Free Radical IJiology & Medicine,

Vol.3, pp. 349-354. 1987 Printedin the USA.All rightsreserved.

0891-5849/87 $3.00+.0O 1987Pergamon JournalsLtd.

DETECTION

OF PHOSPHOLIPID

PEROXIDES

IN B I O L O G I C A L

SAMPLES

FREDERIK J. G. M. VAN KUlJK and EDWARD A. DRATZ* Departmentof Chemistry,MontanaState University, Bor.emun,MT 59717, USA Abstract--Peroxidation of membrane lipids has been hypothesized to play a key role in various types of tissue degeneration and pathology. Lipid peroxides are formed when oxygen reacts with an unsaturated fatty acid chain. Virtually all of the unsaturated fatty acids in biological systems are bound by ester linkages in phospholipids or triglycerides. Phospholipid and triglyceride peroxides are primary products of lipid pcroxidation and have rarely been measured. Most of the commonly used methods for detection of lipid peroxidation are based on detection of malondialdehyde or other chemical species that are derived from oxidized fatty acids. This review prese;~ts an overview of recently developed methods aimed at identifying and measuring oxidized phospholipids and triglycerides which are direct evidence of the occurrence of lipid peroxidation in viva. Keywords--Lipid peroxidation, Lipid autoxidation, Phospholipid peroxides, Calorimetric assay, Enzymatic assay, Ultraviolet absorption spectra, High performance liquid chromatography, Gas chromatography-mass spectrometry

INTRODUCTION Formation of oxygen radicals and lipid peroxidation have been suggested as important mechanisms causing cell damage in many types of tissue degeneration, including heart disease, cancer, and aging.~'2 The lipid peroxidation process involves the oxidation of polyunsaturated fatty acids which are largely esterified in membrane phospholipids. It has been hypothesized that the oxidation of membrane lipids causes an increased permeability of cell membranes. This is thought to lead to edema, disturbance in electrolyte balances, and elevation of intracellular calcium which contribute to malfunction of the cell. Malondialdehyde detection by reaction with thiobarbituric acid (TBA) has been the most frequently used method to measure lipid peroxidation in viva for many years. This test is comlicated by the background interference of several other common biological molecules (which react with TBA). x'e Methods such as iodometric titrations, 5.~ enzyme assays, 7 and ultraviolet absorption spectra s have been primarily applied to oxidized model fatty acids or their methyl ester derivatives. However, membrane phospholipids and triglycerides are thought to be the primary sites of the lipid peroxidation process shice they are the principal deposits of unsaturated fatty acids.

This review will summarize methods aimed at detection of phospholipid and triglyceride peroxides. Methods which have previously been used to study oxidation of free fatty acids were modified in order to apply them to measurements of phospholipid hydroperoxides. The new methods were developed with purified phospholipid substrates that were prepared by photooxidation of fatty acids covalently bound to phosphatidyicholine) Synthesis of phospholipid hydroperoxide standards will be described, followed by other useful methods such as calorimetric reactions, enzyme assays, ullraviolet spectra, and high performance liquid chromatography (HPLC). Finally, powerful new gas chromatography-mass spectrometry (GC-MS) methods developed to determine lipid peroxidation are described.
II. SYNTHESIS OF PHOSPHOLIPID PEROXIDE STANDARDS

*To whom reprint requests and correspodence should be addressed.

Commercially available lipoxygenase cannot be used for synthesis of phospholipid hydroperoxides, since this enzyme is not able to use phospholipids as a substr:ite.~ in some model systems, oxidized phospholipids were obtained by autooxidation reaction.Zt Such reactions are slow, and a considerable amount of the desired products may decompose during the incubation time. The unavailability of well-characterized phospholipid peroxide standards has been an obstacle to the development of improved defection methods. 349

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A procedure for synthesizing phospholipid peroxides was developed in our laboratory, based on the photosensitized production of singlet oxygen. 9 Phospholipid solutions in methanol were illuminated for 12 h with rose bengal photosensitizer and air using the beam from the 150 Watt xenon arc in the sample compartment of a spect1:ofluorometer.9 After illumination, the phospholipid hydroperoxides were extracted into dichloromethane and the rose bengal partitioned into the methanolic phase. The phospholipid peroxide standards were characterized in a number of ways to be described. Thc~ standards can be stored for several months in dichloromethane at -20C. 9 The photooxidation products of a simple phospholipid, (16:0)(I 8: 2) phosphatidylcholine were studied in some detail. The extent of oxidation was limited by the amount of oxygen available. 9 Apalysis of the hydroperoxide groups was carried out with a colorimetric assay (described be!ow) which indicated that 20-25% of the double bonds formed hydroperoxides indifferent preparations. It is known that singlet oxygen reacts with linoleate to yield a mixture of conjugated and nonconjugated derivatives, whereas autooxidation yields exclusively conjugated derivatives.9 The characterization of the different isomers from oxidized phosphatidylcholine was only possible with a GC-MS method that allowed identification of the individual products by their specific fragmentation patterns. 9

A standard curve obtained with stock solutions of cumene hydroperoxide showed that the absorbance increased nonlinearly above 20 nmol peroxide/sample, z~ It was also found that the degree of nonlinearity increased with larger total amounts of lipid present. We proposed that some of the iodine produced in the reaction with the peroxides part{tions with the double bonds of the phospholipids. This dependence on lipid concentration is a limitation of the coiorimetric method, which is most accurate at low lipid levels. The enzyme assay described below does not appear to be influenced by lipid levels and is therefore ihore reliable under circumstances where high amounts of lipids are present.

IV. ENZYME ASSAYS

III. COLORIMETRIC DETERMINATIONS

The amount of hydroperoxide in a sample can be quantitated by colorimetric determination of peroxide functional groups. 5.* Asakawa and Matsushita 6 reported a technique which we modified as described in detail elsewhere. 12 Briefly, the phospholipid hydroperoxides were reduced with potassium iodide in the presence of an anhydrous aluminum chloride catalyst, hydrochloric acid was added, and a starch solution yielded a blue complex with the iodine produced. Absorbance was measured at 570 nm, and calibration was performed with cumene hydroperoxide solutions of known concentrations. In order to apply this method to phospholipid hydroperoxides, several modifications of the method from Asakawa and Matsushita were necessary. In their method hexane was added at the initial peroxide reduction step to remove the hydroxy derivatives. When phospholipid peroxides are analyzed, the hexane was omitted since it causes, turbidity that decreases the reliability of the absorbance measurements. Furthermore, all volumes were reduced tenfold to gain a corresponding increase in sensitivity. Additional minor modifications are described elsewhere.12

The activity of glutathione peroxidase (GSH-Px) was measured under conditions whei'e the rate was limited by the amount of peroxide substrate present according to the method Heath and 'rappel7 modified to use pH 7.4, 0.75 mM glutathione, and ! .'-aM EDTA.~S This enzyme converts hydroperoxides to hydroxy derivatives, producing stoichiometric amounts of oxidized glutathione. The oxidized glutathione is converted back to the reduced form by glutathione reductase with the stoichiometric consumption of NADPH. Free fatty acid hydroperoxides or cumene hydroperoxides could be determined directly by this method. However, it was found that phospholipid hydroperoxides are extremely poor substrates for GSHPx. Therefore, an extra incubation step with phospholipase A2 was added, in order to release the fatty acid hydroperoxides which are excellent substrates for GSH-Px. js The essential role of phospholipase As in reduction of phospholipid hydroperoxides by GSH-Px was recently reviewed. ~3 The total change i.n optical density at 340 nm, caused by the consumption of NADPH in the GSH-Px peroxide reduction, allows determination of the number of nmoles of lipid p~oxides. The total amount of phospholipid hydroperoxides detected nzymatically agreed with the values found by colorimetric determination to within 1% under low lipid levels conditions where the colorimetric assay gives accurate results. The enzyme assay can be used efficiently to determine peroxide values of phospholipid peroxides in membranes after phospholipase As incubation with a sensitivity of ! nmole, whereas the coiorimetric assay is applicable for measurement of peroxide values of phospholipid hydroperoxides dissolved in organic solvents with about the same sensitivity. A disadvantage of the enzymatic method is that the incfibation required for A2 action could possibly change the peroxide value under some conditions.

Forum V, ULTRAVIOLETABSORPTIONSPECTRA Spectrophotometry has often been used for determination of conjugated diene products of lipid peroxidation at 234 nm. 3.s These measurements are complicated by the high molar absorption of many organic solvents in the ultraviolet range. Measurements were carried out in spectral grade dichloromethane in cuvettes with a path length of I ram. Spectra of oxidized phospholipids are very similar before and after reduction of the hydroperoxide group. 9 This implies that optical density at 234 nm measures total conjugated dienes without distinguishing between hydroperoxides, hydroxy derivatives, and other decomposition products containing conjugated dienes. In addition, it should be noted that ultraviolet spectra are not sufficient for quantitation of the oxidation products obtained from reactions with singlet oxygen because the nonconjugated products produced by singlet oxygen have essentially no absorbance at 234 nm. This direct spectrophotometric method has been explored to measure the conjugated diene content in tissues. However, antioxidants are required to avoid additional oxidation during extraction of the lipids and manipulation of the samples, jz EDTA or desferal are usually used to inhibit iron promoted decomposition of lipid peroxides, and butylated hydroxy toluene (BHT) is used to inhibit formation of lipid peroxides during such procedures. BHT introduces a problem because the aromatic structure in this compound absorbs strongly in the ultraviolet range. The BHT absorption interferes with the measurement of dienes, which limits the accuracy of this method. So far we have used this method only to show the presence of dienes in phsopholipids that were photooxidized in methanol. In such a model system we did not employ antioxidants and the singlet oxygen reactions and sample preparations were carried out in pure spectral grade solvents.9
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gradient system. The use of an appropriate isocratic HPLC system, presented in more detail elsewhere, allows up to six analyses per hour. 9 When mixtures of oxidized phospholipids from tissues are analyzed, the different oxidized products run as a partially resolved group of peaks on HPLC just before and after the photooxidized (I 6:0)(18: 2) phosphatidylcholine. Therefore, HPLC can be used as an efficient, low resolution cleanup step that does not discriminate between hydroperoxides and hydroxy derivatives. Since the sensitivity of the detection is limited to about 10 nmole of oxidized lipids, we do not use this method for detection of small samples of tissue. Instead, we measure lipid peroxidation by identification of the products with a mass spectrometer, and the different oxidized species are separated by gas chromatography.
Vll. GAS CHROMATOGRAPHY-MASS SPECTROMETRY

A.

New techniques

VI. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Previously, Crawford et aI.14 reported a method for separation of oxidized dilinoleoyl phosphatidylcholine from the nonoxidized precursor phospholipid. The HPLC system employed a uBondapack C~s column with aqueous methanGl as a solvent. Our method was modified to use a CIS-J0 #recolumn with 16% loading, and 100% methanol containing O. 1% ammonium acetate as a mobile phase. 9 If ammonium acetate is used, the hydroperoxides and hydroxy derivatives give much sharper peaks than previously reported. ~.~sHowever, it was not possible to separate the hydroperoxides from the hydroxy derivatives in this HPLC system, as also described by Ursini et al. ~6 for a linear solvent

The final part of this review deals with the methods for direct identification and measurement of lipid peroxides by GC-MS. Virtually all of the GC-MS analysis that have been carried out on oxidized lipids are only appropriate for free fatty acids. Only Hughes et al. ~7applied HPLC and GC-MS methods to measurement of membrane phospholipid oxidation products. However, their method was based on an enzymatic liberation of the oxidized fatty acids from the phospholipids, which required an additional purification step prior to methylation with diazomethane. We recently presented novel GC-MS methods that are based on a one-step transesterification in organic solvents at room temperautre, which liberate the oxidized fatty acids and simultaneously converts them to fatty acid methyl esters9 or fatty acid pentafluorobenzyl (PFB) esters ~s for gas chromatographic separation. The incubations in organic solvents provide high product recovery as shown by studies with standard phospholipid peroxides. Further derivatization was similar to that reported for model studies of fatty acid methyl ester oxidation. B. Transesterification to form methyl esters

Tissue samples or phospholipid hydroperoxides were extracted by the method of Bligh and Dyer t9 modified to use dichioromethane instead of chloroform. The hydroperoxides were reduced with sodium borohydride in methanol, and reextracted into dichioroinethane. The extract was dried over sodium sulfate, evaporated to a small volume (ca. 50 ~ul), 20/tl of transesterification reagent, 0.2 M m-(trifluoromethyl
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Forum sought. Strife and Murphy found that reaction of free fatty acids with pentafiuorobenzylbromide produced pentafluorobenzyl esters which provide an enhanced sensitivity when GC-MS is employed with negative ion chemical ionization (NICI). 26,29This derivatization procedure would require an additional chemical or enzymatic step in our assay to liberate the fatty acid esters from phospholipids. Therefore a one-step procedure was developed which converts phospholipids directly into fatty acid PFB esters. Preparation of pentafluorobenzylester samples for GC-MS is similar to the method described for methyl esters. Transesterification is instead carried out in dichloromethane with 20% (v/v) pentafluorobenzyi alcohol and i % (w/v) sodium pentoxide in a total volume of about I00 ill. ~s Samples are incubated for 30 min at room temperature. It is useful to incubate at 60C when triglycerides are to be transesterified for analysis. We have shown that under these circumstances, a complete conversion of phospholipids or triglycerides to fatty acid PFB esters occurred. This reaction has broad utility and is also applicable for formation of other esters, such as benzyl esters that are useful derivatives for analysis of fatty acids by HPLC. 3 PFB esters are much more stable derivatives for GC-MS analysis than methyl esters. When analyzed as methyl esters, there was a severe loss of oxidation products from 20:4 and 22:6 on the GC column as evidenced by lower recoveries on longer columns. Sample loss did not occur on longer columns when PFB ester derivatives were formed of these compounds. The greater stability of the PFB esters of phospholipid oxidation products allowed a more sensitive detection and NICI provided an additional advantage factor of 20-100 with PFB esters, relative to the most optimum El conditions.ZS A combination of these factors improves the overall sensitivity about a thousandfold to 10 pg of oxidation product for the PFB esters on the gas chromatograph column.~S

phenyl)trimethyl ammonium hydroxide in methanol was added, and the samples were incubated for 30 rain at room temperature. This procedure provides quantitative conversion of oxidized and nonoxidized phos pholipids or triglycerides to fatty acid methyl esters) .2 Dry pyridine and BSTFA were added to form TMS derivatives of the alcohol functions. The technique described above was first applied to photooxidized (16:0)(18:2) phosphatidylcholine.9 Equal amounts of the conjugated and nonconjugated linoleate peroxides were found after reaction of the phospholipid with singlet oxygen. This ratio of conjugated and nonconjugated products is in agreement with Thomas and Pryor2~ and Terao and Matsushita. 22 in contrast, Frankel et al. 23and Clements et al. 24have reported that 66% conjugated and 33% nonconjugated products when linoleate is photooxidized. The possible causes of this disagreement were discussed previously, and additional experi.ments are required to fully resolve this point. 9 The GC-MS method described above uses electron ionization and has a sensitivity of about 10 ng of oxidation products on the column, 9 We have shown that this sensitivity is sufficient to detect and study the lipid peroxide content of adipose triglycerides in vitamin E and selenium deficient rats. 2~ Large amounts of oxidation.products were found in the adipose of antioxidant deficient animaisl whereas negligible amounts were found in the supplemented animals it was also shown that only conjugated linoleate peroxides were present, which suggests that autooxidative processes have taken place in these animals. In addition, practically no oxidized oleate was found, even though oleate was the predominant fatty acid present. This potentially important finding has been discussed in some detail elsewhere.'~ It should be emphasized that the GC-MS method as described is semiquantitative. For quantitation, isotopically labeled compounds can be used for internal standards. For example, oxygen-18 substitution at the fatty acid carboxyl oxygen was used by Strife and Murphy 26 and Leis et al.2~ for quantitative "analysis of lipoxygenase metabolites of arachidonic acid. Boeyroans et al. 2s used octadeuterated arachidonic acid, and we investigated the GC-MS fragmentation patterns of photooxidized vinyl perdeuterated docosahc~aenoic acid. 9

D. Detection of aldehydes as pentafluorobenzyl oximes

A rapid derivatization technique was employed for analysis of aldehydic products of lipid peroxidation, which introduces the PFB molecule into these compounds for high sensitivity analysis.3! 4-Hydroxyalkenals have been reported by Esterbauer ~2to be very biologiC:ally active molecules. 4-Hydroxyalkenals were converted to PFB oxime derivatives and yielded essentially the same sensitivity as the PFB esters when detected by GC-MS under NICI conditions. Furthermore, it was possible to synthesize a suitable internal standard for 4-hydroxynonenal that carries two deu-

C. Transesterification to form pentafluorobenzyl esters.,

Since more sensitivity was desired to analyze trace amounts of lipid peroxidation products in small samples, a more sensitive derivatization approach was

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terium labels. Results on the quantitative analysis of 4-hydroxyalkenals in various tissues will be published separately.
Viii. . CONCLUDING F,EMARKS

The GC-MS assays presented provide powerful alternatives to classical methods for measurement of lipid peroxidation in vivo. The new methods described are not only more specific, but also give information on the type of oxidation reactions that have taken place. A major thrust of the methodology is based on detection ofpho:;pholipid peroxides, which are formed first when radicals and oxygen attack membrane phospholipids. Alternatively, the methodology can be used to measure peroxidized triglycerides that tend to build up in adipose stores. 2s These approaches are complemented with a GC-MS method to determine major aldehydic secondary oxidation products that is described briefly. Combined measurement of phospholipid peroxides and 4-hydroxyalkenals by GC-MS may provide information about turnover of oxidized phospholipids in vivo. The high sensitivity of GC-MS techniques appears to provide routes to study physiological levels of lipid peroxidation products in small biological samples.
Acknowledgements--This work was supported by grants from The Netherlands Organization for the Advancement of Pure Research (ZWO) (to FJGMvK) and the National Institutes of Health (to EAD).
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Forum E. A. Occurrence of 4.hydroxyalkcnals in rat tissues determined as pentafluorobenzyl oxime derivatives by gas chromatographymass spectrometry. Biochem. 8lophys. Res. Comm. 139: 144149; 1986. 32. Estcrbauer, H. Aldchydic products of lipJdperoxidation. In: McBrien, D. H. C.; Slater, T. E, editors. Free radicals, lipid peroxidation and cancer, Now York: Academic Press; 1982: 101-128.