A molecular defect of myelination

Vol. 15, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A MOLECULAE DEFECT OF MYELINATION John S. O'Brien University

Received

March

of Southern California, School Los Angeles, California

17, 1564

A molecular a rare

mm, sulfates

hereditary

large

Frozen

separated

methanol

2rl

into

separated

column

into

column

on a analytical the purity

closely taminated

with

acids fatty

(21-26 acids.

carbons) In this

is presented

and the pathogenesis

(2,3).

but report

to

of MLD.

The quantity

and sulfate

Paper

and that

analyses The results

values compounds

indicating were 484

chloroform-

from

each

cerebrosides,

lipid

(1)

and a combination

or with

a silicic

was determined

(1,3,4)

was used

of phosphorus, of these that absent

serine

of a Florisil

column column

cerebroside

phosphatides,

a combination

acid

chromatography

content.

abnormal

using

with

extracts

and ethanolamine

of each

as were

theoretical

The lipid

a silicic

of MLD were

and extracted

(DEAE) cellulose with

two cases

ceramides,

phosphatides

balance.

the

(1).

gangliosides,

column

from

matter,

cholesterol,

of each fraction

hexose

cerebroside

and sphingomyelins

hypothesis

lobes

and white

a diethylaminoethyl

of a DEAE cellulose silicate

the frontal

previously

and choline

with

and a working

grey

sphingomyelins,

phosphatides,

acid,

from

as described

were

fatty

of these

defect

in which

cerebrosides

chain

depleted

leucodystrophy

ANDMETHODS

tissue

manually

formation

matter,

long

the biochemical

MATERIALS

sulfates,

of very

are nearly

in metachromatic

of myelin white

are documented

explain

tissue

disorder In normal

lipids

findings

partially

has been found

proportions

in KLD these these

defect

accumulate.

contain

of Medicine

by weighing to evaluate

glycerol,

analyses

the fractions or undetectable,

acid-

fatty

corresponded were

uncon-

The fatty

Vol. 15, No. 5, 1964

acid

BIOCHEMICAL

compositions

liquid

of the three

chromatography

acetals

after

the

AND BIOPHYSICAL

glycerolphosphatides

(GLC) of fatty latter

hydrolysis

(3,5).

determined

by GLC (6).

acid

acid

were

methyl

had been selectively

The fatty

RESEARCH COMMUNICATIONS

esters

isolated

compositions

determined

by gas-

and aldehyde by mild

dimethyl-

alkaline

of the sphingolipids

were

RESULTS The lipid with

a normal

of total

fold

control

lipid,

gangliosides while

compositions

of a similar

cholesterol, were

cases

were

matter

phosphatides,

diminished. decreased.

and ceramides

from

age are given

choline

somewhat

in Case 2 they in both

of grey

each patient in Table

1. The quantities

sphingomyelins,

In Case 1 cerebrosides Cerebroside

were

sulfates

moderately

compared

and were

were

increased

increased

3-4

increased.

TABLE1 LIPID

COMPOSITION OF FRONTAL LOBE GREY MATTER Case 1 Case 2 Normal

Age 9 11 Total lipid 34.80 39.35 Protein 65.20 60.65 Water 83.34* 84.88 Cholesterol 7.01 5.94 Ethanolamine Phosphatides * 9.05 Serine Phosphatides * 2.64 Choline Phosphatides 6.91 8.15 Sphingomyelins 2.37 2.09 Cerebrosides 3.50 1.26 Cerebroside Sulfates 1.60 1.42 Ceramides 0.63 0.75 Gangliosides * 0.28 All values except water are expressed tialues affected by formalin fixation

In white tides, side

sphingomyelin, sulfates

Cerebrosides were

matter

moderately

were were

total

9 47.80 52.20 85.79 7.21

diminished

9 53.50 46.50 79.91* 10.00

9.61 2.68 8.99 2.77 1.91 0.41 0.48 0.40 as percent

lipid,

11 50.30 49.70 82.05 8.10

9 74.13 25.87 77.35 13.24

* 9.10 * 2.87 7.70 7.60 2.94 2.41 6.41 2.62 12.60 5.33 0.70 1.26 * 0.20 of dry weight.

12.Ik 5.13 8.84 4.93 10.67 3.95 0.50 0.44

cholesterol,

and cerebrosides increased

WRITE MATTER Case 1 Case 2 Normal

were

three-fold

485

markedly

three

glycerolphospha-

decreased

in Case 1 and 1.4 fold

to 25% of normal

increased.

all

in Case 2 while

while

cerebra,

in Case 2. ceramides

Vol. 15, No. 5, 1964

BIOCHEMICAL

In MID, matter

the

fatty

acid

glycerolphosphatides

sphingolipids. larger

proportions

of aldehydes dilution

matter

normal.

of myelin (7-10

fold)

acids

with

21-26

carbons

alpha

hydroxy

acids

since

14-20

carbons

being

striking

diminution

sulfates

from Mu) white

toward

larger

small

compared

by glial

length

(Table

in the proportion

proportions

of long

3).

a shift

of shorter

chain

acids.

or sphingomyelins

fatty

acids

fatty

and

chain

fatty

acids

with

The diminution

the

longer

Ceramides

chain

a

was a striking

the longer

acids;

showed

reflect

containing

2 and 3).

also

to cerebrosides

There

containing

of the fatty

proportions

unsubstituted

for

(Tables

the diminution

matter

cells.

both

was specific

matter somewhat

and probably

and sphingomyelins

or increased

to the chain

of the grey

and smaller

small

and cerebrosides

normal

the greater

were

of the grey

contained

acids

2) involving

The defect

sphingomyelins were

fatty

of cerebrosides

acids*.

those

glycerolphosphatides

matter

(Table

compositons

as were

changes

in MLD white

diminution

acid,

the

These

aldehyde

normal

of polyunsaturated

than

proportional

and fatty

were

In white

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fatty

showed

acids.

However, (Table

the fatty

also

in fatty

was

a

Cerebroside

acid

composition

this

shift

was

3).

DISCUSSION The biochemical the fatty for

acids

longer

side

in MID appears

of sphingolipids

chain

cerebrosides,

defect

acids

ceramides

(21-26

beyond carbons)

to be a failure

18 carbons. and effects

and sphingomyelins

to elongate

The defect the fatty

much moreso

than

is acids

those

specific of of cerebro-

sulfates. The nature

shown that fatty palmitic

of the defect

two systems

acids, acid

exist

is not

in the

brain

1) a de novo system which (7,B)

and 2) a chain

siThe proportions of total hydroxy sulfates from grey or white matter

completely for

acids were

486

It

the biosynthesis

condenses

elongation

understood.

8 acetate

system

which

has been

of saturated units attaches

to form 2 acetate

in cerebrosides and cerebroside equal to the control.

Vol. 15, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE2 FATTY

(As percent of total

ACID

ACIDS OF SPHINCCLIPIDS 11 unsubstituted fatty acids in each lipid) GREXMATTER

Sphingomyelin Cerebroside Cerebroside Sulfate Case 1 Case 2 Normal Case 1 Case 2 Normal Case 1 Case 2 Normal

14:0 1.6 16~1 tr 16:0 10.3 18:l 3.0 18:0 76.7 2O:l tr 20:o 2.0 22:l tr 22;o 0.3 23:l tr 23:o if9 2411 24:0 2.0 2511 0.9 2510 0.3 2611 tr 26:0 tr 14-20 93.6 21-26 6.4

1.3 tr 17.0 2.9 72.0 tr 1.3 tr 0.7 tr tr 1.7 1.0 tr tr tr tr 94.5 5.5

1.4 tr 9.4 2.2 78.6 tr 1.2 tr 0.5 tr tr 2.9 1.5 0.8 tr tr -92.8 7.2

10.3 tr 20.4 23.0 31.0 tr 0.5 tr 2.0 0.4 0.8 5.0 3.5 1.0 0.7 0.5 0.3 84.8 15.2

2.8 3.7 26.3 23.6 29.6 tr 0.5 0.3 0.1 tr 0.1 1.0 0.6 0.2 0.1 0.3 tr 85.6 14.4

4.7 tr 23.4 21.3 34.3 tr tr tr tr tr tr 4.4 2.1 0.4 tr tr -83.7 16.3

10.8 0.4 17.5 1.8 16.4 tr 1.0 0.3 2.1 0.5 ‘2.1 15.0 11.5 7.4 3.0 8.2 1.7 47.9 52.1

4.7 0.9 18.7 8.5 24.3 tr 1.6 1.1 1.6 0.4 1.4 14.3 14.3 2.9 1.2 2.6 0.5 58.7 41.3

17.2 2.2 20.0 5.0 13.1 0.4 0.2 tr 0.8 tr 1.2 13.0 5.3 3.2 1.3 3.8 tr 57.9 42.1

ACID

WHITEMATTRR Cerebroside Cerebroside Sulfate Sphingomyelin Case 1 Case 2 Normal Case 1 Case 2 Normal Case 1 Case 2 Normal

14x0 1611 16rO 1811 1810 2O:l 2010 22:l 22:o 23:l 23:0 24.1 24:0 25:l 25:0 26:l 26x0 14-20 21-26

3.0 0.8 10.3 3.8 50.0 0.1 0.9 0.1 0.9 0.1 1.0 16.5 3.4 2.5 1.5 3.0 tr 72.6 27.4

1.0 0.3 9.8 1.2 73.8 tr 1.2 -0.8 tr 0.6 7.2 1.7 0.8 tr 1.0 8::6 12.4

7.8 0.5 6.7 1.3 30.5 tr 0.9 0.4 1.4 0.5 1.6 25.2 6.5 3.2 1.4 2.3 tr 57.5 42.5

0.7 1.5 18.1 32.8 26.4 1.3 0.5 0.1 0.3 0.3 0.8 6.8 1.9 1.1 0.3 1.2 tr 84.1 15.9

0.8 2.2 22.8 19.0 25.9 0.3 0.3 0.3 0.6 0.2 0.7 7.3 6.2 2.2 0.3 0.2 0.2 78.0 22.0

units at a time to make longer chain fatty appears to be exclusively

involved

acids (9,lO).

in MLD. It 487

0.8 tr 8.3 3.3 7.9 0.1 0.4 0.4 1.8 0.8 3.1 40.0 11.3 7.0 3.5 7.4 1.0 22.7 77.7

0.1 0.9 9.4 4.7 7.2 tr 1.8 0.8 2.7 0.9 3.2 27.3 17.9 6.6 5.5 9.8 0.9 25.5 74.5

1.2 1.0 4.3 2.0 8.2 -1.6 -3.3 tr 4.0 27.3 19.2 7.6 3.7 9.6 2.8 22.7 77.3

The latter

4.6 0.4 3.5 1.5 3.1 tr 0.5 tr 1.8 0.6 3.3 38.7 16.1 9.0 5.0 9.8 1.8 13.4 86.6

system

is not obvious from our knowledge

Vol. 15, No. 5, 1964

BIOCHEMICAL

of these

systems

affected

more than

have a fatty that it

this is

acid

lipid

with

those

how the

lipid

21-26

fatty

of another.

composition class

the only

acids

however,

AND BIOPHYSICAL acids

of one sphingolipid

The fact

which

most

accumulates

in KLD containing

that

nearly

in MLD via

RESEARCH COMMUNICATIONS

cerebroside

approaches

sulfates normal

a compensatory

appreciable

can be

suggest

synthesis

proportions

since

of fatty

carbons.

TABLE3 COMPARISON OF CONCENTRATIONS OF SPHINGOLLPIDS IN WHITE MATTER IN MLD AS A FUNCTION OF FATTY ACID CHAIN LENGTH

PATTY ACID

SPHINGOMYELIN Case 1 Case 2

CEREBROSIDE CEREBROSIDE SULFATE Case 1 Case 2 Case 1 Case 2

14:o 16tl 16:0 18:l 18rO 20x0 2110 2221 22LO 2311 23:0 2411 2410 25:l 25:0 2611

23 96 92 175 98 59 -15 38 12 37 39 31 47 64 --

6 28 72 47 118 68 --28 -18 14 13 12 -21

54 185 294 613 206 77 92 15 10 60 16 10 10 10 5 10

27 118 167 160 91 19 44 19 8 20 5 5 15 8 -2

5 543 643 267 754 86 --364 380 233 169 267 176 263 241

30 245 141 152 304 353 --212 -140 81 137 97 84 113

14-20 21-26

82 30

75 14

232 13

97 8

456 206

194 102

Values are expressed as percent of the normal concentration for Only unsubstituted fatty acids each compound in white matter. are included. The hydroxy acids of cerebrosides and cerebroside sulfates showed a similar decline with increasing chain length.

The pathogenesis major

forces

carbon of this for

involved

interactions force

interaction

since with

of MLD may be explained in the cohesion

(11).

Fatty

they

present

adjacent

acids

as follows.

of membranes in complex

a large molecules.

surface Long

488

is

that

lipids

One of the due to carbon-

are major

of available chain

fatty

sources

carbon acids

impart

atoms

Vol. 15, No. 5, 1964

greater the

stability

larger

than

the number

addition,

only

to extend

across

with

long

chain

acids

fatty

acids

that

(13,14),

can be made.

lipids

containing

This

very

to white

of cerebrosides leads

It elongation

white

is our

prediction

system

is

matter

has recently lobes

also

effected

one case of infantile

is

findings almost

long

exclus-

isolated

fatty

acid

acids myelin.*

chain

of myelin

chain

these

synthesis

chain

recently

stable

that

of unstable

fatty

we have

containing

before

deficient

long

disorders

point

stable and

to myelin

sphingolipid

in other

sphingo-

a uniquely

localized

very

to the author's of long

chain

one case of globoid-cell

Gauchers

for

formation found

fatty

a

acids

in

disease.

a deficiency of MID,

it

or to formation

the

by very

membrane.

In MID,

(15).

of this

has been brought

in two cases

provided

by the are

containing

that

of sphingolipids

reported

acids

enough

cross-linking

of sphingomyelins

supported

matter

long

This

requirement

since

In

and interdigitate

must be synthesized

of myelination

from Niemann-Picks It

other

in its

acids

fatty

(12).

the myelin

acids is

leaflet

3) and more specifically

In confirmation

deficiency

Addendum.

chain

from white

to cessation

and breakdown. marked

fatty

and sphingomyelins

either

the

chain,

cohesion.

or more are

layer

quantities

hypothesis

(Table

by ultracentrifugation

fatty

sufficient

long

matter

with

the carbon

the

bimolecular

is unique

chain

these

longer

18 carbons

to stabilize

myelin

and that

the

in the opposite leaflet

long

containing

with

of the myelin

may help

very

since

and the greater

acids

bimolecular

myelin

ively

fatty

of lipids

of the

cerebrosides

acids

chain

the center

containing

membrane

short

of interactions

We postulate lipids

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

saturated

the fatty

of one side

BIOCHEMICAL

disease

attention fatty

that acids

Svennerholm from

frontal

leucodystrophy

and

(16).

ACKNOWLEDGEMENTS We are indebted to Dr. Allen this study and for providing

*Analysis progress)

of myelin isolated may shed further

Cracker for his kind encouragement throughout the case material. This project was supported

from MID by ultracentrifugation light on this point. 489

(now

in

Vol. 15, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by Grant #NB-04136-OlAl from the National Institute of Neurological Diseases and Blindness, Grant #H&08429-01 from the National Heart and Project 61605, Los Angeles County Hospital Attending Staff.

Institute,

REFERENCES 1) O'Brien,J.S., Fillerup,D,L., and Mead,J.F.,.T.Lipid Res.5:109,1964. 2) Rouser,G., Baumann,A.J., Kritchevsky,G., Heller,D. and O'Brien,J.S. J.Am.Oil Chemists'Soc. 38:544,1961. 3) O'Brien,J.S.,Fillerup,Dx.,and Mead,J.F.,J.Lipid Res. In Press. Baumann,A.J.,Nicolaides,N. and Heller,D.,J.Am.Oil 4) Rouser,G., Chemists' Sot. 38:565,1961. 5) Farquahar,J.W.,rLipid Res.3:21,1962. 6) O'Brien,J.S. and Rouser,G.,Anal.Biochem. In Press. 7) Wakil,S.J.,Ann.Rev.Biochem.31:376,1962. 8) Brady,R.O. J.Biol.Chem.235:=99,1960. 9) Fulc0,A.J. and Mead,J.F~.Biol.Chem.236:2416,1961. 10) Hajra,A.K. and Radin,N.S.,J.Lipid ResT270,1963. 11) Salem,L., Can.J.Biochem.Physiol. %:1287,1962. 12) Vandenheuvel,F.A.,J.Am.Oil Chemists' Soc.~:455,1963. 13) Waelsch,H.,Sperry,W.M. and Stoyanoff,V.A.,J.Biol.Chem.135:291,1940. 14) Davison,A.N. and Dobbing,J., Bi0chem.J. 75: 565,196O. 15) O'Brien,J.S., Sampson,E.L. and Connerton,S.R.M., Unpublished data. 16) Svennerholm,L.,Brain Lipids and Lipoproteins and the Leucodystrophies, J.Folch-Pi and H.J.Bauer eds., p 104, Elsevier Pub. Co.Ltd. N.Y. 1963.

490