Reactivity of singlet molecular oxygen with cholesterol in a phospholipid membrane matrix. A model for oxidative damage of membranes

BIOCHEMICAL

Vol. 75, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REACTIVITY OF SINGLET MOLECULAR OXYGEN WITH CHOLESTEROL IN A PHOSPHOLIPID MEMBRANE MATRIX. A MODEL FOR OXIDATIVE DAMAGE OF MEMBRANES.” :g:: >%:* Kunihiko Suwa, Tokuji Kimura and A., Paul Schaap Department of Chemistry, Wayne State University Detroit, Michigan 48202 Received

February

28,1977

Summary : Photooxidation of cholesterol in liposomes with hematoporphyrin sensitization has been studied. With liposomal samples in which the hematoporphyrin is incorporated in the membrane, the yield of the characteristic singlet oxygen product, 3fl-hydroxycholest-6-ene 5a-hydroperoxide, was approximately 6 times greater than that observed in the samples in which the hematoporphyrin was outside the membrane. Small amounts of 3@-hydroxyradical autoxidation products, were cholest-5-ene 7a- and 7p-hydroperoxides, formed in both samples. Photolysis of a dispersion of cholesterol in an aqueous solution of hematoporphyrin gave no singlet oxygen products. It is concluded from these results that endogenous singlet oxygen when formed in the phospholipid membrane has a sufficiently long lifetime to effect oxygenation of cholesterol; whereas exogenous singlet oxygen generated outside the membrane is quenched by solvent before appreciable diffusion into the membrane can occur,, Introduction Singlet reactive

in various

The

involvement

of various

damage

oxygen

species

reactions. tion

molecular

(10,)

chemical

is receiving (1,2)

of singlet

biologically

important

increasing

and biological

oxygen

attention

(3- 7 ) oxidation

in the photosensitized

substrates

as a

inactiva-

and in the photooxidative

of membranes

because

(8 - 11) has been considered. However, questions con1 the possible importance of O2 in biological systems have been raised 1 of the extremely short lifetime of O2 in water (2 ps) (12). As the

lifetime

of ‘02

cerning

generated

in lipophilic

substrates markedly

in non-aqueous membranes

incorporated from

a solution,

several

and the ‘02

lipid

also

will

is significantly should

in the membranes.

of the sensitizer matrix

media

have

enhanced

As the lipid

factors

substrate

exhibit

longer,

0 1977

and the rate

to be considered.

by Academic Press, Inc. in any form reserved.

of reproduction

oxygen

reactivity

toward

matrix

such as the spatial

differs arrangement

of diffusion

We have therefore

:I: This work was supported by a grant from the National (CA 15874). A. Paul Schaap gratefully acknowledges Research Fellowship, 1974-78. *:+To whom correspondence may be addressed.

Copyright Ail rights

singlet

Institutes an Alfred

of lo2

in the

investigated

the

of Health P. Sloan

785 ISSN. 0006291

X

Vol. 75, No. 3, 1977

reaction chosen

of

1

BIOCHEMICAL

0

with cholesterol 2 as the substrate for this

an important

component

which

yields

characteristic

14).

The

peroxide 7~ and

reaction

Ha -via the “ene” 7&hydroperoxides,

in a phospholipid study

of biological singlet

of 102with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

two reasons:

membranes, oxygen

cholesterol

reaction

matrix.

and radical

radical

IVa and IIIa,

not only

but it is also

yields

while

Cholesterol

is cholesterol a substrate

autoxidation

principally

was

products

(13,

the 5c+hydro-

autoxidation

gives

the epimeric

respectively.

HO

Ha, IIb,

R=OOH R=OH

IIIa,

R1=H;

G HO radic> autoxidation

11% 9 R =H 2:;; 1 ; IVa, R1=OOH; R2=H

HO R1 Materials

IVb,

R1=OH;

R2=H

and Methods

Synthetic dipalmitoyl lecithin, cholesterol (recrystallized) and hematoporphyrin were purchased from Sigma. [4-14C] cholesterol (20 pCi/ml) was obtained from Amersham Searle. Silica gel H and precoated Ql thin layer chromatography plates were purchased from Merck and Quantum Ind., 3j3-hydroxycholest-6-ene 5a-hydroperoxide (Ha), 3p-hydroxyrespectively. cholest-5-ene 7a-hydroperoxide (IVa) and cholest-5-ene-7-one-3fi-ol were synthesized (l3,15,l6). Cholest-6-ene-3& 5c+diol (Ilh) and cholest-5-ene-38, ~CWdiol (IVb) were obtained by reduction of the corresponding hydroperoxides (13). A mixture of epimeric IIIb and IVb was prepared by reduction of 7-ketocholesterol with NaBH4. ethanol (2:l v/v) solution containLiposomal sample 1: A chloroform-n-l4 C] cholesterol (0.48 PCi) was ing dipalmitoyl lecithin (13.0 pmoles) and [4added to a 50 ml round-bottomed flask and the organic solvent removed by evaporation under reduced pressure. To the dried lipid film, 10 ml of 0.145 M K-phosphate buffer (pH 7.4) was added and the lipids were thoroughly agitated with a Vortex mixer until the lipid film was no longer detectable on the sides of the flask (17). Liposomal sample 2: Hematoporphyrin in 0.145 M phosphate The buffer (pH 7.4) was added to a liposomal sample prepared as above. concentration of hematoporphyrin was comparable to that in sample 3. Liposomal sample 3: Hematoporphyrin (0.5 mg) was added to the chloroformmethanol solution containing the same amount of dipalmitoyl lecithin and nonlabeled and labeled cholesterol. This sample was then treated as in sample 1. The 1iposoma.l preparation was dialyzed at ambient temperature against 1 1 of a solution containing 0.075 M KC1 and 0.075 M NaCl (isotonic solution) for 15 to 18 hr, in order to remove hematoporphyrin that had not been incor-

786

BIOCHEMICAL

Vol. 75, No. 3, 1977

0

Figure

1.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

9 6 DIALYSIS

3

12 TIME

15 (hrr)

16

21

24

Effect of dialysis on the hematoporphyrin concentration in the liposomal preparations. Ten ml of a free solution of hematoporphyrin and of liposomal samples 2 and 3 were each dialyzed against I 1 of isotonic solution; at the times indicated, a 0.5 ml aliquot of the sample was taken for assay of the hematoporphyrin concentration. The retention of the sensitizer in the samples is shown in Figure 1 as a percentage of initial concentration. The concentration of hematoporphyrin in the liposomal samples was determined by extracting an aliquot of the samples with methanol-chloroform (9:l v/v). The absorbance at 498 nm was measured and the conce4n tra -1 ion c-sfl culated using an extinction coefficient of 1.12 x 10 M cm . x-x, buffer solution of hematoporphyrin; 0-0, sample 2; o-o, sample 3.

z;$d

@o the liposomes. Liposomal sample 4: Cholesterol (6.5 pmoles) - C] cholesterol was dissolved in 1.0 ml of acetone; 9.0 ml of 0.145 M K-phosphate buffer (pH 7.4) was added to the acetone solution to make a cholesterol dispersion. Appropriate amounts of hematoporphyrin were added to the dispersed sample.

The irradiations were carried out for 20 min at ambient temperature with a Sylvania 500-W tungsten-halogen lamp. Samples were contained in Oxygen was bubbled Pyrex vessels and placed 15 cm from the light source. through the samples during the irradiations. A soft glass or Corning UVcutoff filter was placed between the light source and the samples. Results Location

of hematoporphyrin:

porphyrin

were

the extent

of hematoporphyrin

(Fig.

Free

1).

and

subjected

Liposomal

to dialysis

hematoporphyrin

Discussion

against

incorporation was

easily

787

samples

the isotonic into

containing solution

the lipid-membrane

dialyzed

through

hematoto determine space

the dialysis

tube.

Vol. 75, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

TABLE Formation

of Cholesterol

Hematoporphyrin (nmoles/ml)

Sample 1

3

4

I.

Hydroperoxides

in Photooxygenated

Cholesterol hydroperoxide formation (%)

0 Oa 14.1 17.1 24.1 18.0" 26.6a 13.8 16.8 18.0 26,8a 31.Za 26.6=

2

RESEARCH COMMUNICATIONS

Samples

Cholesterol retained (%)

2.0 1.1 3.9 6.5 8.6 3.6 5.2 24.6 19.0 28.7 22.9 23.3 2.9

98.0 98.9 96.1 93.5 91.4 96.4 94.8 75.4 81.0 71.3 77.1 76.7 97.1

The procedure for preparing samples 1, 2, 3 and 4 was given in Methods. Each sample con&ted of dipalmitoyl lecithin (13.0 pmoles), cholesterol (6.5 pmoles), [4 C]cholesterol (0.48 /Ki) and various amounts of hematoporphyrin. Each liposomal sample was irradiated by a tungsten-halogen lamp for 20 min at room temperature. Following the irradiations, the reaction mixture was repeatedly extracted with chloroform-methanol solution (2:l v/v). The pooled extract was evaporated to dryness in vacua -and the resulting residue was dissolved in 0.5 ml of the chloroform-methanol solution. An aliquot (0.05 ml) of this solution was applied to a thin layer plate (silica gel H, 0.25 mm) and developed in ether-cyclohexane (9:l v/v) under saturated air for 60 min at room temperature. Visualization of the products on the tic plate was performed by spraying 50% H2S04 and heating. Cholesterol hydroperoxides were determined by spraying N, N, The yield of cholesterol hydroperN’, N’-tetramethyl-p-phenylenediamine. oxides i s expressed as a percentage of the total radioactivity recovered in each segment of the products in the tic. All values are the mean of duplicate assays and experimental errors are less than 10% of the values Typical counts for the experiment were approximately 10, 000 in the table. at 100%. aA UV-cutoff filter was used for the other experiments.

More 2Z”

than C.

90 percent

When

(liposomal

of free

hematoporphyrin

sample

Z),

for

tube

suggesting

to the surface

of the liposomal

reactions;

hematoporphyrin was

a considerable

in the dialysis

these

added

was

filter

The

9 hrs

at

preparation

molecules dialyzing

was used

within

of hematoporphyrin

that hematoporphyrin

788

dialyzable

to the liposomal

amount

membranes.

a soft glass

was were profile

retained adsorbed

of liposomal

BIOCHEMICAL

Vol. 75, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE NaBH4

Reduction

Sample Hematoporphyrin nmoles/ml Cholesterol IIb IIIb IV-b Unknown Products Unknown Products Origin

B A

Products

II.

of the Photooxygenated

Samples

1

2

3

0

26.6

26.8

31.2

26.6

94.5 0.4 1.0 1.5 0.6 1.5 0.7

90.3

74.8 14.0 1.0 4.0 4.4 1.5 0.5

75.5 13.7 1,4 3.0 4.2 1.6 0.7

92.7 0.5 1.4 1.5 1.4

2.4 1.5 1.5 1.7 1.7 0.9

4

2.3 0.5

The liposomal preparations (samples 1, 2, 3 and 4) and conditions for the photo chemical reaction were the same-f3 those described in the legend for Table I except an increased amount of [4 C]cholesterol (1.92 /Xl). For the reduction of the samples, 1 mg of NaBH4 was added into an aliquot (0.2 ml) of the final extracts (0.5 ml) and the organic solvent was evaporated after reduction. To the residues the same volume of chloroform was added and 0.06 ml of the extracts was placed on tic plates. The samples were developed under the same solvent system at 4 to 6’ C for 120 min. Each product is expressed as a percentage of the total counts recovered from the tic plate. All values are the mean of duplicate experiments; the errors in experiments are less than 10% of the values reported in the Tables. Total counts re29940 2 6.6, 24392 + 6.7, covered from tic plate were 42502 (cpm) f. 1.7X, 25893 t 0.2 and 41203 + 7.8 in samples 1, 2, 3 and 4, respectively. Reduction of the unreacted cholesterol gave the following analysis: cholesterol (94.9%), cholest-6-ene-3@, 5a-diol(IIb) (0.40/o), cholest-5-ene-3@, 7a-diol(IVb) (1. OS), cholest-5-ene-38, 7fi-diol(IIIb) (0.9%), unknown products A (1.6%), unknown products B (0.9%), and origin (0.3%). Total counts recovered was 49700 cpm.

sample

3, in which

similar

to that

phyrin

of sample

betweensamples into

liposomes.

hematoporphyrin

of sample 3 was 2 and The

of hematoporphyrin. resulted

in less

5c$ 7c+, examined

2 except always

3 indicates value

was

Decreasing incorporation

Cholesterol

was

Hydroperoxide

that

higher

incorporated

the amount than

that

the amuunt

of sample

of hematoporphyrin Formation:

(Table

preparation

789

2.

into The

The

difference incorporated concentration lecithin

the liposomes.

formation

liposomal without

was

hematopor-

of cholesterol-dipalmitoyl

in the various

A liposomal

of non-dialyzed

10% of the initial

and 7&hydroperoxides I).

the liposomes,

of hematoporphyrin

approximately the ratio

into

of cholesterol

samples

sensitizer

was (sample

1)

Vol. 75, MO. 3, 1977

Figure

was

2.

as the control.

somewhat

dependent

reaction conversion

However,

of cholesterol

contrast

19.0

to 28.7%

conversion

formed

of sample

was

reduction

obtained.

than

(Table

that

analyzed

II, Fig.

2).

The

IVa and IIIa,

were

not attempted

radical

in only

to identify

products

A were

seen in all

observed

in a reduced

sample

principal

conversion hydroperoxides

The

primary

diols

products

following

7@-hydroperoxides

At the present

products

A and B.

reduced

after

of

starting

material,

790

various

product

samples the

of

2 and 3.

7a- and

amounts.

the unknown

3 in

of the photooxidation 1 by the reaction of O2 with

products, small

with

significant

Samples:

IIa formed

autoxidation

formed

vary

of cholesterol

in samples

The

sample

the amount

as the corresponding

3 is the 5cr+hydroperoxide

cholesterol.

may

observed

of the Photooxidized were

liposomal

the

These

8.69,.

Although

the membrane

in the

concentration

only

with

4, the amount

less

Reduction

was

2 was

present

sensitizer

obtained

3, we consistently

considerably

of the photooxidations

of sample

into

In sample

NaBH4

NaBH4

to those

incorporated

to hydroperoxides.

at the highest

was

in sample

of hematoporphyrin

to the hydroperoxides

in sharp

preparations

of hydroperoxides

even

are

hematoporphyrin

have

The yield

on the concentration

mixture.

results

AND RIOPHYSICAL RESEARCI’ COMML”x”CA”Ot’S

Radioactive scans of thin layer plates of NaBH reduction products of samples 2 and 3. Aliquots (80 ~1) of the fina 4 reduced extracts of samples 2 and 3 were chromatographed on precoated Ql silica gel plates with ether-cyclohexane (9:l v/v) at 4-6” C for 120 min. Scan a, sample 2; scan b, sample 3. 1, Unknown products A; 2, unknown products B; 3, cholest-5-ene-3& ‘la-diol(IVb); 4, cholest-5-ene-3fi, 7p-diol(IIIb); 5, cholest-6-ene-3& 5&diol(IIb); 6, cholesterol (I).

taken

which

BIOCHEMICAL

time

.we

Unknown

photoreaction cholesterol,

and also in the

Vol. 75, No. 3, 1977

same

Significant

amount.

sample

3.

oxygen

with

peroxide

Kulig

The

results

shown

results

and IVa.

membrane aqueous

are

matrix medium

quenched oxygen ponent, is rapidly importance

with

in accord (hydrophobic

dispersed no oxidation

generated

in lipid

cholesterol, quenched

with

with

medium)

solution

of cholesterol. membrane high

matrix

efficiency,

H 0 molecules. 2 membrane damage.

in biological

the cholesterol only

of liposomes

in a much longer

lower

lifetime

cholesterol (sample

yield of IIa. 1 of 02 in the in the

and the sensitizer 4),

the ‘02

is rapidly

In conclusion,

singlet

can oxidize

the membrane

exogenously This

minor

with

to the lifetime

whereas

by solvent

the photolysis

of I to IIa with

When

in aqueous

that

with

compared

the membrane.

Scr-hydro-

products.

photooxidation

the expected

in

of singlet

product,

incorporated

2) resulted

produced

the reaction

2 indicate

conversion

In contrast, (sample

outside

separately

II and Fig.

high

B were

to the major

was

gave

that

as minor

the sensitizer

membrane

products

reported

in addition

in Table

hematoporphyrin

These

were

(13) have

gives

3 in which

of IIIa

adsorbed

of unknown

60(- and 6&hydroperoxides

in the liposomal amounts

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

amounts

and Smith

cholesterol

Ha,

of sample

BIOCHEMICAL

type

molecular com-

generated

of reaction

may

lo2 have

References 1.

2.

3. 4. 5. 6. 7. 8. 9. 10. 11.

Schaap, A. P. (1976) “Singlet Molecular Oxygen, ” Benchmark Papers in Organic Chemistry, Vol. 5, C. A. VanderWerf, ed., Dowden, Hutchinson, and Ross, Stroudsburg, Pennsylvania. Foote, C. S. (1975) “Photosensitized Oxidation and Singlet Oxygen; Consequences in Biological Systems, ” in Free Radicals in Biology, W. A. Pryor, ed., Academic Press, New York. Nakano, M., Noguchi, T., Sugioka, K., Fukuyama, H., Sato, M., Shimizu, Y., Tsuji, Y., and Inaba, H. (1975) J. Biol. Chem., 250, 2404-6. Arneson, R. M. (1970) Arch. Biochem. Biophys. 136, 352-360. Goda, K., Chu, J. W., Kimura, T. and Schaap, A. P. (1973) Biochem. Biophys. Res. Commun., 52, 1300-1306. King, M. M., Lai, E. K., and McCay, P. B. (1975) J. Biol. Chem., 250, 6496-6502. Allen, R. C., Yervich, S. J., Orth, R. W., and Steele, R. H. (1974) Biochem. Biophys. Res. Commun., 60. 909-917. Girotti, A. W. (1976) Photochem. Photobiol., 24, 525-532. Anderson, S. M., Krinsky, N. I., Stone, M. J., and Clagett, D. C. (1974) Photochem. Photobiol., 20, 65-69. Takahama, U. and Nishimura, M. (1975) Plant and Cell Physiol., 16, 737-748. Lamola, A. A. , Yamane, T . , and Trozzolo, A. M. (1973) Science, 179, 1131-1133.

791

Vol. 75, No. 3, 1977

12. 13. 14. 15. 16. 17.

Merkel, Kulig, Smith, Chem. Schenck, Chem., Schenck, Chem. Kinsky, L. L.

BIOCHEMICAL

P. B. and Kearns, M. J. and Smith, L. L. L., Teng, J. I., 38, 1763-1765 G. 0.) Gollnick, 618, 194-201. G. 0.) Neumzller, 618, 202-210. S. C., Haxby, J., M. (1968) Biochim.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

D. R. (1972) J.Am. Ghem.Soc., 94, ‘7244-7253. L. (1973) J. Org. Chem., 38, 3679-3642. M. J. and Hill, F. L. (1973) J. Org. Kulig, and Neumiiller,

K., 0.

A.

and Eisfeld,

Kinsky, Biophys.

0. A. W.

(1958) (1958)

C. B., Demel, R. A. Acta 152, 174-185.

792

Liebigs Liebigs

Ann. Ann.

and VanDeenen,