Title: Studies on in vivo and in vitro myelin sulfated galactocerebroside biosynthesis in central nervous system tissue
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00098938/00001
 Material Information
Title: Studies on in vivo and in vitro myelin sulfated galactocerebroside biosynthesis in central nervous system tissue
Physical Description: x, 74 leaves. : illus. ; 28 cm.
Language: English
Creator: Sprinkle, Terry Joe Curtis, 1942-
Publication Date: 1974
Copyright Date: 1974
Subject: Central nervous system   ( lcsh )
Biochemistry and Molecular Biology thesis Ph. D
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Thesis: Thesis -- University of Florida.
Bibliography: Bibliography: leaves 67-73.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00098938
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000582558
oclc - 14124878
notis - ADB0935


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In memoUy o6 my grandfatheA,

CuAtis M. SpLinkfe,

an able teacheA and educator' 6ot cve,
o.,tiy yeacc and a source od
inZpitatilon to me and many otheAt s-tudents.


The author is indebted to the faculty of the Department

of Biochemistry who have provided much of the foundation and

support conducive to scientific endeavor. Special recognition

and gratitude is due Dr. Murray Bornstein, Edith R. Peterson,

Dr. George Collins, Dr. William Luttge, and Dr. Jerald

Bernstein and many others for their helpful discussions in

the formative stages of this work.

The author is especially grateful for the seemingly infi-

nite patience, constructive criticism and encouragement of Dr.

Owen M. Rennert during the entire course of this work. Dr.

Rennert has not only served as Supervisory Chairman, but also

as an example of communicating the sense of excitement surroun-

ding scientific discovery. The author is also grateful to his

committee members Dr. Charles Allen and Dr. John Tsibris of

the Department of Biochemistry, and Dr. Carl Feldherr of the

Departments of Pathology and Biochemistry for their helpful

comments and criticisms during many hours of discussion.

Deep appreciation is expressed for the encouragement given

the author by his father and mother, wife Ann, and children,

Joe, David, and Ashley.












SUMMARY . . . .

. . . . . . . . iii

. . . . . . . . v

. . . . . . . . .vi

. . . . . . . .viii

. . . . . . . . ix

. . . . . . . . . 1

. . . . . . . . . 8

. . . . . . . . .21

. . . . . . . . .55

. . . . . . . . .64

BIBLIOGRAPHY . . . . . .

. . .67

. . . . . . . . 74




1 Tissue culture growth medium. . . . . . .12

2 Tissue culture medium content of phenylalanine,
phenylpyruvate, phenyllactate and phenyl-
acetate before and after additions of these
compounds . . . . . . . . . ... 14

3 Crude DNA and protein estimation in vitto
in the presence and absence of phenylpyruvate
and phenylacetate . . . . . . .... 42

4 Effect of bilateral intracerebral injections
of phenylalanine, phenylpyruvate, phenyllac-
tate, phenylacetate, a-ketobutyrate, a-
ketoisocaproato, and a-ketoisovalerate upon
brain sulfated galactocerebroside synthesis . .50



35 -
1 In vivo age profile of SO injected
i.p. and incorporated into CD-1 mouse
whole brain lipid-soluble radioactivity.

.2 Distribution of lipid-soluble radioac-
tivity from i.p. administered 35S04= in
CD-1 mouse cerebrum, cerebellum and
spinal cord as a function of age . . .

3 Extent of the contribution of 35S-meth-
ionine sulfur to the in vive synthesis
of sulfated galactocerebrosides in CD-1
mice . . . . . . . . .

4 In vitto incorporation of 35S04 into
myelin-specific sulfated galactocerebro-
sides in CD-1 mouse spinal cord cultures. .

. . 22

. . 25

. . .27

Myelin isolation and extraction . . . . .31

6 The effect of phenylalanine, phenyl-
pyruvate, phenyllactate and phenylacetate
upon sulfated galactocerebroside synthesis
in CD-1 mouse spinal cord culture at high
(a) and low (b) concentrations . . .

7 The effect of phenylpyruvate removal and
replacement with control growth medium
in CD-1 mouse spinal cord culture upon
net sulfated galactocerebroside synthesis

8 Thin layer chromatography of radioactive
myelin-specific sulfated galactocerebro-
sides isolated from cultured CD-1 mouse
spinal cord . . . . . . . .

9 Radiochromatogram of isolated in witlo
synthesized sulfated galactocerebroside


. . .37

10 Brightfield photography of mouse CD-1
spinal cord grown in the presence of
control, phenylpyruvate, and phenylalanine
medium. . . . . ... . . . ... .40

. . 32

. . .35



11 In vivo synthesized myelin in CD-1
mouse spinal cord at 5 days post-
partum and 20 days postpartum. . . . ... 43

12 In vivo whole brain sulfated galacto-
cerebroside synthesis with concomitant
bilateral intracerebral injections of
saline (control a) phenylalanine (b),
and phenylpyruvate (c). . . . . . .45

13 In vivo whole brain sulfated galacto-
cerebroside synthesis with concomitant
bilateral intracerebral injections of
saline (control a), a-ketobutyrate (b),
and a-ketoisovalerate (c) . . . . ... .48

14 The effect of bilateral intracerebral
injections of phenylpyruvate, phenyl-
alanine, a-ketobutyrate, a-ketoisocaproate,
and a-ketoisovalerate upon adult mouse
kidney sulfated galactocerebroside synthesis. .52

15 The effects of phenylpyruvate and a-
ketoisocaproate upon release of 14C02 from
1-14C labeled pyruvate in CD-1 mouse
brain homogenates, and by phenylpyruvate and
a-ketoisovalerate in adult mouse kidney
homogenates . . . . . . . . .53



KB a-ketobutyrate, sodium salt

KIC a-ketoisocaproate, sodium salt

KIV a-ketoisovaierate, sodium salt

MEM minimal essential medium (MEM, Eagle-Earle)

PA phenylacetate, sodium salt

Phe phenylalanine

PLA phenyllactate, sodium salt

PSLP pure solvents lower phase

PSUP pure solvents upper phase

SBSS-X7 Simm's balanced salt solution (X7)


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



Terry Joe Curtis Sprinkle

June 1974

Chairman: Dr. Owen M. Rennert
Major Department: Biochemistry

The in vivo incorporation of radioactive sulfate into

CD-1 mouse whole brain, cerebrum, cerebellum, and spinal cord

sulfated galactocerebroside as a function of age was determined.

Maximum incorporation occurred at approximately 13 days post-

partum, after a rapid onset beginning at day 5-8. The ratio

of peak incorporation in spinal cord to cerebellum to cerebrum

was approximately 12:3:1 respectively, based upon wet weights.

Synthesis of sulfated galactocerebroside was studied

in vitLo in mouse spinal cord culture as a possible model sys-

tem for the investigation of phenylketonuria and its related

metabolite effects upon the synthesis of a specific component

of myelin that is formed during early myelination events.

Inhibition of net synthesis was observed to the extent of

70 percent at 500 uM phenylpyruvate (PPA) and 50 percent at

1000pM phenylalanine (Phe) compared to controls. No signifi-

cant effect was observed at either 300 pM phenyllactate (PLA)

or 250 pM phenylacetate (PA). PPA was found to be only slightly

inhibitory at 50 IM. The inhibitory effect of PPA was shown

to be reversible when control medium was added back at 21 days

in vitro.

The extent of L-methionine sulfur contribution to

sulfated galactocerebroside synthesis in whole brain in vivo

was extremely low, only 2600 dpm per gram wet weight after

injection of 0.57 pCi L-methionine ( 35S) at 40 Ci/mM.

Intracerebral bilateral injections were made into CD-1

mice 8-180 days old with Phe, PPA, a-ketobutyrate (a-KB),

a-ketoisocaproate (a-KIC), a-ketoisovalerate (a-KIV), PLA and

PA, followed by i.p. radioactive sulfate. Sulfated galacto-

cerebroside synthesis was significantly reduced (all p < .05)

with the first five compounds in mice 8-15 days old, but not

with PLA and PA. In adult mice 40-180 days old PPA resulted

in a significant decrease in synthesis (p < .01), although

Phe, a-KB, and a-KIC did show some reduction.

Adult mice also reflected a decrease in their kidney sul-

fated galactocerebroside synthesis with PPA (p < .02), a

lesser reduction with Phe, a-KB, and a-KIC, and no change

with a-KIV.

The effect of PPA as a structural analog of pyruvate was

examined in CD-1 mouse kidney and brain with 1- C pyruvate,
measuring 1CO release. Both systems, using homogenates,
showed decreased 14CO2 release. It is suggested that decreases

observed in sulfated galactocerebroside synthesis may be due

to decreased ATP production required for the synthesis of PAPS,

decreased bound galactocerebroside as a result of decreased

availability of acetate for synthesis, and general deficits

related to oxidative metabolism.




Myelin membrane serves as a barrier to extracellular

migration and transport of many compounds, including small

ions, for example K [1], as well as large compounds. It

demonstrates a high electrical resistance, and a low capaci-

tance [2], and the significance of myelin in the increased

velocity of conduction in myelinated fibers was experimentally

determined in 1966 by Kuffler [3].

Mouse CNS myelin in particular has been shown to contain

approximately 25 percent protein, 25 percent phospholipids,

30 to 35 percent cholesterol, and 16 percent sphingolipids.

Several functions or possible roles have been attributed to

sulfated galactocerebrosides within the latter sphingolipid

class of myelin lipids. Sulfated galactocerebrosides as a

class are considered to be one of the most stable myelin

lipids [4,5]. They have been implicated in cation transport

across the myelin membrane [6], assigned a protective role in

protease response to myelin basic protein [7], and shown to

have a stablizing effect upon myelin membrane [8]. In addition,

sulfated galactocerebrosides have shown different patterns

in various malignant and virus transformed cells [9], and

have been recently shown by Kreps et al. [10] to be associated

with the specialized function of electrogenesis.

Several methods are available for the biochemical study

of myelin metabolism, including direct isolation, purifica-

tion, and characterization of CNS myelin; .i vivo methods,

usually with postpartum myelin forming animals [11,12], and

in vitfo systems either to assay various enzymes, or by cul-

turing techniques that have recently been made available to

study CNS tissue [13-21]. Three of these methods have been

applied to study specific effects of phenylketonuria and maple

syrup urine disease metabolites upon sulfated galactocerebro-

side synthesis in mouse CNS tissue.

Metabolism of Sulfated Galactocerebrosides

The synthesis of sulfated galactocerebroside has been

shown to proceed according to the following series of reactions:

SO4 + ATP -- APS + PP. (1)


PAPS + galactocerebroside (3)

sulfated galactocerebroside + PAP.

The first two reactions have been shown to occur in the soluble

phase from 105,000 g centrifugation of whole brain homogenates

[22,23]. The active sulfate donor PAPS then transfers the

sulfate to the acceptor molecule, galactosylceramide (galacto-

cerebroside) to form sulfated galactocerebroside (sulfatide).

Reaction (3) has been shown to be catalyzed by a cerebroside

sulfotransferase enzyme found in the microsomal fraction

[24-28]. The synthesis of in viatu sulfated galactocerebro-

side yields the 3-position sulfated product, identical to the

natural one [29].

In 1971, Hammarstr6m [30] proposed, based upon GC-MS

data, that synthesis of galactocerebroside proceeded via

acylation of psychosine (O-galactosylsphingosine). A subse-

quent paper [31], however, showed that such acylation could

occur non-enzymatically and, in fact,proceeded faster in the

absence of enzyme. Current evidence [32] supports the cera-

mide pathway primarily, and little or no firm evidence sup-

ports the psychosine pathway to date. The reaction sequence

for the synthesis of sulfated galactocerebroside is therefore

ceramide galactocerebroside sulfated galactocerebroside.

The relevant enzymes in the sequence have been purified and

characterized [33-37]. The enzyme responsible for the trans-

fer of UDP-gal to HFA-ceramide is present in glial cells, and

has been shown by Arora and Radin [38] to be virtually absent

from neurons. Reduced activity of this enzyme can lead to

reduced sulfated galactocerebroside synthesis and impaired

myelin formation as evidenced in msd/u and Jimpy mouse neurol-

ogical mutants [39,40].

Considerable interest has been generated recently in

sulfated galactocerebroside synthesis and degradation in

sulfatide lipidosis [41] (metachromatic leucodystrophy). An

accumulation of sulfate galactocerebroside occurs not only

in the white matter and gray matter, but also in the kidney.

This suggests, but does not prove, that the kidney and brain

enzyme are similar or identical. Not much is known about

the relationship of sulfated galactocerebroside synthesis

in kidney to that in brain. It is clear that the temporal

appearance of the cerebroside sulfotransferase enzyme and its

activity in kidney are vastly different than in brain. Few

comparative studies, however, have looked at chemical com-

pounds that influence brain sulfated galactocerebroside syn-

thesis and relate the findings to the kidney.

Biochemical studies on myelin-specific sulfated galacto-

cerebroside synthesis and other myelination events were de-

layed for some time due to lack of adequate isolation proce-

dures for the subcellular fractionation of CNS myelin from

other structures.

Myelin Isolation and Characterization

Myelin has been isolated from central nervous system

tissue by subcellular fractionation techniques, largely by

ultracentrifugation [42-58]. Subcellular component marker

enzymes such as 2'-, 3'-cyclic nucleotide, 3'-phosphohydrolase

and various chemical compounds such as cerbrosides and

sphingomyelin have been used to monitor the isolation proce-

dures and to estimate the purity of the fractions obtained

[59-66]. Cholesterol, sphingomyelin, plasmalogens, ccrebro-

sides, and sulfated galactocerebrosides (sulfatides) have

been shown to be heavily enriched in white matter, particularly

in the myelin sheath. Experiments carried out by Norton and

Autilio [45] and Cuzner et al. [44] indicate that approxi-

mately 40 to 50 percent of cerebral white matter (dry weight)

is myelin. Such myelin isolated by ultracentrifugation com-

monly contains over 70 percent by dry weight lipid. Virtually

all of the remaining dry weight can be accounted for as pro-

tein and proteolipid protein.

Prior to the early 1960s, it was widely held that myelin

was metabolically inactive and that it had a relatively con-

stant composition at any age in the life of a given animal.

Several papers have appeared since that time showing develop-

mental, regional, compositional, and metabolic differences in

myelin isolated from various species at different ages [67-79].

Recent advances in subcellular fractionation of myelin

membranes, the establishment of reliable markers for the frac-

tions obtained, the development of improved in vitto culturing

techniques, and the availability of suitable radioactive

compounds have made possible the solutions to problems in

the biochemistry of the nervous system that were elusive in

the recent past. In the present work attempts were made to

observe and relate the effects of various metabolites in both

in vitro and in vivo systems upon synthesis of CNS myelin

sulfated galactccerebroside.

Present Research

Several major objectives were defined in the present

research. The first was to determine the developmental course,

onset, and extent of incorporation of radioactive sulfate

into sulfated galactocerebrosides in the developing cerebrum,

cerebellum, spinal cord, and whole brain of CD-1 mice. The

data obtained served as an in vive control for comparison in

subsequent experiments. Secondly, the effects of phenylala-

nine and the structurally related compounds,phenylpyruvate,

phenyllactate and phenylacetate,upon brain sulfated galacto-

cerebroside synthesis in vituto were examined using CD-1 mice

spinal cord cultures.

Brain analyses of some phenylketonurics, that died of

non-neurological causes as adults indicate for the most part

a myelin complement of normal composition, suggesting recovery

or reversibility from any inhibitory effects upon sulfated

galactocerebroside biosynthesis [80]. It seemed, therefore,

very important to determine the reversibility or non-reversi-

bility of any severe inhibitory effect observed when cultures

were grown in the presence of these metabolites in an in vit.o


The role and extent of methionine sulfur in the early

synthesis of sulfated galactocerebroside in mice was investi-
gated using 3S-labeled methionine.

Another objective in the present study was to relate the

in vitAo results back to the intact animal. Intracerebral

injections were made into young mice prior to the time of

rapid myelination to adulthood using selected phenylalanine-

related compounds and (-ketoacids related to conditions in

which CNS myelin deficits have been noted [81-88]. The mice

received i.p. 35SO4 and the whole brains were subsequently

extracted for sulfated galactocerebrosides.

Attempts were made to relate the brain sulfated galacto-

cerebroside synthesis effects to kidney synthesis based upon

the known differences in the temporal appearance of the rele-

vant transferase enzyme.

Finally, a possible explanation is proposed and tested

to explain a reasonable basis for the observed inhibitory

effects of phenylpyruvate and structurally related compounds

upon sulfated galactocerebroside synthesis in mouse kidney and



Experimental Animals

The experimental animals in the following experiments

were CD-1 mice obtained from Charles River. Litter sizes were

limited to 8 to 12 pups. Litters outside this range were not

used unless otherwise stated in order to control or reduce

many of the variables introduced into experiments due to

nutritional state, stage of maturation, and general develop-

ment discussed by some authors [89,90]. The animals were

fed commercial lab chow and given water ad tibitum. Animals

were sacrificed by cervical dislocation, decapitation, or

over-etherization, depending upon the experiment.


Radioactive Na235SO4 35S-L-Methionine, [Methyl-3H]
thymidine and 1- C-L-leucine were obtained from New England

Nuclear. Phenylpyruvate,L-beta phenyllactic acid,and phenyl-

acetic acid were obtained from Nutritional Biochemicals

Corporation. Alpha-ketobutyrate, a-ketoisocaproate, and

a-ketoisovalerate were obtained as their sodium salt from

Sigma. All acids in the following experiments were used as

their sodium salt. Reagent or analytical grade chemicals

were obtained from various commercial suppliers.

Sulfated galactocerebrosides were obtained commercially

(Applied Science Laboratories, Inc.) or isolated from CD-1

mouse brain by Florisil chromatography [91], DEAE chromato-

graphy [92] and thin layer as described subsequently in

Methods and Materials.

Radioactive Sulfate Isolation and Counting

Radioactive 35S located in sulfated galactocerebroside,

produced in subsequent experiments, was isolated by thin

layer chromatography[93,94]and Florisil chromatography [92]

and released by hydrolysis in 1 N hydrochloric acid overnight

at 105C in a 15 ml capped centrifuge tube. An equal volume

of 1M BaCl2 was added, followed by 4 ml of 1 mg/ml K2SO4

solution. The mixture was centrifuged for 15 minutes at

500 g. The precipitate was transferred to a scintillation

vial with 2.0 ml of distilled water added in portions. The

centrifuge tube was then rinsed with 13 ml Aquasol (New

England Nuclear), which was then added to the scintillation

vial for counting as a light gel. Recoveries were approxi-

mately 95 percent for the precipitation step, and 85 percent

was obtained for the counting efficiency. A Packard Tri

Carb model 3345 scintillation counter was used to count the

radioactive samples. This procedure served as additional

confirmation of the identity of the sulfated galactocerebro-

side produced in the in vitao and methionine experiments.

Determination of Phenylalanine-Related Metabolites in
Tissue Culture Growth Media by Gas-Liquid Chromatography

Phenylacetate and phenyllactate were estimated by gas-

liquid chromatography after extraction from growth medium in

which spinal cord cultures were to be fed. One ml of growth

medium was acidified to pH 1 with concentrated HC1, and an

equal volume of saturated NaCl was added. The aromatic acids

were then extracted into diethyl ether or ethyl acetate. The

15 ml screw-capped tubes were centrifuged at 1000 g for 10

minutes after each extraction. Three combined extractions

of 5.0 ml each were taken to dryness at 370C under a steam

of nitrogen. Methyl esters were prepared by the addition of

2.0 ml ethereal diazomethane and 2 drops of methanol. After

20 minutes at room temperature, the solvent was removed under

vacuum, and the sample was dissolved in an appropriate volume

of ethyl acetate for injection. Chromatography was carried

out on an F&M 402 gas chromatograph equipped with dual

flame ionization detectors with 6 ft x 3 mm i.d. glass col-

umns packed with 3 percent OV-17 liquid phase on 80/100 mesh

Chromosorb G (Applied Science Laboratories, Inc.). Instru-

ment parameters were: air 400 cc/min, hydrogen 50 cc/min,

nitrogen 75 cc/min, detector 2750C, injection port 2500C,

temperature 1600C isothermal, or linearly programmed 120 to

1800C. No methyl phenylpyruvate was detected, and no

appreciable 2, 4-dinitrophenylhydrazone could be isolated

from 1 ml medium. Peak areas were measured by triangulation

for quantitation.

Fluorometric Analysis of Phenylalanine in
Tissue Culture Growth Medium

Phenylalanine levels were measured in serum and growth

medium by the method of McCaman and Robins [95] using a

Turner model 111 fluorometer. Aliquots of 200 pl of serum

or media were combined with 200 pl of 0.6 N TCA, mixed well,

and centrifuged after 10 minutes. Samples of 50 pl were

withdrawn for analysis.

In Vitto Growth Medium

The growth medium for the spinal cord culture experiments

consisted of either Medium A or Medium B, whose compositions

are listed in Table 1. Growth medium B is a modification

of one developed by Edith R. Peterson and used in the

laboratory of Dr. Murray Bornstein. Explants of CD-1 mouse

cord were grown in a lying drop position in Maximow chambers

contained in vertical level racks in a Hotpack incubator at


Isolation of Mouse CD-1 Spinal Cord
for Ia Vtz'io Studies

Fourteen and one-half-day pregnant CD-1 mice were sacri-

ficed by cervical dislocation and the abdomen was soaked in

95 percent ethanol for 10 minutes. Embryos were carefully

removed using sterile stainless steel scissors and forceps

and transferred to a 100 x 15 mm petri plate containing Eagle-

Earle Minimal Essential Medium (MEM) supplemented to



A. 6.0 ml SBSS X7
4.0 ml Fetal Calf Serum
0.12 ml 50% glucose
0.10 ml 200 mM glutamine
50 units/ml penicillin

B. 5.1 ml Minimal Essential Medium
(MEM ,Eagle Earle)
1.0 ml Chick Embryo Extract
3.4 ml Human Cord Serum
0.12 ml 50% glucose
50 units/ ml penicillin
0.10 ml 200mM glutamine
.0 ug / ml ascorbate

approximately 600 mg percent glucose and 100 units/ml peni-

cillin. Further dissection was done under a binocular micro-

scope using only Dumont #5 stainless steel forceps and #11

surgical blades mounted in #5 stainless steel handles. The

spinal cord was carefully dissected out using an initial

dorsal and dorsolateral approach and stripped of adjacent

tissue and coverings. Ganglia were removed. The spinal cord

was then bathed in freshly prepared growth medium and cut

into sections less than 1 mm thick forexplantation. Care

was exercised throughout to minimize trauma to the cord, and

the distal portions of each cord were discarded. If sections

were over 1 mm in thickness, a large necrotic zone was found

and very thin sections resulted in failure to myelinate.

Two-tenth mm background grids were therefore used to obtain

uniform optimum sections. Hemisections of cord were trans-

ferred after each cord was sectioned into freshly prepared

growth medium using a wide-bore pipette that had been fire

polished and sterilized. Sections were then transferred to

collagen-coated coverslips that had been preconditioned in

medium as described above. Cultures were routinely fed 100

p1 medium 2 to 3 times a week, and kept in Maximow chambers

at 350C in the lying drop position. Additions to the growth

media of phenylalanine, phenylpyruvate, phenyllactate, and

phenylacetate were made and are listed in Table 2.

Isolation of Central Nervous System Myelin
by Ultracentrifugatian

Myelin was extracted from brain tissue in these experiments











E a

-o -c

a) o -










__ -

n <
. *
- a


- C.


- 0






- o

- 0






- C




.c .c
o C-

using a modified procedure of Cuzner and Davison [44,55].

Osmotic shock of the myelin after preliminary isolation was

carried out to remove loosely bound proteins. Myelin iso-

lated in this manner was used as cold carrier myelin in in

vitto experiments and as a source of reference sulfated


Collagen Matrix Preparation

Rat tails stored at -200C from six 250-gm Sprague-Dawley

rats were soaked in 95 percent ethanol for 10 minutes. The

skin of each tail was dissected free and the silvery white

tendons were carefully dissected out and transferred to dis-

tilled water for rinsing. After rinsing with several changes

of distilled water, the tendons were extracted into 500 ml

sterile 1:1000 acetic acid in deionized water for 24 to 48

hours and then centrifuged in 250-ml sterile bottles at

10,000 g for 1 hour in a Sorvall RC 2-B centrifuge at 40C.

The upper layer was carefully decanted off into sterile 100

ml bottles for storage. The viscous collagen solution was

stable at 4 to 100C for several months.

Immediately before use, 10 ml collagen solution was

dialyzed against 1800 ml sterile water in autoclaved dialysis

tubing (1/4 to 1/2 inch diameter) overnight at 40C. A water

change was made the next morning with 1 Z of sterile water.

Dialysis was continued until the collagen became viscous

such that an inverted dialysis bag produced retarded air

bubble flow upward. Over-dialysis produced a gel that was

difficult to use for a growth matrix. Dialyzed collagen

solution was added to a 7/8-inch coverslip or other suitable

growth surface and solidified to a gel by exposure to ammonia

vapor. The collagen-coated surface was then neutralized by

several rinses of sterile .01 to .02 percent phenol red

solution and preconditioned in Eagle-Earle MEM supplemented

to 600 mg percent glucose and 100 units/ml penicillin. The

collagen matrix was conditioned for at least 48 hours prior

to use in the proper growth medium.

Undialyzed collagen solution, lyophilized, contained

approximately 66 to 70 percent protein by weight as determined

using the Lowry procedure [96] and contained approximately

300 to 500 1g protein/ml in the solution.

DNA and Protein Synthesis and Isolation
from Mouse Spinal Cord Cultures

Cultures of CD-1 mouse spinal cord were labeled with

1 pCi/ml each of thymidine [methyl- H] at a specific activity
of 20 Ci/mM and 1- C-L-leucine at a specific activity of

53.5 mCi/mM. Cultures were rapidly rinsed in cold Sinm's

balanced salt solution (SBSS-X7) supplemented to 600 mg

percent glucose. Cultures were pooled, homogenized in 1 ml

0.9 percent saline at 40C using a Teflon-glass homogenizer

and 3.5 ml cold TCA was added to a final concentration of 5

percent. DNA and protein were then isolated by centrifuga-

tion for 20 minutes at 13,500 g and 40C. The pellet was

resuspended in cold 5 percent TCA twice, spun down as above,

solubilized in NCS solublizer (New England Nuclear) and

counted in toluene-based scintillation fluid.

Lipid Extractions

Lipids were extracted by the Folch-Pi, Lee, and Sloan-

Stanley procedure [97]. A 0.2 volume of 0.37 percent K2SO4

was used to form the two-layer system, and after centrifuga-

tion at 500 g for 10 minutes, the upper layer was aspirated.

The lower phase was brought up to a volume of 8.0 ml with

PSLP. The solution was then rinsed 2 to 4 times with half

volumes of PSUP until the last rinse top layer contained

fewer counts than twice background. The two-phase system

was briefly centrifuged (5 min) after each rinse. The final

lower phase was transferred to glass scintillation vials,

where the solvent was removed by heat. When the vials were

almost dry, they were placed under a gentle stream of nitrogen.

Ten ml Aquasol was added and the samples were counted on a

Packard Tricarb model 3345 scintillation counter.

Ii Vio Synthesis and Distribution of Sulfated
Calactocerebroside Radioactivity in Whole Brain, Cerebrum,
Cerebellum and Spinal Cord

CD-1 mice at various ages were injected i.p. with 0.57

pCi Na2 35SO4 at a specific activity of 850 mCi/mM and specific

regions of brain were isolated and extracted for lipid soluble

counts as described above.

Methionine 3S Sulfur Incorporation into Whole Brain
Sulfated Galactocerebroside-Specific Radioactivity

CD-1 mice of various ages were injected i.p. with 0.57

pCi 3S-L-methionine at a specific activity of 40 Ci/mM.

Whole brain was extracted 24 hours later for lipid soluble

radioactivity after sacrifice by over-etherization. After

lipids were extracted as described above, the extracts were

dried under vacuum at 600C, redissolved in a small volume of

2:1 chloroform-methanol, and subjected to TLC as described


Thin Layer Chromatography

Thin layer sulfated galactocerebroside separations were

done on Silica Gel G plates with or without binder on plates

100, 250, or 500 microns thick. Solvent systems used were:

(a) 75:25:4 (chloroform-methanol-water),

(b) 65:25:4 (chloroform-methanol-water), and

(c) 5:4 (chloroform-methanol) or (b) and (c).

Plates were developed in equilibrated paper-lined TLC tanks


In Vivo Sulfated Galactocerebroside Synthesis
in the Presence of Added Compounds

CD-1 mice of various ages were injected intracerebrally

and bilaterally under ether anesthesia with 8.0 ug of either

PPA, Phe, PLA, PA, a-KB, a-KIC, or a-KIV as their sodium salt.

Control animals received 0.9 percent saline. Within 15

minutes of the intracerebral injections of 1 pl volume,

each mouse received 0.57 pCi/mM Na235SO4 i.p. at an activity

of approximately 215 mCi/mM. Sulfated galactocerebroside

counts were extracted from whole brain and kidney after

periods up to 24 hours as described under Methods and Materials,

and combined brain extracts were counted as lipid soluble

counts. Two-dimensional TLC was carried out on random brain

extracts to be sure that the differences in observed counts

did, in fact, reflect differences in sulfated galactocerebro-

side synthesis, and not some other lipid soluble sulfur-con-

taining compound. In each and every case, the changes in

counts reflected a corresponding change in sulfated galacto-

cerebroside-specific counts. All kidney extracts were sub-

jected routinely to two-dimensional TLC in the presence of

25 pg cold carrier sulfated galactocerebroside as described

in Methods and Materials, and visualized by exposure to iodine

vapor. The iodine was allowed to sublime off and the spots

were counted in Aquasol.

Measurement of 14C02 Release from 1-14C
Labeled Pyruvate in Mouse Kidney and Brain Homogenates

Brains and kidneys from five adult CD-1 mice (40 to 180

days of age) were homogenized in 0.25 M sucrose. One ml

brain and 1 ml kidney homogenate containing 13 and 25 mgpro-

tein, respectively, as measured by the biuret reaction, were

added to 4.0 ml Krebs phosphate buffer at pIH 7.4 in 25-ml


reaction flasks. Each flask was gassed with oxygen, fitted

with a Hyamine well that would hold approximately 0.6 ml

and incubated capped at 37C. Five minutes later, radioac-
tive substrate at 300,000 cpm and 1 x 10 M, and PPA, KIC

or KIV at various-concentrations were added in a volume of

1 ml, injected into the flasks. After gently shaking the

flasks for exactly 30 minutes, 1 ml 1 N H2SO4 was injected

into each flask. One hour later, the 0.4 ml Hyamine from

each CO2 trap was quantitatively transferred to 10 ml toluene-

basedOmnifluor scintillation fluid and counted.


In Vivo Synthesis of Sulfated Galactocerebrosides
in Whole Brain, Cerebrum, Cerebellum, and Spinal Cord

The incorporation of radioactive sulfate into sulfated

galactocerebrosides has been studied by several authors in

rats and mice [24-28,70,97-98]. A maximum rate of accumula-

tion of sulfated galactocerebrosides has been shown to occur

approximately 17 days after birth in rats [97,99-100] and

in mice [101]. Little is known about the regional distri-

bution of the radioactivity, since most previous studies have

utilized whole brain extracts.

In this study, CD-1 mice were used in order to determine

accurately the onset, period of maximum incorporation, and

the regional distribution of that sulfated galactocerebroside

radioactivity as a function of age in the cerebrum, cere-

bellum, and spinal cord. The results of i.p. injection of

Na2 35S04 and subsequent isolation of lipid-soluble sulfated

galactocerebroside counts in whole brain are shown in Figure

1. It has been shown by several authors [27,97,100] that

sulfur radioactivity from Na35 SO4 injected during the period

of rapid myelin synthesis is found specifically in sulfated

galactocerebrosides to the extent of approximately 90 per-

cent or more. This fact was confirmed in the present


100- T


14 0 -


5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23/28/40

Figure 1. In viv( age profile of SO4 injected i.p. and
incorDorated into CD-1 mouse whole brain lipid-
soluble radioactivity. Over 90 percent of the
radioactivity was found specifically in sulfate
galactocerebrositde,;j therefore lipid-soluble
counts in this and subsequent experiments 2e
taken to reflect synthesis of that co-pFund
directly unless st-- d- otherwise. Fiv- mire
were each inject-d with 0.57 UCi/gm body weight
of Na235SO4 at an activity of 850 mCi/mM for each
data point as described in Methods and Materials.
Values are reported as means s.e.m.

experiments by TLC in several-solvent systems, and by column

chromatography as described in Methods and Materials. Lipid

soluble counts were considered, therefore, to reflect sul-

fated galactocerebroside counts directly unless stated other-


A rapid onset of incorporation of 3SO4 was found be-

ginning on or about days 8 to 11 postpartum. At least two

enzyme systems must be fully functional by this time; the

PAPS generating system [26,89,97] involving the cytosol sul-

furylase and kinase steps, and the relevant membrane-bound

transfer enzyme, cerebroside sulfotransferase [24]. Maximum

incorporation of 35SO= occurred between 12 and 14 days after

birth. Adult animals incorporated 3504 at a level com-

parable to 5-day-old animals. Recent evidence [70,102]

supports the existence of two brain sulfate pools, one large

pool associated with sulfated galactocerebrosides with turn-

over half-times on the order of 9 months or a year, and

another very small pool for the synthesis of sulfated galac-

tocerebrosides with a turnover half-time on the order of 2 1/2

days. Evidence suggests that the latter pool is involved in

the outermost myelin layer synthesis. Presumably the incor-

poration observed in the adult animals reflects a composite

of continuing new stable myelin sulfated galactocerebroside

synthesis and that of the more metabolically active outer

membrane regions. Each data point shown in Figure 1 repre-

sents the mean of five animals, extracted independently as

described in Methods and Materials. The peak of incorporation

in whole brain occurred in this experiment at 13 days post-

partum (dpp) and reached a level of 100,000 dpm/gm wet

weight brain. Litter sizes in these experiments were limited

to 8 to 12 pups per litter, for reasons cited earlier.

The regional distribution of the sulfated galactocere-

broside radioactivity in the cerebrum, cerebellum, and spinal

cord as a function of age are shown in Figure 2. All injec-

tions were made at 0.57 pCi/gm body weight, and at the same

specific activity so that the data are directly comparable.
35 -
The age at which maximum incorporation of SO4 oc-

curred into sulfated galactocerebroside in CD-1 mice was

exactly the same (13 dpp) for cerebrum, cerebellum, and spin-

al cord under the present experimental conditions. The levels

of incorporation in cerebrum, cerebellum, and spinal cord at

day 13 were found to be approximately 32,500, 100,000, and

380,000 dpm/gm wet weight tissue, respectively. These results

correlate well with histological studies showing increases

in stainable myelin during this period. Cerebrosides and

sulfated galactocerebrosides are considered by many authors

as a result of these studies to be among the most reliable

indexes of myelin mass in the central nervous system [55,76,


Methionine Contribution to Sulfated
Galactocercbroside Synthesis

The specific contribution of methionine sulfur to sul-

fated galactocerebroside synthesis is shown in Figure 3. The

Figure 2. Distribution of lipid soluble radioactivity
as sulfated galactocerebroside from i.p.
administered 35SO4 in CD-1 mouse cerebrum,
cerebellum, and spinal cord as a function
of age. Details are described in the text.
Each point represents average of 5 animals.
Results are expressed as means + s.e.m.



6 7 6 5 0 II 12 13 I 11 16 17 IA 9 20 21 02 23/60





4 0-

5 6 7 8 9 I0 II 12 13 14 15 16 17 18 I9 20 21 22 23/60


5 6 7 8 9 10 II 12 I I IS I 7IT IB 19 20 21 22 23/6


_ 2,000-
1,800 "
2 1,600

E 1,200.

I 1 I '
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 28

Figure 3. Extent of the contribution of 3S-methionine
sulfur in the in vivo synthesis of sulfated
galactocerebrosides in CD-1 mice. Each point
is the mean of 4-5 mice. Each animal recieved
0.57 vCi/gm body weight of 35S-L-methionine at
a specific activity of 40 Ci/mM. Lipid ex-
tracts from each age animal were combined and
subjected to 2-dimensional TLC to separate
sulfated galactocerebrosides from methionine-
containing lipids,

open circles indicate the dpp age of the mice at the time of

injection. The results are expressed as mean dpm 3S as

sulfated galactocerebroside per gm wet weight whole brain.

Radioactive product was obtained completely separated from

methionine-containing lipids. The delay in peak incorporation

compared to Figure 1 probably represents the increase in

time required to oxidize the sulfur to 3SO04.

Elevated levels of methionine have been noted in the

blood of homocystinurics as well as elevated levels of homo-

cystine [108] and questions have been raised as to the ef-

fects of that methionine upon sulfate utilization in brain.

Subcutaneous injections of methionine in rats followed by

i.p. Na235 O4 have been shown to result in a marked decrease

of label incorporation into sulfated galactocerebrosides com-

pared to controls [100]. It is apparent in Figure 3 that only

2600 dpm 3S was incorporated at the peak (day 14) in CD-1

mice using i.p. injections. Direct intracerebral injections

produce similar results. It was, therefore, concluded that

the methionine sulfur atom was nc utilized extensively in

sulfated galactocerebroside synthesis under the present

experimental conditions. However, one cannot exclude the

possibility that methionine may influence the sulfate avail-

able for synthesis, particularly if the levels in blood re-

mained elevated, by effecting entry of sulfate into the


In Vituo Synthesis of Sulfated Galactocerebrosides
in Mouse Spinal Cord Cultures

Mouse spinal cord cultures were grown in Maximow cham-

bers as described in Methods and Materials in the presence

of Na235SO4. After various periods of time, cultures were

harvested and extracted for sulfated galactocerebroside radio-

activity in myelin. The results, shown in Figure 4, indi-

cate increased incorporation of radioactivity into myelin

through 32 days in vitto. Non-radioactive myelin used as

carrier was isolated and purified according to the flow dia-

gram in Figure 5.

The Effects of Phe, PPA, PLA, and PA Upon In
Vitto Sulfated Galactocerebroside Synthesis

The in vitto system was then utilized to determine the

effects of phenylalanine, phenylpyruvate, phenyllactate and

phenylacetate upon 5S incorporation into myelin-specific

sulfated galactocerebroside as a system in which to study

conditions where general myelin deficits have been observed.

Additions of these compounds were made to control medium

according to the levels listed in Table 2. Marked inhibition

was found in the case of 1000 UM Phe and 500 IM PPA as shown

in Figure 6a, and little or no inhibition was observed at

any of the concentrations used of PA or PLA (Figure 6a or
6b). The reduction in SO4 incorporation into sulfated

galactocerebrosides was approximately 50 percent at 1000 iM

Phe, and 70 percent at 500 pM PPA. The results suggest, but

L 280-

u 200-
E 160-



40- (20)

16 17 18 19 20 2 1 22 23 24 25 26 27 28 29 30 31 32


Figure 4. In vitto incorporation of SO4 into myelin-
specific sulfated galactocerebrosides in CD-1
mouse spinal cord cultures. Hemisections of
spinal cord were grown in the presence of ap-
proximately 100,000 cpm of Na 3 SO4 at an
activity of 850 mCi/mM for 3 days before har-
vesting. Cultures were harvested at various
time intervals and co-migrated with purified
cold carrier myelin and extracted for sul-
fated galactocerebroside-specific radioactivity.




homogenize in 5 vol, bring to 10 vol

0.32M sucrose, 0.001 M EDTA

0.003M Na2 HPO4 ,7.2 15 min at

13,500 g and 4C

25 vol 0.8M sucrose, 0.001 M

EDTA, 0.006M Na2HPO4. 60 min,

40, 000 g, 4C

floating layer

supernatant and pellet

dilute to 20 vol distilled

water. 20 min at 13.500 g,

250C, repeat above step

myelin fraction

extract with CHC.I3 MeOH

myelin lipids

Figure 5. Myelin isolation and extraction. Ninety-five
to 99 percent of the preparation by weight was
soluble in 2:1 chloroform-methanol, and the
myelin obtained was used as cold carrier, in
the previous experiment and in subs-; t
experiments, to isolate sulfat- ''actocere-
broside reference standaz

280- .0
I- (21)
D 240-

S 200- .0

160- (20) (

120 -.-

8o / .*;.'.......... (2

40- (20) .

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32


Figure 6. The effect of phen-lal.nine, :hanyln':ruvate,
phenyllactate, and 'phenyl-acette upon sulrated
galactocerebroside synthr;3is ..n CO-1 xouse
spinal cord c.ltur e .t higi (a' and low (i;)
concentrations. Cop:ion':s ;:er added at the
time of explanation -, cronce-nrations shown
in Table 2. The cultures were -rown various
lengths or time and rnyclin-spc:iLic prod:rct
was isolated as described in methodss and
Materials. The numbi r o:- cul:-e rcprese-,ted
by each data point iare :--.cated in parentheses.




480- 10OiM PHE

440- (17)
*- 50uM PPA
360 ...0(18)


, 240 .oo
280- 0

2 1-6 .......
200- / 0
E (17)

120- /."
80- 4


7 1 l 1 1 1 1 1 1 1 1 1
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32


Figure 6 continued

do not prove, that phenyllactate is not appreciably oxidized

under the present experimental conditions. It may also be

possible to correlate the level of PPA in the Phe curve with

the observed effects in order to obtain better insight into

the nature of the inhibition.

The Effect of PPA Removal upon Sulfated
Galactocerebroside Synthesis In Viteo

Spinal cord cultures grown in the presence of 500 pM

PPA were rinsed at 21 days in culture with SBSS-X7 at 600

mg percent glucose and fed control medium out to 32 days in

culture. At 26 and 32 days in culture, spinal cord explants

were removed from the incubator and extracted as described

previously. The results in Figure 7 indicate a 74 percent

recovery of control values at 32 days in culture. Radioac-

tive product in these experiments was chromatographed in one-

and two-dimensional TLC systems in order to offer additional

confirmation of the structure. Figure 8 illustrates such an

isolation in a 75:25:4 solvent system. Visualization was

done by exposure to iodine vapors. Silica Gel was scraped

from the plates and counted in Aquasol. The radioactivity

corresponded to the position of authentic carrier. The dis-

tances from the origin are indicated in mm. The myelin-speci-

fic radioactivity obtained co-migrated with authentic sul-

fated galactocerebroside as shown in Figure 9 in 75:25:4,

65:25:4 (chloroform-methanol-water), and 5:4 chloroform-meth-

anol systems, as well as inbenzene-methanol (7:3),and by




360 (17)



3 240

160- MEDIUM (16)

120 )

o8 (21)
40- (20)
40 (20)

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Figure 7. The effect of phenylpyruvate removal and
replacement with control growth medium in
CD-1 mouse spinal cord culture upon net sul-
fated galactocerebroside synthesis. PPA
was added at the time of explanation.













6 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Figure 8. Thin layer chromatography of radioactive
myelin-specific sulfated galactocerebrosides
isolated from cultured CD-1 mouse spinal cord.
Twenty cultures (17 days in vitto) were grown
as described previously in Figure 4, homo-
genized in sucrose and co-migrated with ap-
proximately 10 mg unlabeled puified myelin
by ultracentrifugation. The S-labeled
product was isolated by 2:1 chloroform-
methanol extraction and then chromatographed
on Silica Gel G plates using a 75:25:4
chloroform-methanol-water solvent system.

Figure 9. Radiochromatogram of isolated in vitro
synthesized sulfated galactocerebroside.
The R values correspond to authentic
sulfated galactocerebrosides run under
the same conditions (75:25:4, 65:25:4,
and 5:4 in order of increasing R, values
in chloroform-methanol-water systems)
cold carrier myelin extract was co-
chromatographed. Similar results were
obtained by 2-dimensional TLC and column
chromatography. The solvent front (SF)
and origin are indicated.


Rf 0.19

0 I U) 0

Rf 0.25

W 0


Florisil and DEAE chromatography.

Mouse spinal cord cultures grown in the presence of

1000 pM Phe and 500 pM PPA were clearly distinguishable from

control cultures in being more sparsely myelinated as

observed under phase contrast and brightfield microscopy

(Figure 10). Cultures grown in the presence of PLA and PA

were virtually indistinguishable from control cultures grown

under the same conditions. A crude estimation was made of
protein synthesis using 1- C-L-leucine and of incorporation

of thymidine [3H-methyl] into DNA in the culture system to

compare the control cultures to the PPA-treated cultures.

A typical such estimation is shown in Table 3. Any reduc-

tion seen in protein synthesis or incorporation of thymidine

into DNA was less than 10 percent, and not significantly

different from controls.

The Effects of Intracerebral Phe, PPA, PLA, PA,
a-KB, a-KIC and a-KIV Injections Upon In Vivo Sulfated
Galactocerebroside Synthesis in Brain

Phenylalanine, phenylpyruvate, phenyllactate, and phenyl-

acetate injected intracerebrally and bilaterally as described

in Methods and Materials produced effects upon sulfated

galactocerebroside synthesis not unlike those obtained in the

in vitto experiments. No significant reduction of incorpora-

tion of 3S into sulfated galactocerebrosides occurred when

8 pg (each injection) of either PLA or PA was injected bi-

laterally and followed by Na2 35SO4 The total sulfate

Brightfield photography of mouse CD-1
spinal cord grown in the presence of
control (top) phenylpyruvate (center),
and phenylalanine (bottom) medium. The
[PPA] was 500 PM and [Phe] was 1000 pM.
(400X) Cultures were 28 days of in viitto

Figure 10.


Crude DNA and Protein Estimation in vitro in the

Presence and Absence of Phenylpyruvate and


Control cord cultures

PPA treated (500uM)

PA treated (250uM)

3H (PM)







Figure 11. In vivo synthesized myelin in CD-1 mouse
spinal cord at 5 days postpartum (top)
and 20 days postpartum (bottom).





.:, C .

c~ I ~

-a;, L~:~~Yr

>i1=XLch I'~ iL

galactocerebroside content 24 hours after injection has been

shown to be for all practical purposes the same for control

and treated animals, as shown by sulfate content after hy-

drolysis by the rhodizonate method described in Methods and

Materials. The results of Phe and PPA treatment upon whole

brain sulfated galactocerebroside synthesis are shown in

Figure 12. Both compounds produced a significant reduction

(all p < .05) in synthesis in animals 8 to 15 days old. Phe

showed less of an effect in adult animals than PPA, and PPA

always produced a greater effect in any age mouse used.

Alpha-ketobutyrate and a-ketoisovalerate injection re-

sults are shown in Figure 13. In each case, as might be

expected, the results were more severe during the period of

rapid myelin synthesis, a-KIV injected into adult animals,

followed by Na2 35SO4 produced control levels of labeled product.

The test statistic used was the t-test. The results from

all the above experiments are summarized in Table 4.

The Effects of Intracerebral PPA, Phe, a-KB, a-KIC, and a-KIV
Injections Upon In Vcou Sulfated Galactocerebroside
Synthesis in Kidney

Mouse kidney synthesizes sulfated galactocerebrosides in

vivo [109] as does the rat system discussed by McKhann and Ho

[97]. Because of the developmental studies depicting the

late appearance of the relevant cerebroside sulfotransferase

enzyme in a rodent system described by McKhann and Ho [97],

and because of the high activity of the enzyme in kidney, one

In vivo whole brain sulfated galactocere-
broside synthesis with concomitant bi-
lateral intracerebral injections of saline
(control a), phenylalanine (b), and
phenylpyruvate (c) as described in the
text. An average of 4 animals were used
for each data point. Results are expressed
as means + s.e.m.

Figure 12.

30,000- CONTROL





5, 000

8 11 13 15 40-180

30,000- PHE


g i


o 10000-


8 II 13 15 40-180






'a rh F "

8 II 13 15 40-180


In vivo whole brain sulfated galactocere-
broside synthesis with concomitant bi-
lateral intracerebral injections of
saline (control a), a-ketobutyrate (b),
and a-ketoisovalerate (c), as described
in the text. An average of 4 animals
were used for each data point. Results
were expressed as means + s.e.m.

Figure 13.








8 II 13 15 40-180


a KB






8 II 13 15 40-180

30,000 a KIV




l rh
IO, 00-

8 11 13 15 40-180


>0 4

a) >HH4a

a > a 11 W

1c J-l *4Q O

43 0 -4-

aa old a)

c a)a a) 0

a)6 a) 4r, I I

-'0 10

:H >( 4-) 0
$0 (0 *l ll

H m-40
a- 4 0 0 a

- 0 0
S) .-I u 1)

c I o d a) z

a odc
Hn o r3 u (M

S0 *r .-I 0

0 r d 0 f a)

-4 0 -4 H-O
4>1 U 3-4 )

r0 04 0 30.)
I 0 c:0 t
*H 0 a, -
a0 0c cn (.

4- 3E r41

5 -Y o m
0 1 14 4+1

4-1 *Q p 0 *
'- 4-) r 1)n0 (ui 11
4rl 0 d 4-1 w H

nlH4i (D r32 } 1
(U10JHW1 go
a4) r-1 0 m
-r 1 e aP) ni o ) a

M aH U 4- w)

4 r J 11 >1 4) 0i

4 4411 4 -, (U 0 r-
(U4- 1 dl r 4S

44 10 Ln -1
wa r-i4 c (OM i

VI V o Vl VI V- VI

0 0 *0 '*0
: + V + V 1 V +1 V + IA

r- n o ^) 3"

H 0


14 V V VI V V S H

SiN n r-,i N C C. H

H n o Hr ^ 0 0 ~ o 0
f \rD

HO 10 IH H oH

H ^ ^. H ~ H

II a + V I V V

HT O N / ^ 0 0H
(T O' I r^ '

[~ e c^" 4- '"-

H- H Ho NMO H- "
0o o -o -o *'

cr' o a ^ '-i o I a

CM m IN 0 -

H~ H
( dC p) in Ho r i
N cr> 0 o3 I
h N

i--i o r^ ~ u

" -i cr \o ' "

i- i- i-( COIY

(ddp) 33Vi %

might expect the young adult mouse to be a sensitive system

in which to study various chemical effects upon sulfated galac-

tocerebroside synthesis in relation to effects observed in


Adult mice kidneys were removed from animals that had

received bilateral intrecerebral injections of PPA, Phe,

a-KB, a-KIC, and a-KIV, followed by Na235O4 as described

previously. After delay period of a few hours, decreased

synthesis was found in the kidney. In Figure 14, the signifi-

cant decrease in incorporation of Na235SO4 into lipid-soluble

sulfated galactocerebrosides in PPA-injected animals is

readily apparent. Conversely, a-KIV seemed to have no effect.

Phe, a-KB, and a-KIC-treated animals reflected a moderate

(nonsignificant at the .05 level) reduction compared to con-

trol values. A possible interpretation of these results is

presented as they relate to brain synthesis in general dis-


In Vitro Formation of 1CO, from 1- C-pyruvate
in the Presence of PPA and a-KIC in
Mouse Brain, and of PPA and a-KIV in Mouse Kidney

-3 -2
In whole brain homogenates, 1 x 103 and 1 x 102 M phenyl-

pyruvate produced 28 percent and 47 percent reductions (Figure
14 14
15), respectively, in the release of 14CO2 from 1- C pyru-

vate under the experimental conditions described in Methods
and Materials. a-KIC at 1 x 10 M reduced by 52 percent the
amount of CO2 released in control homogenates. Such effects







The effect of bilateral intracerebral injections
of phenylpyruvate, phenylalanine, a-ketobutyrate,
a-ketoisocaproate, and a-ketoisovalerate upon
adult mouse kidney sulfated galactocerebroside
synthesis. Three adult animals 40 to 180 days
of age represent each data point and the re-
sults are expressed as mean cpm 5S as sulfate
galactocerebroside per gram wet weight of kidney
s.e.m. Each animal received 8 ig bilaterally
and intracerebrally in a volume of 1 Hl of each
compound shown, followed by 0.57 pCi Na235S04
per gm body weight as described in Methods and
Materials. Lipid-soluble sulfated galactocere-
broside-specific counts are indicated.



Figure 14.





The effects of phenylpyruvate and a-ketoisocaproate
upon release of C02 from 1-14C labeled pyru-
vate in CD-1 mouse brain homogenates, and by
phenylpyruvate and a-ketoisovalerate in adult
mouse kidney homogenates. The homogenates
were compared and incubated as described in
Methods and Materials. Results are reported
as mean cpm as 1 C02 s.e.m. for each homo-
genate, with n = 3.

Figure 15.

L__ ~1 _

in vivo would be potentially devastating upon energy produc-

tion and precursor molecule availability for brain lipid

biosynthesis. The effects would be compounded first by the

heavy demands for ATP and reducing equivalents by the brain

during the early spurt of myelin-precursor synthesis, and

secondly by the fact that levels of several of the compounds

above are unfortunately maintained, and are available to the

brain and other organs in several clinical conditions.

In the kidney homogenates, a-ketoisovalerate at 1 x 10-

M released 108 percent of control levels of 14CO2 from 1- 14C-
pyruvate, but PPA at 1 x 10 M produced a 42 percent reduc-
tion in 142 release as shown in Figure 15.
tion in CO. release as shown in Figure 15.


Decarboxylation of Pyruvate and Structurally Related Compounds

In the experiments of Burton et al. [109], synthesis

of sulfated galactocerebroside and galactocerebroside was

paralled by incorporation of labeled galactose,and PAPS syn-

thesis has been observed very early in the newborn rat [110].

It has been suggested as a result of these, and other experi-

ments by one author [97] that the availability of galactocere-

broside may be one of the major limiting factors in the syn-

thesis of sulfated galactocerebrosides in the intact animal.

The levels of inhibition observed in the decarboxylation of
1- C pyruvate in this work suggest that the substrate avail-

able for sulfation would probably be significantly reduced

in vivo upon continuous exposure to the compounds demonstrating

the inhibition, and secondly that ATP production would be

reduced during a critical period of nervous system development.

One might expect these effects to bear consequences upon

subsequent myelination events as well, such as further cere-

broside synthesis and deposition into myelin.

One might predict first, that in the presence of chroni-

cally elevated PPA levels in mice for instance, the endogen-

ously bound cerebroside would be reduced, and secondly that

such a reduction would be demonstrable in an endogenous

cerebroside sulfotransferase assay as described by Balasubra-

manian and Bachawat [111]. A second question to be answered

is whether or not PPA is actively decarboxylated in the present
in vitro assay system. Synthesis of 1- C phenylpyruvate and

incubation with the kidney and brain homogenates would pro-

vide a clue as to the exact nature of the inhibition observed.

A differential sensitivity of pyruvate dehydrogenase to

inhibition by KIC, based upon developmental age, has recently

been reported by Bowden et al. [112] in a chick embryo system.

The level of inhibition increased sharply at a constant con-

centration of KIC beginning at day 12. The important point

here is that the susceptibility of the pyruvate dehydrogenase

system to inhibition by KIC was rapidly rising or high during

early myelination. This observation may prove to be more

universal in terms of other animal systems as well when inves-

tigated more thoroughly. Both KMV and KIC have been shown in

rats to be inhibitory upon decarboxylation of pyruvate in

brain and liver, and PPA has been recently shown to be in-

hibitory in rat brain, but not in liver. PLA has no signifi-

cant effect in either brain or liver [113,114]. Results of

this study showed no significant inhibition of decarboxyla-

tion in kidney homogenates using adult mice in the presence of
1 x 10 M KIV, but found a significant decrease at only

1 x 10 M PPA. Both PPA and KIC were inhibitory in CD-1 mouse
brain homogenates at 1 x 10 M. Of the branched-chain

a-keto acids tested in an in vitio culture system by Silber-

berg [115], only KIC inhibited the formation of myelin at

1 x 10 M, and it was cytotoxic at 3 x 103 M. The present

experiment's mortality rates exceeded 50 percent, to as high

as 100 percent, when mice 11 to 15 days of age were injected

intracerebrally with the same amount of KIC given the mice in

the other treatments. (Mortality rates in all other treat-

ment classes were negligible.) The few mice that survived

during that period contained less than 1000 dpm as sulfated

galactocerebroside per gm wet weight of brain. Such devas-

tating effects in sulfated galactocerebroside net synthesis

would also be expected to lead to impaired synthesis and in-

corporation of cerebroside, basic protein, cholesterol, and

other important elements into myelin membrane, and to produce

permanent functional deficits.

Relationship of Various Sulfated Cerebroside
Metabolism Enzymes

A galactocerebroside sulfotransferase has been isolated

from rat kidney by McKhann and Ho [97] that has similar prop-

erties to the brain enzyme as to pH optimum, specificity,

and location within the cell. It is not clear, however, if

the two enzymes are identical. Additional links exist be-

tween kidney and brain sulfated galactocerebroside synthesis

that emphasize the need for additional comparative biochemical

studies of the two structures. Sulfated galactocerebrosides

present in such large amounts in the myelin of metachromatic

leukodystrophy patients also accumulate up to 70 times nor-

mal amounts in the kidney [116]. It has been suggested by

Cumar and co-workers [117] and by McKhann and Ho [97] that

the kidney sulfatase enzyme for sulfated galactocerebroside

and ceramide dihexoside sulfate cleavage is one and the same

enzyme. Present evidence to date indicates that arylsul-

fatase A and cerebroside sulfatase are similar if not iden-

tical [118], and histochemical evidence indicates that

arylsulfatase A is located very near the myelin sheath in

both the CNS and PNS [119]. Further isolation and purifica-

tion of these enzymes will aid in clarifying their roles

in sulfated galactocerebroside metabolism in brain, kidney,

and other structures.

Metabolism and Transport of Sulfated Galactocerebroside

Considerable interest has been generated in the assess-

ment of alternative pathways for the synthesis of sulfated

galactocerebrosides. A generalized scheme showing both major

proposed pathways are shown below:


I gal gal


}f s304

FA--fatty acid

In earlier experiments of Brady [120,121], psychosine

was acylated by acyl-CoA to form galactocerebroside. Morell

and co-workers [122,123] demonstrated synthesis of galacto-

cerebroside from ceramide and UDP-galactose, and could not

acylate psychosine. A recent paper claims enzymatic syn-

thesis of cerebroside, however, from galactosylsphingosine

and stearoyl CoA [124] in embryonic chicken brain. Apparently

the major pathway in the synthesis of cerebroside is the

ceramide pathway. The significance of the psychosine path-

way in the synthesis of cerebrosides and sulfated galacto-

cerebrosides is currently under investigation in several


A sulfated galactocerebroside-containing lipoprotein

fraction was characterized in 1968 by Herschkowitz and co-

workers from rat brain [125], and it was suggested that sul-

fated galactocerebroside is transported from its site of

synthesis in the microsomal fraction to the myelin membrane

by water-soluble lipoproteins very unlike serum lipoproteins.

Little is currently understood about the specific mechanism

of transport of myelin precursors from their sites of

synthesis to the site of incorporation, and studies of how

these processes are controlled are just beginning.

Myelination Sequence

Myelination in the mouse CNS is preceded by a period of

rapid proliferation of oligodendroglial elements and cell

membrane at 10 to 11 days of age. Little myelin can be

detected at this time [126]. The whole brain DNA content

has been shown by Matthieu and co-workers [69] to equal

essentially adult values by 12 days postpartum. The large

increase in DNA just prior to 12 days reflects mainly oli-

godendroglial proliferation [127].

Rapid proliferation of glia is proceeded by the forma-

t.ion of a compact myelin membrane. The current experiments

have shown peak incorporation of 3SO4 into sulfated galacto-

cerebroside myelin precursor molecules in whole brain, cere-

brum, cerebellum, and spinal cord to occur at this time in

CD-1 mice. In vivo experiments in this work indicate that

mice treated with PPA, Phe, PLA, PA, a-KB, a-KIC, and a-KIV

still followed precisely this rigid myelination sequence.

It is reasonable to assume that deficient synthesis within

this period would compound the error in subsequent formation

of compact myelin, and lead to impaired function. Dobbing

[128] pointed out the concept of vulnerable periods in

developing brain and it seems applicable in the interpreta-

tion of the observed inhibition of the incorporation of 35SO4

into sulfated galactocerebrosides in the present experiments.

Route of Injections in CD-1 Mice

In the bi vivo experiments of Chase and O'Brien [100]

in rats, subcutaneous injections of PPA at 5 gm/kg/24 hours

for 18 days followed by i.p. 3S-sulfate resulted in control

values of sulfated galactocerebroside synthesis when brains

were extracted 24 hours later. Phenylalanine administered

in the same manner and at the same dosage produced approxi-

mately a 50 percent decrease in incorporation of 3S sul-

fate into sulfated galactocerebrosides. In preliminary in-

jections of both i.p. and intracerebral injections of PPA

in CD-1 mice in the current experiments, significant reduc-

tions in 3S lipid-soluble counts were observed in each

case. It is suggested,therefore, that in the rat subcu-

taneous injections PPA did not reach the blood or that

adequate blood levels were not maintained for some reason

in the brain. The exact reasons for the difference in results

are not clear at this time.

Tissue and Organ Culture of Mouse Brain

Synaptogenesis, differentiation of neurons and glial

elements, and myelinogenesis have been recently studied in

cerebrum, cerebellum and spinal cord culture [17]. The re-

quirement for basic protein in early myelin synthesis has

been demonstrated utilizing antibody to the protein in an

in vit-io culture system [129] by Bornstein and Raine. In

the presence of antibody myelination was arrested. After

removal of the antibody and rinsing the cultures, myelination

continued. This is further evidence to suggest that myelina-

tion (at least in some stages) proceeds in an orderly timed

sequence, perhaps involving obligate steps for further

synthesis and deposition of myelin into compact mature membrane.

Rabbit EAE sera have been shown to contain two dissimilar

antibody specificities to cerebroside and encephalitogenic

protein. Sera from these rabbits produce demyelination in

CNS cultures, given at sufficient levels, and oligodendroglial

differentiation and myelin formation are inhibited in mouse

spinal cord cultures. Guinea pig serum to basic protein did

not inhibit myelin formation in the cultures, but sulfated

galactocerebroside synthesis was inhibited using rabbit anti-

cerebroside antibody.

Based upon results obtained in the in vitfo brain cul-

ture system, those from the in vivo intracerebral injections,

and from the experiments on the brain and kidney homogenates,

it is consistent with these results to suggest that inhibition

of synthesis of sulfated galactocerebroside in these systems

was at least related to the degree of inhibition of pyruvate

decarboxylation. One would then predict that even in transient

conditions in which 2-keto acids inhibitory to pyruvate decar-

boxylation were elevated in blood that sulfated galactocerebro-

side synthesis would be reduced, and that should such elevated

levels occur during early myelin maturation, irreversible loss

of function could result. An important consideration in ass-

essing the reversibility of recovery as observed in sulfated

galactocerebroside synthesis in the PPA experiments in vitro is

discussed by Anderson, Rowe, and Guroff [130] in their paper on

behavioral changes in rats with experimental PKU, and that

is reversibility or recovery from low levels of synthesis


during a critical period of development does not a pAioti

confer the brain with immunity from permanent functional

damage, even if the apparent structural recovery seems to be

complete upon further development [128].


Maximum incorporation of radioactive sulfate as Na2 35SO4

into sulfated galactocerebrosides in CD-1 mouse cerebrum,

cerebellum, and spinal cord occurred at approximately 13

days postpartum in vivo, after a rapid onset beginning at

day 5 to 8.

In vitto synthesis of sulfated galactocerebroside in

mouse spinal cord culture was shown to be inhibited by 1000

pM Phe and 500 iM PPA to the extent of 50 percent and 70

percent, respectively. PLA at 300 pM and PA at 250 pM had

no significant effect. The PPA and Phe-treated cultures at

high concentrations of 500 pM and 1000 uM, respectively,

demonstrated delayed myelination compared to controls of the

same age in vitto, and such cultures were more sparsely

myelinated as seen under phase and brightfield microscopy.

The inhibitory effect of PPA at 500 pM upon sulfated galacto-

cerebroside synthesis was demonstrated to be reversible when

control medium replaced the PPA-containing medium.

The contribution of methionine sulfur to sulfated

galactocerebroside synthesis was investigated, and was

found to demonstrate peak incorporation at approximately 14

days postpartum at a level of 2600 dpm per gram wet weight

of whole brain when injected i.p., 0.57 uCi at 40 Ci/mM.

Bilateral intracerebral injections of Phe, PPA, a-KB,

a-KIC, and a-KIV at 8 Pg each followed by i.p. Na2 35SO4

significantly reduced (all p < .05) incorporation of 35SO4

into sulfated galactocerebroside during the period 8 to 15

days postpartum. PLA and PA injected near the age of maximum

incorporation however, resulted in no significant effect
35 -
upon incorporation of S04- into product. In adult mice

40 to 180 days old only PPA had a significant effect of

decreased incorporation at the rejection level of .05 (p < .02).

KIV-treated animals produced control amounts of sulfated

galactocerebroside, while Phe, a-KB and a-KIC did show a


Adult CD-1 mice also reflected a decrease in their kid-

ney sulfated galactocerebroside synthesis with PPA (p < .02)

at lesser reduction with Phe, a-KB, and a-KIC, and no change

with a-KIV.

The effect of PPA as a structural analog of pyruvate

was investigated as to its ability to increase or decrease

the in vi-Lo decarboxylation of pyruvate in kidney and brain

homogenates. PPA reduced the amount of 14C02 produced from

- 1C pyruvate by 28 and 47 percent at 1 x 10 M and
1 x 10 M, respectively. In the kidney homogenates, PPA

at 1 x 10 3M produced a 42 percent reduction in 1CO2 re-

leased, but a-KIV produced control values under the same


It is suggested that such severe reductions in CO2

release from pyruvate in the kidney and brain homogenates

in the presence of PPA in the present study, along with the


results of the bilateral intracerebral injections of the

other compounds tested in vivo, could result in decreased

PAPS synthesis (requiring ATP), result in a decrease in

endogenously bound galactocerebroside substrate, and result

in related oxidative metabolism deficits largely accounting

for the observed decreases of incorporation of 35S4 into

sulfated galactocerebroside in vivo and in vitrto in CD-1

mouse kidney and brain.


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Terry Joe Curtis Sprinkle was born on November 19, 1942

in Washington, D.C. and attended public school in Miami, Florida.

He graduated from Nbrth Miami High in 1960 and subsequently

received a Bachelor of Science in chemistry in 1966 and a

Master of Education in science education in 1970 from the

University of Florida. During this period, the author was

employed by the Department of Soils, the U.S.D.A. Laboratory

as a chemist, and as a research assistant at the Pesticide

Research Laboratory, all in Gainesville, Florida. The author

was then employed for some 2 1/2 years as a full-time research

chemist for the Veterans Administration under Thomas Newcomb,

M.D., in the Neurochemistry Section. Upon completion of the

Master's Degree in 1970, he then entered the Department of

Biochemistry under a predoctoral NIH traineeship.

The author is married to the former Ann Calvitte Hinson

of Gainesville, Florida, and has three children, Joe, David,

and Ashley Anne. The author is a member of Lambda Chi Alpha.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.

O. M. Rennert, Chairman
Professor of Biochemistry

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.


C. M. Allen, Jr. )
Associate Professor of Biochemistry

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.

J. M. Tsibris
Assistant Professor of Biochemistry

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.

C. M. Feldherr
Associate Professor of Pathology

This dissertation was submitted to the Graduate Faculty of
the Department of Biochemistry in the College of Arts and
Sciences and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.

June, 1974

Dean, Graduate School

3 1262 08553 3247

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