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302 MICROSOMAL ELECTRON TRANSPORT AND CYT

P-450

4 /-~

tween the amount of reduced pyridine nucleotide added and the amount

of formaldehyde formed indicate that the N-demethylation reaction

represents only one of several pathways for the oxidative metabolism of

the particular substrate or that the reaction leadirlg to formaldehyde

liberation is partially uncoupled, e.g., associatefl 'ith hydrogen peroxide

formation. From the results shown in Fig .

.9 { 2 .2

mol of NADPH can be

calculated to be required for the li.b.l}t1'bn of

1

mol of formaldehyde

from ethylmorphine; for example, lWProximately 55 of the reducing

equivalents remain unaccounted

fef.

Furthermore, it can, be d~onstrated that semicarbazide is not

generally required as a tiajiPfug agent for formaldehyde, since no loss of

formaldehyde occurs ay3'n over a time period of more than 15 min.

Semicarbazide shoulcVbe u~d, however, if conditions, such as the

presence of cyanide/leading lo\he formation of

cyanohydrines,

require

its presence a~/n aldehyde tr~~ing agent. Semi carbazide does not

interfere with lineN-demethylation reaction of ethylmorphine nor does it

attenuate tJ:rt intensity of the forJation of DDL during the Nash

reaction. ly -,

9

D. T;lMowry,

Chem. Rev.

42, 189 (1948).

10

M.A. Correia and G. J. Mannering, Mol. Pharmacal. 9,455 (1973).

[30]Microsomal ipi Peroxidation

By JOHNA. BUEGEand STEVEND. AUST

u

Lipid peroxidation is a complex process known to occur in both

plants and animals. It involves the formation and propagation of lipid

radicals, the uptake of oxygen, a rearrangement of the double bonds in

unsaturated lipids, and the eventual destruction of membrane lipids,

producing a variety of breakdown products, including alcohols, ketones,

aldehydes, and ethers.' The peroxidation of linoleic acid alone results in

the formation of at least 20 degradation products. Biological membranes

are often rich in unsaturated fatty acids and bathed in an oxygen-rich,

metal-containing fluid. Therefore, it is not suprising that membrane

lipids are susceptible to peroxidative attack.

Lipid peroxidation usually begins with the abstraction of a hydrogen

atom from an unsaturated fatty acid, resulting in the formation of a lipid

1

H. W. Gardner,

J.

Agric. Food

Chon.

23, 129 (1975).

2 H. W. Gardner, R. Kleiman, and D. WeisJeder, Lipids 9, 6 (1974).


2/9

[30]

MICROSOMAL LIPID PEROXIDA nON

303

radical. 3 The rearrangement of the double bonds results in the formation

of conjugated dienes. Attack by molecular oxygen produces a lipid

peroxy radical, which can either abstract a hydrogen atom from an

-...J adjacent lipid to form a lipid hydroperoxide, or form a lipid endoperox-

ide. The formation of lipid endoperoxides in unsaturated fatty acids

containing at least 3 methylene interrupted double bonds can lead to the

formation of malondialdehyde as a breakdown product.

H 0 0-0

F\Fv,\---L;==v=\/\

--;=v-\;=\ ~ ~

o O 0-0 / I

M--~ =1~=+R

H H IV

V-\

1)

-

Microsomes isolated from liver have been shown to catalyze on

NADPH-dependent peroxidation of endogenous unsaturated fatty acids

in the presence of ferric ions and metal chelators, such as ADP or

pyrophosphates. Microsomal membranes are particularly susceptible to

lipid peroxidation owing to the presence of high concentrations of

polyunsaturated fatty acids. Poyer and Mct.ay have demonstrated that

both microsomal membranes and phosphate buffer contain sufficient

contaminating iron to facilitate NADPH-dependent microsomal lipid

peroxidation. The m for Fe

3

- in the NADPH-dependent peroxidation of

washed microsomes is 1.6 f M 4 The function of ferric ion chelators is

believed to prevent the binding of iron to components of the microsomal

membrane and to prevent the precipitation of Fe(OH)3'

Pederson

et al,

have demonstrated that an antibody to the microso-

mal flavoprotein, NADPH-cytochrome c reductase, inhibits microsomal

NADPH-dependent lipid peroxidation by over 90. Furthermore, a

reconstituted lipid peroxide-forming system composed of purified

NADPH-cytochrome c reductase, Fe3- chelated by both ADP and

EDTA, and either isolated microsomal lipidor lipoprotein particles 7

promotes NADPH-dependent lipid peroxidation, thus suggesting the

involvement of this enzyme in NADPH-dependent microsomal lipid

peroxidation. The mechanism involved in the initiation of peroxidation

in the NADPH-dependent microsomal system does not appear to

3 W. A. Poyer and J. P. Stanley, J. Org . Chem, 40,3615 (1975).

4 L. Ernster and K. Nordenbrand, this series, Vol. 10. p. 574.

5

J. L. Poyer and P. B. McCay, J.

Bioi. Chem .

246,263 (1971).

6

T. C. Pederson,

J.

A. Buege, and S. D. Aust ,

J.

Bioi. Chern, 248,7134 (1973).

7 T. Noguchi and M. Nakano, Biochim, Biophvs Acla 368,446 (1974).


3/9

. -..J.

\

304

MICROSOMAL ELECTRON TRANSPORT AND CYT P-450

[30)

.

involve either superoxide or hydrogen peroxide, since neither superox-

ide dismutase 7-9 nor thymol-free catalase 7.8 cause inhibition

o f pe rox ida

tion. However, enzymically reduced iron may play an important role in

both the initiation and propagation of NADPH-dependent microsomal

lipid peroxidation.

Microsomal membrane lipids, particularly the polyunsaturated fatty

acids, undergo degradation during NADPH-dependent lipid peroxida-

tion. The degradation of membrane lipids during lipid peroxidation has

been suggested to result in the production of Ag-type singlet oxygen,

which is detected as chemiluminescence.v The disruption of mem-

brane integrity resulting from the breakdown of the lipid constituents has

been implicated as the cause for the decrease of glucose-6-phosphatase

activity during NADPH-dependent lipid peroxidation.P Detergent dis-

ruption of microsomal membranes causes a similar decrease in activity

for glucose-6-phosphatase. Cytochrome

hS13

and cytochrome P-450

I4

are

also inactivated during NADPH-dependent lipid peroxidation, although

the mechanism of inactivation appears to involve the destruction of the

heme group rather than the loss of membrane integrity. The loss of

cytochrome P-450 during lipid peroxidation parallels the loss of drug-

metabolizing activity. Levin et al.t suggested that the rapid loss of

linearity of microsomal drug metabolism may be due to the NADPH-

dependent peroxidative destruction of cytochrome P-450. It has been

observed that some drug substrates undergoing hydroxyla.ion inhibit

lipid peroxidation, suggesting that lipid peroxidation and drug metabo-

lism compete for reducing equivalents from a common electron-trans-

port component. However, recent findings indicate that some drug

substrates are very effective antioxidants, whereas others are converted

to antioxidants once they are hydroxylated by the drug-metabolizing

enzyme system. 15

The antioxidants butylated hydroxytoluene. and o-tocopherol' have

been shown to abolish NADPH-dependent microsomal lipid peroxida-

tion

in vitro.

In addition, the

in vivo

administration of antioxidants such

8

T. C. Pederson and S. D. Aust, Biochim. Biophys Acta 385, 232 (1975).

9 K. Sugioka and M. Nakano, Biochim. Biophys Acta 423.203 (1976).

10 H. May and P. B. McCay, J. Bioi. Chem. 243,2288 (1968).

11

M. Nakano, T. Noguchi, K. Sugioka, H. Fukuyama, M. Sato, Y. Shimizu, Y. Tsuji, and

H. Inaba, J.

Bioi. Chem,

250, 2404 (1975).

12 E. D. Wills,

Biochem.

J. 123,983 (1971).

13 A. L. Tappe) and H. Zalkin,

Nature London)

185,35 (1960).

14 W. Levin, A. Y. H. Lu, M. Jacobson, R. Kuntzman, J. L. Poyer, and P. B. McCay,

Arch. Biochem. Biophys. 158,842 (1973).

15 T. C. Pederson and S. D. Aust,

Biochem. Pharmacol.

23, 2467 (1974).

16 T. K. Shires, Arch. Biochem. Biophys, 171,695 (1975). .

11 H. May and P. B. McCay, J. Bioi. Chem. 243, 2296 (1968).


4/9

: :

.[30]

MICROSOMAL LIPID PEROXIDA TION

305

x..

as o-tocopherol' and promethazine subsequently decrease the suscep-

tibility of isolated microsomes to NADPH-dependent lipid peroxidation.

NADH will not replace NADPH in promoting the rapid peroxidation

. of lipid in intact microsomes in the presence of ADP-Fe

3

-.

However, in

the presence of both ADP-Fe

3

+

and EDTA-Fe,3+ NADH is just as

effective as NADPH in promoting microsomal lipid peroxidation. Peder-

son et al. demonstrated that purified microsomal NADPH-cytochrome

hs reductase promotes the rapid peroxidation.9J extracted microsomal

lipid in a reconstituted system containing NAIJH and Fe

3

+ chelated by

both ADP and EDTA.

Nonenzymic peroxidation of microsomal membranes also occurs and

is probably mediated in part by endogenous hemoproteins and transition

metals. Conditions that lead to the disruption of microsomal membranes,

such as homogenization and repeated freezing and thawing, enhance

autocatalytic lipid peroxidation .. Hatefi and Hanstein' have demon-

strated that destabilization of microsomal membranes by exposure to

chaotrophic agents results in an increased rate of autoxidation, which is

probably promoted by components of the microsomal electron-transport

~Llin. Conversely, treatment of microsomal membranes with glutaralde-

hyde to decrease the mobility of membrane lipids by forming cross-links,

inhibits lipid peroxidation.: It has been suggested that iron-sulfur

proteins and cytochromes initiate autoxidation by catalyzing the homo-

lytic scission of preexisting membrane lipid hydroperoxides, resulting in

the formation of lipid

radicals.v

O'Brien and Rahimtula have re-

cently demonstrated that microsomal cytochrome P-450 interacts with

exogenous lipid hydroperoxides to promote oxygen uptake and the

production of lipid peroxide breakdown products. High (1.0 mM

concentrations of transition metals also promote autoxidation in micro-

somes, especially in the presence of reducing agents, such as ascorbate

or cysteine. 24

Recent evidence suggests that lactoperoxidase-catalyzed iodination

of microsomal membrane proteins occurs concurrent with increased

membrane lipid peroxidation.

25.26

Both lipid peroxidation and the de-

~

18

T. F. Slater,

Biochem.

J. 106, 155 (1968).

19 Y. Hatefi and W. G. Hanstein,

Arch. Biochem. Biophys.

138,73 (1970).

20 A. A. Barber, H. M. Tinberg, and E. J. Victoria, Nutr. Proc. Int . Congr .. Sth Int.

Congr, Ser. No. 213, p. B9 (1971).

21 W. G. Hanstein and Y. Hatefi, Arch. Biochem. Biophys, 138, 87 (1970).

22 R. M. Kaschnitz and Y. Hatefi,

Arch. Biochem. Biophys.

171,292 (1975).

23

P.

J.

O'Brien and A. Rahimtula,

J.

Agric. Food Chem. 23, 154 (1975).

24

E. D. Wills,

Biochim. Biophys. Acta

98, 238 (1965).

25

J. A. Buege and S. D. Aust,

Biochim.

Biophys.

Acta

444, 192 (1976).

26 A. F. Welton and S. D. Aust,

Biochem. Biophys. Res. Commun.

49,661 (1972).

; :F

= I \ ~

\ ..

I

\


5/9

306

MICROSOMAL ELECTRON TRANSPORT AND CYT

P-450

[30]

'-..-/

struction of cytochrome P-450 during enzymic iodination were abolished

by addition of 0.001 BHT to the reaction mixture.

Other conditions that accelerate microsomal lipid peroxidation in-

clude exposure of the microsomes to 'Y-radiation, light in the presence of

photosensitizers, hyperbaric pressure, hyperoxia, ozone, nitrogen ox-

ides, and radical initiators, such as dialuric acid.

Three assayable species are produced during microsomal lipid perox-

idation, including malondialdehyde, lipid hydroperoxides, and lipids

containing conjugated dienes. The detection of each of these products is

described below. The reaction conditions required for NADPH-depend-

ent microsomal lipid peroxidation have previously been described by

Ernster and Nordenbrand.

t..

The Thiobarbituric Acid Assay

Malondialdehyde, formed from the breakdown of polyunsaturated

fatty acids, serves as a convenient index for determining the extent of

the peroxidation reaction. Malondialdehyde has been identified as the

product of lipid peroxidation that reacts with thiobarbituric acid to give a

red species absorbing at 535

nm.

Reagent

Stock TCA-TBA-HCI reagent: 15 w/v trichloroacetic acid;

0.375 w/v thiobarbituric acid; 0.25

N

hydrochloric acid. This

solution may be mildly heated to assist in the dissolution of the

thiobarbituric acid.

Procedure.

Combine 1.0 ml of biological sample (0.1-2.0 mg of

membrane protein or 0.1-0.2 /Lmol of lipid phosphate) with 2.0 ml of

TCA-TBA-HCI and mix thoroughly. The solution is heated for 15 min

in a boiling water bath. After cooling, the flocculent precipitate is

removed by centrifugation at 1000

g

for 10 min. The absorbance of the

sample is determined at 535 nm against a blank that contains all the

reagents minus

the

lipid. The malondialdehyde concentration of the

sample can be calculated using an extinction coefficient of

1.56

x 10

5

M

-128

cm .

Iodometric Assay

Reduction of iodide by peroxides is a convenient method for deter-

mining the amount of lipid hydroperoxides present in a membrane

21

W. G. Niehaus,

Jr.

and B. Samue1sson, Eur.

J.

Biochem. 6, 126 (1968).

2B E. D. Wills,

Biochem. J.

113, 315 (1969).


6/9

[30]

MICROSOMAL LIPID PEROXIDATION

307

sample. The procedure is based on the ability of

1-

to reduce hydrope-

roxides by the following reaction' :


7/9

- -

~.

-

308


[30]

Diene Conjugation Assay

Lipid peroxidation is accompanied by a rearrangement of the polyun-

saturated fatty acid double bonds, leading to the formation of conjugated

dienes, which absorb at 233 nm. Therefore, lipid peroxidation can be

assayed by recording the increase in absorbance of extracted membrane

lipids at 233nm.

Procedure.

Membrane lipids are extracted and taken to dryness as

described for the iodometric assay. The lipid residue is dissolved in 1.5

ml of cyclohexane, and the absorbance at

23 3

nmis determined against a

cyclohexane blank. In fully peroxidized lipids, the absorbance at

23 3

nm

stands out as a distinct peak. In partially peroxidized lipids, the diene

conjugation peak is obscured by end absorption of the nonperoxidized

lipid and extracted contaminants. For such partially peroxidized lipids,

the diene conjugation peak can be obtained as a difference spectra

between partially peroxidized lipid arid an equivalent amount of nonpe-

roxidized Iipid. ' The approximate amount of hydroperoxides produced

can be calculated using a molar extinction coefficient of 2 5 2 x 10

4

M 1 32

General Comments

The thiobarbituric acid assay is the most frequently used method for

determining the extent of membrane lipid peroxidation in vitro. It is not,

however, a suitable assay for the study of lipid peroxide levels in vivo.

Malondialdehyde is readily metabolized in vivo and in tissue suspen-

sions. A mitochondrial aldehyde oxidase is partly responsible for its

metabolism; In addition, malondialdehyde reacts with tissue compo-

nents to form cross-linked lipofusion pigments, thus decreasing its

intracellular concentration. Therefore, the in vivo malondialdehyde

concentration is not likely to reflect peroxidative events occurring within

biological membranes.

35

Hemoproteins and transition metals associated with biological mem-

branes enhance the color formation in the thiobarbituric acid assay by

promoting the formation of oxy and peroxy radicals from the metal-

catalyzed breakdown of hydroperoxides during the heating of the

membrane with the TCA-TBA-HCl reagent. Pure lipid emulsions and

31

R. O. Recknagel and A. K. Ghoshal, Exp. Mol. Pathol, 5,413 (1 6).

32

P. J. O'Brien,

Can.

J.

Biochem.

47,485 (1 9).

33

Z. Pacer, A. Veselkova, and R. Rath,

Experientia

21, 19 (1965).

~I\-:J\.

Horton and L. Packer,

Biochem.

J.

116, 19P (I970).

35

J.

Green,

Ann. N. Y. Acad. Sci.

203, 29 (1972).


8/9

:

~

130]

MICROSOMAL LIPID PEROXIDATION

309

liposomes are particularly susceptible to enhanced, metal-catalyzed

autoxidation. Wills

36

demonstrated that the addition of 0.5m FeCI

3

to

linolenic acid emulsions resulted in a 5-fold increase in the absorbance at

535nmafter heating the lipids with thiobarbituric acid. We have recently

confirmed this finding using liposomes derived from microsomal lipid.

However, the addition of 0.01% BHT to the TCA-TBA-HCI reagent

just prior to use abolishes the metal-catalyzed autoxidation of lipids

during heating with the thiobarbituric reagent.

Malondialdehyde production during the peroxidation of microsomal

membranes varies among different types of tissues, thus making it

difficult to accurately compare the extent of lipid peroxidation. This is

caused in part by the different amounts of polyunsaturated fatty acids

present in the microsomal membranes from different tissues. Since only

unsaturated fatty acids with 3 or more methylene-interrupted double

bonds can ultimately form malondialdehyde, variation in malondialde-

hyde production may be a reflection of the lipid composition rather than

the susceptibility to lipid peroxidation.

Tissue aldehydes and sugars also react with thiobarbituric acid to

produce a chromophore absorbing at 535 nm. Both acetaldehyde and

sucrose interfere with the detection of malondialdehyde when present in

millimolar quantities. Huber et al.

38

have modified the thiobarbituric

acid assay by reducing the temperature in the heating step from 100to

80 to avoid interference from sucrose present in the buffers. Despite

these problems, the thiobarbituric acid assay remains a useful tool in

monitoring lipid peroxidation

in vitro

owing to its sensitivity and

simplicity.

The direct measurement of lipid hydroperoxides has an advantage

over the thiobarbituric acid assay in that it permits a more accurate

comparison of lipid peroxide levels in dissimilar lipid membranes.

However, its use is limited by the fact that lipid hydroperoxides in

biological membrane are transient species that are exposed to factors

that catalyze their breakdown. It has been demonstrated that NADPH-

dependent peroxidation of microsomal membranes results in a rapid

increase in lipid hydroperoxides, followed by a sharp decrease, presum-

ably caused by the increased breakdown of hydroperoxides.

In vitro,

transition metals, particularly in their reduced state, and hemoproteins

36 E. D. Wills,

Biochirn. Biophys. Acta

84,475 (1964).

37

T. F. Slater, Free Radical Mechanicms in Tissue Injury, p. 34. Pion Limited,

London, 1972.

38

C. T. Huber, H. H. Edwards, and M. Morrison,

Arch. Biochern. Biophys.

168, 463

(1975).

39

B. K. Tam and P. B. McCay,J. Bioi. Chern. 245,2295 (1970).


9/9

'---../

~

-

-----./

310


[31]

facilitate the decomposition of hydroperoxides.Vr'' Addition of metal

chelators affords some protection against metal-catalyzed hydroperoxide

decomposition in biological membranes, and should be present during

the isolation and storage of membranes to be assayed for hydroperoxide

levels using the iodometric assay. The iodometric assay remains a

particularly useful tool in measuring hydroperoxide levels in lipid

emulsions and liposomes, where metal-catalyzed decomposition of hy-

droperoxides is minimized.

The detection of conjugated dienes in unsaturated lipids is a sensitive

assay that can be used to study both in vivo and in vitro lipid

peroxidation. Rao and Recknager detected increased lipid peroxidation

in the liver microsomal membranes of rats exposed to carbon tetrachlo-

ride by recording the increased absorbance of the extracted membrane

lipids at

233

nm. Diene conjugation has also been used to study

NADPH-dependent in vitro peroxidation of microsomes and the autoxi-

dation of purified lipids. When purified lipids are used as the lipid

source, direct spectrophotometric analysis of water-lipid emulsion can

be performed without the need to extract the lipid into organic solvents.

Other methods for measuring microsomal lipid peroxidation have

been described elsewhere and include the measurement of oxygen

uptake; the loss of unsaturated fatty acids; the change in membrane

turbidity, the appearance of fluorescent products.f and the evolution

of ethane.

43

4

V

.0

-- IU

o c..

: > C Il

:> < , ::

V

Or < B

N

Zg,

~O

~J

Cl t--

Q~

01

::r:~

E < ; ; a

~I

~

beuge & aust

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