Free-radical damage to lipids, amino acids, carbohydrates and nucleic acids determined by thiobarbituric acid reactivity

0020711X/82/070649-05SO3.00/0 Copyright 0 1982 Pergamon Press Ltd

fnt. .I. Biochem. Vol. 14, pp. 649 to 653. 1982 Printed in Great Britain. All rights reserved

FREE-RADICAL DAMAGE TO LIPIDS, AMINO ACIDS, CARBOHYDRATES AND NUCLEIC ACIDS DETERMINED BY THIOBARBITURIC ACID REACTIVITY Division

JOHN M. C. GUTTERIDCE of Antibiotics, National Institute for Biological Standards Holly Hill, Hampstead, London NW3 6RB, U.K. (Receiced

and Control,

22 October 1981)

Abstract-l. The thiobarbituric acid (TBA) reaction, widely applied to the detection of autoxidation in polyunsaturated fatty acids, can be used to measure free-radical damage to amino acids, carbohydrates and nucleic acids. 2. In all of these systems malondialdehyde (MDA) is predominently formed from intermediate precursor molecules which break down during the acid-heating stage of the TBA test. 3. The acid reagent used to bring about these decompositions appears to be critical.

MATERIALS AND METHODS

INTRODUCTION the last 40 years, the thiobarbituric (TBA) reaction has been used for the detection During

acid

of oxidative deterioration in bulk lipids and foods with a high lipid content. More recently it has been increasingly applied to complex biological materials to detect and measure oxidative changes that may take place by either predominantly enzymic reactions such as prostaglandin biosynthesis, or non-enzymic freeradical peroxidations. In both cases TBA reactivity has been successfully applied as a useful marker of oxidative changes in polyunsaturated fatty acid content three-carbon-compound malondialdehyde The (MDA) forms a characteristic chromogenic adduct with two molecules of TBA (Sinnhuber et al.. 1958). This has led to the widespread belief that the TBA test detects the formation of MDA in biological systems. Most of the MDA detected in the TBA test is, however,

formed

from

peroxidic

precursors

which

de-

compose under the acid-heating conditions of the reaction (Gutteridge ef al., 1974: Porter er al.. 1976). MDA is not exclusively derived from polyunsaturated fatty acids. Recently, two structurally different antitumour antibiotics have been shown to release an intermediate from DNA which gives rise to MDA during the TBA test (Kuo & Haidle, 1973; Gutteridge. 1979; Kappen & Goldberg, 1978). Radiation and irondependent damage to amino acids and carbohydrates can also release intermediates which form an MDATBA adduct under the acid-heating conditions of the test (Kapp & Smith 1970: Ambe & Tappel, 1961: Morre & Morazzani-Pelletier, 1966; Gutteridge 1981). Many variations of the TBA test are now applied to biological materials. These differ in the type of acid used for colour development as well as the heating time allowed for the reaction. The present study was undertaken to examine the acid conditions necessary to bring about maximum decomposition of the intermediate molecules which form MDA during the TBA test.

The fatty acids linolenic acid 99% pure, arachidonic grade IV, docosahexaenoic grade II were from Sigma, London Ltd. Linoleic acid puriss Koch Light Ltd. 2-deoxy-DRibose. DNA calf thymus type I. glucuronic acid. 2-hydroxypyrimidine, 2-methyl indole from Sigma London Ltd. Bleomycin sulphate Lundbeck Ltd. l.l.3.3-tetramethoxypropane Aldrich Chemical Co. Ltd. All other chemicals were of AnalaR grade where available and obtained from BDH Ltd. Diethyl ether contained copper gauze and not phenolic antioxidants to prevent peroxide formation during storage. All reactions were carried out in glass tubes open to the air.

(A) Autoxidation of fatty acids. 100 mg of each fatty acid was dispersed in 5 ml of buffer (0.024 M phosphate, 0. I5 M sodium chloride pH 7.4). The tubes were left at room temperature under normal fluorescent laboratory lighting in glass tubes and shaken each day. Arachidonic and docosahexaenoic acids were left for 3 days. linolenic acid 4 days. and linoleic acid 6 days under these conditions. (B) Lipoxidase treatment of the fatty acids. Peroxides were prepared from the four fatty acids by a modification of the method of Egmond et ul. (1976) using soybean lipoxidase. Following incubation in a borate buffer pH 9.0. and an oxygen enriched atmosphere. resulting peroxides were extracted into diethyl ether. This was removed by evaporation and the residue dissolved in borate buffer and stored under nitrogen (Armstrong et crl.. I98 I ). (C) Autoxidation of bovine brain phospholipid powder in air. Phospholipid powder was allowed to autoxidize in air on the bench at room temperature for 3 days. During this time it changed from a white powder to an orangeyellow coloured product. This was prepared as a 5 mg, ml suspension in 0.15 M NaCI. (D) Autoxidation of phospholipid liposomes. Liposomes were prepared as previously described (Gutteridge. 1977) at a phospholipid concentration of 5 mgiml in 0.15 M NaCI. These were allowed to autoxidize under ditions for 3 days at room temperature. Extruction

of lipid

(A) Autoxidation buffer by shaking

This was repeated 649

o.xidtrtion

products

hyperoxic

con-

protlucls

were extracted

with an equal volume

4 times and the pooled

from the 5 ml of diethyl

ether

ether.

extracts

650

JOHN M. C. GUTTERIDGE followed by addition of 2.Og Dowex resin. The tube was incubated at 37°C for 1 hr with constant shaking. The yellow-coloured supernatant was removed, after centrifuging and 0.2 ml applied to a column of Sephadex G-10 (70 x 5 mm). 2 ml fractions were collected after elution with water pH 7.4 (distilled water adiusted with sodium hydrogen carbonate). These were diluted l/200, then 0.5 ml of this, together with 0.5 ml phosphate-saline buffer, were taken for TBA reactivity.

evaporated to dryness under nitrogen. The residue was dissolved in 1 ml of ethanol and stored under nitrogen at - 2O‘C. (B) Lipoxidase samples were similarly extracted by diethyl ether, the extracts evaporated to dryness under nitrogen and the residue dissolved in borate buffer pH 9.5 (Armstrong er al., 1981). These were stored under nitrogen at -2O’C. Iron-dependent carbohydrates

free-radical

damage

to

crmino

acids

and Development

Iron-dependent free-radical

damage to DNA

Deoxyribonucleic acid (DNA) was prepared as a 1 mgiml solution in sodium chloride 0.15 M and stored at 4’C. 0.5 ml of this solution was added to the phosphate saline buffer previously used, followed by the addition of 0.1 ml of 20 mM ferrous ions. In a similar way, buffered DNA was incubated with 0.1 ml of bleomycin sulphate 1mgjml and 0.1 ml ferrous ions 0.5 mM. All tubes were incubated for 15 min at 37’C. Preparation

A 53Znm. Development

of MDA

1. Relative

TBA reactivity

of oxidized

lipids with different

TBA reactivity

Buffer pH 3.5

TCA 28% w/v

of I-methyl

indole reucrivir)

2-Methyl indole (0.1 g) was dissolved in 100 ml ethanol and the volume made to 125 ml by the addition of hydrochloric acid (Scherz er al., 1967). This reagent was stored in a dark bottle at 4-C. Sample reactivity was developed in exactly the same way as for TBA reactivity except that 1.0 ml of methyl indole reagent was added instead of TBA and l.Oml of 257” HCI was added as the only acid. The tubes were mixed, heated for 5 min at IOO’C. cooled, and the absorbance read at 550 nm.

1,1,3,3-Tetramethoxypropane (TMP) was prepared as a 2 mM stock solution in-di&lled water. MDA-w& prepared by hydrolysing TMP using Dowex 5OW-X8 in the H+ form. 5 ml of TMP was mixed with 5 ml distilled water

Table

of 7BA reactivity

2-Thiobarbituric acid was prepared as a 17, solution in 0.05 M sodium hydroxide. Acid reagents used were 25yj0 v/v hydrochloric acid (HCI), 28”” w/v trichloracetic acid (TCA), glacial acetic acid and acid buffer pH 3.5 (prepared from 0.1 M potassium hydrogen phthalate and 0.1 M HCI). Lipid extracts were diluted 1 in 20 with 0.15 M saline and 0.1 ml 12, TBA reagent and l.Oml of one of the acid reagents. For the amino acids, carbohydrates and DNA, the total sample was obtained following incubation with iron as described in sections 3 and 4, and was used for TBA reactivity by adding I.0 ml of TBA reagent and 1.0 ml of acid reagent. All tubes were mixed and heated for 15 min at 100°C. The resulting TBA chromogen(s) was recorded at

A 5 mM solution of the amino acids and carbohydrates was prepared in distilled water treated with chelex-100 resin. Homocysteine was used as an ultrasonicated suspension in water. 0.5 ml of each sample was added to 0.5 ml of 0.024 M phosphate, 0.15 M saline pH 7.4. Iron-dependent damage was initiated by the addition of 0.1 ml 10 mM ferrous ions. followed by incubation at 37~C for 15 min. Appropriate blanks were included from which iron was omitted.

HCl 25% v/v

A532

acid reagents

nm

2-Methyl

indole

reactivity

A550

Glacial acetic acid

Phospholipid

(bovine

brain)

(1)

0.81

1.00

2.04

1.47

1.08

Phospholipid

(bovine

brain)

(2)

0.64

0.27

1.76

0.46

0.40

Linoleic

acid

(A)

0.19

0.12

0.12

0.14

0.20

Linoleic

acid

(E)

0.11

0.05

0.08

0.04

0.10

Linolenic Linolenic

acid acid

(A)

0.93

0.49

0.39

0.31

0.22

(E)

0.82

0.33

0.24

0.21

0.17

1.30

0.48

0.50

0.36

0.27

Arachidonic

acid

Arachidonic

acid

(A)

0.40

0.17

0.12

0.11

0.16

Docosahexaenoic

arid

(A)

0.87

0.37

0.24

0.29

0.23

Docosahexaenoic

acid

(E)

0.91

0.56

0.24

0.32

0.29

(E)

nm

(A) Fatty acids autoxidized in aqueous dispersion, (E) Fatty acids whose oxidation was enzymically catalysed with lipoxidase. Results are shown as relative absorbance readings at 532 nm when the same volumes of fatty-acid-oxidation products were heated under the same conditions with TBA but using different acids. The results are expressed as a mean of 3 separate assays: (1) bovine brain phospholipids oxidized in air for 3 days as a dry powder; (2) bovine brain phospholipid liposomes oxidised in buffer 7.4 for 3 days.

Determination

of free-radical damage by TBA reactivity

651

Table 2. Relative TBA reactivity of products resulting from iron-dependent damage to amino acids and carbohydrates TBA

Buffer pH 3.5

reactivity

VA 28% w/v

HCl 25% v/v

0

A532

nm

Glacial Acetic acid

Glycerol

0.03

0.03

0.03

Deoxyribose

0.40

1.04

0.90

1.27

0

0.01

0

0

0.08

0.02

0.05

0.12

0.10

0.02

0.06

0.56

0

0.01

0.04

0.16

0

0.02

0

0.15

0.01

0.02

0.02

0.12

Proline

0

0.01

0.06

0.11

Arginine

0

0.01

0.04

0.07

Galactose Glucuronic Glutamic

acid acid

2-Aminobutyric

acid

Homocysteine Methionine

Relative TBA reactivity of products resulting from damage by 0.9 mM ferrous ions to 2.27mM aminoacids and carbohydrates. Expressed as a mean of 3 separate assays.

RESULTS

The oxidation products derived from unsaturated lipids showed greatest TBA reactivity when buffered to pH 3.5. However, oxidation of a mixture of phospholipids showed maximum TBA reactivity with 25% hydrochloric acid and least TBA reactivity when buffered to pH 3.5 (Table 1). Products resulting from iron-dependent damage to amino acids and carbohydrates were, except from deoxyribose, most TBA reactive in the presence of glacial acetic acid (Table 2). Of the carbohydrates, glycerol and galactose showed no detectable TBA reactivity after incubation with iron. The presence of iron interfered with the methyl indole reagent and meaningful results could not be obtained in these experiments. Release of TBA-reactivity material from DNA in the presence of high ferrous ion concentrations was

negligible. However, when the DNA-binding and metal-chelating glycopeptide bleomycin was added, considerable quantities of TBA-reactive material were released when only traces (0.04mM) of ferrous ion was present (Table 3). Bleomycin in the absence of ferrous ions showing no DNA-damaging activity. MDA is not a stable compound and is usually prepared by the hydrolysis of its tetra-ether derivatives. Complete hydrolysis of 1,1,3,3-tetramethoxypropane (TMP) yields one molecule of MDA and 4 molecules of methyl alcohol. Acid hydrolysis is, however, often accompanied by problems of MDA polymerization (Gutteridge, 1975). Isolation of monomeric MDA can be achieved by treatment of TMP with Dowex resin followed by column chromatography on Sephadex. This MDA should then react directly and rapidly with TBA regardless of the type of acid used in the test. Fraction number 3 eluting from the column fulfills this requireinent (Table 4).

Table 3. Relative TBA reactivity of DNA derived products and 2-hydroxypyrimidine TBA Buffer pH 3.5

DNA

+ Ferrous

ions

DNA + Blecmycin + ferrous ions 0.04 2-Hydroxypyrimidine

reactivity

TCA 20% w/v

HCl 25% v/v

A532

nm

Glacial Acetic acid

1.8 mM

0.01

0.05

0.07

0.08

mM

0.36

0.43

0.48

0.59

5.60

3.18

1.56

5.60

2.5 mM

Relative TBA reactivity of DNA 0.45 mg/ml incubated with iron at high concentration (1.8 mM) in the absence of bleomycin and at low concentration (0.04 mM) in the presence of bleomycin (0.09 mgjml). Expressed as a mean of 3 separate assays.

JOHN M. C. GUTTERIDGE

652 Table 4. Relative

TBA reactivity of MDA in different

TBA reactivity

A532

actd reagents

2-Methyl

nm

reactivity

Buffer pH 3.5

28% w/v

TCA

25% VJV HCl

Glacial

Indole As50

nm

acetic Acid

1,1,3,3-Tetramethoxypropane (TMP)

4 fl

MDA from hydrolysed TN? separation on Sephadex

0.06

0.20

0.20

0.29

0.11

following G-10

Fraction

2

0.36

0.94

0.82

0.86

0.62

Fraction

3

1.16

1.28

1.28

1.28

1.28

Fraction

4

0.70

1.16

1.16

1.15

0.89

Fraction

5

0.07

0.26

0.25

0.25

0.14

Relative TBA reactivity of MDA samples eluted from Sephadex G-10 after hydrolysis of TMP with DOWEX resin, 5 ml of TMP was diluted with an equal volume of water for hydrolysis. After application of 0.2 ml to the column eluted fractions were diluted 1 in 200 for subsequent TBA reactivity. Results are a mean of 2 separate assays.

DISCUSSION

The TBA reaction is frequently used to measure free-radical mediated oxidative changes to polyunsaturated fatty-acid containing lipids as well as other lipid classes such as squalene (Kwon & Olcott, 1966) and the polyenoic antibiotics (Gutteridge & Thomas, 1980). When a selection of polyunsaturated fatty acids were subjected to enzyme-catalysed oxidation and autoxidation both showed maximum TBA reactivity at pH 3.5. Lipid peroxides formed by lipoxidase catalysed oxidations are mostly hydroperoxides, whereas autoxidation tends to produce a more complex mixture of hydro, cyclic and endoperoxides: the latter being important precursors of MDA in the TBA test (Pryor et al., 1976). The time-dependent formation of these intermediates during autoxidation can also influence relative TBA reactivity (Gutteridge, 1978). Autoxidation of phospholipids, both as a powder and in aqueous dispersion, as part of an “organized” liposomal membrane, was detected with TBA most effectively using 25’:;, HCI. Unlike the fatty acids, these showed least TBA reactivity at pH 3.5. Ferrous ions, or ferric ions in the presence of a superoxide-generating system, can bring about the release of TBA-reactive intermediates from several amino acids and carbohydrates (Gutteridge, 1981; Halliwell & Gutteridge, 1981). This damage was shown to be hydroxyl-radical dependent (Halliwell & Gutteridge, 1981) a species known to be formed during high energy irradiation of water. Indeed, several previous reports have indicated that TBA-reactive products and MDA are formed following irradiation of amino acids (Ambe & Tappel, 1961) and carbohydrates (Kapp & Smith, 1970; Morre & MorazzaniPelletier, 1966). Glycerol and galactose did not yield TBA-reactive products under the iron-dependent conditions described here, but upon irradiation galactose has been reported to form TBA-reactive (Morre &

Morazzani-Pelletier, 1966) and glycerol. methylindole-reactive products (Scherz, 1968). Early observations of the TBA test described the reactivity of certain pyrimidines (Kohn & Liversedge, 1944) which was later confirmed in a more detailed study (Shepherd, 1948). Renewed interest has centred around the finding that the DNA-binding antitumour antibiotics, bleomycin and neocarzinostatin both release bases, together with TBA-reactive products from DNA (Kuo & Haidle, 1973; Kappen & Goldberg, 1978). The intermediates responsible for the TBA reactivity mediated by bleomycin have recently been shown to be base-propenals which breakdown during the TBA test to release MDA (Giloni et al., 1981). Preparation of MDA standards by the hydrolysis of compounds such as 1,1,3,3-tetramethoxypropane can give rise to a complex mixture of hydrolysis products (Marnett & Tuttle, 1980) and MDA polymers (Gutteridge, 1975; Gutteridge et al., 1977). A simple technique for isolating monomeric MDA would appear to be the chromatographic separation of products following hydrolysis with Dowex resin. Methyl indole proved to be a useful additional reagent for detecting MDA-forming intermediates, particularly on thin-layer chromatograms. Its use, however, appeared to be limited to reactions in which iron salts were not used for the generation of free radicals. In conclusion, TBA reactivity is essentially a reaction involving the decomposition of intermediate molecules to MDA during the TBA test conditions. In this respect, both the type and strength of acid used is important to achieve maximum liberation of MDA.

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