio­ sid es. Suzuki (16) described a cell-free system for the incorporation of D-glucose into gangliosides, and Kanfer et al. (17) reported the incorporation of N-acetylneuraminic acid into a monosialo ganglioside. Mcllwain (18) recently discussed some of the possible physiolog­ ical roles of gangliosides in brain tissue. Thin layer Chromatography of gangliosides As mentioned earlier, many workers who investigated the proper­ ties of gangliosides and related materials developed various isolation procedures and thin layer chromatographic solvent systems for examining the homogeneity of their preparations. Unfortunately, many of the early reports did not adequately describe the thin layer chromatograph­ ic system used to test the homogeneity of the isolated gangliosides. In 1960, Yfeicher (19) described the use of n-propanol/N NH^O U/ water (6/^1)* for the separation of gangliosides on a thin layer chromatogram. A year later, KLenk and Gielen (20) isolated four fractions from a ganglioside mixture. Two of these fractions migrated as individual spots on a thin layer chromatogram when irrigated with n-butanol/pyridine/water (3/^1).