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). KLenk and Gielen arbitrarily iden­

tified each distinct chromatographic band with a letter, A through D,

*A11 solvent ratios are reported v/v/v. with ganglioside A having the fastest rate of migration in this TLC system. A later report from KLenk*s (21) laboratory indicated that frac­ tion B was further subdivided into two fractions B and B • However, 1 2 the authors failed to describe the TLC properties of either of these subfractions.

During the same year, Wagner et al. (22) reported ,the use of chloroforn/ methanol/ water (60/35/8) for the separation of ganglio­

sides. Subsequently, other workers(23, 24) reported separating sam­ ples of mixed gangliosides into five components with the aid of this

solvent system. Mlieldner (25), in 1962, reported resolving eight

components from his preparation of mixed gangliosides with the use

of this TLC solvent system.

Meantime, Kuhn et al* (26) and Svennerholm (27) used another

solvent system consisting of n-propanol/water (7/3). Although these

workers each independently assigned arbitrary designations to the

components which were resolved by their TLC system, both assignments

were based upon the ganglioside pattern developed by this solvent

m ixture.

During 1963, Wherrett and Cumings (28) examined mixed ganglio­

sides from various sources in several TLC systems and concluded that

the system which resolved the mixtures into the greatest number of

components consisted of chloroforn/methanol/ 2.5 N NH^OH (60/35/8).

By the end of 1963, several solvent systems for resolving ganglio­

sides with TLC were described in the literature, but information

relating composition and structure of purified gangliosides to their

TLC characteristics was for the most part missing. Prior to 1964, Kuhn and co-workers described the thin layer chromatographic behavior of one trihexose-trisialo molecule ( G jy ) , two trihexosedisialo molecules (G j j j ) (Gn)» one trihexose-monosialo molecule (G j) and a dihexose-monosialo molecule (Gq ) in the TLC sol­ vent n-propanol/water (7/3), Svennerholm et al, (27) confirmed these

TLC characteristics,

Johnson and McCluer (23) isolated a group of similar, and per­ haps identical, gangliosides and reported their TLC properties in the

solvent chloroforn/methanol/water (60/35/8), More recently, Kuhn

et al, (9) reported the composition, structures and TLC properties

of four minor gangliosides. This report included the position of

these minor gangliosides relative to the major gangliosides described

earlier. Data were recorded for two solvent systems, n-propanol/

water (7/3) and chloroforrr/methanol/water (60/35/8),

In the n-propanol/water system, a monohexosamine-dihexose-disialo

ganglioside (G*Qffj-r u ) migrated between Gjj and Gjjj while a dihexose-

disialo ganglioside (G*Lac) migrated between Gj and Gj j . T h is same

order did not hold for the other solvent system. In this case, G'QNrrll

migrated between Gj and Gjj while G*£ moved in between Gj and G^.

At about this same time, Tettamanti and co-workers (10) reported

the composition and TLC properties of gangliosides they isolated

from pig brain. They presented photographic data of TLC plates showing

spots which corresponded to a heretofore unreported monohexosamine- s tetrahexose-disialo ganglioside which was well resolved from Kuhn*s

Gjv in propanol/NH4Of/water {6/2/1) but which was not resolved in the

propanol/water (7/3) system. This paper rather vividly illustrated 8 profound effect which different solvent systems have on the mo­ bilities of the various gangliosides. It further raised the question of whether any single solvent system is capable of demonstrating homogeneity of a ganglioside preparation.

The value of these TLC data was limited, however, by the fact that the various investigators used different solvent systems for the TLC assays and only rarely cross referenced their preparations to sol­ vent systems used by other investigators. This lack of cross re­ ferencing made it difficult to determine if an investigator reported a new ganglioside or whether the analytical data was in disagreement with previously reported data.

Ganglioside structures

In 1963, Johnson (12) made an extensive review of the data pertaining to the ganglioside structures. At that time, Johnson

states, "The failure to recognize at this time a basic structural

formula for gangliosides had been due in large part to the wealth

of conflicting data present in the literature." However, since

1963 data from different laboratories confirmed the structure of

several ganglioside species. Most investigators now accept data presented by Kuhn and Megandt (29) for the structure of the mono­

sialo ganglioside. This molecule is now believed to have the fol­

lowing structures

N-acylsphingosine-(l*l)-Glu-(4*1)Gal-(4*1)-GalNac-(3«l)-Gal 3 T 2 NANA 9

This ganglioside has been isolated from both human and calf brain.

Klenk and Gielen,s (21) data for ganglioside Bj conform with the structure given by Kuhn and Wiegandt (30) for their ganglioside

Gj j . This ganglioside reportedly has the second NANA residue at­ tached by a (2-»3) linkage to the terminal galactose residue of Gj.;

A second disialo ganglioside Gj j j reported by Kuhn and Wiegandt

(30) had the second NANA residue attached to the first NANA residue of Gj by a (2*8) linkage. These same workers (30) also described

a trisialo ganglioside Gj j j which combined the linkages described

in Gj j and Gjn* ^

Johnson and McCluer (31) reported on periodate studies of

isolated ganglioside preparations and confirmed the proposal for

Kuhn's Gj j j but disagreed with the proposal for Kuhn's Gjy. Johnson

and McCluer (31) found that one mole of galactose was destroyed

during the periodate oxidation and concluded that the terminal

galactose did not have a NANA moiety attached to it as suggested

by Kuhn. These data then suggested the existence of isomeric tri­

sialo molecules analogous to the isomeric pair of disialo gangli­

osides previously described. Johnson and McCluer suggested that

perhaps the third NANA residue was attached to the galactosamine

residue in this trisialo molecule.

The ganglioside which accumulates in the brain of patients

with Tay-Sach's disease was elucidated and confirmed by several

groups of workers (23,29,31,32). This ganglioside was identical

with Kuhn's Gj except that the terminal galactose residue was ab­

se n t. 10

Four other gangliosides which occur in minor amounts in

normal human brains were recently studied by Kuhn and Wiegandt

(9)* GQai was identified as

N-acyl sph i ng o s i ne-C 1*-1)-G al- ( ) -NANA.

G, was identified as Lac N-acylsphingosine-(l«-l )-Glu-(4

to the first NANA through a (2-»8) linkage. Likewise, G*GNrr n

was identical with the ganglioside described earlier as Kuhn*s

Gq except that it contained another mole of NANA. Non-availability

of material prevented these investigators from determining the

position of linkage for the second NANA residue.

All of these structure proposals were postulated from partial

hydrolysis studies, enzymatic hydrolysis data, and permethylation

d a ta .

Klenk (8) reported a ganglioside which contained only galactose,

NANA, sphingosine, and fatty acids. He reported the following struc­

ture for the sphingosinyl-hexose skeleton*

N-acylsphingosine-(l*-l)-Gal-(3<-l)-Gal-(3«-l)-Gal

He also found that two moles of NANA were removed with neuraminidase

to obtain the above skeleton molecule. No data was presented to

elucidate the points of attachment of these NANA residues.

A summary of reported ganglioside structures and the nomen­

clature symbols used by several workers in this field are presented

in Figure 1. Figure I

STRUCTURE AND NOMENCLATURE SUMMARY OF BRAIN GANGLIOSIDES

C er: Hex Nac: Glu: Gal : NANA: (Hax) Struct ura Nomenclature

Kuhn Svm norhdm Johnson Tottomont# Klonk •tot. •t al. a • t ol. • t Ol. Molar Ratio MeCluor

1:0:0: l: I: {l) Cor(l*l)Gol (3*2) NANA GAL

i:o:i:i:i:(e) Cor(hM)6lu (4*»l)Gol<3'»2) NANA LAC M3

i: o: i: i: 2: (2) Cor(l«-l) Glu(4**l)Gol(3«-2) NANA (8*- 2) NANA G,1’LAC

i: i: i: i: i:(2) Cor(l*-l) Olu(4*-l)Gol(4*“ 0 GolNoc G0 1 SGNTrll ®M2 NAN* i: f: i:i:2:(2) Car(i*l)Giu(4*l)Gal(4— DGalNac 2} NANA (Unk)NANA

Car(l*l)Ghl(4*l)Gal(4<*l)GalNac (3 * 0 Sol '".i- 2) NANA ®I 1-G l a i:i: i: 2:2:0) Car(l*l)Gly (4*1) G ol(4*l) GOINOC (3*1) Gal(3*2) NANA 'J- 2-G l b 2) NANA 1:1:1:2:2: (3) Car(l*l)Glu(4«-l)e*l(4*l)8*INao(3*l) Sal 3-G "Dlb 2) NANA ( 8 * 2 ) NANA 1:1:1:2:s :(s) C t r ( l * l ) G lu ( 4 * l) S a l ( 4 * I ) Gal N a c ( 3 * I) Gal ( 3 * 2 ) NANA

2) N A N A (i« -2 ) NANA

1; 0:0:3; 2: c 3) Car(l*l) Gal(3*1) G al(3*l) Gal (Unh)NANA

i : i :-:-: i :( 3 ) T T T b

i : i :-:-:2 :(4) mo

r : i : - > : 4 :(3) IZ b NANA 1:1:12:3: (3) Card* I) Glu(4*1) Gal(4*l) GalNac(3*l) Gal

2) NANA ( 8 * 2 ) NANA 12

The pyranose ring forms of the glucose and galactose moieties of gangliosides were determined by Klenk (20). He also presented data to support a beta linkage for these hexoses in the gangliosides.

The structure of NANA was determined by Kuhn (33).

The fatty acid composition of gangliosides was reported by several investigators (26,34,35,36,37).S te a ric acid was reported to comprise 85 percent Id 95 percent of the total, with palmitic, arachidic, and behenic the only other acids present in greater than 1 percent amounts.

The sphingosine base composition of brain gangliosides was studied by Sambasivaro and McCluer (38). They reported that a C sphingosine exists uniquely in brain gangliosides. This base com­ prises about 50 percent of the ganglioside sphingosine in all species studied. This aspect of gangliosides was recently reviewed by Carter et al. (7).

It should be emphasized here that the brain gangliosides are • apparently unique since they contain only trace amounts of unsaturated fatty acids, if any at all, and apparently exclusively contain a

C^q sphingosine base.

Gas-liquid chromatography of carbohydrates

Mclnnes et al. (39) first reported the GLC analysis of car­ bohydrates in early 1958. They reported the separation of methyl­ ated hexoses. Following this work, several workers adapted a var­ ie ty of polyhydroxy compounds to GLC an aly sis (40, 41, 42, 43,

44, 45). Derivatives other, than the methylated sugars were reported 13 as being useful for GLC analysis. Hedgley (46) reported using the isopropylidine derivatives while others (47, 48, 49) reported the GLC analysis of acetylateehsugars and acetylated glycosides.

More recently, the trim ethyl-silyl ethers were found to be useful derivatives for carbohydrate analysis (50, 51, 52, 53, 54,

55, 56, 57, 58). The largest contribution to this field was by

Sweeley and co-workers (54) who reported GLC properties of the

TMS-ether derivatives for almost 100 polyhydroxy compounds. These included glycosides, aldoses, ketoses, amino sugars, oligosaccharides up to and including stachyose, ascorbic acid, methyl ester of neur­

aminic acid and N -acetylated amino sugars.

The use of acetals and ketals was recently reported by Jones

and co-workers (59, 60).

During the last year, several reports appeared in the liter­

ature regarding quantitative assays of carbohydrates by GLC methods.

Sweeley and Walker (55) were the first to report the ratio of fru-

dbse to galactose from glycolipid samples. Richey (56) et al.

described a quantitative GLC method which gave data with 10 to 15

percent reliability. Wells (57) also reported a GLC assay method

for sugar from sera and urine samples.

Perry (61) developed a GLC method for the determination of

hexosamines which gave excellent results. EXPERIMENTAL

Instrumentation

The instrum entation used fo r th e GLC assays was th e 5000

series equipment manufactured by Barber-Coleman, Rockford, Illinois,

The detection system was the hydrogen flame detector sold by the same

company. The recorder was equipped with a disc integrator for mea­

suring the areas under the chromatogram peaks.

The column used for the hexose assays was a 0,25" xl08"

stainless steel tube packed with 3 percent SE-52 on Anakrom ABS,

110-120 mesh. The tube was fitted with Swagelok fittings at each

end in order to secure an in je c tio n septum on th e fro n t end and to

complete the connection to the detector at the exit end. The car­

rier gas entrance tube consisted of a stainless steel capillary

tube silver soldered into the wall of the column about 1,5" below

the injection septum, A sufficient length of this capillary tube

was inserted into the column before soldering so that it terminated

about 0,1" below the injection septum. The septums were secured

from F and M scientific Corporation, Avondale, Pennsylvania,

All other equipment used throughout this investigation was

well known laboratory equipment and will not be further described.

14 M aterials and standards

The Anakrom ABS support and SE-52 gum were purchased from

Analabs, Hamden, Conneticut. Swagelok fittings were purchased

from Ohio Valve and Fittings, columbus, Ohio. The ^-methyl-D-glucopyan- oside was purchased from Nutritional Biochemicals Corporation, Cleve­

land, Ohio. It was checked for GLC purity and used as purchased. f2 -methyl-D-glucopyranoside was obtained from Calbiochem, Los Angeles,

California, checked for GLC purity, and used without further puri­

fication. Theo(-methyl-D-galactopyranoside was synthesized according

to a procedure described by Mowery (62). The purity of this crystal­

line monohydrate of methyl-galactoside was checked by melting point,

(m.p. - 107.5°-108.5° uncorrected with 111 reported) and specific

rotation [fOiT = +176.2°with a value of -+178°reported).

C, H analysis by Crobaugh Laboratories, Charleston, West Virginia

gave 39.93a and 8.0% respectively - (theoretical C-39.63a, H-7.63a).

This product, after silanation, gave a single GLC peak which was

indistinguishable from a sample obtained from the laboratory of

M. L. Wolfrom at Ohio State University.

TMS and HMJSz were purchased from K and K Laboratories,

Plain View,N.Y. and were redistilled before use. The pyridine and

carbon disulfide were also redistilled before use. The pyridine

was kept dry by storing over WOH.

A reference standard of

D-glucopyranose was synthesized according to the procedure of

Hedgley and Overend (53). This compound was purified by fractional o vacuum d is tilla tio n * b .p . - 108 uncorrected, at 0.1 mm Hg (reported b .p . - 114 - 115° at 0.23 mm Hg) and CrOp -

+85.4° in CC1 (reported CfO© ~ 4-88.4° in CC1 ) (53). 4 4 The aquacide II and NANA were purchased from Calbiochem,

Los Angeles, California and used as obtained. The D-mannitol was

obtained from the same source, checked for purity by GLC analysis

and used w ithout fu rth e r p u rific a tio n .

The fatty acid methyl ester standards were obtained from

Applied Sciences Laboratory, State College, Pennsylvania and used

as received. The D-galactosamine-HCl was purchased from Sigma

Chemical Co., St. Louis. Missouri, and used without further puri­

fic a tio n .

D-galactose was supplied by Matheson, Coleman and Bell Co.,

Norwood, Ohio and used as received. D-glucose was purchased from

Mallinckrodt, Cleveland, Ohio and used as received.

The galacto-cerebroside was purchased from Light and Co.

Ltd., Colnbrook, England. This material was checked for purity

by TLC analysis, C, H, N analysis by Crobaugh Laboratories Charleston,

West Virginia, and by total hexose assay. C,H, N, analysis reported

68.8#, 11.2# and 1.93# respectively.

HG-1 ganglioside standard was prepared in this laboratory.

Purity was established by component analysis and the percent weight

determined; NANA -19.8#, Hexose - 31.4#, GalNac-14.2# with theoretical

values of 19.8#, 34.6# and 14.2# respectively.

Silica Gel G was purchased from Brinkmann Instruments, Great

Neck, N.Y. and was used as purchased for TLC plates. Anasil S from

Analabs, Camden, C onneticut, was washed fre e of fin e p a r tic le s by suspension and decantation in methand. prior to packing columns*

All other chemicals were standard ACS reagent grade materials and used w ithout fu rth e r p u rific a tio n .

Preparation of mixed aanaliosides

Fresh brain was homogenized in two volumes of acetone and the

insoluble residue allowed to settle. The supernatant was decanted through 8 to 12 layers of cheese cloth and the solvent squeezed

out by hand. The tissue was re-extracted with two volumes of ace­ tone and dried under a stream of nitrogen. The acetone powder was extracted twice with two volumes of anhydrous diethyl ether

for 30 minutes each time and the residue dried under a stream of

n itro g en .

The dried powder was extracted for 72 hours in a soxhlet

extractor with chloroforn/ methanol (l/2). This extract was taken

to dryness with a rotary evaporator connected to a water aspirator.

The chloroforn/ methanol residue was dissolved or suspended

in Folch lower phase (63) at a concentration of 10 percent and

equilibrated with Folch upper phase made with 0.1 percent saline.

A three tube counter current distribution was performed with Folch

solvents. The upper phases were combined and filtered through

medium sintered glass filters to remove remaining interphase mat­

erial. This solution was transferred to a dialysis tube and covered

with Aquacide II until the volume was reduced to about one-third o of the original. This operation required about four days at 4 C.

The solution was dialysed exhaustively against distilled water, 18 passed over a Dowex 50 x 2(H+) column and ly o p h ilized .

The dried material was re-distributed with the Folch solvent system as previously described, concentrated, dialysed, passed over another Dowex 50 column, and lyophilized. This dry powder was used as mixed gangliosides.

Isolated ganqlioside preparation

Sufficient Anasil S, which had been suspended in methanol to remove the fines as described previously, was dried overnight at

120° and suspended in sufficient chloroforn/ methanol/ water (60/

35/8) to make a thin slurry. This slurry was poured into a column tube until the column bed had a length to diameter ratio of 20

to 1.

A sample of mixed gangliosides, about 1/250 of the weight

of the Anasil used for the column bed, was dissolved in a minimum

of chloroforn/ methanol ( 2 /l) and carefully loaded on the top of

the bed. After the loading solution drained to the top of the column

bed, the eluant reservoir was attached to the column and filled with

chloroforn/methanol/water (60/35/8). The flow rate was adjusted

to about 2 ml./minute and fractions collected until resorcinol

positive material was no longer eluted from the column. If frac­

tionation of gangliosides having a greater TLC mobility than HG-1

was desired, the column was equilibrated and eluted with chloro­

forn/ methanol/ water (65/30/5) until ganglioside HG-1 was obtained,

then eluted with chloroforn/methanol/water (60/35/8).

Each fraction was tested for sialic acid by spotting a small 19 amount on a TLC p la te and spraying w ith reso rcin o l spray reag en t.

The resorcinol positive fractions were assayed for homogeneity by TLC analysis in four solvent systems. All fractions which appeared to be homogeneous and had the same TLC mobility characteristics were combined, dried by evaporation, dissolved in water, filtered through sintered glass filters and lyophilized. All fractions which were not homogeneous were combined, used as mixed gangliosides, and rechromatographed.

The lyophilized samples were dissolved in a minimum of meth­ anol, filtered, and the ganglioside precipitated by adding two volumes of diethyl ether. This precipitate was redissolved in a minimum of methanol and the ganglioside crystallized in the cold.

This crystallized product was again checked for TLC homogeneity and used as a pure sample.

Qualitative analytical methods

Thin layer chromatographic procedure

The b asic methods of TLC have been reviewed by Mangold (64).

Throughout this investigation, a slurry of 30 gm. of Silica Gel

G in 60 ml. of water was applied to clean glass plates (8” x 8") with the use of an adjustable applicator. All plates were spread with uniform 250 micron thick layer. The TLC plates were allowed to air dry for 10-20 minutes, further dried by heating at 110°

for 20 minutes, and finally cooled. When the plates were cool,

they were washed by sitting them in a presaturated chamber of methanol/ diethyl ether (4/l) and the solvent allowed to ascend 20 the full length of the plate. After the wash, the plates were air dried and stored in a dessicated box.

Before the plates were spotted, they were activated by heating o 0 at 130 -135 for 90 minutes and air cooled. The ganglioside samples were usually dissolved in sufficient chloroforn/ methanol (2/l) to attain a concentration of approximately 1 mg0/ml. Occasionally methanol or water solutions were also spotted. The solutions were applied to the adsorbent layer with micro pipettes (2 - 25 pi) in

1 cm. streaks. These spots were thoroughly dried by a stream of warm air from a hair dryer.

After the spotting operation was completed, the edges of the adsorbant layer were scrapped so that they were even and parallel.

These plates were placed in the pre-equilibrated chamber for ascen- tion of the solvent. Equilibration of the chamber was accomplished with a sheet of filter paper, which was wetted with about 150ml.

of the desired solvent. After the solvent was added to the chamber,

a glass plate was placed on top to seal the chamber and the closed

chamber was allowed to set for at least 1 hour before the plate was

in se rte d .

In order to attain maximum resolution of mixtures, the solvent

was allowed to ascend to within 1-0.5 inches of the top of the plate

before it was removed from the chamber. After the plates were

removed from the chambers they were thoroughly dried before they

were sprayed with the development reagent.

Four solvent systems were used to establish chromatographic

purity of individual ganglioside preparations, and to establish a 21 tentative identification. These solvent systems were (60/35/8) chloroforn/ methanol/ water (c/llty^O), (60/35/8) chloroforn/ methanol/ 2.5 N NH^ OH (C/n/NH^), (7/3) n-propanol/water (Pt/I-^O), and (6/2/l) n-propanol/water/ concentrated NH^ OH (Pr/NH^).

The developing reagent used routinely was the resorcinol-

Cu++ reagent prepared according to Svennerholm (65). After the plates were sprayed, a clean glass plate was laid over the adsorbant layer, held in place by clamps, and the color developed by heating at 130 - 135° for 20 minutes.

If non-sialic acid containing material was suspected, the plates were sprayed with bQ% sulfuric acid and heated slowly on a hot plate for development.

N-acetylneuraminic acid

The sialic acid residue of gangliosides was identified from a solution of the ganglioside which had been treated with neuramin­ idase from Cl perfrinqes • The solution was spotted onto a TLC plate and resolved with the solvent n-propanol/ ammonia/ water

(6/ 2/ 1). Authentic NANA was spotted as a comparison standard.

The color was developed on the plate with the Cu*+ resorcinol reagent as previously described.

Hexosamine

One sample of a purified ganglioside preparation was subjected to qualitative identification of the hexosamine. Standard paper

chromatographic techniques were used to confirm the presence of a 22 hexosamine* Final identification of the hexosamine was accomplished by means of GLC techniques* The basic method fo r hexosamine an aly sis as described by Perry (61) was used* A pure sample of D-galactosamine was used as the comparison standard*

Hexose

The id e n tific a tio n of th e hexoses in gangliosides was accomp­ lished by two independent methods* The ganglioside was hydrolysed according to the conditions described by Johnson (12) and the sample dried. Part of this sample was treated according to standard paper chromatographic methods with a solvent of ethylacetate/pyridine/ water (10/4/3). Part of the sample was treated according to Sweeley*s

(54) method and subjected to GLC a n a ly sis. Pure D-glucose and D- galactose were used as standards in both methods.

Fatty acids

All purified ganglioside preparations were assayed for quali­ tative differences by GLC techniques* Each sample was treated with anhydrous HCl-methanol according to the conditions described by Sweeley(55)* After the methanolysis, the reaction vials were

opened and the volume doubled with absolute methanol. This solu­

tion was passed over a short (0.5 x 2 cm.) Dowex 50X4 (Hf) form

resin, bed. The eluate and one column volume of methanol wash

tolution were collected and evaporated to about one ml. The methyl

esters of the fatty acids were extracted from this methanol sol­

ution with three, 3 ml* extractions of n-heptane* The n-heptane 23 extract was evaporated to dryness, the contents dissolved in one ml, of carbon disulfide and portions injected onto the GLC column for separation. The column was a 0,25" x 108" stainless steel tube packed with 3 % SE-52 on Anakrom ABS, 110-120 mesh. Operating temperature was 210° C, with 100-120 ml,/minute carrier flow rate.

Nitrogen was used for the carrier gas and a hydrogen flame ioniza­ tion unit provided the detection system. Identifications were made by comparison with authenticated standards.

Quantitative analytical methods

N-acetylneuraminic acid •

Ganglioside NANA was determined by the resorcinol method of Svennerholm (65) as modified by Miettinen and Takki-Luukkainen

(6 6 ), Dichromatic readings were taken a t 580 and 450 m illim icrons to compensate for the galactose interference. The NANA content was calculated according to the method published by focCluer et al.

(69), Pure NANA was used to periodically check the reliability

of the method,

Hexosamine

Gangliosides were subjected to hydrolysis according to the

conditions described by Johnson (12) and the hexosamine determined

in the hydrolysate according to method B of Svennerholm (72),

N-acetylgalactosamine and galactosamine hydrochloride were used

as standards. 24

Hexpses

Total hexose values were obtained by two independent methods.

The first method employed a phenol-sulfuric acid assay (68) of the

ganglioside hydrolysate. The sample was hydrolysed according

to the conditions described by Johnson (12). Because galactose

was used as the standard for this procedure, all values were re­

ported as galactose equivalents. It should be noted however, that

with a galactose to glucose ratio of 2, only about a 3 percent

error was involved when pure galactose was used as the standard

because the reported relative extinction coefficient for glucose/

galactose was 1.1 (68) for this method.

The second method of obtaining the total hexose content was

to add the values obtained for glucose and galactose as determined

by the GLC method. The GLC method is fully described in the

following section of this manuscript.

Glucose and galacto se

Samples subjected to GLC analysis for glucose and galactose

were treated basically in accordance with the procedure described

by Sweeley et al. (54, 55). However, several modifications were

made which expanded the capability of the method to include the

determination of percentage composition data. The final procedure

is described in detail below.

A known amount of an aqueous ganglioside solution, containing

approximately 1 mg. of ganglioside, was transferred into a five

ml. g la ss v ia l. A known amount of standardized aqueous mannitol 25 solution, about 0.1 mg. mannitol, was added to the contents of the vial. The contents were mixed and then lyophilized. The contents of the vial were dissolved in two ml. of a 0.5 N anhydrous HCl-methanol and the vial sealed by fusion. This sealed vial was incubated for twenty-four hours at 75° - 80° C.

After incubation, the vial was cooled to room temperature, opened and two ml. of anhydrous methanol added to the contents.

The entire solution was passed over a short (0.25 x 2.0 cm.) column 2 of Dowex 50 x 4 (H+ ) • The eluate and one column volume of meth­

anol wash were collected and the volume was reduced to about one

ml. with the aid of a water aspirator.

The concentrated solution was extracted three times with 3

ml. portions of redistilled n-heptane. The n-heptane fraction was

either discarded or saved for fatty acid analysis.

The methanolic solution was taken to dryness with the aid of

a water aspirator connected to a Roto-Evap shaker. Five ml. of

dry pryidine was added to the dried residue in the tube and the o contents shaken in a 40 C. water bath for twenty minutes to

dissolve the carbohydrates. After the residue was dissolved,

0.2 ml. of HWDz and 0.1 ml. of TMCS were added and the contents

vigorously shaken for another ten minutes.

This resin was pre-extracted for 16 hours with methanol

in a Soxhlet extractor. 26

After this last period of shaking, the pyridine and excess reagents were removed by evaporation with the aid of the water aspirator. The dry contents of the tube were dissolved in one ml. of redistilled carbon disulfide and re-evaporated to dryness.

When the smell of pyridine was no longer detected, the residue was re-dissolved in carbon disulfide and appropriate portions injected onto the GLC column for separation.

The conditions for this separation were as follows* column size, 0.25" x 108" stainless steel tube packed with 3$ -

SE-52 on Anakrom ABS 110-120 mesh; temperature, 18 C.; flow rate,

100 m l./m inute; c a r rie r g as, nitro g en .

The amounts of glucose and galactose were determined by measuring the total area under the peaks representing the glucose, galactose and mannitol, and performing the necessary calculations. The peak areas were determined with the aid of a disc type integrator at­ tached to the recorder portion of the equipment or by triangulation.

After determining the total area representing the individual hexoses and the standard, the percentage of glucose and galactose were calculated according to the following formulae*

% glucose-- (area g lu) x (1.30) x (0.928) x (mg. mann) x 100 (area mann.) x (mg. sample)

% galactose _ (area aal) x (1.43) x (0.928) x (mg. mann.) x 100 (area mann.) x (mg. sample) 27

The ratio of galactose to glucose was determined either by dividing the percent galactose by the percent glucose or by the following formulas

galactose _ (area gal) x 1.10 glucose (area glu)

More frequently, the data was desired in the form of micromoles/mg*

and was calculated according to the following formulae: urn, glu — (area glu) x (1.30) x (0,928) x (mg. mann) mg. sample (area mann.) x (l80) x (mg. sample)

um. gal. _ (area gal.)x Cl. 43) x (0.928) x Cmg. mann.) mg. sample (area mann,)x (180) x (mg. sample)

The derivation of these formulae are shown in the Results

and Discussion section of this manuscript.

Sphingosine

Sphingosine was determined by the method described by Lauter

and Trams (73). Ganglioside HG-1 was used as the standard. RESULTS AND DISCUSSION

Glucose and g alacto se assay by GLC

Glucose and galactose are the only reported hexose components in brain gangliosides, and determination of the glucose to galactose ratio is a fundamental criterea in characterizing ganglioside preparations. Such data are also quite useful in judging the homogeneity of such preparations.

Generally, to obtain a percent hexose value, nonspecific color­ imetric assays such as anthrone and phenol-sulfuric acid methods are employed, Johnson and McCluer (23) and Klenk et a l. determined glucose/galactose ratios in acid hydrolysates of gangliosides by paper chromatographic techniques, followed by elution and subse­ quent colorimetric analysis of the separated sugars, Wolfe et al.

(69) determined the total hexose by the anthrone method and the O glucose measured independently with "Glucostat" . The galactose was determined by d iffe re n c e . However, these authors reported

consistantly low glucose values, and galactose to glucose ratios

of 3.0 in contrast to 2.0 reported by others (23, 29), Sweeley and

Walker (55) reported a GLC method for obtaining hexose ratios but

did not attempt to obtain absolute percentage values.

3 A commercial preparation for enzymatic determination of free

glucose. 28 29

Hydrolysis conditions for quantitatively liberating hexoses

from gangliosides are troublesome* It is very difficult to obtain

conditions which yield complete liberation without destruction.

Kuhn (29) resorted to formic acid hydrolysis followed by mild hydrolysis with HC1. The GLC technique described by Sweeley

appeared to offer the possibility of quantitatively converting

the ganglioside hexoses to the glycosides by methanolysis and

direct determination of glucose and galactose* This technique

also had the potential of being able to detect any different car­

bohydrate moieties which may be present in a ganglioside preparation.

A preliminary examination indicated that Sweeley*s (54) relative

retention time data were reasonably well duplicated with the GLC

instrumentation facilities available in this laboratory* Data

summarized in Table 1 demonstrated that while relative retention

times were not exactly duplicated, the basic method was reliable

enough to consider developing a quantitative method for the glucose-

and galactose.

While working with the silanation procedure described by

Sweeley et al. (54), it was noted that unless large amounts of

the glycosides were used, relative to the amount of pyridine,

the pyridine tailing caused severe difficulty in measurement of

the peak areas. In order to be capable of assaying sub-milligram

amounts of hexose, attempts were made to reduce or eliminate

this sever tailing of pyridine. Attempts to eliminate the pyridine

from the reaction mixture by using dioxane or tetrahydrofuran TABLE 1

Relative Retention Times of Some Carbohydrate TMS Derivatives

Compound Rel. Ret.* Rel. R e t.2 time tim e (Exp.) (Sweeley) (54)

-Methyl-D-arabinopyranoside 0.26 0.22

c< -Methyl-D-xylopyranoside 0.42 0.34

/ 3 -Methyl-D-xylopyranoside 0.45 0.37

oi, -Methyl-p-mannopyranoside 0.67 0.64

o£, -Methyl-D-galactopyranoside 0.79 0.78

o< -M ethyl-D-glucopyranoside i . oo 3 1.00

^-MethylHD-glucopyranoside 1.07 1.16

D-mannitol 1.23 1.31

These relative retention times were determined at 181° C. using 3$ SE-52 as the liquid phase on Anakrom ABS, 110-120 mesh* The carrier gas was nitrogen at 50 ml./minute.

*These values were calculated from data taken at 140°C. The liquid phase was 3% SE-52. 3 Retention time was 15.8 minutes. 31 as the solvent resulted in reaction rates so slow that the procedure was impractical* Addition of pyridine to these solvents increased the reaction rate, but when sufficient pyridine was added to make the reaction rate fast enough to consider it practical for the procedure, the pyridine tailing resulted in unsatisfactory chroma­ tograms. This problem was finally solved by removing the pyridine after the silanation reaction was complete. The TIvS-ether deriva­ tives were then soluble in any one of a wide variety of organic so lv en ts.

The solvent of choice was carbon disulfide because it yields practically no signal from a hydrogen flame.

Sweeley*s data (54) indicated that a better separation could be obtained with the glycosides than with the free sugars. There­ fore, the glycosides were selected as the derivatives to be used for the assay.

A study was undertaken to identify the TMS derivatives which corresponded to the chromatographic peaks. An authentic sample of

After the retention time of this compound was established, a

sample of *C-methyl-D-glucopyranoside was processed according to the proposed assay procedure and chromatographed. The resulting peak had the same retention time as the tetrasilanated sample.

When the identity of this peak was established, and confirmed

Sweeley*s report, the other relative retention time data reported

by Sweeley (54), were considered sufficient for identifying the 32

/3 -glucoside, iK-galactoside and /3-gfclactoside TMS derivatives#

At th is tim e, i t became evident th a t i f q u a n tita tiv e data was to be obtained, the -galactoside and -glucoside peaks had to be chromatographically resolved. The operating conditions

reported by other workers (54) for resolving these derivatives did not, in this investigator's hands, give adequate resolution when the columns supplied with the GLC equipment were used.

After repacking of these columns failed to increase the resolution,

a stainless steel column was made which incorporated a carrier

gas inlet design suggested by Sweeley (70) (see EXPERIMENTAL,

Instrumentation section). Initial testing of this column showed

improved resolution although the separation was still considered

inadequate.

A systematic study of the parameters affecting resolution

was undertaken to determine if the maximum resolution was attained

with these operating conditions. Using p<-methyl*^-glucoside

as a model, the effect of flow rate on retention time was recorded

at various temperatures and is summarized in Figure 2.Conditions

which resulted in retention times greater than thirty minutes were

considered impractical for the analysis and were not recorded.

The effect of temperature and flow rate on column efficiency

was studied. The column efficiency was estimated by the theoretical

plate calculation as discussed by Szymanski (71). From the data,

summarized in Figure 3, it was concluded that decreased flow rate

resulted in increased column efficiency at all temperatures studied.

The effect of temperature, shown in Figure 4 was not as predictable Retention Time, min 0 3 25 20 20 30 40 eeto Tm vru Flow Rote versus Time Retention l w ae m Ng/min ml Flow Rate, 60 0 5 iue 2 Figure 70 80 90 00 140° C 140° 181C ° 171°C 5° C 150° 160° C 160° CO GO 2400

Theoretical Plates 2000 2200 1400 1800 1200 1600 0 0 0 0 0 0 80 70 60 50 40 30 20 hoeia Pae vru Fo Rate Flow versus Plates Theoretical lw ae ml l^/min Rate, Flow iue 3 Figure =171°& C =151°C • 11 C =161 0 =8 C =181 □ Legend 40 ° C 40 0 9 100 34 2200 2400

Theoretical Plates 2000 1800 1600 1400 200 140 hoeia Pae vru Temperature versus Plates Theoretical 5 170 150 Temperature, ° C ° Temperature, 160 iue 4 Figure 180 100 ml/min 60ml/min 40ml/min 20 ml/min 80 80 ml/min 190 36 since the efficiency was variable between 140° and 170° C. but then o o increased markedly between 170 and 180 C. From th ese d ata, new operating conditions of 180 C, and 50 m l./m inute flow r a te were selected. These conditions resolved the two peaks under consideration.

Although the precision of the instrumentation is usually not a limiting factor in an analytical procedure, instrumental precision was evaluated before investigating the other chemical operations proposed for the final procedure. The precision was evaluated by determining the average deviation in measuring the ratio of two peak areas. A single sample containing two components was injected three times, the peak areas measured and the ratio calc u la te d . The average d ev iatio n was then evaluated fo r a number of different samples. These data;tabulated in Table 2, indicated that such measurements could be made with an average deviation less than three parts per hundred. Therefore, it was concluded that sample data would be accepted if three successive injections resulted in the average deviation of peak area ratios was less than three parts per hundred.

The common methods for quantitative GLC analysis include the technique of quantitative dilution and injection followed by measurements of the peak areas which are then related to mass from a standard mass-area curve. A second technique involves adding a known amount of an internal standard, followed by measure­ ment of the two peak areas, and direct mass calculations. Although this second method requires that a relative mass response factor 37

TABLE 2.

GLC Precision Data

Sample Area Mean D eviation Avg. Dev. Avg. Dev, No. R atio (X) (M ( la) in parts n per 100

9-26-3-1 0.939 0.020 0.967 0.008 0.972 0.959 0.013 0.014 1.4

9-26-3-2 1.11 0.050 1.06 0.000 1.02 1.06 0.040 0.030 2.8 9-27-63-1 0.982 0.029 0.941 0.012 0.935 0.953 0.018 0.020 2.1

9-27-63-2 0.891 0.015 0.873 0.003 0.865 0.876 0.011 0.010 1.1 9-30-63-1 0.899 0.021 0.860 0.018 0.876 0.878 0.002 0.014 1.3

10-11-63-1 1.09 0.020 1.03 0.040 1.08 1.07 0.010 0.020 1.9

10-11-63-2 0.860 0.006 0.878 0.006 0.872 0.872 0.000 0.004 0.5

1 -1 4 -6 4 -5 2.56 0.030 2.49 0.040 2.53 2.5 3 0.000 0.020 0 .9 38 be determined before th e amount of sample can be calcu lated from the relative areas and the known amount of standard, this method appeared to be the method of choice for the most accurate quan­ titative GLC methods reported in the literature. This technique was selected as the technique of choice for the analysis under consideration.

The selection of a standard compound had to meet certain requirements. First, it had to elute either before or after the glycoside derivatives under consideration. Second, it should not be a material expected to be present in the normal range of samples to be analyzed. Next, it would be preferable if it had chemical properties similar to the material being assayed, and finally it would be desirable if it were chemically more stable than the mater­ ial being assayed. In considering materials which met these require­ ments, mannitol was selected over a group of readily available pentose glycosides mainly because of its marked chemical stability.

Although it was expected that the relative mass response for c^-methyl-D-glucopyranoside and -methyl-O-galactopyranoside would be unity for the flame ionization detector, subsequent data did not confirm this prediction. Known amounts of pure o<-methyl-

D-glucopyranoside and o<.-methylHD-galactopyranoside were processed through the silanation procedure and subjected to analysis. The relative mass response, K3, was calculated according to the formula

K - (mg. cral.) (area g lu .) 3 ~ (mg. g lu .) (area g a l .; . 39

T his value was determined to be 1*10 t 0*03 as shown in T able 3.

The derivation of this formula follows*

If mass<=< peak area

Then mass = k (peak area) (eq. l)

Somg0 gal. ■= k (area gal.) (eq. 2) 3 and mg. glu. ■= k^(area glu.) (eq. 3)

Dividing (eq. 2) by (eq. 3) mg. gal. - ka(area gal.) mg. glu. ~ k^ (area glu.) (eq. 4)

By defining k _ IC and rearranging (eq. 4), kb K - (mg. gal) (area glu.) (eq. 5) (mg. glu)(area g a l.)

Thus, is a relative mass response factor and not an absolute

mass response factor.

Since the value of Kg did not equal unity as expected, a value

was determined for /^-methyl-D-glucopyranoside relative to

D-galactopyranoside. This value duplicated K3 as shown in Table 4.

From these data, it was concluded that the configuration about

carbon number one resu lte d in no d e tectab le e ffe c t on the mass

response with this detector. Since it was not the primary purpose

of this investigation to study such relative mass responses, this

area was not pursued beyond the limits of the problem. It was noted

however, that the data of Richey et al. (56) indicated a difference

in mass response for glucose and mannose w ith th e beta io n izatio n 40

TABLE 3

Kg Data

Sample Area q lu Wt. qal No. Area gal Wt. glu K3

9-27-63-1 1.05 1.06 1.11 1.06 1.12 1.09 1.15

9-27-63-2 0.871 1.33 1.16 0.853 1.13 0.871 1.16

9-30-63-1 1.04 1.06 1.10 1.08 1.15 1.04 1.10

10-11-63-1 1.00 1.06 1.06 0.980 1.05 1.03 1.07

10-11-63-2 0.512 2.13 1.09 0.529 1.13 0.521 1.11

1-14-64-5 2.56 0.421 1.08 2.49 1.05 2.53 1.11

=: mean - avg. dev. K3 x 1.10 t 0.03 41

TABLE 4

K4 Data

Sample Area qlu Wt. qal No. Area gal Wt. glu k4

1-9-64-1 1.02 1.09 1.11 1.01 1.09 1.02 1.11

3-25-64-2 0.759 1.49 1.13 0.731 1.09 0.761 1.13

3-25-64-3 1.84 0.596 1.10 1.84 1.10 1.84 1.10

k4 ^ 1.11 + 0.01 n -vT mean £ avg. dev. 42 d e te c to r.

The relative mass response factor for oi,-methyl-D-galacto- pyranoside to D-mannitol, K^, was determined in a similar manner

(see Table 5) and found to be 104 3 t 0.03. Combining Kj and Kp a value for the relative response factor of o( -methyl *3)-glucopyr a no­

side to D-mannitol, K^, was calculated to be 1.30. This value was checked d ire c tly and found to be reproducible.

Sweeley and Walker (55) reported recently on a procedure for

obtaining glucose to galactose ratio data from glycolipids with

a GLC method. In this paper they described conditions for con­ verting the hexose moieties to the corresponding methyl glycosides.

Although these authors inferred that the conversion was quantitative,

they offered no direct evidence to substantiate this. A pure

galacto cereb ro sid e sample was selected as a model compound to

confirm or disprove this proposal. Two cerebroside sanples were

treated in accordance with the described methanolysis conditions

(55) and then processed according to the previously described assay

procedure. The percent g alacto se values of 24.2% and 24.6% were

within the experimental tolerances and agreed well with the theoret­

ical value of 23.6%. These data indicated that the methanolysis

conditions did yield complete conversion to the methyl glycosides

with no detectable destruction. The percent galactose was calculated

according to the formula % galactose ^ (area gal.) (1.43) (0,928) (mg. mann.) (100). (area mann.) mg. sample)

The derivation of this formula follows* 43

TABLE 5

Data

Sample Area mann. Wt. Gal. No. Area g a l. Wt. mann. K1

11-5-64-5 1.76 0.804 1.42 1.77 1.43 1.78 1.43

11-5-64-6 1.78 0.804 1.43 1.77 1.42 1.74 1.40

11-4-64-4 1.84 0.750 1.38 1.85 1.39 1.85 1.39

11-4-64-2 1.46 1.02 1.49 1.45 1.47 1.43 1.46

1.43 ± 0.03

mean £ avg. dev. 44

From (eq. 5)

k3 r (ma. gal.) (area glu.) (mg. g lu .) (area g a l.) (eq. 5)

an analogous equation can be written for

_ (mg. gal.) (area mann.) (mg. mann.) ( area g a l.) (eq. 6)

Rearranging and solving fo r (mg. g a l.)

(mg. g a l.) - (K^ ) (area g a l.) (mg. mann.) (eq. 7) (area mann.)

However, this equation yields mg. of galactoside. This

can be converted to mg. of galactose by multiplying the

equation by the gravimetric factor Mol. Wt. galactose Mol. Wt. g a la c to sid e .

Thus, (eq. 8) (mg. g alacto se)-. (K ) (area g a l.) (mg. mann) (orav. fa c to r) 1 (area mann.)

Substituting in values for and the grav. factor,

dividing by the sample weight and multiplying by 100,

the final equation is obtained*

% galactose - (1.43) (area gal.) (mg. mann.) (0.928) (100) (area mann.) (mg. sample) (eq. 9)

Raffinose was considered as another model for testing the procedure since it contains both glucose and galactose. However, repeated analysis always resulted in high glucose values. It was noted that the methanolysis solution of raffinose was always yellow and it was thought that the fructose was degrading into products which interferred with the glucose determination. As a qualitative test of this proposal, sucrose was processed without the mannitol 45 standard. The methanolysis solution developed the same yellow color observed with the raffinose solutions but the chromatogram indicated only a glucose response without any indication of interference.

As a result of these experiments, raffinose was discarded as a model for testing the procedure. No explaination is offered for the ob­ served data except that apparently, fructose can not be assayed by this method, - -

During their studies, Sweeley and Walker (55) noted, while attempting to get hexose ratio data from the gangliosides, that some degradation products of hexosamine interferred. In order to eliminate this problem, the methanolysate was passed over a short

Dowex 50 X 4 (H+) column before proceeding with the remaining steps of the procedure. Analysis of ganglioside HG-1 gave excellent agreement for the galactose and glucose percentage values, with 21.4# and 10.2# respectively, compared to the theoretical values of 21.4# and 10.9# respectively.

As a final modification of the procedure, the mannitol standard was added to the glycolipid sample prior to methanolysis. Data from both the galactocerebroside and ganglioside HG-1 confirmed the prediction that mannitol was not degraded by these conditions.

A typical chromatogram is shown in Figure 5.

For final evidence of the method*s reliability, several ganalioside preparations were analysed for the hexose content by th is GLC method and by th e p h en o l-su lfu ric acid assay of acid hydrolysates. The excellent agreement of the data from these two methods are tabulated in Table 6. Detector Response a Lqi Crmtga o Hxs Assay Hexose of Chromatogram LiquidGas Time iue 5 Figure 0-Me-Glu Mannitol 47

TABLE 6

Summary of Hexose Analysis

Sample Phenol-H2S 04 GLC GLC galactose GLC glucose jjn/mg. ym/mg. ym/mg. yrr/mg.

HG-1 1.75 1.83 1.22 0.61

HG-2 1.53 1.50 0.97 0.43

HG-4 1.51 1.69 1.14 0.55

H3-5 1.52 1.55 1.02 0.53

HG-6 1.08 1.14 0.78 0.36

B3-1 1.51 1.66 1.14 0.52

BG-2 1.35 1.49 0.99 0.50

BG-4 1.27 1.27 0.84 0.43

BG-5 0.99 1.01 0.61 0.40 48

During this investigation of gangliosides, it was convenient to express the hexose data in terms of micro-moles per milligram of sample. The equations used to calculate this data directly from the GLC charts were derived as follows*

The mg. hexose was calculated from a general form of

(eq. 8) which may be w ritten as

mg. hexose- fK) (area hex.) (mg. mann.) (arav. factor) area mann. (eq. 10)

and since pm. hexose is simply mg. hexose divided by mol. wt,

pm. hexose _ (K) (area hex.) (mo. mann.) (gray, factor) (area mann. ) (130) (eq. 11)

Therefore, pm. hex./mg. sample can be calculated by dividing

(eq. 11) by mg. sample. Thus

Jim. hexose^(K) (area hex.) fma. mann.) (arav. factor) (area mann.) (180) (mg. sample)

(eq. 12).

Therefore the formulae for calculating ypi. glucose and j*n. galactose are as follows*

pm. glu. - (area glu.) (1.30) (0.928) (mg. mann.) (area mann.) (180) (mg. sample)

pm. gal. (area gal.) 1.43) 0.928) (mg. mann.) (area mann.) 180) (mg. sample)

Gangliosides analysis

The major interest in this laboratory has been, and continues to be, the role of the gangliosides in the central nervous system.

During the la st three or four years, the major effort was expended toward the isolation and characterization of the individual components 49 of this closely related family of glycolipids.

During the course of this investigation, nine different prepar­ ations were isolated for the purpose of characterizing them and studying their various properties* Four of these preparations were isolated from a sample of calf brain mixed gangliosides (BG- series) while five fractions were isolated from a sample of human brain mixed gangliosides (H3-series). To characterize these pre­ parations, each sample was quantitatively analysed for NANA, hexose, hexosamine, sphingosine, glucose, and galactose, according to methods described earlier. These data are summarized in Tables

7 and 8. In addition to these assays, each preparation was qual­

ita tiv ely analysed for the fatty acid components, fhese data

are discussed later under a separate heading.

After calculating the molar ratio data of the components, these preparations were placed in one of three catagories, dependent

upon the NANA content. The least complex group included HG-1

and BG-1. Both of these gangliosides yielded data which supported

a monosialo ganglioside molecule and appeared identical to each

other in the TLC assays. These samples were assumed to have the

basic structure described earlier as Kuhn*s Gj since both the

analytical data and the TLC properties conform to literature data.

I t should be noted however, that BG-1 showed minor contamination

by other gangliosides as indicated by the analytical data and the

extra spots observed with TLC assays.

The second catagory included HG-2, BG-2, HG-4 and BG-4.

The analytical data indicated that each of these gangliosides were 50

TABLE 7 .

Ganglioside Component Analysis Data

Sample NANA Sphing GalNac Hexose Gal Glu pm/mg pm/mg pm/mg pm/mg ym/mg pm/mg

HG-1 0.64 0.65 0.65 1.8 1.2 0.61

HG-2 1.0 0.48 0.52 1.5 0.97 0.49

HG-4 1.1 0.50 0.50 1.5 1.1 0.50

HG-5 1.2 0.43 0.41 1.5 1.0 0.53

HG-6 1.2 0.37 0.32 1.1 0.78 0.38

BG-1 0.64 ---- 0.50 1.5 1.1 0.52

BG-2 0.90 0.41 0.46 1.4 0.99 0.50

BG-4 0.88 0.47 0.44 1.3 0.84 0.43

BG-5 0.88 0.23 0.31 0.92 0.57 0.37 51

TABLE 8.

Component Molar Ratios of Gangliosides

Sample NANA Sohina GalNac Hex Gal Glu Glu Glu Glu Glu

HG-1 1.0 1.1 1.1 2.9 2.0

HG-2 2.0 1.0 1.1 3.1 2.0

HG-4 2.2 1.0 1.0 3.0 2.2

HG-5 2*2 0.81 0.77 2.8 1.9

HG-6 3.1 0.97 0.85 2.8 2.0

BG-1 1.2 — 0.96 2.9 2.1

BG-2 1.8 0.82 0.92 2.8 2.1

BG-4, 2.0 1.1 1.0 3.0 2.0

BG-5 2.4 0.62 0.83 2.5 1.5 52 diaialo molecules, while the TLC data subdivided these into two sub-groups, HG-2 and BG-2 were identical to each other and had the fastest mobility properties whereas HG-4 and BG-4 moved iden­ tic a lly but markedly slower than HG-2 and BG-2, On the basis of these TLC characteristics and the analytical data, the faster moving disialo gangliosides were assumed to have the structure proposed by Kuhn as Gjj and the other two disialo gangliosides were assumed to have the structure reported by Kuhn as Gj j j ,

The third group included HG-5, HG-6 and BG -5, ^ince each of these preparations yielded somewhat different analytical data, they will be discussed separately.

The analytical data of HG-6 strongly suggests that the molecule is a trisialo ganglioside having the same basic structure as HG-1, but containing two additional moles of NANA, At the present time, sufficient sample is not available for extensive analysis so the con­ firmation of the components in this molecule will have to await further isolation and subsequent analysis,

BG-5 was assigned to the trisialo group even though the glu­ cose value is high. At the present time, it is the opinion of this author that this fraction contains some extraneous material which interferred in the hexose assays. Since this preparation corres­ ponded chromatographically with the trisialo ganglioside isolated and characterized by Johnson (12), it was not further purified at this time,

HG-5 was subjected to extensive analysis and the significance of these data is not fully understood at the present time. The 53 hexosamine, sphingosine, and NANA data resulted in molar ratios indicative of a trisialo ganglioside. When the total hexose was determined from an acid hydrolysate of HG-5, the data indicated that i t contained four moles of hexose. Since other workers (10) reported a tetra-hexose containing ganglioside, this was not an un­ reasonable possibility. However, when these investigators reported a tetra-hexose ganglioside, they did not assay for the galactose to glucose ratio. Therefore, it seemed particularly important to obtain these data from our sample. Subsequent analysis by

GLC for the hexose data resulted in the anomolous value of 2.3 for the galactose to glucose ratio.

When repeated analysis confirmed all of these data, samples of this preparation were hydrolysed and the hydrolysate subjected to qualitative hexose analysis with standard paper chromatographic techniques. These assays continued to confirm the presence of only glucose and galactose. TLC assay of the neuraminic acid residue confirmed the presence of only NANA, and galactosamine was con­ firmed as the hexosamine residue by GLC and paper chromatography.

Specific color reactions for deoxy and keto sugars were all negative.

From this information, it was postulated that the preparation was a mixture of gangliosides species which were not resolved by any of the TLC systems used to study this ganglioside fraction.

In view of th is information, an attempt was made to resolve the postulated mixture by re-isolating another preparation of the

HG-5 fraction. Another portion of the same mixed gangliosides, from which the first HG-5 fraction was isolated, was processed over an Anasil S column. The fractions showing the TLC mobility characteristics of the fifth spot in the mixed gangliosides were pooled and purified as described e a rlie r. This time however, the purification was followed by NANA analysis after each purification step. When the NANA percentage value reached 37-38%, subsequent re-crystallization failed to increase the NANA content. At this point, the preparation was assayed for the other components, only to find that they all agreed with the previous preparation except the galactose to glucose ratio which changed to 1.9. In each case, when the preparation was assumed to have a fatty acid to sphingo­ sine ratio of 1.0 and a percent fatty acid calculated, the sum of the components totaled between 94 percent and 105 percent. At this point, it was decided that this type data would not resolve the problem and a different technique was needed to offer evidence either for or against the postulate that the preparation was a mixture.

Neuraminidase data

In order to establish a basis for comparison of the inter­ mediates in the neuraminidase catalysed hydrolysis of the gangli­ osides fractions, a sample of the trisialo ganglioside isolated by Johnson was used as a model. When th is material was treated with the enzyme, in 0.05 M phosphate buffer, pH 5.35, an intermed­ iate spot which had the TLC characteristics of HG-4 was found to be the only detectable spot before the molecule was converted to the final product. The final spot traveled with the same mobility as 55 the HG-1 and was resistant to further action of the neuraminidase*

These observations were in complete agreement with those made by

Johnson when he characterized this preparation* Furthermore, it was in complete agreement with the reports of Kuhn et a l*

The treatment of HG-6, postulated above to be a trisialo isomer of the molecule isolated by Johnson, resulted in two intermediate spots, one corresponding to HG-4, and one under this spot which was unidentified. Again, the final product appeared to be neura­ minidase resistant and had the TLC characteristics of HG-1* Thus,

it was concluded that HG-6 was a trisialo ganglioside* Furthermore, based on the conversion of HG-6 to H3-4, it was postulated that this isomeric trisialo ganglioside differs from the molecule isolated by Johnson, probably by having the third NANA residue attached to

a different carbohydrate residue* Yfhen Johnson and McCluer (23) treated their trisialo ganglioside with periodate, they concluded

that the point of attachment of the third NANA residue was not

on the terminal galactose moiety as postulated by Kuhn for the

trisialo molecule which was isolated in his laboratory* The final

clarification of this point must be based upon more extensive studies

than the amount of material available at the present time allows*

Since the BG-5 appeared to be primarily a trisialo ganglioside

with TLC characteristics similar to Johnson*s trisialo molecule,

it was predicted that the neuraminidase treated material would

produce an intermediate pattern similar to the other trisialo gang­

liosides described above. When this experiment was accomplished,

close examination of the spot corresponding to HG-1 appeared to 56 have two components* One of these components developed a much brighter and more stable blue color with the resorcinol than the other*

Otherwise, these two materials were not resolved in the C/l^NHg solvent system. When this enzymatic hydrolysate was subjected to

TLC analysis in the solvent the component yielding the intense blue spot had only migrated a little above the position of

HG-2. At the present time, no data is available concerning the nature of this unusual product of the enzymatic hydrolysis* It

should be noted, however th at the quantity of th is component was

about equal to the component having the TLC mobility similar to HG-1.

The treatment of HG-5 resulted in an even more complex pattern

of intermediate and final products. A product migrating like HG-4

and one migrating like HG-3 were present in approximately equal

quantities during the early stage of hydrolysis, when the hydroly-

sare was assayed with C/jv/NH as the solvent. As the hydrolysis 3 continued, two spots appeared in the area of HG-1. When the reaction

had proceeded to perhaps the half-way point, the quantity of the

two components which migrated in the area of HG-1 were no longer

resolvable and appeared as one spot. Although sone difficulty

was encountered in getting HG-5 completely converted to the products

which migrated as HG-1, on one occasion this was accomplished.

When this plate was oversprayed with sulfuric acid and heated, no

further spots developed. Attempts to resolve the spot, which

migrated like HG-1, in other solvent systems were not successful.

Although defin itiv e evidence is not available at the present

time, one working hypothesis which appears to fit the analytical data and the neuraminidase data, postulates the existence of two tetra-hexose trisialo gangliosides which are mixed in different proportions in the two preparations of HG-5. One of these molecules would contain a third mole of galactose as the extra hexose residue whereas the other would contain a second mole of glucose as the extra hexose residue. Such a mixture would result in'constant total hexose data but different galactose to glucose ratio s depending upon the amount of each component present in any given preparation.

Furthermore, if it were assumed that such monosialo-tetrahexose gangliosides would migrate similar to HG-1 and were only marginally

resolvable, it could account for the observed spots found during en­

zymatic hydrolysis. Since such a monosialo ganglioside has not yet

been reported in the literature, there is no evidence available to

confirm or deny such a postulated TLC mobility. Until such time

as material is available to isolate the intermediates produced

during neuraminidase treatment and characterize them, the final

answer to these data must remain in iMs present state of speculation.

Fattv acid data

The fatty acid methyl esters are formed as one of the products

when the gangliosides are subjected to methanolysis for the hexose

determination. The hexose procedure required that these esters

be removed prior to analysis. As a matter of routine, the methyl

esters were saved when they were extracted from the methancfysate.

The n-heptane solutions of the fatty acid esters were concentrated

and subjected to qualitative analysis by GLC. 58

Although the peak areas were not measured, i t was estimated that stearic acid constituted about 90% of the fatty acid compo­ sition, with archidic comprising the large majority of'the remainder in all preparations examined.

TLC properties of gangliosides

As mentioned earlier, the TLC characteristics of the ganglio­ side preparations were used as a means of identification and as a c rite ria of homogeneity. This technique is valid for drawing such conclusions only when it can be demonstrated that the TLC properties are indeed unique for a given single component. With th is in mind, a ll of the characterized ganglioside samples avail­ able in this laboratory were used to study the ganglioside TLC characteristics in the four solvent systems previously described.

Before studying the TLC characteristics of the purified sample, a mixture of at least six components which migrated with or below

HG-1 and a mixture of at least five components which migrated above

HG-1, in C/ft/NH , were spotted and resolved in the other solvent systems. The patterns of resolution observed with these solvents are recorded in Plate I. These patterns compared favorably with the patterns described by the investigators routinely using these solvents. These plates provided the basis for deciding that we could probably reproduce the separations which other workers observed.

After establishing this basis for comparison, all of the purified ganglioside preparations were subjected to analysis in the same TLC Patterns of Mixed Gangliosides c/WNHj

Figure I Figure 2

*

m

Figure 3 Figure 4 VA Plate I VO 60 solvent systems* Data for the HG- series are recorded in

Plate II. The BG- series were compared to the HG- analogs in a ll solvent systems to establish any differences which may exist between gangliosides obtained from different species.

By comparing the TLC behavior of gangliosides reported in the literature, with the observations made during this study, the gangliosides of Kuhn et a l., Svennerholm et al. and Johnson and

McCluer were compared, and assignments made to relate the symbolism

of these authors to the spots which fit these data. In doing this,

it was hoped that part of the confusion existing in the literature would be clarified. The final conclusions of these studies were

recorded schematically in Plate III.

After completing these studies, several postulates were made

for predicting the migration of the different gangliosides in the

different solvent systems. One of these postulates considered the

ratio of NANA to hexose content, another considered the percent

NANA while still another considered the molecular weight as the

determining factor. All of these proposals failed at least once

in predicting the relative mobility characteristics of the pure

gangliosides studied.

Certainly one of the reasons that these hypothesises failed

was that not enough data was available to evaluate the effect of

structure on the mobility characteristics. The fact that structure

plays an important role in these phenomena is illustrated by the observation that HG-2 and HG-4 are easily separated in all of these

solvent systems in spite of the fact that they are isomeric disialo

gangliosides. TLC Patterns of Individual Gangliosides

Figure I Figure 2

Figure 4 GANGLIOSIDE TLC MOBILITIES

(1) (2) ( 3 ) ( 4 ) ( 2) ( 4 )

HG-A -...////-I HG-B ^ZJ^LL HG-C ®LAC UNK HG-D G o FM ®M2 UNK HG-E HG-C W W W HG-1 ® I l- G ®Ml 252 H G - la G'l a c FM HG-1 l-G

UNK W W W H G -2 ® n 2 - G G Olo H G -2 Gn 2 - G UNK H G -3 H G -3 H G -4 3 -G

H G -4 6 m 3 - G ®Dk HG-S 4 -G HG-G ~y / / H G -5 Gj j C?) 4 - G GT,(?)

H G -6

H G -7 (f

NANA NANA

Figure I Figure 2

P r /N H , ( 1 ) ( 2 ) (3) (4) P r / H f i (1 ) ( 2 ) (3 ) ( 4 )

J /m 'T T T UNK HG-C 6 LAC Gm b tmrrn HG-DFM ®or r ®M2 UNK HG-1 a 6 lac HG-C 6 LAC G M9 HG-1 1-G ®MI ((Mi H G -2 ? x 2 -G GDIs HG-D G o FM 6 M2 HG-1 l-G //} I U ) H G -3 '///////, GX Gmi u u r m H G -la G lac

H G -2 G j i 2 - G ®DI« mm. H G - 3 H G -4 Gttt 3 - G ® D l b '//////! ntu/nH G - 4 Qn r 3 - G G oib u m n H G -5 G jy (7) 4 - G Gt i (?) '///////) H G -5 6 3 1 (?) 4 - G Gt |(T) /////// HG-6 mum H G -6

NANA NANA

Figure 3 ('\ ^ ",ch McC,u,f Figure 4 (2) Kuhn of. ol. (3) Johnson a M c C lu a r (4) Svtnnsrholm o l ol. Plate HI 63

While sufficient data is presently not available to successfully determine the controlling factors involved in the separation phenomena observed in the different solvents, certain conclusions can be arrived at regarding the reliability with which identification of the gang­ liosides, and the degree of homogeneity, can be ascertained from this type analysis. In regards to the identification of a ganglioside based upon its TLC mobilities, this investigation demonstrated that while HG-5 appears to have the same TLC characteristics as the trisialo ganglioside isolated by Johnson, the analytical data does not support th is id en tification. Although th is case may be an exceptional one, an examination of the charts in Plate III reveals a number of cases where different gangliosides migrate at the same rate in one or more solvent systems. One case in point is that HG-2 and HG-3 can not be resolved in the Pr/H 20 system and perhaps only marginally resolved in the Pr/NHg system. Thus, it becomes imperative that other information be considered before absolute identification is made.

The data from these studies further indicate that at least in one case, HG-5 the TLC assays failed to demonstrate that the prep­ aration was a mixture, while the analytical data and the neuraminidase data suggest that the sample is indeed a mixture.

This type of information certainly leads to the question of defining the term "chromatographically homogeneous." Theoretically, the term homogeneous implies that the entire population of molecules 64 in the sample is identical with respect to content and structure.

From a practical view point, however, th is term is used to denote that the population appeared identical within the limits of detection of the system used for assay. Certainly this is the limits of the term as used in conjunction with the TLC assay. For the most part, the gangliosides have been considered homogeneous when the popula­ tion contained the same number of moles of glucose, galactose, hexosamine, NANA, sphingosine and fatty acid per molecule, although the fatty acid content may be heterogeneous. This modified term was accepted in this area of investigation simply because techniques for sub-dividing these molecules into more homogeneous populations has not yet been demonstrated.

Although the TLC systems is the most powerful tool available to the investigators in this field for gaining information about this family of closely related glycolipids, it is this authors

opinion that frequently an unjustified amount of emphasis has been placed upon the term "chromatographically homogeneous.”

One of the areas where extreme caution must be exercised in

interpretation of such TLC patterns is in the area of comparing

ganglioside patterns from pathological tissue with those of normal

tissue. Certainly, differences can be demonstrated, but unless

other data are available, caution must be exercised in identifying

the pathological component.

In conclusion then, these studies have shown that while the

TLC technique is invaluable in the study of gangliosides, the un­

predictable mobilities of tese components requires that before 65 more than a very ten tativ e id entification be made of a "homogeneous” component, other data must be available. Even then, i t is possible that an identification may be wrong due to the mixtures which are possible within this family of glycolipids. SUMMARY

Gangliosides were isolated by employing a hot chloroform- methanol (l/2) extraction of an acetone-ether powder of human brain tissue. The lipid residue was subjected to multiple part­ itioning in Folch solvents using 0.1% saline. The upper phases were collected, combined, exhaustively dialysed, passed over a

Dovyex 50 (H+ ) bed and lyophilized. This material was further purified by repeating the Folch partitioning and all subsequent operations. The resulting powder was used as mixed gangliosides for further fractionation. Similar preparations were obtained from Wilson Laboratories as a source of calf brain gangliosides.

Individual gangliosides were obtained by fractionation on an

Anasil S column using chloroform-methanol-water as the eluting media. Using this technique, fractions labeled HG-1, HG-2, HG-4,

HG-5, and HG-6 were obtained from the human mixed gangliosides

and fractions labeled BG-1, BG-2, BG-4 and BG-5 were obtained

from the mixed calf gangliosides. Each of these preparations was

subjected to component analysis.

HG-1 and BG-1 were monosialo-gangliosides with BG-1 containing

trace amounts of other ganglioside contaminants. HG-2 and BG-2

were disialo-gangliosides as were HG-4 and BG-4. The HG-2 and

BG-2 displayed identical TLC mobilities but were easily distinguished

from HG-4 and BG-4 in a ll TLC systems studied. BG-5 assayed

as a trisialo-ganglioside and appeared as a homogeneous spot in the

66 67

TLC analysis. However, the hexose ratio data indicated that the

preparation was probably a mixture. HG-5 also appeared as a homo-

genious spot in all TLC systems used, but the analytical data and

enzymatic studies led to the conclusion that this preparation

was a mixture. The total hexose data indicated that the material

was a tetra-hexose-trisialo ganglioside but the hexose ratio data

indicated the presence of only three hexose residues. Likewise the

neuraminidase studies suggested that the material was mainly converted

to a neuraminidase resistant, monosialo-ganglioside which had

TLC mobilities similar to HG-1. HG-6 analysis yielded data indicating

the presence of a trisialo-ganglioside which could be distinguished

by TLC analysis from the trisialo-ganglioside isolated by Johnson(l2).

A modification of a GLC procedure for glucose and galactose

described by Sweeley and Walker (55) was demonstrated to yield

reproducible quantitative data for the ganglioside preparations.

The addition of mannitol as an internal standard allowed the calcu­

lation of absolute percentage as well as the ratio of these hexoses

in gangliosides.

The TLC characteristics of these preparations and two other minor

gangliosides were studied in for' solvent systems; chloroform-methanol-

water (60/35-8), chloroform-methanol-2.5N ammonia (60/35/8), n-propan-

ol-water (7/3), and n-propanol-con. ammonia-water (6/2/l). These

preparations were related to the gangliosides reported by other

investigator in so far as possible from the data available in the

literature. Their relative mobilities in all four TLC solvent sys­

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73, Lauter, C., Trams, E. J. Lipid Res., 3, 136 (1962). AUTOBIOGRAPHY

I, Ronald Jack Penick was born in Dover, Ohio, on Noverber 15,

1933* I attended the New Philadelphia, Ohio, public schools and graduated from high school in June 1951. I attended Miami University at Oxford, Ohio,from September 1952 through June 1958. I received a B. A. degree from this institution in June, 1956, and a M. Sc. degree in August 1958.

I was commissioned as a Second Lieutenant in the United States

Air Force Reserves upon graduation in 1956. In July 1958, I was called to active duty and in July 1961, I accepted a regular commis­ sion in the Air Force. In September, 1962, I entered the Graduate % School of The Ohio State University and registered for a program directed toward the Ph. Do degree. This period of graduate study was by direct assignment through the Air Force Institute of Technology,

Wright-Patterson A. F. B., Ohio.

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