Lipid peroxidation in hemolysate of rabbit erythrocytes

Vol. 105, March

No.

BIOCHEMICAL

2, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

30, 1982

Pages

LIPID

PEROXIDATION

IN

Yohnosuke Department

Received

of

February

Kobayashi

Pediatrics,

3,

HEMOLYSATE

OF RABBIT

and

Hiroshima Hiroshima 734,

539-545

ERYTHROCYTES

Tomofusa

Usui

University Japan

School

of

Medicine

1982

Summary - Incubation of hemolysate of rabbit erythrocytes with Zn'+ induced The combined use of superoxide dismutase and catalase lipid peroxidation. and histidine inhibited the reaction but mannitol did not, suggesting that the active oxygen species, probably singlet oxygen, arising in the interaction between superoxide and hydrogen peroxide is the responsible factor. a promoting one at low and an inhibAscorbate had biphasic effects, i.e., and a-dl-tocopherol also suppressed lipid itory one at high concentrations, peroxidation. This reaction was sensitive to high NaCl concentrations, and protein in the reaction mixture had a non-specific inhibitory action. Production of

peroxidation

oxygen a nutier hemolysis of

of

lipid

which produce

Materials -__

peroxide

damage

species of

to

be

studies

and

to

hemolysate

of

lipid

peroxide. in

relation

been

used

erythrocytes

8-12).

to

the

fatty

in

this

as

target

during

This

characteristics functions

of of

(l-7). and

the

various

evaluated as

scavengers

indexes

a method

cells

reaction

active So far

stress

intact

outcome

and

describes of

the

acids,

cells

oxidative

instead

reflect

reaction

communication

erythrocytes Some

to

polyunsaturated

formation

rabbit

considered

implicated

malondialdehyde (2,

has

cellular

intimately have

peroxidation

particularly oxygen

lipid

is are

by

used

to

discussed, of

active

species. and

methods

Superoxide dismutase (Bovine blood, 3,00Ou/mg protein) and catalase (Bovine liver, 30,000 Sigma units/mg protein) were purchased from Sigma Chem. co. D-(-)-mannitol, u-dl-tocopherol, Tween 20, D-histidine, u-ascorbic acid and other reagents were products of Nakarai Chem. Co. Bovine albumin powder (Fraction V from bovine plasma) was from Armour Pharmaceutical Co. Enzymes were inactivated by autoclaving for 20 min. Heparinized blood was drawn from male rabbits, weighing about 2 kg, which had been fed a regular diet and water ad lib. Three rabbits were used interchangeably, as all of them exhibited the same tendency with little individual variation. Following centrifugation, plasma and buffy coat were removed, erytt Abbreviations used : HEPES, N-hydroxyethylpiperazine'N'-ethanesulfonic pH 7.4; Zn-HEPES, 17mM HEPES containing l.OmM ZnCl,.

acid,

0006-291X/82/060539-07$01.00/0 539

Cop.vri~ghl 0 1982 b-v .4cademic Prerr, Inc. ,411 nyhrs a./ reprodwrion tn anv ,/km wwrved.

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

rocytes were washed three times with saline and suspended to contain log of hemoglobin/lOOml. They were stored in small aliquots at -80°C without any effect on subsequent determination. Before use they were thawed and treated in ice by sonication with a Heat Systems Cell Disruptor Model W-225R (Ultrasonics, Inc.) for 60 set at 60 watts. One-tenth of a milliliter of the hemolysate was used without further centrifugation in a final reaction volume of 0.5ml of 17ti HEPES buffer containing the constituents described in each experiment. Lipid peroxide was measured by a slight modification of the fluorometric method of Yagi (13) with thiobarbituric acid. The lipid peroxide concentration of the sample was determined in terms of malondialdehyde (n moles/g of hemoglobin) based on the values of tetraethoxypropane as a standard. c\-dl-Tocopherol was prepared as an emulsion, 1 part of Tween 20 to 4 parts of a-dl-tocopherol (w/w). Results Incubation to

yield

was

an

of

any

hemolysate

further

increase

increase

in

to

be

necessary,

rating

one

of

the

CuCl,

CuC12,

those

tested,

the

following

aliquots Fig.

ZnCl,

there

was

that

remained

condition,

the

was

specimen, sate

at

was

then

incubated

in

Fig.

lB,

92.4mM

of

NaCl

at

inconstant

were

made

in

kept

in in

and

this by

an

ice

varying of

assayed

of

the

presence

the bath

lipid for

the

lo-?

to

was

of

Consequently l.OmM

ZnCl,.

for

peroxide.

lipid

37'C

linearly in

154mM In

absence to

length NaCl almost

and

in

a

NaCl

in

latter ZnCl,.

2 '/L

In

hours,

of of

in

the of

value

Among lipid

at

peroxide

was

10e7M.

CaCl,.

limited

same

MnC12,

incubated

or

of

peroxide

FeCl,,

incubation.

concentrations lipid

incorpo-

suspension

period

time

by

increased

cell

pos-

increase

augmentation

was

there

were

containing

hemolysate

incubation double

NaCl

but

ions

by

HEPES

failed

metal

of an

37°C

level,

FeC12,

in

17mM

intact

affected

almost

mixture:

at

basal

an

enhancement

in

during

the

induce

30.8mM

of

the

contaminant

to

intervals

that

4 hours

concentrations

and

while

to

some

made

for

above

resulted

peroxide

not

alone

reaction

ZnC12

lipid

production

the

time. Zn-HEPES.

completely

control HemolyAS suppressed

concentration.

superoxide

autoclaved

the

certain

was

sufficient

were

containing

experiments

which

Both each

value

was

shown

less

unchanged

subsequent

which

the

As

consistently

at

manner,

Zn-HEPES

peroxide

and

Zn-HEPES

removed

time-dependent

the

CaClz,

in

1A shows

lipid

into

determinations

were

in

attempts

l.OmM

Hemolysate

buffer

water.

following

MgC12,

and

HEPES

undeionized

tulated

peroxide,

in

dismutase enzyme

also

and

catalase

inhibited

the 540

inhibited reaction

lipid to

almost

peroxidation, the

same

but degree

Vol. 105, No.

BIOCHEMICAL

2, 1982

01’ 0

Fig.

(Fig.

2).

than

when

claved In

1

order

they

either

to

were

one was

plasma

behave

as

an

inhibitor

plasma

albumin,

in

3 tlmdhr)

0-

4

combined, used

30.8 NoCl

marked,

was

an

The

61.6 924 123.2 concentrotlon(mM)

the

the

protein in

amount

adjusted

catalase

be in

this

was

of

system

both

to experiment,

the

was

shown).

sum

of

yielded

autoenzymes.

the

and

not

greater

and

inhibited

(data

equal

hypotonic in ZnOrdinates

native

effect

reaction

manner to

used

the

in saline

effect

between

specific

15.4

(B). hemoglobin.

nonspecifically

incorporated a dose-dependent

(e)

suppressive

difference

indicating

in

and

the

however,

alone.

whether

albumin

dismutase

was

more

determine

bovine

superoxide

2

RESEARCH COMMUNICATIONS

Time course of lipid peroxidation of hemolysate saline in Zn-HEPES and of intact cells in isotonic HEPES (O)(A) and effect of NaCl on the reaction indicate malondialdehyde expressed in n moles/g Experimental details are given in the text.

When

enzymes

1 Incubation

AND BlOPHYSlCAL

reaction, found

Bovine the

proteins approximately

the samevalue as the autoclaved enzymes.

-.

0 None

Fig.

1

MO

yD CAT

CAT

BPA

Effects of superoxide dismutase(SOD), catalase(CAT)and bovine plasma albumin(BPA)on lipid peroxidation of hemolysate of rabbit erythrocytes. Ordinate indicates percent increment of the lipid peroxide value above the basal value in the hemolysate which was kept in an ice bath for the same length of time, and abscissa additions to the reaction mixture. SOD, 6,000~ (Zmg protein); catalase, 6,000 Sigma units (0.2mg protein); bovine plasma albumin, 2.2mg. Total height of the middle three columns shows the lipid peroxide levels with autoclaved enzymes and hatched portions those with native enzymes. respectively.

541

to

of

Vol. 105, No. 2, 1982 Table

1

BIOCHEMICAL

Effects of peroxidation

Additions complete

AND BIOPHYSICAL

ascorbate, a-dl-tocopherol, in hemolysate of rabbit

to system

RESEARCH COMMUNICATIONS and histidine erythrocytes

Percent above

increment

of

the

level

None 5

u-dl-tocopherol

1.0

18 150

0.01

115

(mM)

1.0

O(

0.1

31(

0.01

100(100)

1.0

31

0.5

51

0.25

90

MDA*: Malondialdehyde. ** : Figures in parentheses used

no

effects

of

demonstrable

press with

and

ing

effects

the

removed,

on

cell

the

with

lipid

significant

the

after

fuged

specimen,

basal

level.

basal

level

The by

centrifuged

value

of had

was

Tween

both

shown

tested

itself

the

decreased

by

compared

with

one of

35,000xg

in

but

had

found

20 was

to

sup-

examined

inhibitory

0.1%

gave

cell

The

25%.

a 100% a 32%

X-100

to

60

was

When

only

triton

for

supernatant

hemolysate.

centrifuged addition

at and

uncentrifuged

incubation; the

acid

also

are

and

enhanc-

0°C

to

concentration.

fractions,

peroxide

20

effect

Ascorbic

was

membrane

the

system,

acid

was

Tween

20 only,

a-dl-tocopherol.

radical, As

)**

Tween

n-ascorbic

shown).

this

with

0 100)

corresponding

hydroxyl

ti-dl-tocopherol.

hemolysate

compared

of not

in

depending

values

the

a-dl-tocopherol,

(data

peroxidation

Sonicated

and

histidine,

of

a scavenger

without

indicate

emulsification

effect

lipid

remove

for

Mannitol,

1.

MDA*

0

0.1

Histidine(tnV)

Table

basal

100

Ascorbate(mM)

The

on lipid

min

similarly

increase

the

assayed

membranes

difference

rise

at

were

became in

above hemolysate

more

the

uncentri-

the

corresponding decreased

the

40%.

Discussion The lipid

present

peroxidation

study during

shows

that

incubation

hemolysate at

37°C 542

of

rabbit

in

the

erythrocytes presence

of

induces Zn'+

at

lOW

Vol.

105,

NaCl

No.

concentrations.

catalysts in

lipid

lipid

that

additional

ions

tested,

(1,

peroxidation

in

metalsare was

to

be

inhibit

to

the

reaction.

The is

its

hemoprotein

thus

allowing

the

essential of

interpreted

and

of

are

absence

Among

required,

(17),

iron,

15).

alone

precipitation

peroxidation

14,

buffer

be

RESEARCH COMMUNICATIONS

especially

5-7,

for

found

the

ions,

3,

HEPES

required

zinc

appears

strongly

metal

peroxidation

on ly

therefore

AND BIOPHYSICAL

Transitional

for

crease

to

BIOCHEMICAL

2, 1982

as the

an

in-

indicating

various

most

plausible

(16),

which

reaction

metal role is

to

known

proceed

uninhibited. The

additive

gests

that

oxide

anion

inhibitory

the

active

and

rocyte

hemolysate.

oxygen

scavenger,

port of

the the

and

hydrogen

be

in

the

involved

of

process.

not

only

This but

also

effects

on

involved

applicable

to

This the

but

process.

autoxidation

also

and

Recently of it the

reaction This proceeds

to

persistence

of

also

finding faster

sensitive

a loss

the

present

albumin

which

is

in at

line low

with ionic 543

is

that strength.

of

and

most

likely

indicates

not

only

native inhibitory

is

intimately

suggested

that

is

catalase

due

to

and

of

Amano

the

released

explanation

concentrations, Pederson

that

reaction.

non-specific

same

propaactivity

the

have

to

the

inhibitory

superoxide

the

high

material

in

of

dismutase

NaCl

basis

initiation

that

(15)

heated

sup-

On the

inhibits

have

of

mannitol

study

finding

whether

to

of

the

of

Gutteridge

activity

eryth-

a singlet

participates

proteins

determined

effect

super-

rabbit

histidine,

superoxide

superoxide be

of

related.

oxygen

in

sug-

between

defined

5),

catalase

acid-reactive

previous

and

heat-inactivated

is

causally

reduction,

Halliwell

remains

be

plasma our

an

that

purified

tetrazolium

of

al.(G)

2,

bovine

other

effect

reports (1,

with

peroxidation

such

showed

several

the

thiobarbituric et

and

interaction

of

singlet

heated

compatible

nitroblue

effect Cu'+,

to

but

enzymes

(18).

inhibitory free

is

boiled

of

and

when

enzyme

observation

also

Svinger

contrast

dismutase heated

may

while

the

in

determinations

initiation,

dismutase

in

absence

peroxidation

In

superoxide

apparent

assays,

lipid

formed

inhibitory

oxygen

hydroperoxide of

superoxide

results the

the

their

propagation

gation

peroxide

singlet

of

of

species

Moreover,

that

results

lipid

oxygen

and

view

effect

is albumin.

which and (19)

Aust

(1)

also

studied

inhibit that the

Vol. 105, No. 2, 1982 effect

of

maximal

salt

concentration

rate

to

Ascorbate narrow

range

trations

of

be at has

of

is

property in

42 been

ascorbate

essential ascorbate

respiratory

burst

aggregating

agents

one

of

in and

non-specific

and

of

of

to

of E,

the vitamin

scavenger

0.66mM

the

systems

Wills

concentrations.

higher in 17,

(17)

and

(21) of

cause

inhibit

the

which marked

for

Higher

assumes

that

an

to

peroxidation free

This

been but is

concenopti-

ratio, dual

shown also

not

in

depression

only

leukocyte

potentiated

platelet

response lipid

20).

has

its

a relatively

process.

mitochondria action

found

ferrous/ferric

reaction

lymphocyte E in

at

autoxidation

to to

and

(3,

appropriate the

this

reduction

peroxidation

and

demonstrated

leukocytes, block

to

the

lysis

Vitamin

normal

role

keep

limited

been

lipid

0.1

maintenance

23).

to

promote

a decline

reaction,

to

not

has

inhibitory the

the

RESEARCH COMMUNICATIONS

tetrazolium with

i.e.,

the is

(22,

tetrazolium,

as

to

swelling

nitroblue

The

NaCl,

shown

for

metabolism

(24).

53mM

necessary

ascorbate-induced

oxygen

nitroblue

inhibit is

of

to

AND BIOPHYSICAL

on

concentrations,

mum concentration which

BIOCHEMICAL

by of

response

the to

phytohemagglutinins may

thus

be

regarded

radicals.

Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific (Project No. 56570363) from the Ministry of Education, Science and Japan and a Grant-in-Aid from the Japan Medical Research Foundation. are grateful to Dr. Alice S. Cary for assistance in preparing this

Research Culture of Authors manuscript.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Pederson, T. C., and Aust, S. D.(1972) Biochem. Biophys. Res. Commun. 48, 789-795. Fee, J. A., and Teitelbaum, H. D.(1972) Biochem. Biophys. Res. Commun. 49, 150-158. and Aust, S. D.(1973) Biochem. Biophys. Res. Commun. 52, Pederson, T. C., 1071-1078. and Hartmann, H-J.(1973) FEBS Lett. Zimmermann, R., Flohe, L., Weser, U., 29, 117-120. Gutteridge, J. M. C.(1977) Biochem. Biophys. Res. Commun. 77, 379-386. Svingen, B. A., O'Neal, F. O., and Aust, S. D.(1978) Photochem. Photobiol. 28, 803-809. F.(1979) Biochem. Biophys. Res. Commun. 89, 1239-1244. Nakano, K., and Obo, Dodqe, J. T., Cohen, G., Kayden, H. J., and Phillips, G. B.(1967) J. Clin. Invest. 46, 357-368. Invest. Mengel, C. E., Kann, H. E., Jr., and Meriwether, W. D.(1967) J. Clin. 46, 1715-1723. T. L.(1971) Brit. J. Haematol. 20, 95-111. Stocks, J., and Dormandy, Fee, J. A., Berqamini, R., and Briggs, R. G.(1975) Arch. Biochem. Biophys. 169, 160-167. Vertongen, F., Heyder-Bruckner, C., Fondu, P., and Mandelbaum, 1.(1981) Clin. Chim. Acta 116, 217-222. Yagi, K.(1976) Biochem. Med. 15, 212-216.

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Vol. 105, No. 2, 1982

14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Talcott, R. E., Shu, H., and Wei, E. T.(1979) Biochem. Pharmacol. 28, 665671. J. M. C.(1981) EEBS Lett. 128, 347-352. Halliwell, B., and Gutteridge, Oelshlegel, F. J., Jr., Brewer, G. J., Knutsen, C., Prasad, A. S., and Schoomaker, E. B.(1974) Arch. Biochem. Biophys. 163, 742-748. Wills, E. D.(1969) Biochem. J. 113, 315-324. Amano, D., Kagosaki, Y., Usui, T., Yamamoto, S., and Hayaishi, 0.(1975) Biochem. Biophys. Res. Commun. 66, 272-279. Amano, 0.(1976) Ann. Paed. Jap. 22, 99-107. Gibson, D. D., Hornbrook, K. R., and McCay, P. B.(1980) Biochim. Biophys. Acta 620, 572-582. Hunter, F. E., Jr., Scott, A., Hoffsten, P. E., Guerra, F., Weinstein, J., Schneider, A., Schutz, B., Fink, J., Ford, L., and Smith, E.(1964) 3. Biol. Chem. 239, 604-613. Bigley, R., Gerhardt, N. B., Eigner, T., Dehlinger, A., Niedra, J., and Stankova, L.(l978) J. Reticuloendothel. Sot. '24, l-7. Kraut, E. H., Metz, E. N., and Sagone, A. L., Jr.(1980) J. Reticuloendothel. Sot. 27, 359-366. and White, J. G.(1978) Am. J. Pathol. Repine, J. E., Rao, G., Beall, G. D., 90, 659-674.

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