[38]
CHEMILUMINESCENCE
ASSAY FOR LIPID HYDROPEROXIDES
371
[38] A s s a y o f L i p i d H y d r o p e r o x i d e s U s i n g H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y with I s o l u m i n a l Chemiluminescence Detection B y YORIHIRO YAMAMOTO, B A L Z FREI, a n d BRUCE N . AMES
Introduction Lipid peroxidation has been implicated in several diseases such as atherosclerosis, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, postischemic reoxygenation injury, and aging. Lipid peroxidation proceeds by a free radical chain mechanism and yields lipid hydroperoxides as major initial reaction products. Therefore, it would be desirable to detect and identify lipid hydroperoxides in biological tissues. The thiobarbituric acid assay, which measures an aldehydic breakdown product (malondialdehyde) of lipid hydroperoxides, has served as the most commonly used method for measuring lipid peroxidation. However, the application of the assay to biological samples is very problematic, since a variety of biological compounds such as deoxyribose, many other carbohydrates, sialic acid, prostaglandins, and thromboxanes also react with thiobarbituric acid and interfere with the assay. 1 There are methods for the detection of lipid hydroperoxides themselves2,3 rather than their breakdown products, but these methods, too, cannot be applied unreservedly to biological samples, because of interference by antioxidants present in biological tissues and/or lack of sensitivity. Warso and Lands 4 have developed a method for the detection of lipid hydroperoxides based on activation of prostaglandin H synthase by hydroperoxides. However, this method, like most other methods for measuring lipid peroxidation, is unable to distinguish between different classes of lipid hydroperoxides. Furthermore, the specificity of this assay remains to be established. The luminol chemiluminescence assay for the detection of hydrogen peroxide is well known for its picomole-level sensitivity. 5-7 Microperoxl j. M. C. Gutterdige, Free Radical Res. Commun. 1, 173 (1986). T. F. Slater, this series, Vol. 105, p. 283, and references therein. 3 R. Cathcart, E. Schwiers, and B. N. Ames, Anal. Biochem. 134, 111 (1983). 4 M. A. Warso and W. E. M. Lands, J. Clin. Invest. 75, 667 (1985). 5 D. T. Bostick and D. M. Hercules, Anal. Chem. 47, 447 (1975). 6 W. R. Seitz, this series, Vol. 57, p. 445. 7 B. Olsson, Anal. Chim. Acta 136, 113 (1982).
METHODS IN ENZYMOLOGY, VOL. 186
. Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
372
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[38]
idase (a heme fragment of cytochrome c) has been shown to be the most effective catalyst for this assay. 7,8 We have adapted the luminol-microperoxidase assay to organic hydroperoxides and obtained a sensitivity similar to that for hydrogen peroxide? Since isoluminol gave better results than luminol in terms of signal-to-noise ratio, we used isoluminol as the substrate in our assay. The reaction sequence leading to the emission of light from isoluminol in the presence of lipid hydroperoxides (LOOH) and microperoxidase is assumed to be as follows: LOOH
microperoxidaseLO"
LO" + isoluminol (QH-) ---->LOH + semiquinone radical (Q ~) Q ~ + O2 ~ quinone (Q) + 02 ~ Q = + 02 = --~ isoluminol endoperoxide --~ light (kmax 430rim)
(1) (2) (3) (4)
Reactions (1) 1° and (2) are not well established, but it is known that production of the semiquinone radical is essential for the emission of light from isoluminol. Reactions (3) and (4) have been well studied. 11A2 Analysis of biological samples with the isoluminol assay can be hampered by antioxidants, which quench the production of chemiluminescence by scavenging intermediary radicals in the above reaction sequence [Eqs. (1)-(4)]. We have eliminated this problem by separating biological antioxidants from lipid hydroperoxides by high-performance liquid chromatography (HPLC) and using the isoluminol assay as an on-line postcolumn chemiluminescence detection system. 9 HPLC-Isoluminol
Chemiluminescence
Assay
Equipment is arranged as outlined in Fig. I. Antioxidants and hydroperoxides in the sample are separated by HPLC, using one of the chromatographic conditions described below, and then mixed with a reaction solution containing isoluminol (6-amino-2,3-dihydro-l,4-phthalazinedione; Sigma, St. Louis, MO) and microperoxidase (Type MP-1 l, Sigma) in a special mixer (Model 2500-0322, Kratos, Westwood, N J ) . The reactions leading to the emission of light take place in a mixing coil made from a H. R. Schroeder, R. C. Boguslaski, R. J. Carrico, and R. T. Buckler, this series, Vol. 57, p. 424. 9 y . Yamamoto, M. H. Brodsky, J. C. Baker, and B. N. Ames, Anal. Biochem. 160, 7 (1987). lo T. A. Dix and L. J. Marnett, J. Biol. Chem. 260, 5351 (1986). 11 j. Lind, G. Mer6nyi, and T. E. Eriksen, J. Am. Chem. Soc. 105, 7655 (1983). 12 G. Mer~nyi, J. Lind, and T. E. Eriksen, J. Phys. Chem. 88, 2320 (1984).
[38l
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
373
sample HPLC mobile phase
~
pump A
=
injector column
UV detector reaction solution (isoluminol, microperoxidase)
---
pump B
~ mixer mixing coil
fluorometer FIG. 1. Schematic diagram of the HPLC-isoluminol chemiluminescence assay.
a piece of HPLC tubing. The emitted light is measured in a fluorometer (Model FS 970 equipped with a 10-~1 flow cell; Schoeffel Instrument Corporation, Westwood, NJ) used as a photon detector with the excitation source turned off and in the absence of an emission filter. The time constant and the supplying voltage for the fluorometer are set at 4 sec and 1160 V (58% of full range), respectively. The flow rates of pump A and B (Fig. 1) are 1.0 and 1.5 ml/min, respectively. The length of the mixing coil is 45 cm (inner volume -92/zl). The reaction solution is prepared as follows: 100 mM aqueous borate buffer (38.14 g of sodium tetraborate decahydrate per liter) is prepared, and the pH is adjusted to 10 with sodium hydroxide. Isoluminol (177.2 mg, final concentration 1 mM) is dissolved in 300 ml of methanol and 700 ml of the above borate buffer, and then 25 mg of microperoxidase is added. For the analysis of the hexane phase of plasma (see below), the reaction solution is composed of isoluminol and microperoxidase in 700 ml of methanol and 300 ml of borate buffer instead. All solutions as well as the HPLC mobile phases are kept in brown bottles in order to minimize light-induced generation of chemiluminescence-producingmaterial. Several useful HPLC conditions for the separation of various classes of lipid hydroperoxides and biological antioxidants are summarized in Table I. For example, condition III is useful for the separation of neutral lipid hydroperoxides such as hydroperoxides of cholesterol, triglycerides, and cholesteryl esters. Condition V can be used for the separation and quantitation of hydrogen peroxide, free fatty acid hydroperoxides, and phospholipid hydroperoxides. Under all chromatographic conditions the coefficients of variation (standard deviation/mean value) for the chemi-
374
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[38]
TABLE I H P L C CONDITIONS AND RETENTION TIMES OF HYDROPEROXlDES AND ANTIOXIDANTSa HPLC condition: Guard column/colum#: Mobile phase:
I LC18 d
II LC18DB c e
III LC18DB c f
IV LCSi g
V LCNH2 h
Retention times as monitored by chemiluminescence (min)
Compound i Hydrogen peroxide tert-Butyl hydroperoxide Linoleic acid hydroperoxide Cholesterol hydroperoxide PCI8:2-OOH PEI8:2-OOH Trilinolein hydroperoxide Chl8:2-OOH
3.0 3.4 2.5 6.2 -----
a-Tocopherol ~/-Tocopherol Ascorbate Urate
10.4 9.2 2.1 1.9
3.1 3.0 -3.9 3.2 3.1 2.2 3.6 2.2 3.1 1.5 4.8 7.1 4.0 2.3 3.5 6.3 -14.2 7.4 4.6 -2.6 5.1 -5.9 2.1 ND -8.0 2.1 ND Retention times as monitored by U V (rain) 12.9 11.2 2.0 1.8
4.8 4.5 2.3 2.0
2.0 2.0 1.4 1.2
3.3 3.3 9.9 8.7
a Flow rate = 1.0 ml/min; - - , not eluted within 30 min; ND, not determined. Adapted from Yamamoto et al. 9 b The 5-/~m analytical columns (25 cm × 4.6 mm i.d., except LCSi: 3/zm, 15 cm × 4.6 mm i.d.) were purchased from Supelco (Bellefonte, PA), with the corresponding guard columns (5 p.m, 2 c m x 4.6 mm i.d.). c DB, Deactivated for basic compounds. d Methanol. e Methanol containing 0.01% triethylamine. Methanol-tert-butanol (50 : 50 by volume). g Acetonitrile-tert-butanoi-water (55 : 30 : 15 by volume). h Methanol-40 m M sodium phosphate, monobasic (95 : 5 by volume). The combination of LCNH2 and LCSi (both 5 p.m, 25 cm x 4.6 mm i.d.) in series using methanol-40 m M sodium phosphate, monobasic (90 : 10 by volume) gave similar and more reproducible retention times. PC18:2-OOH, Dilinoleylphosphatidylcholine hydroperoxide; PE18:2-OOH, dilinoleylphosphatidylethanolamine hydroperoxide; ChI8:2-OOH, cholesteryl linoleate hydroperoxide.
luminescence peak areas of the hydroperoxides listed in Table I are less than 3% (n -> 3). Table II summarizes the sensitivity of the assay to various hydroperoxides relative to the sensitivity to linoleic acid hydroperoxide under chromatographic conditions II-V. The assay is less sensitive to hydrogen peroxide and tert-butyl hydroperoxide than to other hydroperoxides.
[38]
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
375
TABLE II SENSITIVITY OF HPLC-IsOLUMINOL CHEMILUMINESCENCE ASSAY TO VARIOUS HYDROPEROXIDES RELATIVE TO SENSITIVITY TO LINOLEIC ACID HYDROPEROXIDEa
HPLC conditionc Compoundb
II
III
IV
V
Hydrogen peroxide tert-Butyl hydroperoxide Linoleic acid hydroperoxide Cholesterol hydroperoxide PC18"2-OOH PE18:2-OOH Trilinolein hydroperoxide ChI8:2-OOH
0.57 0.57 1.00 0.84 1.44 1.04 ---
0.39 0.39 1.00 0.52 --1.18 0.75
-0.80 1.00 0.91 1.13 1.16 1.04 1.54
0.53 0.53 1.00 0.82 0.92 0.83 --
Sensitivity is defined as the ratio of peak area to amount of hydroperoxide. From Yamamoto et al.9 b For abbreviations see footnote i of Table I. c See Table I. The c h e m i l u m i n e s c e n c e r e s p o n s e relative to the amount o f linoleic acid h y d r o p e r o x i d e is linear o v e r a range of 1 to 1000 pmol. Other hydroperoxides such as hydrogen peroxide and tert-butyl hydroperoxide also give good linear relationships. The detection limit of the assay for linoleic acid h y d r o p e r o x i d e is a b o u t 1 pmol. Application of Assay to Analysis of H u m a n Blood Plasma In h u m a n blood plasma, the m a j o r lipids are unesterified fatty acids, phospholipids, cholesterol, triglycerides, and cholesteryl esters. ~3As evident f r o m Table I, it is v e r y difficult to separate all hydroperoxides of these lipids f r o m the p l a s m a antioxidants ascorbate, urate, and a - t o c o p h erol in one c h r o m a t o g r a p h i c step. Therefore, plasma is separated into two phases b y extraction with methanol and hexane prior to analysis with the H P L C - i s o l u m i n o l chemiluminescence assay. A n a l y t i c a l P r o c e d u r e s . F o r extraction, 0.5 ml of heparinized plasma is added to 2 ml o f H P L C - g r a d e methanol and mixed vigorously. This is followed b y the addition o f I0 ml o f hexane ( H P L C grade; Aldrich, Milwaukee, WI). (Before use the hexane should be washed with water in order to r e m o v e trace a m o u n t s o f hydroperoxides. A b o u t I volume of H P L C - g r a d e w a t e r is added to I0 volumes of hexane in a brown bottle ~3B. Frei, R. Stocker, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 85, 9748 (1988).
376
[38]
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE TABLE III RECOVERY OF LIPID HYDROPEROXIDES ADDED TO PLASMA PRIOR TO EXTRACTION WITH METHANOL AND HEXANEa Recovery (%) Compound b
Aqueous phase
Hexane phase
Linoleic acid hydroperoxide PCI8:2-OOH Cholesterol hydroperoxide ¢ Trilinolein hydroperoxide Chl8:2-OOH
72 58 10 0 0
0 0 14 56 56
Lipid hydroperoxides were added at 10 tzM 1.0 min prior to extraction. Adapted from Frei et al. I~ b For abbreviations see footnote i of Table I. c Cholesterol hydroperoxide is degraded rapidly in plasma lB. Frei, R. Stocker, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 85, 9748 (1988)].
and stirred vigorously overnight. The water is allowed to settle for several hours before the upper-phase hexane is used for extraction.) The methanol-hexane extract of plasma is vortexed for 1 min and then spun at 1000 g for I0 min at 4°. Nine milliliters of the upper, hexane phase is collected and dried under vacuum in a rotary evaporator. Dried hexane extracts are dissolved in 0.45 ml of methanol-tert-butanol (50 : 50 by volume). One hundred microliters of this solution (corresponding to 100/zl of plasma) is subjected to HPLC using condition III (see Table I). Under this chromatographic condition hydroperoxides of cholesterol, triglycerides, and cholesteryl esters, and the antioxidants ~- and 3,-tocopherol, all of which are extracted into the hexane phase of plasma, are well separated, z4,~5 The aqueous methanol phase of extracted plasma is passed through a 0.2-/zm filter (Type ARCO LC 13, Gelman Science, Ann Arbor, MI), and 20/zl of this solution (corresponding to 4 ~1 of plasma) is subjected to HPLC using condition V (see Table I). This chromatographic condition is used since it allows good separation of hydrogen peroxide, free fatty acid hydroperoxides, phospholipid hydroperoxides, ascorbate, and urate, all of which are extracted into the aqueous methanol phase of plasma. ~4,~5 The recoveries of lipid hydroperoxides added to plasma at a concentration of 10/zM prior to biphasic extraction with methanol and hexane and analysis as described above are given in Table III. The detection limits for lipid hydroperoxides in plasma extracting into the aqueous t4 y . Yamamoto and B. N. Ames, Free Radicals Biol. Med. 3, 359 (1987). t5 B. Frei, Y. Yamamoto, D. Niclas, and B. N. Ames, Anal. Biochem. 175, 120 (1988).
[38]
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
377
methanol phase and the hexane phase are about 0.03 and 0.01 /~M, respectively. 15 In order to assess the purity of all reagents and materials used, for each set o f analyses a blank (0.5 ml o f water) should be extracted and then carried through the entire analytical procedure as described above. After each set o f H P L C runs, a calibration is performed under the respective chromatographic condition, using linoleic acid hydroperoxide as a standard. This calibration is necessary since the sensitivity of the assay varies slightly, depending mainly on the freshness of the microperoxidaseisoluminol solution. Figures 2A and 2B show typical chemiluminescence chromatograms
A hexane extract
ubiquinol-10
a- and y-tocopherols B aqueous methanol extract
I H202 urate ascorbate t
0
r
r
5
,
r
10
i
15
Time (rain)
FZG.2. Typical chemiluminescence chromatograms of the hexane extract (A) and aqueous methanol extract (B) of human blood plasma from a healthy subject. The evaporated hexane phase was dissolved in methanol-tert-butanol (50:50 by volume) and analyzed under chromatographic condition III (Table I). The aqueous methanol phase was analyzed on an LCNH2 column using condition V (Table I). Chemiluminescence was recorded at 0.02/~A.
378
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[38]
of the hexane phase and the aqueous methanol phase, respectively, of extracted human blood plasma from a healthy subject. 14,15 No chemiluminescence-producing compounds are observed that comigrate with spiked standards of hydroperoxides of linoleic acid, dilinoleylphosphatidylcholine, cholesterol, trilinolein, or cholesteryl linoleate (see Table I). However, the hexane extract (Fig. 2A) contains two chemiluminescenceproducing compounds, one of which has been identified as ubiquinol-10 (see also below). In the methanol extract (Fig. 2B) a chemiluminescenceproducing compound comigrating with hydrogen peroxide is observed (see below). Both extracts contain plasma antioxidants which produce negative peaks in the chemiluminescence chromatograms owing to quenching of background chemiluminescence. Figure 3 illustrates the successful application of the assay to the analysis of human blood plasma after it has been incubated with activated polymorphonuclear leukocytes (PMNs). Besides a decrease in the plasma level of ubiquinol-10, such incubation led to formation of detectable amounts of triglyceride hydroperoxides, cholesteryl ester hydroperoxides (Fig. 3), and phospholipid hydroperoxides.13
/I I t
I
¢3
I
I--- .o"
3: O O I LU O
il _f 0
5
10
5
Time (min) Fro, 3. Chemiluminescencechromatogramof the hexanephaseof plasmaincubatedwith activated PMNs with (dashed line) and without (solid line) subsequent treatment with sodium borohydride. Chemiluminescence was recorded at 0.02 cA. TG-OOHs, Triglyceride hydroperoxides; CE-OOHs, cholesteryl ester hydroperoxides.
[38]
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
379
Interference Hydrogen peroxide can be produced by autoxidation of ascorbate during workup and analysis. 15Thus, the assay is not suitable for the quantitation of hydrogen peroxide in biological samples containing ascorbate. The assay is not sensitive to endoperoxides, hydroxyalkenals, alkenals, quinones, aldehydes, and alcohols. 15 However, ubiquinols produce chemiluminescence in the assay (see Fig. 2A). ~5 Using chromatographic condition III, the assay yields a chemiluminescence signal for ubiquinol-10 that is 47% of the signal for an equimolar amount of linoleic acid hydroperoxide; ubiquinols-6,-9, and -I0 give chemiluminescence peaks with retention times of 4.2, 6.1, and 7.2 rain, respectively. ~5Naturally occurring ubiquinols-7 and -8, too, produce chemiluminescence in the assay, as do other hydroquinones. Human plasma contains about 0.7 /zM ubiquinol-10 (Fig. 2A). 15 We also occasionally observed trace amounts of ubiquinol-9 (see also Ref. 16). Chemiluminescence signals produced by ubiquinols and other hydroquinones cannot be eliminated by treatment with reducing agents, in contrast to the chemiluminescence signals produced by hydroperoxides. Therefore, in order to distinguish between lipid hydroperoxides and ubiquinols, the evaporated hexane extract of plasma is dissolved in 0.5 ml of methanol-tert-butanol (50 : 50 by volume) and incubated with 0.5 ml of a freshly prepared solution containing 10 mg/ml of sodium borohydride, triphenylphosphine, or stannous chloride in methanol. The sample is incubated for 60 min at 4° in the dark, and then 1 ml of methanol and 0.5 ml of water are added, followed by 10 ml of hexane. After extraction and centrifugation of the sample, 9 ml of the hexane phase is collected and analyzed as described above. Figure 3 demonstrates the usefulness of this procedure for the identification of chemiluminescence-producing compounds generated in plasma by incubation with stimulated PMNs: the chemiluminescence peaks of triglyceride hydroperoxides and cholesteryl ester hydroperoxides are eliminated, in contrast to the chemiluminescence peak produced by ubiquinol-10. Besides ubiquinol-10, a second compound producing chemiluminescence appears in the hexane phase of plasma (compound X in Fig. 2A). This compound elutes from the LC18DB column near the solvent front. It is absent in unextracted plasma, 15and thus seems to be produced artifactually during the analytical procedure. The nature of compound X as well as the reason for the increase of its chemiluminescence signal following sodium borohydride treatment (Fig. 3) are unknown. ~6S. Vadhanavikit, N. Sakamoto, N. Ashida, T. Kishi, and K. Folkers, Anal. Biochem. 142, 155 (1984).
380
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[39]
As mentioned earlier and illustrated in Fig. 2, antioxidants such as ascorbate, urate, and tocopherols give negative peaks since they quench background chemiluminescence. The background chemiluminescence most probably arises from trace amounts of hydroperoxides present in the HPLC mobile phase. The negative peaks provide only qualitative, but not quantitative, information, since this type of response is dependent on the background chemiluminescence. Conclusion
Detection of microperoxidase-isoluminol-dependent chemiluminescence on-line to high-performance chromatographic separation of oxidants and antioxidants is a rapid, sensitive, and selective assay for lipid hydroperoxides in biological samples. Possible interference by naturally occurring ubiquinols and other hydroquinones can be disclosed and corrected for by reductive treatment, for instance, with sodium borohydride. The assay has been successfully applied to the analysis of normal human blood plasma, 15 pulmonary edema fluid from patients with adult respiratory distress syndrome, 17 and traumatic spinal cord tissue of rats. ~s In addition, the assay has proved useful in in vitro studies on bile 19 and human blood plasma. 13,2° When used with the appropriate precautions, the assay allows for the detection, identification, and quantitation of lipid hydroperoxides in body fluids and tissues and thus should contribute to our understanding of diseases associated with oxidative stress. 17 C. E. Cross, T. Forte, R. Stocker, S. Louie, Y. Yamamoto, B. N. Ames, and B. Frei, J. Lab. Clin. Med. (in press). ts M. Lemke, B. Frei, B. N. Ames, and A. I. Faden, Neurosci. Lett. 108, 201 (1990). 19 R. Stocker and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 84, 8130 (1987). 2o B. Frei, L. England, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 86, 6377 (1989).
[39] D e t e r m i n a t i o n of M e t h y l L i n o l e a t e H y d r o p e r o x i d e s b y 13C N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r o s c o p y By E. N. FRANKEL, W. E. NEFF, and D. WEISLEDER
Much work has been published on the hydroperoxides of unsaturated lipids. Significant progress during the last decade in understanding the mechanism of lipid oxidation can be attributed to a large extent to the development of new analytical tools such as the combination of gas chromatography aim mass spectrometry (GC-MS), high-performance METHODS IN ENZYMOLOGY,VOL. 186
Copyright © 1990by AcademicPress, Inc. All rightsof reproductionin any form reserved.
CHEMILUMINESCENCE
ASSAY FOR LIPID HYDROPEROXIDES
371
[38] A s s a y o f L i p i d H y d r o p e r o x i d e s U s i n g H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y with I s o l u m i n a l Chemiluminescence Detection B y YORIHIRO YAMAMOTO, B A L Z FREI, a n d BRUCE N . AMES
Introduction Lipid peroxidation has been implicated in several diseases such as atherosclerosis, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, postischemic reoxygenation injury, and aging. Lipid peroxidation proceeds by a free radical chain mechanism and yields lipid hydroperoxides as major initial reaction products. Therefore, it would be desirable to detect and identify lipid hydroperoxides in biological tissues. The thiobarbituric acid assay, which measures an aldehydic breakdown product (malondialdehyde) of lipid hydroperoxides, has served as the most commonly used method for measuring lipid peroxidation. However, the application of the assay to biological samples is very problematic, since a variety of biological compounds such as deoxyribose, many other carbohydrates, sialic acid, prostaglandins, and thromboxanes also react with thiobarbituric acid and interfere with the assay. 1 There are methods for the detection of lipid hydroperoxides themselves2,3 rather than their breakdown products, but these methods, too, cannot be applied unreservedly to biological samples, because of interference by antioxidants present in biological tissues and/or lack of sensitivity. Warso and Lands 4 have developed a method for the detection of lipid hydroperoxides based on activation of prostaglandin H synthase by hydroperoxides. However, this method, like most other methods for measuring lipid peroxidation, is unable to distinguish between different classes of lipid hydroperoxides. Furthermore, the specificity of this assay remains to be established. The luminol chemiluminescence assay for the detection of hydrogen peroxide is well known for its picomole-level sensitivity. 5-7 Microperoxl j. M. C. Gutterdige, Free Radical Res. Commun. 1, 173 (1986). T. F. Slater, this series, Vol. 105, p. 283, and references therein. 3 R. Cathcart, E. Schwiers, and B. N. Ames, Anal. Biochem. 134, 111 (1983). 4 M. A. Warso and W. E. M. Lands, J. Clin. Invest. 75, 667 (1985). 5 D. T. Bostick and D. M. Hercules, Anal. Chem. 47, 447 (1975). 6 W. R. Seitz, this series, Vol. 57, p. 445. 7 B. Olsson, Anal. Chim. Acta 136, 113 (1982).
METHODS IN ENZYMOLOGY, VOL. 186
. Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
372
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[38]
idase (a heme fragment of cytochrome c) has been shown to be the most effective catalyst for this assay. 7,8 We have adapted the luminol-microperoxidase assay to organic hydroperoxides and obtained a sensitivity similar to that for hydrogen peroxide? Since isoluminol gave better results than luminol in terms of signal-to-noise ratio, we used isoluminol as the substrate in our assay. The reaction sequence leading to the emission of light from isoluminol in the presence of lipid hydroperoxides (LOOH) and microperoxidase is assumed to be as follows: LOOH
microperoxidaseLO"
LO" + isoluminol (QH-) ---->LOH + semiquinone radical (Q ~) Q ~ + O2 ~ quinone (Q) + 02 ~ Q = + 02 = --~ isoluminol endoperoxide --~ light (kmax 430rim)
(1) (2) (3) (4)
Reactions (1) 1° and (2) are not well established, but it is known that production of the semiquinone radical is essential for the emission of light from isoluminol. Reactions (3) and (4) have been well studied. 11A2 Analysis of biological samples with the isoluminol assay can be hampered by antioxidants, which quench the production of chemiluminescence by scavenging intermediary radicals in the above reaction sequence [Eqs. (1)-(4)]. We have eliminated this problem by separating biological antioxidants from lipid hydroperoxides by high-performance liquid chromatography (HPLC) and using the isoluminol assay as an on-line postcolumn chemiluminescence detection system. 9 HPLC-Isoluminol
Chemiluminescence
Assay
Equipment is arranged as outlined in Fig. I. Antioxidants and hydroperoxides in the sample are separated by HPLC, using one of the chromatographic conditions described below, and then mixed with a reaction solution containing isoluminol (6-amino-2,3-dihydro-l,4-phthalazinedione; Sigma, St. Louis, MO) and microperoxidase (Type MP-1 l, Sigma) in a special mixer (Model 2500-0322, Kratos, Westwood, N J ) . The reactions leading to the emission of light take place in a mixing coil made from a H. R. Schroeder, R. C. Boguslaski, R. J. Carrico, and R. T. Buckler, this series, Vol. 57, p. 424. 9 y . Yamamoto, M. H. Brodsky, J. C. Baker, and B. N. Ames, Anal. Biochem. 160, 7 (1987). lo T. A. Dix and L. J. Marnett, J. Biol. Chem. 260, 5351 (1986). 11 j. Lind, G. Mer6nyi, and T. E. Eriksen, J. Am. Chem. Soc. 105, 7655 (1983). 12 G. Mer~nyi, J. Lind, and T. E. Eriksen, J. Phys. Chem. 88, 2320 (1984).
[38l
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
373
sample HPLC mobile phase
~
pump A
=
injector column
UV detector reaction solution (isoluminol, microperoxidase)
---
pump B
~ mixer mixing coil
fluorometer FIG. 1. Schematic diagram of the HPLC-isoluminol chemiluminescence assay.
a piece of HPLC tubing. The emitted light is measured in a fluorometer (Model FS 970 equipped with a 10-~1 flow cell; Schoeffel Instrument Corporation, Westwood, NJ) used as a photon detector with the excitation source turned off and in the absence of an emission filter. The time constant and the supplying voltage for the fluorometer are set at 4 sec and 1160 V (58% of full range), respectively. The flow rates of pump A and B (Fig. 1) are 1.0 and 1.5 ml/min, respectively. The length of the mixing coil is 45 cm (inner volume -92/zl). The reaction solution is prepared as follows: 100 mM aqueous borate buffer (38.14 g of sodium tetraborate decahydrate per liter) is prepared, and the pH is adjusted to 10 with sodium hydroxide. Isoluminol (177.2 mg, final concentration 1 mM) is dissolved in 300 ml of methanol and 700 ml of the above borate buffer, and then 25 mg of microperoxidase is added. For the analysis of the hexane phase of plasma (see below), the reaction solution is composed of isoluminol and microperoxidase in 700 ml of methanol and 300 ml of borate buffer instead. All solutions as well as the HPLC mobile phases are kept in brown bottles in order to minimize light-induced generation of chemiluminescence-producingmaterial. Several useful HPLC conditions for the separation of various classes of lipid hydroperoxides and biological antioxidants are summarized in Table I. For example, condition III is useful for the separation of neutral lipid hydroperoxides such as hydroperoxides of cholesterol, triglycerides, and cholesteryl esters. Condition V can be used for the separation and quantitation of hydrogen peroxide, free fatty acid hydroperoxides, and phospholipid hydroperoxides. Under all chromatographic conditions the coefficients of variation (standard deviation/mean value) for the chemi-
374
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[38]
TABLE I H P L C CONDITIONS AND RETENTION TIMES OF HYDROPEROXlDES AND ANTIOXIDANTSa HPLC condition: Guard column/colum#: Mobile phase:
I LC18 d
II LC18DB c e
III LC18DB c f
IV LCSi g
V LCNH2 h
Retention times as monitored by chemiluminescence (min)
Compound i Hydrogen peroxide tert-Butyl hydroperoxide Linoleic acid hydroperoxide Cholesterol hydroperoxide PCI8:2-OOH PEI8:2-OOH Trilinolein hydroperoxide Chl8:2-OOH
3.0 3.4 2.5 6.2 -----
a-Tocopherol ~/-Tocopherol Ascorbate Urate
10.4 9.2 2.1 1.9
3.1 3.0 -3.9 3.2 3.1 2.2 3.6 2.2 3.1 1.5 4.8 7.1 4.0 2.3 3.5 6.3 -14.2 7.4 4.6 -2.6 5.1 -5.9 2.1 ND -8.0 2.1 ND Retention times as monitored by U V (rain) 12.9 11.2 2.0 1.8
4.8 4.5 2.3 2.0
2.0 2.0 1.4 1.2
3.3 3.3 9.9 8.7
a Flow rate = 1.0 ml/min; - - , not eluted within 30 min; ND, not determined. Adapted from Yamamoto et al. 9 b The 5-/~m analytical columns (25 cm × 4.6 mm i.d., except LCSi: 3/zm, 15 cm × 4.6 mm i.d.) were purchased from Supelco (Bellefonte, PA), with the corresponding guard columns (5 p.m, 2 c m x 4.6 mm i.d.). c DB, Deactivated for basic compounds. d Methanol. e Methanol containing 0.01% triethylamine. Methanol-tert-butanol (50 : 50 by volume). g Acetonitrile-tert-butanoi-water (55 : 30 : 15 by volume). h Methanol-40 m M sodium phosphate, monobasic (95 : 5 by volume). The combination of LCNH2 and LCSi (both 5 p.m, 25 cm x 4.6 mm i.d.) in series using methanol-40 m M sodium phosphate, monobasic (90 : 10 by volume) gave similar and more reproducible retention times. PC18:2-OOH, Dilinoleylphosphatidylcholine hydroperoxide; PE18:2-OOH, dilinoleylphosphatidylethanolamine hydroperoxide; ChI8:2-OOH, cholesteryl linoleate hydroperoxide.
luminescence peak areas of the hydroperoxides listed in Table I are less than 3% (n -> 3). Table II summarizes the sensitivity of the assay to various hydroperoxides relative to the sensitivity to linoleic acid hydroperoxide under chromatographic conditions II-V. The assay is less sensitive to hydrogen peroxide and tert-butyl hydroperoxide than to other hydroperoxides.
[38]
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
375
TABLE II SENSITIVITY OF HPLC-IsOLUMINOL CHEMILUMINESCENCE ASSAY TO VARIOUS HYDROPEROXIDES RELATIVE TO SENSITIVITY TO LINOLEIC ACID HYDROPEROXIDEa
HPLC conditionc Compoundb
II
III
IV
V
Hydrogen peroxide tert-Butyl hydroperoxide Linoleic acid hydroperoxide Cholesterol hydroperoxide PC18"2-OOH PE18:2-OOH Trilinolein hydroperoxide ChI8:2-OOH
0.57 0.57 1.00 0.84 1.44 1.04 ---
0.39 0.39 1.00 0.52 --1.18 0.75
-0.80 1.00 0.91 1.13 1.16 1.04 1.54
0.53 0.53 1.00 0.82 0.92 0.83 --
Sensitivity is defined as the ratio of peak area to amount of hydroperoxide. From Yamamoto et al.9 b For abbreviations see footnote i of Table I. c See Table I. The c h e m i l u m i n e s c e n c e r e s p o n s e relative to the amount o f linoleic acid h y d r o p e r o x i d e is linear o v e r a range of 1 to 1000 pmol. Other hydroperoxides such as hydrogen peroxide and tert-butyl hydroperoxide also give good linear relationships. The detection limit of the assay for linoleic acid h y d r o p e r o x i d e is a b o u t 1 pmol. Application of Assay to Analysis of H u m a n Blood Plasma In h u m a n blood plasma, the m a j o r lipids are unesterified fatty acids, phospholipids, cholesterol, triglycerides, and cholesteryl esters. ~3As evident f r o m Table I, it is v e r y difficult to separate all hydroperoxides of these lipids f r o m the p l a s m a antioxidants ascorbate, urate, and a - t o c o p h erol in one c h r o m a t o g r a p h i c step. Therefore, plasma is separated into two phases b y extraction with methanol and hexane prior to analysis with the H P L C - i s o l u m i n o l chemiluminescence assay. A n a l y t i c a l P r o c e d u r e s . F o r extraction, 0.5 ml of heparinized plasma is added to 2 ml o f H P L C - g r a d e methanol and mixed vigorously. This is followed b y the addition o f I0 ml o f hexane ( H P L C grade; Aldrich, Milwaukee, WI). (Before use the hexane should be washed with water in order to r e m o v e trace a m o u n t s o f hydroperoxides. A b o u t I volume of H P L C - g r a d e w a t e r is added to I0 volumes of hexane in a brown bottle ~3B. Frei, R. Stocker, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 85, 9748 (1988).
376
[38]
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE TABLE III RECOVERY OF LIPID HYDROPEROXIDES ADDED TO PLASMA PRIOR TO EXTRACTION WITH METHANOL AND HEXANEa Recovery (%) Compound b
Aqueous phase
Hexane phase
Linoleic acid hydroperoxide PCI8:2-OOH Cholesterol hydroperoxide ¢ Trilinolein hydroperoxide Chl8:2-OOH
72 58 10 0 0
0 0 14 56 56
Lipid hydroperoxides were added at 10 tzM 1.0 min prior to extraction. Adapted from Frei et al. I~ b For abbreviations see footnote i of Table I. c Cholesterol hydroperoxide is degraded rapidly in plasma lB. Frei, R. Stocker, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 85, 9748 (1988)].
and stirred vigorously overnight. The water is allowed to settle for several hours before the upper-phase hexane is used for extraction.) The methanol-hexane extract of plasma is vortexed for 1 min and then spun at 1000 g for I0 min at 4°. Nine milliliters of the upper, hexane phase is collected and dried under vacuum in a rotary evaporator. Dried hexane extracts are dissolved in 0.45 ml of methanol-tert-butanol (50 : 50 by volume). One hundred microliters of this solution (corresponding to 100/zl of plasma) is subjected to HPLC using condition III (see Table I). Under this chromatographic condition hydroperoxides of cholesterol, triglycerides, and cholesteryl esters, and the antioxidants ~- and 3,-tocopherol, all of which are extracted into the hexane phase of plasma, are well separated, z4,~5 The aqueous methanol phase of extracted plasma is passed through a 0.2-/zm filter (Type ARCO LC 13, Gelman Science, Ann Arbor, MI), and 20/zl of this solution (corresponding to 4 ~1 of plasma) is subjected to HPLC using condition V (see Table I). This chromatographic condition is used since it allows good separation of hydrogen peroxide, free fatty acid hydroperoxides, phospholipid hydroperoxides, ascorbate, and urate, all of which are extracted into the aqueous methanol phase of plasma. ~4,~5 The recoveries of lipid hydroperoxides added to plasma at a concentration of 10/zM prior to biphasic extraction with methanol and hexane and analysis as described above are given in Table III. The detection limits for lipid hydroperoxides in plasma extracting into the aqueous t4 y . Yamamoto and B. N. Ames, Free Radicals Biol. Med. 3, 359 (1987). t5 B. Frei, Y. Yamamoto, D. Niclas, and B. N. Ames, Anal. Biochem. 175, 120 (1988).
[38]
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
377
methanol phase and the hexane phase are about 0.03 and 0.01 /~M, respectively. 15 In order to assess the purity of all reagents and materials used, for each set o f analyses a blank (0.5 ml o f water) should be extracted and then carried through the entire analytical procedure as described above. After each set o f H P L C runs, a calibration is performed under the respective chromatographic condition, using linoleic acid hydroperoxide as a standard. This calibration is necessary since the sensitivity of the assay varies slightly, depending mainly on the freshness of the microperoxidaseisoluminol solution. Figures 2A and 2B show typical chemiluminescence chromatograms
A hexane extract
ubiquinol-10
a- and y-tocopherols B aqueous methanol extract
I H202 urate ascorbate t
0
r
r
5
,
r
10
i
15
Time (rain)
FZG.2. Typical chemiluminescence chromatograms of the hexane extract (A) and aqueous methanol extract (B) of human blood plasma from a healthy subject. The evaporated hexane phase was dissolved in methanol-tert-butanol (50:50 by volume) and analyzed under chromatographic condition III (Table I). The aqueous methanol phase was analyzed on an LCNH2 column using condition V (Table I). Chemiluminescence was recorded at 0.02/~A.
378
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[38]
of the hexane phase and the aqueous methanol phase, respectively, of extracted human blood plasma from a healthy subject. 14,15 No chemiluminescence-producing compounds are observed that comigrate with spiked standards of hydroperoxides of linoleic acid, dilinoleylphosphatidylcholine, cholesterol, trilinolein, or cholesteryl linoleate (see Table I). However, the hexane extract (Fig. 2A) contains two chemiluminescenceproducing compounds, one of which has been identified as ubiquinol-10 (see also below). In the methanol extract (Fig. 2B) a chemiluminescenceproducing compound comigrating with hydrogen peroxide is observed (see below). Both extracts contain plasma antioxidants which produce negative peaks in the chemiluminescence chromatograms owing to quenching of background chemiluminescence. Figure 3 illustrates the successful application of the assay to the analysis of human blood plasma after it has been incubated with activated polymorphonuclear leukocytes (PMNs). Besides a decrease in the plasma level of ubiquinol-10, such incubation led to formation of detectable amounts of triglyceride hydroperoxides, cholesteryl ester hydroperoxides (Fig. 3), and phospholipid hydroperoxides.13
/I I t
I
¢3
I
I--- .o"
3: O O I LU O
il _f 0
5
10
5
Time (min) Fro, 3. Chemiluminescencechromatogramof the hexanephaseof plasmaincubatedwith activated PMNs with (dashed line) and without (solid line) subsequent treatment with sodium borohydride. Chemiluminescence was recorded at 0.02 cA. TG-OOHs, Triglyceride hydroperoxides; CE-OOHs, cholesteryl ester hydroperoxides.
[38]
CHEMILUMINESCENCE ASSAY FOR LIPID HYDROPEROXIDES
379
Interference Hydrogen peroxide can be produced by autoxidation of ascorbate during workup and analysis. 15Thus, the assay is not suitable for the quantitation of hydrogen peroxide in biological samples containing ascorbate. The assay is not sensitive to endoperoxides, hydroxyalkenals, alkenals, quinones, aldehydes, and alcohols. 15 However, ubiquinols produce chemiluminescence in the assay (see Fig. 2A). ~5 Using chromatographic condition III, the assay yields a chemiluminescence signal for ubiquinol-10 that is 47% of the signal for an equimolar amount of linoleic acid hydroperoxide; ubiquinols-6,-9, and -I0 give chemiluminescence peaks with retention times of 4.2, 6.1, and 7.2 rain, respectively. ~5Naturally occurring ubiquinols-7 and -8, too, produce chemiluminescence in the assay, as do other hydroquinones. Human plasma contains about 0.7 /zM ubiquinol-10 (Fig. 2A). 15 We also occasionally observed trace amounts of ubiquinol-9 (see also Ref. 16). Chemiluminescence signals produced by ubiquinols and other hydroquinones cannot be eliminated by treatment with reducing agents, in contrast to the chemiluminescence signals produced by hydroperoxides. Therefore, in order to distinguish between lipid hydroperoxides and ubiquinols, the evaporated hexane extract of plasma is dissolved in 0.5 ml of methanol-tert-butanol (50 : 50 by volume) and incubated with 0.5 ml of a freshly prepared solution containing 10 mg/ml of sodium borohydride, triphenylphosphine, or stannous chloride in methanol. The sample is incubated for 60 min at 4° in the dark, and then 1 ml of methanol and 0.5 ml of water are added, followed by 10 ml of hexane. After extraction and centrifugation of the sample, 9 ml of the hexane phase is collected and analyzed as described above. Figure 3 demonstrates the usefulness of this procedure for the identification of chemiluminescence-producing compounds generated in plasma by incubation with stimulated PMNs: the chemiluminescence peaks of triglyceride hydroperoxides and cholesteryl ester hydroperoxides are eliminated, in contrast to the chemiluminescence peak produced by ubiquinol-10. Besides ubiquinol-10, a second compound producing chemiluminescence appears in the hexane phase of plasma (compound X in Fig. 2A). This compound elutes from the LC18DB column near the solvent front. It is absent in unextracted plasma, 15and thus seems to be produced artifactually during the analytical procedure. The nature of compound X as well as the reason for the increase of its chemiluminescence signal following sodium borohydride treatment (Fig. 3) are unknown. ~6S. Vadhanavikit, N. Sakamoto, N. Ashida, T. Kishi, and K. Folkers, Anal. Biochem. 142, 155 (1984).
380
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[39]
As mentioned earlier and illustrated in Fig. 2, antioxidants such as ascorbate, urate, and tocopherols give negative peaks since they quench background chemiluminescence. The background chemiluminescence most probably arises from trace amounts of hydroperoxides present in the HPLC mobile phase. The negative peaks provide only qualitative, but not quantitative, information, since this type of response is dependent on the background chemiluminescence. Conclusion
Detection of microperoxidase-isoluminol-dependent chemiluminescence on-line to high-performance chromatographic separation of oxidants and antioxidants is a rapid, sensitive, and selective assay for lipid hydroperoxides in biological samples. Possible interference by naturally occurring ubiquinols and other hydroquinones can be disclosed and corrected for by reductive treatment, for instance, with sodium borohydride. The assay has been successfully applied to the analysis of normal human blood plasma, 15 pulmonary edema fluid from patients with adult respiratory distress syndrome, 17 and traumatic spinal cord tissue of rats. ~s In addition, the assay has proved useful in in vitro studies on bile 19 and human blood plasma. 13,2° When used with the appropriate precautions, the assay allows for the detection, identification, and quantitation of lipid hydroperoxides in body fluids and tissues and thus should contribute to our understanding of diseases associated with oxidative stress. 17 C. E. Cross, T. Forte, R. Stocker, S. Louie, Y. Yamamoto, B. N. Ames, and B. Frei, J. Lab. Clin. Med. (in press). ts M. Lemke, B. Frei, B. N. Ames, and A. I. Faden, Neurosci. Lett. 108, 201 (1990). 19 R. Stocker and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 84, 8130 (1987). 2o B. Frei, L. England, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 86, 6377 (1989).
[39] D e t e r m i n a t i o n of M e t h y l L i n o l e a t e H y d r o p e r o x i d e s b y 13C N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r o s c o p y By E. N. FRANKEL, W. E. NEFF, and D. WEISLEDER
Much work has been published on the hydroperoxides of unsaturated lipids. Significant progress during the last decade in understanding the mechanism of lipid oxidation can be attributed to a large extent to the development of new analytical tools such as the combination of gas chromatography aim mass spectrometry (GC-MS), high-performance METHODS IN ENZYMOLOGY,VOL. 186
Copyright © 1990by AcademicPress, Inc. All rightsof reproductionin any form reserved.
