Biochimica et Biophysics Acta, 712 (1982) 1-9 Elsevier Biomedical Press

BBA 51121

RABBIT LIVER MICROSOMAL LIPID PEROXIDATION

THE EFFECT OF LIPID ON THE RATE OF PEROXIDATION

MING TIEN and STEVEN D. AUST

Department of Biochemistry, Michigan State University, East Lansing, MI 48824 (U.S.A.)

(Received November 20th. 1981)

Key words: Cytochrome P-450; Lipid peroxidation; Fatty acid; (Rabbit liver microsome)

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe 3+ Although liver microsomes from both species .

contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid pet-oxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reducfase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid per-oxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.

Introduction

Microsomal NADPH-cytochrome P-450 re-

ductase-dependent lipid peroxidation has been ex-

tensively studied in microsomes isolated from rat

livers. Numerous investigators have demonstrated

that the addition of Fe3+, chelated by ADP (ADP-Fe3+) [l-6], and NADPH [7,8] to micro- somes greatly enhances the rate of lipid peroxida- tion. Peroxidation of microsomal lipid is evidenced

by molecular oxygen uptake, with the concom- mitant formation of malondialdehyde and lipid hydroperoxides.

It is generally thought that NADPH-cyto- chrome P-450 reductase catalyzes electron transfer from NADPH to an intermediate which is a pre- cursor to a more reactive species that initiates lipid

peroxidation [9-l 11. Cytochrome P-450 has also

been proposed to participate in microsomal lipid

peroxidation by reacting with lipid hydroperoxides

to yield free radical products [ 121. These free radi-

cal products can then further react in the propaga-

tion of lipid peroxidation. However, other investi-

gators have questioned the participation of cyto- chrome P-450 in microsomal lipid peroxidation [ 13,141.

Rat and rabbit liver microsomes have been found to contain similar levels of cytochrome P-450

[ 151. It has also been reported that rabbit and rat liver microsomes contain comparable levels of NADPH-cytochrome P-450 reductase [ 151. How- ever, the rate of lipid peroxidation catalyzed by rabbit liver microsomes is much lower than that of rat liver microsomes [ 16- 181. Levin and co-workers

000%2760/82/0000-0000/$02.75 0 1982 Elsevier Biomedical Press

2

[16] found lower rates of lipid peroxidation cata-

lyzed by rabbit liver microsomes as detected by

cytochrome P-450 destruction. Gram and Fouts

[ 171 observed that the microsomal supernatant

fraction of rabbit livers catalyzed lower rates of

lipid peroxidation than similar supernatant of rat

livers when assayed by malondialdehyde forma-

tion.

Mishin and co-workers [ 181 also reported lower

rates of lipid peroxidation catalyzed by rabbit liver

microsomes. The rationale for these findings have

included either the increased content of endoge-

nous antioxidants or structural peculiarities of the

rabbit liver microsomal membrane. To help clarify

this area, we have reconfirmed the relatively slow

rates of lipid peroxidation catalyzed by rabbit liver

microsomes and, in addition, have characterized

the lipid of rat and rabbit liver microsomes to

determine whether possible differences in the com-

position of the lipid could account for the ob-

served rates of lipid peroxidation.

Materials and Methods

Thiobarbituric acid, NADPH, cytochrome c

(Type VI), ADP, bromelain and butylated hy-

droxytoluene were obtained from Sigma Chemical

Co. (St. Louis, MO). Column packing for gas-

liquid chromatography was a product of Supelco

(Bellefonte, PA). All reagents were analytical grade and were used without further purification.

Rat liver microsomes were isolated from 250-

274-g male Sprague-Dawley rats (Spartan Re-

search Animals, Haslett, MI) fed on standard rat

lab chow by the method of Pederson et al. [lo].

Rabbit liver microsomes were also isolated by this

method from 2-kg male New Zealand white rab- bits (Spartan Research Animals) fed on standard

rabbit lab chow. Microsomes were washed by re- suspension in argon-purged, distilled deionized water to a protein concentration of 5 mg/ml and centrifugation at 100000 X g for 90 min. All mi- crosomes were stored in argon-purged 50 mM Tris-HCl at -20°C (pH 7.5 at 37°C). Microsomal lipid was extracted by the method of Folch et al. [ 191 and stored at - 20°C in CHCl JCH ,OH (2: 1). All solvents used in microsomes and lipid isolation and storage were purged with argon and

kept at 4°C to prevent autoxidation of microsomal

lipid. NADPH-cytochrome P-450 reductase (EC

1.6.2.4) was purified from both rat and rabbit liver

microsomes after bromelain solubilization to a

specific activity to 55 units/mg protein [lo]. Microsomal lipid peroxidation reaction mix-

tures contained ADP-Fe3+ (3 mM ADP, 0.15 mM

FeCl,), 0.5 mg/ml microsomal protein and 0.1

mM NADPH in 50 mM Tris-HCl, pH 7.5, at

37°C unless otherwise indicated in the figure

legends. Liposomal lipid peroxidation reaction

mixtures contained 1 pmol lipid phosphate/ml,

0.1 unit NADPH-cytochrome P-450 reductase/ml,

ADP-Fe3+ (1.7 mM ADP, 0.1 mM FeCl,),

EDTA-Fe3+ (0.11 mM EDTA, 0.1 mM FeCl,),

and 0.1 mM NADPH in 50 mM Tris-HCl, pH 7.5

at 37°C. Reactions were initiated by the addition

of NADPH. Iron chelate solutions were made by

the addition of FeCl, to the appropriate chelate

solutions and then the pH was adjusted. All iron

chelate concentrations are expressed as concentra-

tions of FeCl,. Lipid peroxidation was assayed by

malondialdehyde and lipid hydroperoxide forma-

tion [20]. To prevent further peroxidation of lipids

during the malondialdehyde assay, 0.02 vol. of

ethanolic 2% butylated hydroxytoluene was added

to the 2-thiobarbituric acid stock solution [21]. All

rates are initial velocities calculated from the lin-

ear portion of the curve. All reactions were carried

out at 37°C in a metabolic shaking water baths

under an air atmosphere.

The fatty acid composition of the microsomal phospholipids was determined by gas-liquid chro-

matographic analysis of the methyl esters. Methyl esters were prepared by the method of Morrison

and Smith [22]. Chromatography was performed

on a Varian Model 3700 gas chromatograph equipped with a flame ionization detector. The

glass column (6 feet X l/4 inch outer diameter)

was packed with 10% DEGS-PS on 80/100 Supelcoport.

Protein content was determined by the method of Lowry et al. [23]. Total lipid phosphate was assayed by the method of Bartlett [24]. NADPH- cytochrome P-450 reductase activity was measured by cytochrome c reduction [lo]. Cytochrome P-450 content was calculated from its carbon monoxide difference spectrum [25].

Results

Microsomal enzymes and lipid peroxidation in rat

and rabbit liver microsomes

Table1 is a comparison of the specific activity

of NADPH-cytochrome P-450 reductase (mea-

sured by cytochrome c reduction), the specific

content of cytochrome P-450, and the rates of

lipid peroxidation in rat and rabbit liver micro-

somes. The specific activities of the reductase and

the specific content of cytochrome P-450 in micro-

somes from the two species were approximately

equal. In contrast, the rates of NADPH and ADP-

Fe3+-dependent lipid peroxidation catalyzed by

rabbit liver microsomes were only 41 and 38% of

the activity of rat liver microsomes when assessed

by malondialdehyde and lipid hydroperoxide for-

mation, respectively. Extensive studies were per-

formed to assure that the comparative rates of

lipid peroxidation in the rat and rabbit systems

were determined under optimal conditions for each

microsomal preparation. Lipid peroxidation in-

cubation conditions were optimized in regards to

pH, ionic strength, NADPH concentration and

protein concentration (data not shown). The effect

of iron and chelation of iron by ADP were also

examined to optimize the rate of lipid peroxida-

tion. Fig. 1 plots the rate malondialdehyde and

lipid hydroperoxide formation in rabbit liver mi-

crosomes as a function of ADP concentration. The Fe”+ concentration was kept constant (0.15 mM).

ADP:Fe3’ molar ratio

Fig. 1. Rabbit liver microsomal lipid peroxidation as a function

of ADP: FeCl, molar ratio. Microsomal reaction mixtures con-

tained 0.5 mg of microsomal protein/ml, 0.1 mM NADPH.

0.15 mM FeCl, and the specified amount of ADP in 50 mM

Tris-HCl, pH 7.5 at 37°C. Incubation and assay conditions are

described in Materials and Methods. A, Lipid hydroperoxide;

0, malondialdehyde.

The results indicate that the rate of lipid peroxida-

tion is exceedingly sensitive to ADP chelation of

Fe3+. Activity increased rapidly when the molar

ratio of ADP to Fe3+ increased to 20 : 1. No

increase in the rate of malondialdehyde or lipid

hydroperoxide formation was associated with

higher ratios of ADP to Fe3+. A similar lipid

peroxidation profile was seen with rat liver micro-

TABLE I

MICROSOMAL ENZYMES AND LIPID PEROXIDATION IN RAT AND RABBIT LIVER MICROSOMES

Rat and rabbit liver microsomal lipid peroxidation reaction mixtures contained 0.5 mg protein/ml, ADP-Fe3+ (1.7 mM ADP, 0.1

mM FeCl,) and 0.1 mM NADPH in 50 mM Tris-HCI, pH 7.5 at 37°C. Incubation and assay conditions are described in Materials

and Methods. Values are mean* S.E. Values in parenthesis are percentage of the corresponding parameter in rat liver microsomes.

Cytochrome P-450

(nmol/mg protein)

NADPH-

cytochrome P-450

reductase

(units/mg protein)

Lipid peroxidation

(nmol/min per mg protein)

Malon- Lipid

dialdehyde hydroperoxides

Rabbit liver microsomes

(n =6)

Rat liver microsomes

(n=6)

1.06 * 0.09 0.25 -to.03 1.15*0.20 6.250.4

(9651;) (89%) (41%) (38%)

l.lO”O.06 0.28t0.01 2.78r0.08 16.5-t 1.0

somes (not shown). Fig. 2 shows the effect of Fe3+

(chelated by ADP, 20: 1 molar ratio) concentra- tion on the rate of rabbit liver microsomal lipid

peroxidation. Very low rates of lipid peroxidation

occurred in the absence of added Fe3+. The rates

of malondialdehyde and lipid hydroperoxide for- mation increased rapidly with increased ADP-Fe3+

[ADP-Fe? (mM)

Fig. 2. Rabbit liver microsomal lipid peroxidation as a function

of ADP-Fe3+ concentration. Microsomal reaction mixtures

contained 0.5 mg of microsomal protein/ml, 0.1 mM NADPH,

and the specified amount of FeCI, in 50 mM Tris-HCI, pH 7.5

at 37°C. The ADP:FeCl, molar ratio was kept constant at

20 : 1. Incubation and assay conditions are described in Materi-

als and Methods. A, Lipid hydroperoxides; 0, malondialde-

hyde.

0 2 4 6 8 IO 12 14

Time (mid

Fig. 3. Time course of rabbit liver microsomal lipid peroxida-

tion. Microsomal reaction mixtures contained 0.5 mg of micro-

somal protein/ml, ADP-Fe ‘+ (3 mM ADP, 0.15 mM, FeCl,),

and 0.1 mM NADPH in 50 mM Tris-HCI, pH 7.5 at 37°C.

Incubation and assay conditions are described in Materials and

Methods. A, Lipid hydroperoxides; 0, malondialdehyde.

concentration. Maximum rates of lipid peroxida-

tion were obtained when the ADP-Fe3+ con-

centration was greater than 0.12 mM. Again, rat

liver microsomes exhibited a similar lipid per-

oxidation profile.

Fig. 3 shows the time course of malondialde-

hyde and lipid hydroperoxide formation during a

TABLE II

RECONSTITUTION OF RABBIT LIVER MICROSOMAL LIPID PEROXIDATION

Reaction mixtures contained liposomes (1 prnol lipid phosphate/ml) in 50 mM Tris-HCl, pH 7.5 at 37°C. The following additions

were made as indicated: 0.1 mM NADPH, ADP-Fe 3+ (I.7 mM ADP, 0.1 mM FeCI,), EDTA-Fe3+ (0.11 mM EDTA, 0.1 mM

FeCI,), and 0.1 unit NADPH-cytochrome P-450 reductase. Incubation and assay conditions are described in Materials and Methods.

Values are nmoI/min per ml.

Additions Malondialdehyde Lipid

hydroperoxides

None 0.0 1 0.1

NADPH 0.05 0.4

ADP-Fe3+ 0.05 0.3

NADPH, reductase 0.05 0.4

ADP-Fe3+, reductase 0.05 0.3

NADPH, ADP-Fe’+ , reductase, 0.23 0.9

NADPH, ADP-Fe3+, reductase, EDTA-Fe3+ 0.83 7.5

5

rabbit liver microsomal lipid peroxidation reaction

at optimal conditions. The rate of lipid peroxida- tion calculated from Fig. 3 is approximately 40%

of that for rat liver microsomes. These microsomal

lipid peroxidation studies have utilized Tris-HCl as a buffer. Tris-HCl has been shown to inhibit

hydroxyl radical-dependent reactions [26]. The ef-

fect of Tris-HCl on microsomal lipid peroxidation

was investigated and 50 mM Tris-HCl had no

effect of the rate of microsomal lipid peroxidation

when compared to incubations containing 50 mM

NaCl (not shown).

Reconstitution of microsomal lipid peroxidation

Lipid peroxidation was reconstituted using the

reconstitution system of Pederson et al. [lo]. Lipo-

somes made from extracted microsomal phos-

pholipid (1 pmol lipid phosphate/ml) were in-

cubated with 0.1 unit/ml purified protease-solubi-

lized reductase, 0.1 mM NADPH and various iron

chelates (ADP-Fe3+ and EDTA-Fe3+). In the re-

constitution of rabbit liver microsomal lipid per-

oxidation, the incubation of liposomes and re- ductase of rabbit origin with NADPH and ADP-

Fe3+ (0.1 mM) resulted in low rates of lipid

peroxidation (0.23 nmol malondialdehyde/min per ml and 0.9 nmol lipid hydroperoxides/min per ml)

(Table II). The addition of EDTA-Fe3+ (0.11 mM

EDTA, 0.1 mM FeCl,) to this reaction mixture

caused an 8-fold increase in the rate of lipid

hydroperoxide formation and a 3.6-fold increase

in the rate of malondialdehyde formation.

The relative rates of lipid peroxidation cata-

lyzed by rat and rabbit liver microsomal re-

ductases are shown in Table III. As previously

reported [ 181, rat and rabbit liver microsomal re-

ductases are equally active in catalyzing lipid per-

oxidation. Irrespective of the reductase origin, the

rates of malondialdehyde and lipid hydroperoxide

formation in liposomes made from extracted rab-

bit liver microsomal lipid (1 pmol lipid phos-

phate/ml) were consistantly 40 and 43%, respec- tively, of that for liposomes made from extracted

rat liver microsomal lipid (1 pmol lipid phos-

phate/ml). The same relative difference in rate

was observed in microsomal reaction mixtures. To

examine whether the lower rates of lipid peroxida-

tion observed with liposomes of rabbit origin were

due to the presence of antioxidants or other inhibi-

tors in the lipid, liposomes (1 pmol lipid phos-

phate/ml) of rabbit origin were added to a reac-

tion mixture containing rat reconstitution compo-

nents (Table III). To ensure that the liposomes

(containing phospholipids from both rat and rab-

bit liver microsomes) were well intermixed, the

phospholipids (chloroform/methanol suspension)

were mixed prior to liposomes preparation. As

shown in Table III, the rate of lipid peroxidation

TABLE III

RECONSTITUTION OF MICROSOMAL LIPID PEROXIDATION WITH RAT AND RABBIT LIVER MICROSOMAL

COMPONENTS

Reaction mixtures contained ADP-Fe 3+ (1.7 mM ADP, 0.1 mM FeCl,), EDTA-Fe 3f (0.11 mM EDTA, 0.1 mM FeCl,), 0.1 mM

NADPH, liposomes (1 pmol lipid phosphate/ml) made from rat or rabbit microsomal lipid as indicated, and 0.1 unit NADPH-cyto-

chrome P-450 reductase from rat or rabbit microsomes, as indicated, in 50 mM Tris-HCI, pH 7.5 at 37°C. The incubation containing

liposomes made from both rat and rabbit microsomal lipid contained each at an equal molar ratio totalling 2 pmol lipid

phosphate/ml. Incubation and assay conditions are described in Materials and Methods. Values are nmol/min per ml.

Conditions

Source of lipid Source of reductase

Malondialdehyde Lipid hydroperoxides

Rabbit Rabbit 1.19 8.0

Rabbit Rat 1.12 8.2

Rat Rabbit 2.70 18.9

Rat Rat 3.18 18.9

Rat, rabbit Rat 2.90 20.5

Lipid Phosphate (mM) Lipid Phosphate (mM) 6

Fig. 4, Effect of lipid phosphate concentration on reconstituted lipid peroxidation. A. Reaction mixtures contained 0.1 units/ml of rat

microsomal reductasc, ADP-Fe 3t (1.7 mM ADP, 0.1 mM F&J,), EDTA-Fe 3+ (0.1 I tnM EDTA, 0.1 mM F&l,). 0.1 mM NADPH. and liposames (made from extracted rat microsomal lipid) at the specified lipid phosphate concentralmn in 50 mM Tris-HCl, pH 7.5

at 37°C. B. Experimental conditions were as indicated in A except the liposomes were made from extracted rabbit microsomal lipid. Incubation and assay conditions are described in Materials and Methods. MDA, malondialdehyde.

observed with these liposomes were not lower than data show that rat liver microsomal lipid is en-

incubations containing rat reconstituted compo- riched with arachidonic and docosahexaenoic acid.

nents. In contrast, docosahexaenoic acid was undetecta- The effect of lipid concentration (standardized

by lipid phosphate content) on the rate of lipid

peroxidation in the reconstituted system is shown in Fig. 4. Rat liver microsomal lipid was near

saturation at a concentration of 1 pmol lipid phos-

phate/ml (Fig. 4A). However, at an identical con- centration rabbit liver rnicrosomal lipid was still

limiting (Fig. 4R). The rates of malondialdehyde

formation increased linearly with increased rabbit

liver microsomal lipid content, demonstrating no evidence of saturation even at 6pmol lipid phos-

phate/ml.

TABLE IV

FATTY ACID COMPQSITIQN OF MICROSOMAL PHOS-

PHOLIPIDS

Phospholipids were extracted from rat and rabbit liver micro-

somes (5 mg protein). The phospholipids were hydrolyzed and

the methyl esters of the fatty acids were prepared for gas chromatograpbic analyses as described in Materials and Meth-

ods. The instrument conditions were: carrier gas flow rate of 30

ml/m.in, temperature program of l70-220°C at 3”C/min. The

fatty acid composition is expressed as the percentage (w/w) of

pahnitic acid in the specified sample. The values in parenthesis

represent the percentage (w/w) of palmitic acid in each sample.

Values are mg/IOO mg palmitic acid.

Since the results shown in Table III suggested that the lower rates of rabbit liver microsomal lipid peroxidation were not due to antioxidants or other inhibitors, the fatty acid composition of microsomal phospholipids was examined. The re- sults of these analyses are shown in Table IV. The content of each fatty acid is expressed as per- centage of the palmitic acid content (w/w>. The

Fatty acid Rat microsomes Rabbit microsomes

Palmitic loo (19%) Steak 147 OkiC 39 Linoleic 70 Arachidonic 156

Docosahexenocc 25

IO0 (22%)

I09

65

132 49

not detected

B I I 1

5 IO I5

Time (mid

Fig. 5. Analysis of fatty acids lost during microsomal lipid

peroxidation. The rate of fatty acids lost during microsomal

lipid peroxidation was determined by sampling 5-ml aliquots of

reactions mixtures at the times indicated. The aliquots were

analyzed for fatty acid content as described in the legend to

Table IV and in Materials and Methods. The content of the

specified fatty acids are expressed as the percentage (w/w) of

the original content. The number preceding the colon indicates

the carbon chain length; the number following indicates the

number of double bonds present. A. Reaction mixtures con-

tained 0.5 mg/ml of rat liver microsomal protein, ADP-Fe3+

(3 mM ADP, 0.15 mM FeCl,) and 0.1 mM NADPH in 50 mM

Tris-HCI, pH 7.5 at 37’C. B. Experimental conditions were as

indicated in A except the microsomes were of rabbit liver

origin.

7

ble and arachidonic acid was present in much

lower levels in the rabbit liver microsomal lipid.

Linoleic acid accounted for 29% (w/w) of the total

rabbit liver microsomal fatty acids. The fatty acid

content (mg fatty acid/mg protein) of rat liver

microsomes was approximately the same as found

with rabbit liver microsomes. Loss of fatty acids during microsomal lipid

peroxidation was measured by gas-liquid chro-

matography. The results of the analyses are shown

in Fig. 5. The degree of unsaturation of a fatty

acid correlated well with the rate of its disap-

pearance. Although docosahexaenoic acid accounts

for less than 5% (w/w) of the total rat liver

microsomal fatty acids, the rate of disappearance

was greatest (Fig. 5A). The rates of arachidonic

and linoleic acid disappearance were lower; the

saturated fatty acids and oleic acid did not show

any appreciable loss (Fig. 5A). The profile of fatty

acid loss in rabbit liver microsomes was similar to

that of rat liver microsomes (Fig. 5B). These re-

sults suggest that the lower polyunsaturated fatty acid content in microsomal phospholipid is the

limiting factor in rabbit liver microsomal lipid

peroxidation.

Enhancement of microsomal lipid peroxidation To examine further whether polyunsaturated

fatty acids are the limiting component in rabbit

liver microsomal lipid peroxidation, liposomes were

added to the microsomal reaction mixture. Table V

shows that the addition of liposomes (0.2 pmol

lipid phosphate/ml) to the microsomal reaction

TABLE V

ENHANCEMENT OF RABBIT LIVER MICROSOMAL LIPID PEROXIDATION BY PHOSPHOLIPIDS

The complete microsomal reaction mixtures contained 0.5 mg microsomal protein/ml, ADP-Fe3+ (3 mM ADP, 0.15 mM FeCI,) and

0.1 mM NADPH in 50 mM Tris-HCI, pH 7.5 at 37°C. Liposomes were made from extracted rabbit liver microsomal lipid and added

at the indicated lipid phosphate concentrations. Incubation and assay conditions are described in Materials and Methods. Values are

nmol/min per mg.

Conditions Malondialdehyde

Complete, - NADPH 0.15

Complete 1.14

Complete, + liposomes (0.2 pmol/ml) 1.64 Complete, + liposomes (0.5 ~mol/ml) 1.97

Lipid hydroperoxides

0.6

6.2

9.1 11.4

8

mixture resulted in a 44 and 46% increase in the

rate of malondialdehyde and lipid hydroperoxide

formation, respectively. The addition of liposomes

yielding a final concentration of 0.5 pmol lipid

phosphate/ml caused a 74% increase in the rate of

malondialdehyde formation and an 83% increase in the rate of lipid hydroperoxide formation.

Discussion

Previous studies have shown that rabbit liver

microsomes do not catalyze lipid proxidation read-

ily, in comparison to rat liver microsomes [ 16-181.

This present study confirms the previous reports

and shows that the lower rates of lipid peroxida-

tion, when compared to rat liver microsomes, are

not attributable to differences in incubation condi-

tions. Some investigators have attributed the dif-

ferences in rates to the presence of endogenous

antioxidants or inhibitors in rabbit liver mi-

crosomes rather than to a lower specific activity of

NADPH-cytochrome P-450 reductase [ 181. Our

results obtained from a comparative study of six

different preparations of rat liver microsomes with

six preparations of rabbit liver microsomes also

showed that the lower rate of lipid peroxidation

observed for rabbit liver microsomes is not due to

the specific activity of the reductase. It is also

unlikely that the lower rates of peroxidation are

due to antioxidants or other inhibitors since the

addition of rabbit microsomal lipid did not inhibit

the rate of lipid peroxidation of isolated rat micro-

somal lipid catalyzed by rat microsomal reductase.

Low concentrations of rat microsomal lipid (1 pmol

lipid phosphate/ml) were required to produce maximal rates of lipid peroxidation in a system

reconstituted with rat microsomal reductase. In-

creasing rat liver microsomal lipid concentration beyond 1 pmol lipid phosphate/ml did not result

in a significant increase in the rate of lipid per- oxidation. However, increasing rabbit liver micro- somal lipid concentration from 0 to 6pmol lipid phosphate/ml resulted in a linear increase in the rate of lipid peroxidation. This suggests that rabbit

microsomal lipid is not as susceptible to peroxida- tion as rat microsomal lipid.

Gas-liquid chromatographic analysis of the fatty acid composition of microsomal phospholipids re- vealed that rat liver microsomes are enriched in

the polyunsaturated fatty acids, arachidonic and

docosahexaenoic. Rabbit liver microsomes were

found devoid of docosahexaenoic acid, contained

a lower percentage (w/w) of arachidonic acid, and

were enriched in linoleic acid. These analyses also

revealed that the fatty acid content (mg fatty

acid/mg protein) of rabbit liver microsomes were

not significantly different from rabbit liver micro-

somes. These differences in the fatty acid composi-

tion may have two possible effects on lipid per-

oxidation: (1) The rate of initiation may be lower

in rabbit liver microsomes due to lower availability

of polyunsaturated fatty acids. (2) The rate of

chain propagation reactions may be enhanced

when the unsaturated centers of fatty acids are in

close proximity [27]. This configuration may maxi-

mize the possibility of chain reactions. Due to the

greater polyunsaturated fatty acid content, such

might be the case with rat liver microsomes to a

greater degree than with rabbit liver microsomes.

The work of May and McCay [28] with micro-

somal lipid peroxidation demonstrated that the

increased unsaturation of a fatty acid rendered it more susceptible to peroxidation. Our results with both rat and rabbit liver microsomal lipid per-

oxidation are in accord with this observation.

Within 15 min after initiation, 70% of the docosahexenoic acid was lost from the rat liver

microsomal reaction mixture, as well as over 50%

of the arachidonic acid. In the rabbit liver micro-

somal reaction mixture, over 30% of the

arachidonic acid and 15% of the linoleic acid were

lost during this time span. The saturated fatty

acids were not significantly altered in either sys- tem.

Increasing the concentration of polyunsaturated

lipid in the rabbit liver microsomal reaction mix-

ture by the addition of extracted microsomal lipid increased the rate of lipid peroxidation. These

results suggest that the availability of polyun- saturated fatty acids is the rate-limiting step in rabbit liver microsomal lipid peroxidation. Fur- thermore, they indicate that the deleterious effects of lipid peroxidation to membrane-bound proteins and other cellular components may be controlled by the degree of membrane phospholipid un- saturation, in addition to the presence of cellular antioxidants [29], superoxide dismutase [30], cata- lase [31] and other protective enzy-mes [32].

Acknowledgements

Dr. John Bucher is thanked for his assistance during the writing of this manuscript. We would

also like to thank Mrs. Cathy Custer for the pre-

paration of this manuscript. Supported by NSF

Grant Number PCM 79-15328, Michigan Agri-

cultural Experiment Station Journal Article Num-

ber 10135.

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