The distribution of galactitol in tissues of rats fed galactose

Vol. 20, No. 4,1965

m

BIOCHEMICAL AND EIOPHYSICAL RESEARCH COMAMJNlCATlONS

DISTRIBUTION

GALACTITOL IN TISSUES OF RATS FED GALACTOSE

OF

R. Quan-Ma and W. W. Wells Biochemistry

Department,

Pittsburgh,

ReceivedJuly9,

School of Medicine,

Pittsburgh,

Pennsylvania

demonstrated

that galactitol

(1959a)

of rats

fed a 35% galactose-containing

contain

galactitol

hydrate

were

In the course analysis

galactitol

1965a).

cardiac

diet.

galactitol

in the urine

These observations converting

in the urine if

patients

the disease

diets

was not diminished

in the lens

tissues

found to

methods for carbo-

Wells --et al.

who had died with

amounts of galactitol

other

of quantitative

and plasma of galactosemic

of two patients

accumulated

muscle though only in trace

chromatography,

Rats fed 35% galactose-containing

significant

15213

Tbe only

and skeletal

of the development

by gas-liquid

in the urine

in the brain

of

1965

Van Heyningen

amounts.

University

for

several

(Chin,

1964).

(1964a)

discovered

and subsequently (Wells, weeks

et al., excreted

The quantity

the lens of the rata were

of removed.

suggested that tissues other than lens must be capable of

galactose to galactitol.

content of galactitol

Wewish to report the distribution

and

in various tissues of rats fed a 35% galactose-containing

diet for 16 days. Sixteen weanling male albino rata of the Holtzman strain were divided into two equal groups and housed in individual

cages.

The control

group was

fed a diet of the composition, 72.8% sucrose, 18% casein, 4.0% Wesson salt mix,* 5.0% cotton seed oil choline chloride

(supplemented with cod liver

and 0.1% vitamin mix.

expense of an equal amount of sucrose. Biochemicals Corporation,

0.1%

The experimental group was fed a diet

oE the samecomposition except for the substitution

*Nutritional

oil and a-tocopherol),

of 35%of galactose at the

At the end of the 16 day feeding period Cleveland 28, Ohio

486

BlOCHEMlCAL AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

Vol. 20, No. 4, 1965

the

rats

nised

(ether

anesthetieed)

were

The blood

was centrifuged

syringe.

The blood

cells,

plasma,

and leg muscle Plasma

were

of Wells, diluted

were

with

loroacetic

(1965b)

3 volumes acid

and allowed

was re-extracted

were

7-10

times

An aliquot

TCA. analysis. (lg)

with

The brain,

extracted

by the procedure

the Folch

procedure

Aliquots

were

dried

of Wells,

et al.,

vigorously

cells

centrifugation TCA extracts

extract

was free

of

and gas chromatographic and representative

were

weighed,

(1957).

extract

and trimethylsilylated

of blood

The combined

lenses

--et al.

(1964b).

After

the ether

muscle

as the water

intestine

2 ml of 10% Trich-

two hours.

until

pooled

and leg

at 4”.

of the method

with

trimethylsilylation

of Folch,

as well

small

a modification

for

ether

kidney,

intestine

liver,

2 ml of 5% TCA.

before

15 minutes

Two ml of a suspension shaken

diethyl

heart,

small

cells,

to stand

with

was dried

of liver,

were

a hepari-

analyzed.

by the method blood

with

rpm for

kidney,

until

was employed.

the residue washed

heart,

from red

of water

(TCA)

by exsanguination at 3,000

frozen

determined

of sugars

et al.,

brain,

immediately

sugars

For the analysis

lens,

killed

prior

and the lipids

The water of the

samples

washes

residue

from

were

combined.

to gas chromatographic

analy-

sis.

With

The galactitol

concentration

the exception

of the kidney,

no detectable contained cle

acylic the highest

accommodated

/g tissue,

ties

amount

of galactitol

packed

blood

tially

devoid

gas-liquid

from

quantities

1924)],

observed

amount

kidney,

rats. the level

but small

The liver of sensitivity

The gas chromatographic

487

T.

contained

(1959a),

eye lens

and cardiac

mus-

and 18.06moles muscle

of galactitol

the brain,

in

22.32

skeletal

Lesser

even at technique.

Skeletal

in Table

animals

van Heyningen

of the polyol,

total

suaunarieed

the control

body weight.

of the galactose-fed

of the hexitol

with

respresentative

the

to 185mg/lOOg

chromatography

tissue

is

of galactitol.

Assuming

were

cells

the

In agreement

significant

(I)onaldson,

would

tissues

concentration

respectively.

body weight muscle

polyol.

of several

[45.4%

deposited

in

sigoificant

provided records

the

quanti-

intestine

aad plasma

of

sod were

essenby the

of the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No.4, 1965

TABLE1 Free Calactitol

Concentration Tissue

&moles

/g fresh

Calactose-fed

Eye lens*

44.38

Skeletal Muscle Cardiac Muscle

22.32+2.79* 18.Oti3.75

Brain

5.1720.63

Kidney Small Intestine Proximal l/3 Distal 213

3.18AO.91

Blood Cells*

0.67kO.12 none 11

in Tissues

Sucrose-fed none II II I, 0.3~0.10+ none tt II II

1.9tiO.28 l.ltiO.44

Liver Plasma * w + +b

tissue)

Pooled sample of 12 lenses TX 2 ,N=8 Standard deviation, s cf. ,& IiTentitatively identified as sorbitol Concentration is based on umolesfml of packed cells

I 0

20

IO TIME

IN

MINUTES

Figure

1

488

30

of the Bat

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMAWNlCATlONS

Vol. 20, No. 4, 1965

free

sugar

trimethylsilyl

rats

are shown in Figure

and S galactose

ethers 1.

of sorbitol

trace

only a and 0 glucose,

necessarily sis,

(5) and myo-inositol

the finding indicate

the low permeability

and Davies,

plasma strongly reductase

(van Heyningen,

examined in this

seminal

vesicle

due to the toxicity

in a given site

1956) and inhibit

membranes to the sugar polyols

(Le Fevre

Eye lens contain

galactose

1965) a NADPH requiring

Galactitol

of galactose

in the other

formation

in the

1960). is believed

of galactosemic

patients

(Ginsberg

may be a new candidate be an important

by the galactose-l-phosphate

to be

than to the accumulation

galactose-l-phosphate

of phosphoglucomutase

or it may simply

of galactose

demonstrated

rather

For example,

in the

the enzyme aldose

to galactosemics

derivative

in red blood cells

does not biosynthe-

of the sheep (Hers,

of feeding

the action

tissue

though the enzyme has been located

in the tissues.

found to accumulate

In the lower

of galactitol

hypothesis.

report,

plus a

1951) and the absence of galactitol

of a galactose

of the free sugar

disposition

accumulation is the actual

to Y, a!

can be seen.

1959b; Hayman and Kinoshita,

and placenta

The pathogenicity

1957).

(3 and 6), gelactitol

enzyme has not yet been successfully

tissues

bolite

the tissue

this

peaks corresponding

and -myo-inositol

of cellular

supports

enzyme, but this

Sidbury,

sorbitol

1951; Wick and Drury,

and galactose-fed

(7) may be identified.

of galactitol

that

of control

In the upper trace,

(1, 2 and 4), 0 and S glucose

trace

Although

from the kidneys

uridyl

(Schwarz,

--et al.

and Neufeld,

1957;

for

alternate

has been

the toxic

pathway

for

metathe

transferase-less

human. ACKNOWLEDGMENTS Supported by a research grant (H-02458) from the National Institutes of Health, U. S. Public Health Service. Dr. Wells has been the recipient of an Established Investigatorship of the American Heart Association. EEFEEENCES Chin,

T., M. S. Thesis,

Univ.

of Pittsburgh,

(1964)

Donaldson, H. H. 'The Bat” Memoirs of the Wistar Biology No. 6 Philadelphia (1924) p. 133 489

Institute

of Anatomy and

Vol. 20, No. 4,1965

Folch,

J.;

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G. I-I., J. Biol.

Ginsburg, V. and Neufeld E. F., Abstr. Sept. (1957)

Chem.,

226, 497 (1957)

132nd Meeting of the Amer. Chem. Sot.,

Hayman, S. and Rinoehita J. I-I., J. Biol.

Chem., 240, 877 (1965)

Hers, H. G., Biochim. Biophys. Acta, 37, 120 (1960) Le Fevre, P. G. and Davies, R. I.,

J. Gen. PhySiOl.,

Schwarz, v.; Golberg, L.; Eomrower, G. M.; and Holzel, 34 (1956) Sidbury, J. B., Abstr.

2,

515 (1951)

A.,

Biochem. J., 62,

132nd Meeting of the Amer. Chem. SOC., Sept. (1957)

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