Autoxidized and lipoxidase-treated polyunsaturated fatty acids

Bioehtmicu et Biophysrca Acta. 711 (1982) 460-465 Elsevier Biomedical Press

460

BBA 51118

AUTOXIDIZED

AND LIPOXIDASE-TREATED

AUTOFLUORESCENCE JOHN

M.C. GUTTERIDGE

ASSOCIATED a, PHILIP

J. KERRY

POLYUNSATURATED

WITH THE DECOMPOSITION a and DONALD

ARMSTRONG

(Revised

September manuscript

OF LIPID PEROXIDES

h

* Kational Institute for Biological Standards and Control, Ho& Hill, Hampstead, London, Ophthalmolo~, ~n~versi~ of Florida, Coflege of medicine, ~ainesvi~ie, FL 32611 (U.S.A.) (Received

FATTY ACIDS

W W3 bRB

(U.K.i

aird h department

of

11 th, 198 1) received

Kqv words: Autoxidation;

January

Lipoxidase;

25th, 1982)

Autofluorescence;

Lipid peroxide;

Polyunsaturated

fatty acid

Autoxidized and lipoxidase-catalysed oxidation of polyunsaturated fatty acids in aqueous dispersion produced lipid peroxides which were extractable into diethyl ether. These lipid peroxides had little fluorescence associated with them, most remaining behind in the aqueous phase. When lipid peroxide decomposition was accelerated by heating, substantial amounts of autofluorescent material then formed. These products had excitation and emission spectra similar to those of polymerized malondi~dehyde.

Introduction

detects a variety of peroxides and unsaturated aldehydes which decompose under the acid-heating conditions of the test to release malondialdehyde, which then forms a characteristic adduct with thiobarbituric acid, measurable by absorption at 532 nm of fluorimetrically at 553 nm. A re-

The autoxidation of polyunsaturated fatty acids is a complex sequence yielding a variety of primary peroxidic and secondary carbonyl compounds, many of which still remain poorly characterized. Oxidation of fatty acids to peroxides in vivo may occur by either enzyme-catalysed mechanisms or by free-radical-mediated autoxidations. The enzyme-catalysed reactions are more controlled biological events, such as those leading to the formation of prostaglandins and leucotrienes. Autoxidation, on the other hand, particularly of cell membranes, is regarded as a destructive event against which the cell must be protected. These generalizations may, however, not be entirely true. Oxidative deterioration in lipids can be detected by colour reactions specific for peroxide and carbonyl groups or by direct spectrophotomet~ of diene and triene conjugates. For the detection of the carbonyl malondialdehyde, the thiobarbituric acid test has been used most widely. This test OOOS-2760/82/0000-0000/$02.75

0 1982 Elsevier Biomedical

cently described

sensitive

alternative

for the detec-

tion of lipid autoxidation in biological material, is that based on the detection of fluorescent Schiff bases [l]. These bases are formed between secondary carbonyls and available amino groups. Depending on the nature of the amino donor, the resulting Schiff base complex may have either polar or non-polar properties. However, autofluorescence has also been observed to accompany the autoxidation of several polyunsaturated fatty acid compounds even in the absence of amino groups 1231. The purpose of this study was to examine the visible and ultraviolet fluorescence found in autoxidizing polyunsaturated fatty acids.

Press

46.1

Materials and Methods Fatty acid samples: linoleic grade III, linolenic 99% pure, arachidonic grade IV, and grade I, docosahexaenoic grade I and lipoxidase type I were obtained from Sigma Chemical Company, Poole, Dorset. All other chemicals were of AnalaR grade, where available, and were obtained from BDH Ltd., Poole, Dorset. Diethyl ether was purchased containing copper gauze and not phenolic antioxidants (to prevent peroxide formation on storage). Lipoxidase-catalysed oxidation of fatty acids [4,5]. 10 mM fatty acids in 0.1 M borate buffer, pH 9.0, were added to 2.3 mg of lipoxidase. The flask was saturated with oxygen and incubated for 2 h at 4°C. The peroxides formed were extracted twice into 20 ml of diethyl ether and evaporated to dryness under nitrogen. The residue was dissolved in 0.5 ml of borate buffer, pH 9.0, and then sonicated to effect dispersion in an ice-bath. The solution was then stored at -20°C under nitrogen. Autoxidation of fatty acids in aqueous dispersion. 70 mM fatty acids in phosphate/saline buffer, pH 7.4 (0.024 M phosphate/O. 15 M sodium chloride), were separately pipetted into clean, previously unused glass tubes. Each tube was vigorously vortexmixed and left open to the air and exposed to normal fluorescent laboratory lighting at room temperature for the required number of days. After the appropriate time interval, samples were extracted four times, each with 1.0 ml diethyl ether. The pooled extracts were evaporated to dryness under nitrogen and the residue dissolved in 200 ~1 of methanol. Samples were stored under nitrogen at -20°C. Autoxidation of arachidonic acid as an oil-in-air preparation. lo-p1 samples of arachidonic acid were left open to the air and exposed to normal fluorescent laboratory lighting at room temperature from O-4 days. At the end of the required time, the contents were dissolved in 0.1 ml methanol for measurement of autoxidation products as described in the appropriate sections. Thiobarbituric acid reactivity. Extracts from lipoxidase, aqueous autoxidation and oil-in-air samples were prepared as 1: 800 dilutions with phosphate/saline buffer, pH 7.4. 1 ml of this solu-

tion was diluted with an equal volume of potassium hydrogen phthalate/HCl buffer, pH 3.5, followed by 1.0 ml of 1% thiobarbituric acid in 0.05 M NaOH [6]. The tubes were heated for 15 min at lOO”C, cooled, and the absorbance read at 532 nm. Thiobarbituric acid reactivity of the aqueous phase remaining after diethyl ether extraction of the autoxidized products were measured by mixing 0.1 ml of the aqueous phase with 1.0 ml of phosphate/saline buffer, pH 7.4. To this was added 1.0 ml of acid buffer, pH 3.5, and 1.0 ml thiobarbituric acid reagent, followed by heating for 15 min at 100°C. Measurement of fluorescence. 10 ~1 of the extracts dissolved in methanol or buffer were added to 1.0 ml methanol and the fluorescence measured with a Perkin-Elmer Model MPF 4 spectrofluorimeter, using a block standard containing 1O-7 M tetraphenylbutadiene as the reference standard. The standard was set to 100 units under the following conditions: excitation, 350 nm; emission, 440 nm; filter, 430 nm; slits, 10 nm; sensitivity, X 1. Relative fluorescence intensity of the oxidized lipid products were measured at: excitation, 340 nm; emission, 390 nm; filter, 350 nm; slits, 10 nm; sensitivity, X 3 for ultraviolet fluorescence. For visible fluorescence: excitation, 360 nm; emission, 430 nm; filter, 390 nm; slits, 10 nm; sensitivity, X 3; as previously described [2]. Measurement of ultraviolet absorbance (Diene conjugation). 0.1 ml of the dilution prepared for fluorescence was added to 2.0 ml methanol and mixed. This was then scanned using appropriate blanks and scan-difference spectra in a PerkinElmer model 402. Diene conjugation was recorded at 235 nm as a measure of lipid peroxides and triene conjugation at 270-280 nm as a measure of aldehydes [7]. Decomposition of lipid peroxides by heating. 10 ~1 of the extracts were heated at 100°C for 60 min in 20-ml tightly capped glass tubes. After cooling, 1.0 ml of methanol was added and the fluorescence read. Appropriate dilutions were made from these solutions for ultraviolet scans against suitable blanks. Samples for thiobarbituric acid reactivity were prepared separately and diluted 1: 800 with phosphate/saline buffer, pH 7.4, as described in the section on thiobarbituric acid reactivity. Peroxidation of bovine brain phospholipids.

462

Bovine brain phospholipids were prepared as previously described [g]. Phospholipid at a concentration of 5 mg/ml in 0.15 M sodium chloride was peroxidized by the addition of 0.1 mM ferrous ions and incubation for 2 h at 37°C. Resulting fluorescence, due to non-polar Schiff bases, were extracted into chloroform/methanol, 2 : 1. Results

Polyunsaturated fatty acids oxidized in aqueous dispersion, both enzymically and non-enzymically, produced lipid peroxides which could be extracted into diethyl ether (Tables I and II). The ratio of thiobarbituric acid reactivity to diene conjugation (A 235nm) was considerably higher in the autoxidized samples than in the lipoxidase-treated samples. In both systems, linoleic acid formed substantial amounts of lipid peroxide, but showed, as expected, low t~obarbitu~c acid reactivity. There was little fluorescent material present following diethyl ether extraction of the oxidized fatty acid components (Tables I and II). However, when the extracted material was heated in air, the lipid peroxides decomposed, with the formation of substantial amounts of fluorescent material (Tables I and II). The poor response of autoxidized

linoleic acid in the thiobarbituric acid test, under the conditions described, was also reflected in the lower yield of fluorescent material following decomposition of its peroxides (Table II). Examination of the aqueous phase from the autoxidized polyunsaturated fatty acid, remaining after extraction of their peroxides into diethyl ether, showed that detectable quantities of thiobarbituric acid reactive and fluorescent material remained in the aqueous phase (Table III). Autoxidation of bovine brain phospholipids produced large amounts of autofluorescent material characteristic of the Schiff bases with excitation 360 nm, and emission 430 nm wavelength maxima [S]. Oxidized polyunsaturated fatty acid, however, yielded both ultraviolet and visible fluorescent material with excitation, 340 nm; emission 390 nm; and excitation, 360 nm; emission, 430 nm; respectively 121.In spite of the similarities in fluorescent spectra, between the Schiff bases and fluorescence associated with polyunsaturated fatty acid oxidation, the two products must be entirely different since there are no amino groups present in the polyunsaturated fatty acid samples to produce the amino iminopropene structures. Following accelerated decomposition of lipid per-

TABLE I THIOBARBrTURIC ACID REACTIVITY, DIENE CONJUGATION AND FLUORESCENCE FATTY ACIDS BEFORE AND AFTER DECOMPOSITION OF LIPID PEROXIDES

IN LrPOXlDASE-TRF~T~~

Table I shows relative changes in the patterns of thiobarbituric acid reactivity, diene conjugation and fluorescence before and after d~omposition of the lipid peroxides by heating. The peroxides were formed in the pol~nsaturated fatty acid samples by the technique described in Methods and Materials. Ex, excitation; Em, emission. Lipoxidase-treated fatty acids.

Thiobarbituric reactivity

acid

Relative

Absorbance

fluorescence

(A 532 nm)

A 235

nm

A 275 nm Ex. 340 nm; Em, 390 nm

Linoleic acid ( 18 : 2) Linolenic acid ( 18 : 3) Arachidonic acid (20 : 4) Docosabexaenoic acid (22 Changes

following

: 6)

decomposition

Linoleic acid Linolenic acid Arachidonic acid Docosahexaenoic acid

0.08 0.24 0.12 0.24 of the lipid peroxides 0.02 0.03 0.02 0.02

units

1X3) Ex, 360 nm; Em, 430 nm

0.27 0.74 0.21 0.70

0 0.15 0.03 0.15

3 10 5 13

3 7 7 17

0 0.09 0.02 0.07

0 0.05 0.02 0.05

49 122 104 110

48 232 162 243

by heating

463

TABLE II THIOBARBITURIC OF FA’M3’ ACIDS

ACID REACTIVITY, DIENE CONJUGATION AND FLUORESCENCE OF LIPID PEROXIDES

IN AUTOXIDIZED

SAMPLES

BEFORE AND AFTER DECOMPOSITION

Table II shows relative changes in the patterns of thiobarbitu~c acid reactivity, diene conjugation and fluorescence before and after decomposition of the lipid peroxides by heating. The lipid peroxides were formed in the polyunsaturated fatty acid samples by the techniques described in Methods and Materials, Ex, excitation; Em, emission. Autoxidized fatty-acids (2 days)

Thiobarbituric acid reactivity (A S32nm 1

Relative fluorescence units (X3)

Absorbance A 235nm

A 275nm

Ex, 340 nm; Em, 390 nm Linoleic acid ( 18 : 2) Linolenic acid (18 : 3) Arachidonic acid (20 : 4) Docosahexaenoic acid (22 : 6)

0.04 0.43 0.77 0.37

Ex, 360 nm; Em, 430 nm

0.56 0.51 0.74 0.49

0.01 0.30 0.23 0.12

5 11 11 19

3 7 11 23

0.17 0.17 0.25 0.29

0.05 0.08 0.15 0.16

70 135 171 117

52 264 370 330

Changes following decomposition of lipid peroxides by heating Linoleic. acid Linolenic acid Arachidonic acid Docosahexaenoic acid

0.02 0.03 0.04 0.06

oxides, the fluorescent products had. a slightly higher wavelength emission, at 470 nm, similar to that of polymerized malo~dialdehyde (Fig. 1). Autoxidation of arachidonic acid as an oil-in-air preparation from O-4 days (without extraction) indicated a close association between thiobarbituric acid reactivity and diene conjugation. These parameters reached a maximum at 3 days, after which time they began to decrease. As this oc-

curred, fluorescence began to increase (Table IV). Since diene conjugation and most of the observed thiobarbituric acid reactivity comes from lipid peroxides (which decompose during the acid-heating stage of the thiobarbituric acid test to yield malondialdehyde and other carbonyb) it could be expected that as thiobarbituric acid reactivity and diene conjugation (peroxide content) of the samples decreased so carbonyl content (autofluo-

TABLE III AUTOXIDIZED ~LYUNSATU~TED FATTY ACIDS. THIOBARBITURIC IN THE AQUEOUS PHASE AFTER DIETHYL ETHER EXTRACTION

ACID REACTIVITY AND FLUORESCENCE

Polyunsaturated fatty acids were autoxidized in buffer at pH 7.4 for 2 days. The solution was extracted with diethyl ether, which was discarded, and the remaining aqueous phase was used for the above analysis. Fatty acids autoxidized 2 days in buffer

Linoleic acid Linolenic acid Arachidonic acid Docosahexaenoic acid

T~ob~bit~c reactivity (A 532nm 1 0.01 0.04 0.34 0.43

acid

Relative fluorescence units (X 3) Ex, 340 nm; Em, 390 nm

Ex, 360 nm; Em, 430 nm

14 41 48 36

21 82 210 216

464

lNm4TlON

FREE RADICAL PYFA H ABSTRACTION

RFI

[DIENE ~~J~GATI~N]

70.

O2 UPTAKE

-

i

LIPID

PEROXIDES

--

l

-7

I

DECOMPOSITION 50.

CARBONYLS INCLUDING _z--

30.

,’

‘\

,’ TC

,_----___ ,

\ \

,’

.

MDA . .

I’

[TEA-

REMIVE]

OHC-CH,-CHO *.

r’

,’

t

8’

“

\

,’

‘\

t

: ‘\

. RNH2

\

: ::

L-._.

HO

CHO

=H2

OHC’

RN=CH-CH=CHOH

i

330

+ OHC-CH2-C

’

‘CH=C’

I

300

430

4eorh-n . RNH2

Fig. 1. Fluorescence scans of polymerized malondialdehyde prepared from 1.1,3,3-tetramethoxypropane and arachidonic acid lipid peroxides decomposed by heating. RFI, relative fluorescence intensity. -, Polymalondiaidehyde. Excitation, 360 nm; emission, 470 nm; filter, 430 nm; slits, 10 nm; sensitivity, x0.3. - - - - - -, Decomposed peroxide from autoxidized arachidonic acid. ~citation, 375 nm; emission, 470 ; filler, 430 nm; slits, 10 nm; sensitivity, X0.3.

may polymerize in the way suggested. Fluorescence observed in fatty acid preparations can only result from the latter mechanism since there are no amino groups available to form Schiff bases, PUFA, acid.

TABLE

polyunsaturated

fatty acid; TBA, thiobarbituric

RN=CH-CH=CH-NHA

P~LYMALG~~D~ALGWYCE

AMINO-IMINOPROPENE SCHIFF BASE FLUORESCENT

PRODUCTS

Fig. 2. Lipid autoxidation initiated by a free radical leads to hydrogen abstraction and a rea~angement of double bonds to give a conjugated diene structure. FolIowing oxygen uptake peroxy free radicals are formed (LOO.) which further abstract hydrogen from lipid molecules, leading to a chain reaction. The resulting lipid peroxides can then decompose to form carbonyl compounds including malondialdehyde (MDA). Malondialdehyde can cross-link

with amino groups

to form Schiff bases or

IV

AUTOXIDATION

OF ARACHIDONIC

ACID

AS AN OIL-IN-AIR

PREPARATION

OVER A 4 DAY PERIOD

Samples of arachidonic acid autoxidized in air over a period of 4 days. 0 time represents the time the ampouie was first opened. This shows that considerable quantities of lipid peroxide were present in the preparation before autoxidation was intended. Thiobarbitu~c acid reactivity, diene conjugation and fluorescence are shown as a function of time. Ex, excitation; Em, emission. Days autoxidized

0

1 2 3 4

Thiobarbituric reactivity

acid

Absorbance

Relative

fluorescence

units (X 3)

(A 532”l?l)

A 235nm

A 275nm

Ex, 340 nm; Em, 390 nm

Ex, 360 nm; Em, 430 nm

0.65 I .30

0.35 0.53 0.60 0.75 0.46

0.04 0.10 0.10 0.24 0.08

10 12 16 15 26

8 9 10 16 42

1.44 1.46 1.24

465

rescence) would increase (Fig. 2). When malondialdehyde polymerises it has previously been observed that thiobarbituric acid reactivity is lost [9]. Similarly, many of the carbonyls will be oxidized during the heating and lose their thiobarbituric acid reactivity. Discussion Following the autoxidation of polyunsaturated fatty acid in aqueous dispersion, most of the less polar lipid peroxides can be removed by diethyl ether extraction. These peroxides had little fluorescence associated with them; most remaining in the aqueous phase. Both lipoxidase-treated and autoxidized fatty acids formed ultraviolet and visible fluorescence when their peroxides were decomposed. ‘Decomposition of lipid peroxides has been shown to produce epoxides [lo], carbonyls and carboxylic acids [I I]. Ultraviolet and visible fluorescence previously observed in autoxidizing pol~nsaturated fatty acid has been tentatively ascribed to the presence of endoperoxides and the formation of carbonyl polymers [2,3,I2]. The slow formation of these autofluorescent products in polyunsaturated fatty acid, contaminated with lipid peroxides, can occur during storage under nitrogen, Autoxidation of arachidonic acid as an oil-in-air preparation showed a maximum thiobarbituric acid reactivity and diene conjugation at 3 days, after which time they both fell. Accompanying this fall was a rise in fluorescence, which is consistent with the proposal that fluorescence is dependent on the decomposition of primary peroxides to secondary carbonyls. One such molecule released from lipid peroxides and shown to polymerize to form fhtorescent complexes is the carbonyl malondialdehyde [13]. Heating of lipid peroxides under acid conditions, such as occurs during the thiobarbituric acid test, is known to enhance greatly decomposition of the peroxides, particularly the endoperoxides, with the release of malondialdehyde. This approach has been used here to accelerate greatly lipid peroxide decomposition and, hence, increase autofluorescence in the oxidized pol~nsaturated

fatty acid samples. The fluorescent material obtained had spectral properties similar to those of polymerized malondialdehyde prepared from 1,1,3,3-tetramethoxypropane. Malondialdehyde is not derived exclusively from lipids and can arise from intermediate molecules following free radical damage to DNA, amino acids and carbohydrates [ 14,151. Evidence that polymalondialdehyde is formed in vivo has been provided recently in ageing Neurospora crassa and Podespora anserina [ 161, as well as in the autofluorescent lipopigments [ 17f. Polymalondialdehyde and other carbonyl polymers may, therefore, contribute to the autofluorescent pigments observed in biological materials following lipid oxidation and decomposition of the lipid peroxides.

References

6 7 8 9 10

ii 12 13 14 15 16 17

Tappel, A.L. (1975) in: Pathobiology of Cell Membranes (Trump, B.F. and Arstila, A.V., eds.), pp. 145-170, Academic Press, New York Gutteridge, J.M.C., Lunec, J. and Heys, A.D. (1978) Analyt. Lett. 7, 537-544 Gutteridge, J.M.C. (1982) in Ceroid-lipofuscinosis (Armstrong, D., ed.), pp. 351-365, Elsevier Biomedical, Amsterdam Egmond, M., Brunori, M. and Fasells, P. (1974) Eur. J. B&hem. 61, 93-100 Armstrong, D., Hiramitsu, T., Gutteridge, J.M.C. and Nilsson, SE. (1982) Exp. Eye Res., in the press Gutteridge, J.M.C. (1982) Internat. J. B&hem., in the press Thomas, H.V., Mueller, P.K. and Lyman, R.L. (1967) Science, 1.59,532-534 Gutteridge, J.M.C. (1977) Analyt. B&hem. 82, 76-82 Gutteridge. J.M.C. (1975) Analyt. B&hem. 69, 518-526 Hamberg, M. and Gotthamar, B. (1973) Lipids, 8, 737-744 Gutteridge, J.M.C., Lamport, P. and Dormandy, T.L. (1976) .I. Med. Microbial. 9, 105 110 Gutteridge, J.M.C. and Kerry, P.J. (1981) Brit. J. Pharmacol. 74, 918-919P Gutteridge, J.M.C., Heys, A.D. and Lunec, J. (1977) Analyt. Chim. Acta 94, 209-211 Gutteridge, J.M.C. (1979) FEBS Lett. 105, 278-282 Gutteridge, J.M.C. (1981) FEBS Lett. 128, 343-346 Munkres, K.D. (1981) in Age Pigments (Sohal, R.S., ed.), pp. 83- 100, Elsevier/North-Holland, Amsterdam Siakotos, A.N. and Munkres, K.D. (1981) in Age Pigments (Sohal, R.S., ed.), pp. 181-202, Else~er/North-HoIland, Amsterdam