J. Nutr.

Lipid

Peroxidation

Tsutomu

of Liposome

Induced

Sci.

Vitaminol.,

38,

381-390,

1992

by Glucosone

NAKAYAMA,* Munetaka YAMADA, Toshihiko and Shunro KAWAKISHI

OSAWA,

Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Nagoya 464-01, Japan (Received January 29, 1992)

Summary Lipid peroxidation of liposome made of egg lecithin was induced by glucosone (D-arabino-hexos-2-ulose), a secondary product of Maillard reaction or glycation of protein. Lipid peroxidation was assessed with measurement of TBARS (thiobarbituric acid reacting substances), POV (peroxide value), and HPLC measurement of MDA (malondialde hyde). EDTA and DTPA (diethylenetriamine-pentaacetic acid) in hibited the lipid peroxidation assessed by each method described above, indicating involvement of metal ions. The observed reduction of Fe3+ to Fe2+ by glucosone might be a critical step of the lipid peroxidation. Our findings suggest a possible role of lipid peroxidation of low-density lipo proteins (LDL) induced by glucosone in atherosis caused by diabetes mellitus. Key Words lipid peroxidation, glucosone, glycation, Maillard reaction, Amadori compound, TBARS, MDA

LDL atherogenicity is a representative diabetic complication. Recently oxida tive damage of LDL has been suggested as an origin of atherosclerosis (1). In this regard, Hunt et al. suggest that autoxidative glycosylation (glycation) of LDL and the following lipid peroxidation of its lipid moiety may affect cellular interactions of LDL and result in atherosclerosis (2). They observed an increase of TBARS (thiobarbituric acid reacting substances) and POV (peroxide value) in LDL incubated with glucose in the presence of Cu2+ They concluded that the lipid peroxidation was due to the reduction of metal ion and consequent active oxygen formation. Sakurai et al. reported that glycated polylysine or glycated LDL induced lipid peroxidation in the presence of Fe3+-ADP (3, 4). They suggest that Abbreviations: substances;

POV,

pentaacetic

acid;

tos-l-yl)-L-lysine; lar

vesicles; *

To

TBA, peroxide

thiobarbituric value;

whom

small

unilamellar

correspondence

TBARS,

malondialdehyde;

Na-t-BOC-N ƒÃ-fructoselysine, fructoselysine,

SUV,

acid;

MDA,

thiobarbituric DTPA,

acid

reacting

diethylenetriamine

Na-t-butoxycarbonyl-NƒÃ-(1-deoxy-D-fruc

NƒÃ-(1-deoxy-D-fructos-l-yl)-L-lysine; vesicles; should

glucosone, be

addressed

381

MLV, D-arabino-hexos-2-ulose.

.

multi

lamel

382

T. NAKAYAMA

Amadori

compound,

oxidant

the

formation

two

research

products

groups

of

sone

of

attributed

Glucosone

is

degradation both

of

under

glucosone

liposome tried

to

detect

clarify

the

biological

to

of

of

effects

of

Reagents.

same

Wako

thiobarbituric

was

purchased

Co.

from

from

et

(St.

ICN

Bayne

obtained

by

acetic

acid.

al. the

as

expect

formed

in

browning

and

been

D-fructose

implicated

mammalian

studies,

the

as

a

cells,

2 mM

of

in vitro

DNA-synthesis

these

of

liposome

also

induce

that

of

glucosone

in

mechanism

the

lipid

acid.

a model

and

the

presence

of

peroxidation

ascorbic

in

atherogenicity

of

Thus,

system

we

in

order

of

other

urea

were

mechanism

METHODS

for

from

Immuno

catalase

were

(SOD)

U. S. A.).

Glucosone

phenylosazone

NƒÃ-fructoselysine

2

obtained

dismutase

IL,

according

prepared

according

to

N a-t-BOC-NƒÃ-fructoselysine were

reagent

grade

to

(Na-t-butoxycarbonyl the

method

(NƒÃ-(l-deoxy-D-fructos-l-yl)-L-lysine)

of

(2

Japan).

liver

Superoxide

glucose

TBA

(Tokyo,

bovine

(Lisle,

from

and Japan).

Ltd.

from

U. S. A.).

was

chemicals

(Osaka,

Japan

Biological

prepared

purposes

Ltd.

Merck

MO,

N a-t-BOC

hydrolysis

biochemical

E.

and

Fructoselysine

other

AND

Industries,

Louis,

(16).

(17).

All

of

would

egg

-NƒÃ-(1-deoxy-D-fructos-l-yl)-L-lysine) Njoroge

has on

and

spite

by

LDL

Chemical

was

of

Ama

to

oxidative

D-glucose

and

peroxidation

dicetylphosphate Chemical

method

gluco of

reasonable

in

of

protein

structure

purchased

(D-arabino-hexos-2-ulose) the

that

compounds

products

action

In

induced in

made

was

- Hydroxypyrimidine, Sigma

main

expected,

enediol

Pure

acid)

from

reported

glucosone.

Lecithin

from

degradation

degradation

Amadori

(7-10),

(12).

MATERIALS

obtained

the

biological

lipid we

glycation

of

any

an

these

unknown.

causes

peroxidation

role

of

in of

glucose.

growth,

cells

al.

it is

in ƒÁ-radiolysis

its

remains

the

lipid

or

et oxidative

Therefore,

conditions

glucosone,

owing

one

tumor

acid

(13-15),

as (6)

inhibitor

the

degradation

resulting neither

to

Kawakishi

(S).

with

anaerobic

effects

ascorbic

ions

the

Fe3+,

However,

peroxidation

by

Cu2+

Concerning

ascites

biological

metal

and

is a strong

Since

by

formed

(11).

Ehrlich

its

also

lipid

formed

polylysine

N-D-glucoside

agent

cultured

or

aerobic

mutagenic

the

of

chelated

peroxidation.

Recently

presence

produced

LDL

glycation,

lipid

was

the

was

reaction

of

the

compounds.

in

glucosone

the

to

has

Amadori

compounds

that

of

product for

(D-arabino-hexos-2-ulose)

dori

of

initial

responsible

et al.

with and

of was

were

used

5mg

of

70%

trifluoro

without

further

purification. Preparation were

of

dissolved

evaporated make

in with

a

dried

a thin

milliliters

of

sonicated

in an

liposome.

10ml

l0 mM

of

rotary film sodium

ultrasonic

Lecithin chloroform. evaporator

of

(100mg) The and

phospholipid phosphate cleaner

then on

buffer (Bransonic

and

solution

the (pH 220,

in

with

a

inner 7.4)

dicetylphosphate

a round-bottomed vacuum surface was

Branson,

poured

flask

pump of

the into

U. S. A.). J. Nutr.

in

order

flask. the The Sci.

was to Ten

flask

and

resulting Vitaminol.

LIPID

MLV and of

(multi

PEROXIDATION

lamellar

bubbled

with

100W

for

10 min.

filter

final

of

was

for

Dismic-25,

diluted of

the

of solution

0.375%

w/v

bath,

TBA

and

were

calculated

of

and

as

the of

Aust

(18).

graphy

HPLC

to

mixture, of

Under

10%

BHT

these

pyrimidine. Milford,

5ml

of

ODS-5

the

Japan). an

ization

UV

the

Fifty

fructoselysine et

was al.

(20).

5mM

of

at room

phenanthroline

chelate

calculated

by

Vol.

4, 1992

38, No.

(4.6

standardization

w/v

of TCA,

a boiling

water

The

results

was

the

done

mixed

method by

with

of

using

Buege cumene

and

The

was

the of

was

Reduction

buffer

(5mM,

plus

with

10mM

indicated

measured

was

60 min.

2-hydroxy (C

then

into

a Develosil

Chemical

Co.,

was

calculated

18:

adjusted

Ltd.,

carried by

out

standard

2-hydroxypyrimidine.

of

the

was

Detection

MDA

for

give

injected

of and

SEP-PAK and

Nomura solvent.

1ml

solution,

water to

through

was

with

Briefly,

M urea

urea

buffer

sample

determination

phosphate

boiling

passed

of

amount

in

of

measured

(19). 0.12

chromato

peroxidation

was

of

with

phosphate

authentic

by

At

heated reacts

as

liquid both

Shibamoto 0.1ml

i. d.•~250mm;

used

ortho-phenanthroline

temperature.

to

during

and

concentration.

monitored Sodium

(18).

products

solution,

10mM

was

of

Fe2+

formed

microliters

309nm.

height of

in

Aust

high-pressure

peroxidation

mixture

column

peak

Determination

at

0.1ml

(15%

mixture

was

with

Osawa

with

water at

reaction

reaction

mixed

the

volume.

detector

the

incubated

mixture,

15 min

and

according

quantitatively

U. S. A.)

Distilled

with

were

cooling,

reverse-partition

Aichi,

the

MDA

the of

MDA

MA, final

of treated the

1.2 N HCl

solution

After

Waters,

of

method of

conditions,

other

produce

was

solution

for

Buege

(MDA)

of

the

with

to

MDA.

and

amount

0.1ml

to

standard.

acid-hydrolysis

according

reaction

mixed

reaction

TBA

heated of

milliliter

malondialdehyde

The

MLV a cellulose

7.4)

mixture

the of

mixed,

of

peroxide

of

and

FeC13,

of

One

of 2ml

method

(2:1)

the

(HPLC).

liposome

kept

equivalent POV.

at

conditions.

and

were the

Standardization as

Determination

Tien

to

cavity

Japan).

(pH

reaction

milliliter

BHT,

N HCl)

chloroform:methanol

hydroperoxide

10ƒÊl

10%

according

Measurement 5ml

One

of 0.25

aerobic

from

through

Ltd.,

bottle

the

sonicated

liposome

buffer

This

in

and

liposome/ml),

phosphate

Teflon

water

solution

Kaisha,

(1mg/ml). under

the

the

(10mg

sodium

TBARS.

and

treated

Roshi

in

Japan)

of

of

383

a 20ml

placed

Kubota,

change

solution

10mM

times

methanolic

phase filtration

into

was

201M,

Toyo

liposome

indicated

bottle

by

BY GLUCOSONE

transferred

sealed

the

SUV

with

Measurement 10%

with

vesicles)

INDUCED

was

The

(Insonator

liposome.

concentration

40•Ž

30s.

confirmed

(0.2ƒÊm:

Incubation reagents,

We

unilamellar

LIPOSOME

solution

for

sonicator

(small

nitrate

to

vesicles)

N2 gas

a cup-horn-type

SUV

OF

of

at

7.4)

glucosone the

by to

The of

Feet

glucosone the

containing or

or

method

of

0.2mM

of

fructoselysine

absorbance

510nm. sample

Fe3+

according

pH

times

a control

of

Fe2+

due amount

to of

was

the

ferrous

Fe2+

was

384

T. NAKAYAMA

et al.

RESULTS

First enediol with

we structure,

after

in

with

the

initial of

did the

TBA lipid

not

1(a)).

On

examined in

peroxidation Fe3+

in

to

the

role

the

presence

reaction (Fig.

themselves

had

glucosone was

that

trace

1.

metal

As

Increase

glucosone control; •œ,

the

in

the

by

of

fructoselysine.

the

on

TBARS

glucosone

(in

the

absence

glucosone; •£,

same

lipid

in

mixture

liposomes

or of

(1mg fructoselysine liposome); •œ,

ions

were

induced

at

in

Fe3+

ascorbic

liposome/ml)

was 40•Ž.

glucosone; •£,

these in

(a) •›,

acid,

ions of

experiments, results

Fe2+

indicate the

is

lipid

a critical

glucosone,

and

with

1 mM

incubated control; •¡, ascorbic

or

absence

for

to

lipid

Cu2+

metal

the

these

These

with

peroxidation

responsible of

by

of

in

Next,

of of

lipid

liposome

Fe3+.

reduction

rate

Addition the

metal

effect

incubated the

due acid

1(b)).

was

addition

than

ascorbic

(Fig.

enhanced

the

of

of is

enhancing

2).

the

confirmed increase

glucosone

DTPA,

(Fig.

of

also

case

liposome

control

of

Since

the no

1ƒÊM

peroxidation

reaction

solution

the

conditions,

concentrations

and

535nm

degradation

the

autoxidized

peroxidation

glucosone.

SUV acid,

lipid

in

liposome

at its

was

liposome

form

compound

absorbance

This

showed

or

of

the

Amadori

Therefore,

than

glucosone

the

the

the

When

that

incubated an

and/or

1(a)).

slowly

EDTA

as

Under

to

ions

induced

ascorbic

same

on

effective

ions. 1ƒÊM

possibly

of

later.

slower

which

containing

3).

effect

3).

more

peroxidation

Fig.

no

(Fig.

Cu2+

the

(Fig.

of

was

which

glucosone

fructoselysine

metal

mixture

dose-dependently

rate

hand,

of

almost

value

is

increase

described

sample,

of

No that

mixture

its

We

(which

1).

TBA

reaction

control

liposome.

shows

method

the

the

was the

the

other

the

(Fig.

TBA

although

with

glucosone

to

the

compounds,

fructoselysine

step)

HPLC

peroxidation,

(Fig.

we

the

three

of

and

with

contribute of

of

peroxidation

glycation

observed

comparison

activity

acid,

glucosone

results

value

lipid

ascorbic

reaction

products

the

on

glucosone,

formed

to

compared

control acid.

(b) •›,

fructoselysine.

J. Nutr.

Sci.

Vitaminol.

LIPID

Fig.

2.

Effects

induced mM

PEROXIDATION

of

by

3. by

glucosone

at

40•Ž

Concentration

(b)

the

solution

indicated

on

solution or

of (1mg times. •›,

INDUCED

the

(1mg

without

1 ELM of

dependency SUV

for

with with

LIPOSOME

DTPA

SUV

glucosone

glucosone.

Fe3+

and

glucosone.

glucosone; • ,

Fig.

EDTA

OF

metal

increase

of

TBARS

liposome/ml) metal

EDTA; •£,

ions

ion

was

on

the was

2h; Ģ,

385

in

of

incubated 4h; • ,

1

1ƒÊM of DTPA.

TBARS with

4h

with

control; •œ,

with

increase

liposomes

incubated

chelators. •›,

glucosone

liposome/ml) Oh; •œ,

BY GLUCOSONE

in

induced Cu2+

the

absence

(a)

or of

glucosone.

step of lipid peroxidation caused by ascorbic acid (15), we compared Fe3+ reduction rate by glucosone and that by fructoselysine. Figure 4 shows that both compounds reduced Fe3+to Fee, but the reduction rate by glucosone was much higher than that by fructoselysine. Although the TBA method makes it easy to evaluate the extent of lipid peroxidation, its results reflect the total amount of lipid hydroperoxides and secondary degradation products. Thus, we examinedPOV of the reaction mixtures Vol.

38, No.

4, 1992

386

T. NAKAYAMA

Fig. Fig.

4.

Reduction

buffer plus

of

(5mM, 10

pH mM

of

temperature. MATERIALS Fig.

5.

Increase

liposome/ml) cosone; •£,

of was glucosone

Fig. by

glucosone

containing

and

0.2

glucosone

The AND

4

Fe3+ 7.4)

et al.

(•œ)

concentration

mM or of

of

fructoselysine. FeCl3,

Sodium

5 mM

fructoselysine Fe2+

was

5

of

(•›)

phosphate

ortho-phenanthroline was

kept

determined

as

at

room

described

in

METHODS. POV

in

incubated with

liposomes

induced

with 1ƒÊM

1mM of

by glucosone

glucosone. at

40•Ž. •›,

SUV

solution control; •œ,

(1mg glu

EDTA.

to confirm the formation of hydroperoxides of phospholipids. Figure 5 shows that lipid hydroperoxides were accumulated in the liposome during incubation with glucosone under the same conditions as the TBA experiments. Inhibition of EDTA on the increase of POV indicates that metal ions were also responsible for the formation of lipid hydroperoxides. Since the reaction mixtures were heated in a boiling water bath and absorbance at 535 nm was measured in the TBA method, there is a possibility that the TBA value might also reflect other factors formed in the heating process than MDA. We tried to confirm MDA formation in the process with the HPLC method, by which MDA can be selectively detected. Figure 6(b) shows that the control liposome treated with urea showed no peak corresponding to 2-hydroxypyrimidine. The peak of glucosone appeared at 4 min with minor peaks probably due to its degrada tion products (Fig. 6(d)). A sample prepared from the liposome incubated with glucosone gave a new peak corresponding to 2-hydroxypyrimidine (Fig. 6(a) and (c)). This means that MDA and its precursors were formed during the incubation of liposome with glucosone. In the case of liposome incubated with glucosone and EDTA, the height of the corresponding peak was very low, indicating that metal ions participated in the process of lipid peroxidation (Fig. 6(e)). We compared the value of TBARS measured by TBA method with the value of MDA measured by the HPLC method. Since the value of MDA and TBARS of the same sample in J. Nutr.

Sci.

Vitaminol.

LIPID

Fig.

6.

Chromatograms

with

urea

e)

as

indicate

present (c) (1

PEROXIDATION

in

mM),

presence ODS-5 rate,

of described

formation each

liposome

OF

the in of

sample.

(e) of

AND

authentic

(1ƒÊM)

for column

temperature,

with

urea.

(b)

(4.6•~250mm); detection,

at of

untreated (1 mM)

with

urea

15h,

at

treated

min

in

with

liposome for

c,

(1mg/ml), (d)

column,

distilled

(a, MDA

glucosone

(1 mM)

50ƒÊl;

eluents, UV

was

5.5

glucosone

volume,

387

sample

reaction

glucosone

Injection

ambient;

Each Peaks

by

incubated 15h.

BY GLUCOSONE

METHODS.

MDA, with

(1mg/ml)

reverse-partition 1.0ml/min;

treated

INDUCED

2-hydroxypyrimidine

incubated

liposome EDTA

samples

MATERIALS

(a)

(1mg/ml)

LIPOSOME

in

the

Develosil water;

flow

309nm.

Fig. 6(c) were 6.1 and 5.7nmol/ml respectively, the value of TBARS reflected mainly the amount of MDA. These results also show that the HPLC method is effective in studying lipid peroxidation of liposomes. DISCUSSION

The finding of the lipid peroxidation induced by glucosone suggests the relation between glycation and lipid peroxidation. It is already known that ascorbic acid induces lipid peroxidation of liposome made of rat liver and egg yolk-lecithin due to its enediol structure in the presence of Fe3+ (13-15). Since glucosone and fructoselysine can form enediol structure (Fig. 7), it is easy to expect these compounds to induce lipid peroxidation of liposome. Sakurai et al. reported that Vol.

38, No.

4, 1992

388

Fig.

T. NAKAYAMA

7.

The

lysine,

III.

structures

of

the

enediol

et al.

compounds.

I. ascorbic

acid,

II.

fructose

glucosone.

glycated polylysine, which consists of many Amadori moieties, induced lipid peroxidation of liposome in the presence of 60,uMof Fe3+ (3). They assumed active complexes of an Amadori moiety, Fe3+ and oxygen in the glycated polylysine, and ascribed these to lipid peroxidation. They also suggested glycated LDL induced lipid peroxidation of its own lipids with the same mechanism (4). When no metal ions were added to the reaction mixture, lipid peroxidation was not induced by an Amadori compound but by glucosone (Fig. 1). This result indicates reactivity of glucosone is much higher than that of Amadori compounds. Therefore, if Amadori moieties of glycated protein are partly decomposed to lysine and glucosone in the presence of metal ions, the produced glucosone induces lipid peroxidation more efficiently than the Amadori moieties. We think our model system helps in the study of the mechanisms of LDL atherogenicity as a complication of diabetes, because the protein moiety of LDL is glycated much more under diabetic condi tions than under normal conditions (4). Hunt et al. supposed that glucose induced lipid peroxidation of LDL in the presence of Cu2+ (2). If the protein moiety is once glycated, degradation of Amadori products easily occurs in the presence of Cu2+ under physiological conditions (5). Therefore, glucosone might be formed in their model system and initiate lipid peroxidation more efficiently than glucose. The inhibitory effects of EDTA and DTPA on all items examined here shows that trace amounts of metal ions are critical for the lipid peroxidation induced by glucosone (Figs. 2, 5, 6). Although Cu2+ enhanced the lipid peroxidation of the liposomes more efficiently than Fe3+ (Fig. 3), the catalytic effects of Fe3+ seem more important under physiological conditions (2), especially in the lipid peroxida tion of LDL. It is well known that lipid hydroperoxides are decomposed to secondary products in the presence of ascorbic acid and metal ions. If so, there is a possibility that glucosone might not be responsible for the initiation of lipid peroxidation but only for the degradationn process. We can exclude this possibility by the results of POV experiments and confirm the role of glucosone as an initiator J. Nutr. Sci. Vitaminol.

LIPID

PEROXIDATION

OF

LIPOSOME

INDUCED

BY GLUCOSONE

389

of lipid peroxidation, because POV of the liposome also increased during incubation with glucosone (Fig. 5). Miller and Aust indicated that ascorbic acid reduces Fe3+ to Fe2+ and the resulting Fe2+: Fe3+ complex promotes lipid peroxidation of liposome (15). Con sidering that ascorbic acid and glucosone possibly have the same enediol structure (Fig. 7), we propose that the prooxidant effects of glucosone should be ascribed to reduction of metal ions with this structure. Although Amadori compounds can also have the enediol structure (Fig. 7), its reducing properties were much weaker than that of glucosone (Fig. 4), suggesting the reason why fructoselysine did not cause lipid peroxidation in our system. Therefore, it is necessary to investigate the role of glucosone and other enediol products formed from Amadori compounds in the presence of metal ions in order to clarify the mechanism of the lipid peroxidation caused by glycation. Our findings also suggest that various biological effects of glucosone (11, 12) might be related to its prooxidant effects on cell components including lipid bilayers. REFERENCES

1)

2)

Halliwell, metal

ions

Hunt,

J.

4)

T.,

of

8)

9)

27,

11)

in J.

38, No.

(1990):

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and

LDL

radicals

and

186,

catalytic

1-85. glycosylation

modification

presence

of

studies

on

Oxidative

by

and glucosone.

Res.

K.

(1991):

of

Acta,

glycated

degradation

Carbohydr.

Res.,

reaction.

degradation

and

polylysine, 433-439.

Autoxidative

amino-carbonyl

27-33.

generation

Commun.,177,

iron.

glycated

1043,

(1991): O2-

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Biophys.

copper

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