ANALYTICAL BIOCHEMISTRY 99, 249-253 (1979)

A Spectrophotometric Method for the Determination of Lipid Hydroperoxides

M A R K H I C K S A N D J A N U S Z M . G E B I C K I

School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2113, Australia

Received December 27, 1978

A modification of the iodine technique for peroxide determinat ion is described, in which the react ion is carried out in deaerated methanol :acet ic acid solution in a spec t rophotometer cuvet te . Measu remen t s of the triiodide ions released are carried out either at 290 or 360 nm. In this solvent , the molar ext inct ion coefficients are ~290 = 4.41 × 104 and Ea00 = 2.80 x 104 liters mo1-1 cm - t . Calibration with s tandard peroxide solutions showed that 1 mol of I~ is p roduced for every peroxide group added. In its present form, the method can be used to measu re 1 nEq of lipid hydroperoxide .

Formation of lipid hydroperoxides is known to be associated with many deleter- ious changes in biological materials, ranging from spoilage of food to accumulation of harmful pigments in aging organisms (1-3). The exact role of the peroxides in such diverse processes is generally unknown, because of the lack of suitable methods for their detection. Most of the presently used assays are either nonstoichiometric, or not specific for the hydroperoxide group, or require relatively large quantities of oxidized lipids (4-6).

We have reinvestigated a promising variation (7) of the classical technique based on the liberation of iodine by per- oxides (8) with the purpose of increasing its sensitivity and suitability for routine use. The method developed is reasonably speci- fic for the hydroperoxide group, stoichio- metric, sensitive, and uses commonly available reagents and apparatus. Its prin- cipal disadvantage is sensitivity to oxygen, which can however be excluded by fairly simple means.

MATERIALS AND METHODS

Reagents. Analytical grade reagents and solvents were used throughout. All aqueous

solutions were made up in triply distilled water. Potassium iodide was recrystallized twice and iodine was resublimed before preparation of stock solutions.

Solutions of hydrogen peroxide and iodine were standardized with 0.0994 M thiosuifate (9). Lipoxygenase (EC 1.13.11.12) was obtained from Sigma. A crude total lipid extract was prepared from beef liver by the Bligh and Dyer method (10) and stored under liquid nitrogen.

Linoleic acid hydroperoxide. Oxidized linoleic acid was prepared by two methods. In the first the acid was dispersed as stable membranous vesicles (11) in Tris buffer and heated at 60°C for 2 h. Thin layer chromatography (12) confirmed that only the parent acid and its hydroperoxide were present. In the second method, oxidation was catalyzed by a short exposure to lipoxygenase and the pure hydroperoxides isolated by the method of Gardner (13). Concentration of the hydroperoxide was measured in methanol at 234 nm using the molar extinction coefficient of 2.8 x 104 liters mo1-1 cm -i (14).

Peroxide assay. The solvents used in the assay were 1:1 methanol:acetic acid and a saturated solution of KI in methanol. These

249 0003-2697/79/160249-05502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

250 HICKS AND GEBICKI

were prepared on the day of use. High purity nitrogen was passed through meth- anol and the methanol/acetic acid for 20 rain to ensure removal of oxygen. About 20% (w/v) of KI was then added to the methanol and the solution was stirred for a further 20 min under nitrogen to ensure saturation. We have not found it necessary to remove the last traces of oxygen from the nitrogen stream but, for slow-reacting per- oxides, this can be effectively done by passing the gas through a column filled with active manganese (II) oxide (15). Both the iodide and acetic acid solutions were saturated with disodium EDTA.

Peroxide assays were carried out in standard 1-cm quartz spectrophotometer cuvettes fitted with a 7-ram deep Teflon plug. The plug had a capil|ary hole for addition of peroxide solutions, which was normally sealed with a piece of wire covered with Teflon. A 1.8-ml portion of the de- aerated methanol:acetic solution added to the cuvetted was bubbled for 5 -7 min with a presaturated nitrogen. Similarly deoxy-

/ / i , I ~ l i ! I i ' i

!

: : i 1

.6 : i : [ 1 i i = i I i ! _ :.. ~ .~_ . i

1 ~ [ ,7-- , 1 / 'q lN~ i ! P i ., , I v !

~ . i , i ' i i ~ i i..L_i__i__ t I=: l - i ¢:,:~ -I f i!

ill

260 300 3,50 400

W A V E L E N G T H ( n m )

FIG. 1. (A) Spectrum of the I~ ion in methanol: acetic acid (3:1). (B) spectrum of holmium glass.

genated KI solution (1.9 ml) was then added under nitrogen and the plug placed so that there was no head space over the liquid. Loss of a small part of the solution at this stage is not important.

Absorbance of the solution was recorded at 290 or at 360 nm for about 10 min. If it increased by more than 0.005 unit, the solution was discarded. Otherwise between 5 and 100/zl of solution containing between 0.2 and 100 nmol of peroxide was injected through the capillary, which was resealed. After mixing by repeated inversion, absorb- ance of the solution was again recorded until any further change was small and linear with time.

All operations were carried out with the lights directly over the working area switched off.

RESULTS The absorption spectrum of the tri-

iodide ion in the assay solvent (3:1 meth- anol:acetic acid) is shown in Fig. 1. The lower spectrum is that of a standard hol- mium glass, used for wavelength calibra- tion. The peaks at 290 and 360 nm are displaced only slightly from the 287.5- and 353.0-nm maxima reported for aqueous tri- iodide (16). The extinction coefficients at the peak wavelengths were determined with solutions containing accurately meas- ured amounts of iodine (Fig. 2). The ratio I-/Iz was always greater than 3 x 104 to ensure conversion of Iz to Iy. Heaton and Uri (7) have shown that a ratio of 5 is suf- ficient for practically complete formation of I~ in the equilibrium I - + 12 ~-I~. The molar extinction coefficients for Iy were calculated by least squares from readings shown in Fig. 2. At 290 nm, the value was (4.41 + 0.14) × 104 liters mo1-1 cm -1 and at 360 nm it was (2.80 +_ 0.13) x 104 liters mo1-1 cm -1. The limits are the calculated confidence intervals of the slopes (17). The 360-nm extinction coefficient decreased linearly at 0.14% per degree as the tempera- ture rose from 10.5 to 31.6°C.

S P E C T R O P H O T O M E T R I C L I P I D H Y D R O P E R O X I D E A S S A Y 251

.6

z < r n

r~ _0 .'~ - L

E

o

~ 90

o ~ ~b i~ IODINE (pmole.litre-')

FIG. 2. A b s o r b a n c e o f I~ at 290 (O) and at 360 (©) n m as f unc t i on o f the c o n c e n t r a t i o n o f iodine add ed to the iodide a s s a y so lu t ion .

The kinetics of the reaction between 1- and two different peroxides are shown in Fig. 3. Hydrogen peroxide (curve A) reacted within 4 min, while linoleic acid hydroperoxide (curve B) required about 25 min. The initial brief recording established the blank level and stability. Any increase could be attributed to presence of oxygen. This was also the reason for the slow steady increase in absorbance after completion of the reaction, which can be seen in both curves. For accurate measurements, this steady rate is extrapolated back to the time of addition of the peroxide to give the true net absorbance change.

The stoichiometry of the relation between the amounts of peroxide added and I~ pro- duced was established by reacting accu- rately measured quantities of H2Oz with iodide and calculating the amounts of I~ produced from absorbance at 360 nm (Fig, 4). The slope of this line is 0.99, indicating a 1:1 stoichiometry, as suggested by the scheme

H202 + 2I- + 2H + --~ I2 + 2H20

I2 + I - ~ I3-

w i t h the equilibrium displaced to I~ by the excess I-. In similar experiments, known quantities of linoleic acid hydroperoxide were introduced into the iodide solution and the amounts of I~ produced were determined.

In Fig. 5 the upper line shows that, for this peroxide, Beer's law is obeyed within the useful range of the spectrophotometer.

In the lower curve, the I~ absorbance produced by reduction of standard hydo- gen peroxide and linoleic acid hydroperoxide are plotted together. This result confirms the 1:1 stoichiometry for the reaction between iodide and the fatty acid hydroperoxide, the only uncertainty lying in the molar extinc- tion of the latter (14).

To test the response of the assays to conditions commonly used in lipid analysis, a 100-/zl sample of crude liver lipids (50 mg/ml) in 2:1 methanol:chloroform was injected into the assay solution. This volume is the maximum that can be added to the cuvette without significant change in the blank reading. The 5 mg of lipids added remained in solution in the acetic acid: methanol solvent. In other tests, the addi- tion of up to 3% water or chloroform did not alter the molar extinction of the Iy ion or interfere with the assay. In practice, much smaller amounts of lipids, water, or chloro-

E

i i i 0 Z

i

i

! ! i i i ! i i i ~ ~ i ! : ' : ! i i ~}

t I ; F ~i i i

i ' ! i I i / i , ; ! i I : F F ! ~ !

0 lO TIME {min)

FIG. 3. R e c o r d e r t r aces o f the f o r m a t i o n o f I~ m e a s u r e d at 360 nm. C u r v e A, H202; c u r v e B, l inoleic acid h y d r o p e r o x i d e , added 6 m i n af ter s ta r t o f record ing .

252 HICKS AND GEBICKI

25-

20-

-~ 15-

uJ a ~ lO

i

I--- 5

0 10 15 20 2'5

HYDROGEN PEROXIDE (pmole.lilre-')

FIG. 4. Stoichiometry of the reaction between iodide and HzO2 under standard assay conditions.

form are likely to be introduced into the test solution.

The effects of a number of substances which might alter the course or extent of the reaction were also investigated. Butylated hydroxytoluene, riboflavin, nitroblue tetra- zolium, and bilirubin, all at 10/xmol/1 liter did not interfere when hydrogen peroxide was used as the oxidant. Tetranitromethane liberated iodine directly.

DISCUSSION

In this study we have concentrated on developing or improving an analytical method which would be especially suitable for the measurement of small amounts of fatty acid hydroperoxides in biological materials. The main qualities sought were high sensitivity, specificity for the hydro- peroxide group, and an exact stoichiometry.

Of the many chemical assay techniques based on the reduction of the fatty acid hydroperoxides, only the iodine method possessed the potential to fulfill our require- ments. However, the commonly used procedures suffer from two disadvantages: low sensitivity, particularly when the liber- ated iodine is estimated by thiosulfate titration, and interference from oxygen. The titration has been eliminated in some recent work by colorimetric estimation of

the I2. In one modification (18), the color of iodine-starch complex was measured at 560 nm. While the potential sensitivity is good, the procedure is not simple and the extinction varies with the source of starch (19). Takagi et al. (20) measured the iodine at 358 or at 410 nm and complexed the remaining I- with Cd to achieve protection from oxygen. This test requires a consider- able quantity (at least 20 mg) of lipid.

A different difficulty is inherent in an older spectrophotometric measurement of I~ ion; Heaton and Uri (7) used extinction coefficients measured between 380 and 560 nm, which lie on the steep shoulder of the 360 nm peak (Fig. 1). An inaccuracy in wavelength calibration of only 2 nm intro- duces a 10% error in peroxide estimate at 380 nm.

All these methods need a blank to allow for the release of any Iz in the absence of hydroperoxide. In contrast, the use of continuous recording in the method de- scribed in this paper allows for blank correction on the actual solution in which the reaction is carried out. This makes separate blanks, with their inherent vari- ability, unnecessary. Apart from oxygen, the major interfering substances here are

ua / / ~ A 290 0 / 1.0 n,-

O o

~ - 5 a o_

i

0 o lb ~

PEROXIDE (pmoie l i lre-')

FIG. 5. The relationship between the amounts of H20~ or linoleic acid hydroperoxide reacted and IE produced. Top line (A), linoleic hydroperoxide at 290 nm. Lower line, (Q) linoleic hydroperoxide and (O) H202, at 360 nm.

SPECTROPHOTOMETRIC LIPID HYDROPEROXIDE ASSAY 253

precisely the same as in the standard iodine methods; transition metals, mercap- tans, and solvents such as acetone.

Our technique improves the sensitivity and reproducibility of the spectrophoto- metric determinations by using the measure- ment of I~ at its absorbance maxima. The molar extinction coefficients of 4.41 x 104 and 2.80 x 104 liters mol -a cm -a at 290 and 360 nm, respectively, agree well with values measured in other solvent systems (16). While this study was in progress, an abstract was published which also described the use of iodide in methanolic solutions, but the reported extinctions were much lower; 3.21 × 104 at 290 and 1.83 × 104 at 360 nm (21). Until a full account of this work appears, it is not possible to determine the reason for disagreement with our results.

The limit to the detection by this method is about 0.2-1 nmol of hydroperoxide in the 3.7 ml contained in cuvette, depending on the spectrophotometer sensitivity. This is 20-fold lower than the Heaton and Uri (7) method and about 1000-fold better than the best thiosulfate titrations. It can be calcu- lated from standard data (22,23) that our technique can measure the peroxides in 3/xl of blood, if 10% of the erythrocyte unsat- urated fatty acids in the sample are oxidized.

The demonstration of a 1:1 stoichiometry for the reaction of the fatty acid hydroper- oxide group means that the mechanism of the iodide-peroxide reaction is straightfor- ward and not subject to interference by substances reacting with possible inter- mediates. This is confirmed by the failure of free radical and singlet oxygen scavengers to affect the course of the reaction.

Results obtained with liver lipids show that the method is directly applicable to biological materials. Further studies will be needed to discover whether tissue peroxides can be estimated in some cases without the need for extraction and separation of the lipids and whether the technique can be applied in measurement of nonlipid per- oxides.

ACKNOWLEDGMENTS

We thank the Australian Commonwealth for a post- graduate scholarship held by one of us (M.H.) and the Macquarie University Research Grant for provision of funds.

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