Oxidative modification of glycated low density lipoprotein in the presence of iron

Vol.

177,

No.

May

31, 1991

BIOCHEMICAL

1, 1991

AND

RESEARCH

BIOPHYSICAL

COMMUNICATIONS Pages

OXIDATIVE

MODIFICATION

433-439

OF GLYCATED LOW DENSITY LIPOPROTEIN

IN THE PRESENCE OF IRON

Tamiko

ab

Sakurai

, Satsuki a

b

College

Tokyo

Kimurab,

College

of Medical

Minoru

of Pharmacy,

Received

April

Prefectural

26,

b*

Tokyo,

Care and Technology, Gunma 371,

'Gunma

Nakano

and Hirokazn

Kimura

bc

Japan

Gunma University,

Maebashi,

Japan

Maebashi

Hospital,

Gunma,

Japan

1991

This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by'incubation with glucose (GLDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8 % modification of lysine residues) and 84 % of G-LDL G-LDL incubated with Fe3' was adsorbed on phenylboronate affinity column. for4 h caused asignificantly higher level of lipid peroxidation than U-LDL (untreated with glucose),and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Corresponding to change on SDS-PAGE, increasing with incubation period. G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. (L -tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+. @ 1991 Academic Press, Inc. Much attention

has been

lipoprotein

(LDL)

acetylated

LDL could

macrophages, that

not

endocytosis

cells.

of

Oxidized

receptors

*

but

and

in

to

reacting

substances;

development

of by

by LDL receptors

been

also

lipid-laden

should low

role

of modified

scavenger

reported

in to

cells

It

is

the

of

monocyte-

widely

accepted

formation

be mediated (2).

density

Chemically

receptors

(1).

results

foam

low

atherosclerosis.

of cells

LDL by macrophages

form

LDL,

on the

be recognized

LDL has

To whom correspondence

Abbreviations:

the

focused

of

foam

by scavenger

Oxidation

of

LDL in

be addressed.

density

ADP, adenosine

lipoprotein;

TBARS,

5'-diphosphate

thiobarbituric

sodium

acid

salt. CXlU6-291X/91 $1.50

433

CnpyYght 0 I991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

177,.

No.

vitro

1, 1991

studies

BIOCHEMICAL

is

usually

However,

what

and

Evidences

of

presence

rabbits

by

are

unclear

except

and generates ion

in

of

for

vivo

does

elicits

liver

microsome

and

urged

us to investigate

cell’s

(5).

LDL at

the effects

MATERIALS

were clearly

in

the

of

of glycation

ion.

of

in aorta

LDL?

of WHHL

demonstrated

(4).

however, Recently,

chelates

polylysine

with chelated

liposome

pH (6,7). of LDL on its

the

peroxidation

circulation,

phospholipid

physiological

cuppric

oxidation

by lipid

polypeptide

The glycated

COMMUNICATIONS

of

shown

lipoxygenase of

peroxidation

human

the

B modified

glycation

lipid

vivo

oxidants

endothelial

anion

concentration

which

occurring

RESEARCH

causes

LDL in

apoprotein

non-enzymatic superoxide

BIOPHYSICAL

by low

of oxidized

Naturally

that

ferric

where

deposition (3).

reported

initiated

immunocytochemistry,

extracellular prodncts

AND

we iron with

from

These

rat

results

own lipids.

AND METHODS

Preparation of LDL. LDL (1.019< d < 1.063) was isolated by ultracentriwhich contained 0.4 mg of 2 Na EDTA/ml, fugation from human plasma according to the method described by Schumaker and Puppione (8). The isolated LDL was dialyzed against 67 mM phosphate bnffer(pH 7.4) saturated with N, gas. Protein concentration was determined by Lowry’s method. Purity of LDL was verified by agarose gel (1%) electrophoresis, SDSpolyacrylamide gel (lo%), and immunoassay of apoprotein. LDL sample showed single band on both electrophoretic plate after staining with coomassie brilliant blue staining (for SDS-polyacrylamide gel plate) and Fat Red 7B staining (for agarose gel plate), and it contained about 0.7 mg of apoprotein BJmg of protein. Glycation containing

of LDL. LDL (2mg of protein) in 2 ml of 67 mM phosphate buffer 200 mM glucose and 0.01 % gentamycin was incubated at 37 “c for At the end of incubation, the glucose-treated LDL was dialyzed 3 days. 1mM EDTA containing 67 mM phosphate buffer at 4 “c. The dialyzed sample was reffered to G-LDL, LDL which was incubated without glucose and dialyzed according to the same procedure as described above was referred to IJ-LDL. Prior to use, both samples were dialyzed against 67 mM Amounts of glucose incorporated into phosphate buffer to remove EDTA. acid method after apoprotein B, which had been assayed by thiobarbituric to be approximately hydrolysis of LDL by oxalic acid (6)) were calculated of molecular weight of 500 10 mol/mol of apoprotein B, on the assumption K. This corresponds to modification of 2.8 % of lysine residues in apoprotein B. Incubation conditions. The standard reaction mixture contained 50 pg of Gbuffer at pH 7.4, LDL (or U-LDL) , 60 pM Fe3 ’ - 1mM ADP, and 67 mH phosphate Fe3 b -ADP complex (1: 16.6 M/M) was prepared in a total volume of 100 pl. Reaction was initiated by the addition of as described previously (9). Fe3 +--ADP and cant inued during the time cited at 37 g= with a gentle agitation. a -tocopherol and probucol, which were obtained from Eisai Company Pharmaceutical Co.Ltd., respectively, were dissolved in and Oht suka 434

Vol.

177,

No.

1, 1991

BIOCHEMICAL

AND

before use. These were ethanol concentration was 2 % in the reaction

BIOPHYSICAL

added mixture.

RESEARCH

to

G-LDL

COMMUNICATIONS

so

that

ethanol

Assays. Extent of lipid peroxidation of G-LDL or U-LDL was monitored by (TBARS) (10) . the formation of thiobarbituric acid reacting substance Modification of G-LDL or II-LDL after exposure to Fe3’-ADP was assessed by electrophoresis (see above). Cholesterol in G-LDL or U-LDL was estimated by cholesterol oxidase-phenol method (Wako Pure Chemical).

RESULTS Contents

of glycated

G-LDL 8.5

or

by 0.1

Amicon,

N NaOH cm)

solution

collected

which and

though

U-LDL possessed

lipid

found

that

peroxidation

ml4

mM

pl)

phosphate

mM sorbitol.

retained

on the

was adjusted

affinity

phosphate

by cholesterol

no affinity

of glycated

been

induces

Cu”,

100

is

100 100

estimated

LDL which

peroxidation has

with

contained

glycated

It

with

first

LDL was

pg protein/300

to a phenylboronate

equilibrated

Fig.l,

Lipid

(270

and applied

was developed buffer

same

U-LDL

1x10

column

LDL in G-LDL

column

buffer buffer

The

then

The

with

ml-fractions

contents. column

(PBA-10,

at pH 8.5. and

0.5

to pH

were

As shown

was 84 % in

G-LDL,

LDL by iron glycated

polylysine-Fe3’-complex

as well

of LDL (7) and of microsomal

+SORBITOL

phospholipid

-

TUBE NUMBER

The 100 (270

Phenylboronate affinity chromatography. Column was developed first with 100 mt4 phosphate buffer at pH 8.5. retained LDL on the column was elated with 1OOmM sorbitol-containing mM phosphate buffer at pH 8.5. (1) G-LDL (270 pg/300 pl), (2) U-LDL W/PU

in

to the phenylboronate.

PHOSPHATE

Fin.1.

the

.

435

as

Vol.

177. No. 1, 1991

BIOCHEMICAL

60

30

Fig.2. effect

AND BIOPHYSICAL

90

min

0

RESEARCH COMMUNICATIONS

102030405060 Fe3’ I PM

Lipid pet-oxidation of G-LDL and U-LDL induced by iron (A) and concentrations of ferric ion (B). (A) C-LDL or U-LDL (100 pg/2OOPl) was incubated with Fez+-ADP (60 PM) in 67 ml4 phosphate buffer (pH 7.4) at the indicated time, TBARS was assayed. (1) G-LDL + Fe3+, (2) II-LDL + Fe3+, (3) G-LDL, (4) U-LDL. (B) G-LDL or U-LDL (100 pg/200 Pl) was incubated with various (1) G-LDL, (2) U-LDL. concentrations of Fe3’-ADP for 4 h, at 37°C. of

liposomes

ADP ,

only

the

peroxidation

of

When G-LDL

(6).

which

the

lipid

induced

of

expense

monitored

of

are

peroxidation

a short

decrease

was

lag

time

of

apoprotein

peroxidation

of

U-LDL

but

concentration

in proportion

to the

Denaturation To

samples

shown

in

observed

iron

of apoprotein investigate

exposed

The lipid

of

Time

course

in

which

iron-

up to 4 h Such

may be due

to an

and alkenals)

hand,

a

for

iron-induced

lipid

during

4

With

a fixed

20 h.

h-

iron

concentrations

during

peroxidation

of G-LDL

increased

are

no significant shown

increase

in

in Fig.ZB.

in G-LDL

denaturation

to

lipid

increased

though results

showed

linearly

incubation

additional

the effects

Fe3 +-

24 h thereafter.

other

during

These

inset,

(malondialdehyde

concentration

U-LDL.

at

significantly

increased

without

TBARS.

almost

time

On the

not

examined.

on

increased

products

did

of

Fig. 2A and

long

the

or

obviously

formation

during

of G-LDL or U-LDL, were

was

the

decreased

obviously

4 h-i.ncubation

TBARS,

by

B.

with

Fe3 + -ADP

and rather

peroxidation

modification

incubation,

was incubated

of G-LDL

TBARS value lipid

U-LDL

containing

peroxidation

lipid

after

systems

or

iron

of were

apoprotein

subjected 436

moiety

to

in

SDS-polyacrylamide

G-LDL,

the gel

Vol.

177,

No.

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

12345676

+2OOK 1

+116K * 97K *

66K

+

45K

2

3

4

5

6

7

6

+Ofigir

03 Fig.3.

SDS-PAGE of G-LDL and U-LDL incubated with ferric ion. The standard reaction mixture was used for iron-induced reaction. Native LDL (45 pg/ 100 pl) was also incubated with Cu2+ (5 pM) in 67 mH phosphate buffer. At the end of reaction, equal volume of the treatment buffer solution, which contains 0.125 H Tris-HCl at pH 6.8, 4 % SDS, 20 % glycerol, 10 'R, 2-mercaptoethanol, 0.003 % BPB and 2 mN PMSF was added to an aliquot (30 pl) of each reaction mixture and 10 pl of the treated sample was applied on the gel. The 10 % running gel and 5 I stacking gel were used. Marker (Biorad) contained myosin, B-galactosidase, phosphorylase B, bovine serum albumin and ovalbumin. lane 1; 0 h-Fe3+-exposed G-LDL, lane 2; 4 h-Fe3'-exposed G-LDL (sample A), lane 3; 24 h-Fe3+-exposed G-LDL (sample B), lane 4; 0 h-Fe3+-exposed U-LDL lane 5; 4 h-Fe3'-exposed U-LDL, lane 6; 24 h-Fe3+-exposed U-LDL (sample D) , lane 7; marker, lane 8; 4h-Cu 2+-exposed native LDL (sample C). Fi.q.4. Electrophoretic mobilities of G-LDL or U-LDL incubated with or without ferric ion on agarose gel. G-LDL or U-LDL was incubated under the same conditions as in Fig.3. At the end of reaction, an aliquot (1 pl) of the reaction mixture was run on the gel and stained by Fat Red 78. lane 1; 0 h-Fe3'-exposed G-LDL, lane 2; 4 h-Fez+-exposed G-LDL (sample A), lane 3; 24 h-Fez+-exposed G-LDL (sample B), lane 4; 0 h-Fe3+ -exposed U-LDL lane 5; 4 h-Fe3+-exposed U-LDL, lane 6; 24 h-Fez+-exposed U-LDL (sample D), lane 7; 24 h-non treated GLDL, lane 8; 4h-Cn2+-exposed native LDL (sample C).

electrophoresis

(Fig.3)

LDL,

which

had

least

two

bands

weight

as that

molecular

been

protein

I .

Feat-ADP

for

the

Such

when only

agarose

SDS gel

(protein

24 h (sample

G-LDL

a great

amount

24 h (Sample gel

plate,

H).

and stained

incubated

electrophoresis for

4 h

i.e.,

(protein

one with

II

increased in with

of protein

apoprotein

of

431

for

other

only

showed

with

of 4 h

to Feat-

decrease

of

LDL was also (sample

in U-LDL exposed

the

at

higher

exposed

A to D and others

Red 78,

C-

same molecular

a great

moiety

H was detected

by the Fat

the

with

A),

the

G-LDL

5 pM Cu2+

When samples

(Fig.4).

(sample

I ) and

The protein

change

D).

gel

Feat-ADP

B) significantly

LDL was trace

agarose

to

of untreated

ADP for

However,

to

exposed

on the

weight

observed,

and

were

samples

run

C). to on

B and C,

Vol.

177,

No.

1, 1991

BIOCHEMICAL

AND

BLOPHYSICAL

Fig.5. Effects of a-tocopherol and probucol LDL induced by ferric ion. G-LDL (50 pg/lOO pl) was preincnbated probucol (2) at various concentrations for exposed to 60 pH Fe3’-ADP for 4 h.

which

contained

protein

II

G-LDL or LDL to anode Effects

of

to greater

(Fig.4)

antioxidants

on

RESEARCH

on lipid

COMMUNICATIONS

peroxidation

of G-

with a -tocopherol 15 min at 37 r,

extent,

moved faster

(1) or and then

than

untreated

. the

lipid

peroxidation

of

G-LDL

induced

by

exposed

to

iron When a -tocopherol 60

pM Fe3+-ADP

peroxidation

in

or probncol the

standard

was strikingly

by 7 pM (x -tocopherol

was added reaction

inhibited,

to G-LDL and then mixture

i.e.,

for

4

h,

50 % inhibition

or by 13 pM probncol.

Results

the

lipid

was achieved

were

shown

in Fig.5.

DISCUSSION The present

results

in G-LDL,

but

naturally

occurring

As suggested in

Amadori

to

ferry1

that and

in LDL,

were

indicated

(6))

that

denatured

transition

previously compound

Fe3+,

could

peptide

with

a high

redox

potential.

It

produces

ferry1

type

complex

peroxidation

to

products

438

in

vitro

be coordinated

glycated

lipid

in the

during

from

resulted

apoprotein

and oxidized

metal, Fe3+

both

derived

iron

G-LDL the

not

clearly

and

with could

seems likely,

initiate cause

lipid the

and lipids presence

of a

incubation. endiol

group

be converted therefore, peroxidation,

modification

of

Vol.

177,

No.

1, 1991

apoprotein,

identical

glycated

and lipid

probucol

action

to the

AND

case of

polylysine-Fe3’-complex

apoprotein or

BIOCHEMICAL

on

which the

was

lipid

could

Under

the

added,

conditions

also

of LDL by in

modified,

possessed

probably

COMMUNICATIONS

modification

be oxidatively

exogeneously peroxidation,

RESEARCH

the oxidative

(7).

in G-LDL

BIOPHYSICAL

which

both

a -tocopherol

powerful

inhibitory

on

the

denaturation

of

LDL

in

of

apoprotein. Significantly hyperglycemic modified

LDL

which

may

This

can

to oxidative

be

the

modification

has

first

apoprotein been

reported

phagocytized

Thus,

be a candidate is

of

subjects

lesions.

iron,

patients.

glycation

diabetic

atherosclerotic with

high

by

glycated for

LDL,

promoting

observation

macrophage

accumulates

could

be coordinated

atherosclerosis

for

contributing

from

Oxidatively

(11).

which

plasma

in

diabetic

to the glycation

of LDL.

REFERENCES 1.

Brown,M.S.

and Goldstein,J.L.

(1983)

Annu.

Rev.

Biochem.,

52,

223-

261.

2. 3. 4. 5. 6.

Quinn,M.T. Parthasarathy,S., Fong,L.G. and Steinberg,D. (1987) Proc. Natl. Acad. Sci. USA, 84, 2995-2998. Harberland,M.E., Fong,D. and Cheng,L. (1988) Science, 241, 215-218. Henriksen,T., Mahoney,E.M. and Steinberg,D. (1981) Proc. Natl. Acad. Sci. USA, 78, 6499-6503. Sakurai,T. and Tsuchiya,S. (1988) FEBS Lett., 236, 406-410. Sakurai,T., Sugioka,K. and Nakano,M. (1990) Biochim. Biophys. Acta, 1043,

7. a.

27-33.

Sakurai,T., Kimura,S., Nakano,M. Biophys. Acta, submitted. Schmaker,V.N. and Puppione,D.L.

and Kimura,H. (1986)

Method

(1991)

Biochim.

in Enzymol.,

128,

155-170.

9. 10. 11.

Sugioka,K. and Nakano,M. (1982) Ohkawa,H., Ohishi,N. and Yagi,K. Lyon,J., Baynes,J.W., Patric,J.S., Virella,M.F. (1986) Diabetologia,

Biochim. Biophys. Acta, 713, 333-343. (1979) Anal. Biochem. 95, 351-358. Corwel1,J.A. and Lopes29, 685-689.

439

in