438
ASSAY A N D REPAIR OF B I O L O G I C A L D A M A G E
[45]
ides are very poor activators of the cyclooxygenase in contradiction to a preliminary observation reported earlier. 7 For example, the esterified hydroperoxide in triglyceride and phosphatidylcholine were 1-4% as effective as the nonesterified 15-HPETE in activating cyclooxygenase. However, after nearly complete enzymatic hydrolysis (determined by TLC analysis), the preparations were 72-76% as effective as 15-HPETE.II Any sample likely to contain esterified material should thus be treated with esterases before assay with the cyclooxygenase method, so as to obtain a more valid assessment of the total hydroperoxide content. We find the assay described above useful in quantitating fatty acid hydroperoxide in amounts ranging from 50-200 pmol (-100-300 t~l sample per assay). However, materials other than hydroperoxide may activate cyclooxygenase (e.g., ethanol, acetone); added controls are valuable. Treating samples with glutathione peroxidase selectively reduces the activating fatty acid hydroperoxide and permits measurement of any nonhydroperoxide activator. Comparing the amounts of glutathione peroxidase-reducible and glutathione peroxidase-resistant activator is essential in determining how much of the activator in a sample is hydroperoxide. Acknowledgments This work was supported by grants GM30509 and HL34422 from the United States Public Health Service.
[45] C h o l e s t e r o l E p o x i d e s : F o r m a t i o n a n d M e a s u r e m e n t By G. A. S. ANSAm and LELAND L. SMITH
The isomeric cholesterol 5,6-epoxides 5,6a-epoxy-5a-cholestan-3fl-ol (cholesterol a-oxide, 1) and 5,6/3-epoxy-5/3-cholestan-3/3-ol (cholesterol /3-oxide, 2) are found in human tissues and foods and on bioassay exhibit diverse toxic effects in oitro.~,2 Both are formed together from cholesterol (cholest-5-en-3/3-ol, 3) by many defined oxidants, including air oxidation, 1,3 and by the actions of soybean lipoxygenases and of liver microsomal lipid peroxidation systems in oitro. 4 Both are formed by an un1 L. L. Smith, "Cholesterol Autoxidation." Plenum, New York, 1981. 2 L. L. Smith, Chem. Phys. Lipids 44, 87 (1987). s j. Gumulka, J. S. Pyrek, and L. L. Smith, Lipids 17, 197 (1982). 4 L. Aringer and P. Eneroth, J. Lipid Res. 15, 389 (1974).
METHODS IN ENZYMOLOGY, VOL 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[45]
CHOLESTEROLEPOXIDES
No C 7
.o~¢.+>/
: "" O
439
ceM7
.o-~x..--%/
O
1
2
3
characterized in vivo epoxidation system in mammals, 5 and the 5a,6c~epoxide (1) is formed by a liver microsomal enzyme in vitro. 6 Of all the recognized oxidation products of cholesterol formed by attack on cholesterol of defined active oxygen species, the 5,6-epoxides (1 and 2) are the most broadly formed, being formed by ground state dioxygen (302), electronically excited (sing,let) dioxygen (IO2), peroxide (O22+), ozone (O3), dioxygen cation (O2+), and hydroxyl radical (HO .). The ratio of product 5,6-epoxides (1:2) favors the 5/3,6/3-epoxide (2) in most cases, the 5a,6ot-epoxide (1) predominating only in oxidations involving the specific 5a,6ot-epoxidase, HO., and peracids. 3 In new experimental systems generating the 5,6-epoxides it is of importance that the products be properly identified and the 5,6-epoxide ratio (1:2) ascertained. The 5a,6aepoxide (1) alone has been posed as the 5,6-epoxide formed in biological systems and as the active agent in numerous bioassays but without adequate supporting evidence. Sources
The 5a,6a-epoxide (1) is commercially available from several sources, including Sigma Chemical Co. (St. Louis, MO), Research Plus Inc. (Bayonne, NJ), and Steraloids Inc. (Wilton, NH); the 5fl,6/3-epoxide (2) is also available from Research Plus Inc. The 5a,6a-epoxide (1) is readily prepared from cholesterol by peracid oxidation according to simple directions. 7 Synthesis of the 5fl,6fl-epoxide (2) from cholesterol8 and from 5acholestane-3fl,5,6fl-triol triacetate 9 also provides ready access. The limited commercial availability of the 5fl,6//-epoxide (2) is probably responsible for the lack of attention given this isomer in prior studies. Commercial and synthetic samples may contain significant amounts of the isomeric 5,6-epoxide, and the identity and purity of all reference samples 5 A. Sevanian, J. F. Mead, and R. A. Stein, Lipids 14, 634 (1979). 6 T. Watabe and T. Sawahata, J. Biol. Chem. 254, 3854 (1979). 7 L. F. Fieser and M. Fieser, "Reagents for Organic Synthesis," Vol. I, p. 136. Wiley, New York, 1967. s S. G. Levine and M. E. Wall, J. Am. Chem. Soc. 81, 2826 (1959). 9 A. T. Rowland and H. R. Nace, J. Am. Chem. Soc. 82, 2833 (1960).
440
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[45]
should be determined by appropriate chromatography and ready reliable means, generally including melting point, optical rotation, and proton nuclear magnetic resonance (NMR) spectra. The literature gives data in ranges: 5oL,6a-epoxide (1), mp 139-148 °, [OdD - 4 0 to -48.5 °, 8n 2.90 (d, J = 3.3-4. I Hz, 6fl-proton); 5fl,6fl-epoxide (2), mp 130-136 °, [a]D +8 to +11.5 °, 8rl 3.05 (d, J = 2.1-3.0 Hz, 6a-proton). ~ The isomers exhibit minor differences in infrared and mass spectra that are useful for confirming the identity of pure samples, but they are not useful for analysis of mixtures.l°-~z Characterization of the 5,6-epoxides as the 3/i-acetate, 3flbenzoate, or 3fl-trimethylsilyl ethers should also be considered in special cases. Recovery o f 5,6-Epoxides
Typically, total lipids from tissues, biological fluids, in vitro incubations, etc. may be extracted with chloroform-methanol (2: 1, v/v) using the classic methods of Folch et al. ~3or Bligh and Dyer ~4or with hexane-2propanol (3:2, v/v) according to Hara and Radin. 15 For foods and tissues containing phospholipids, preliminary removal of phospholipids from neutral lipids is necessary; for this purpose chromatography on silica gel using hexane-diethyl ether (9: 1, v/v) may be used to elute hydrocarbons, triacylglycerols, and other neutral lipid esters, followed by hexanediethyl ether (5:3, v/v) to elute sterols and oxysterols. Polar phospholipids are retained on the column. For oxysterols recovered from in vitro incubations in which massive amounts of triacylglycerols and phospholipids are not involved, direct thin-layer chromatography (TLC) of the recovered total lipid fraction may provide satisfactorily resolved 5,6epoxide preparations for analysis. The column fractionation should be monitored by TLC even though TLC does not resolve the isomeric 5,6-epoxides. Silica gel-coated chromatoplates irrigated with benzene-ethyl acetate (3:1, v/v) in three ascending irrigations or with cyclohexane-diethyl ether (9: 1, v/v) resolve the mixed 5,6-epoxides from other oxidized cholesterol derivatives. The 5,6-epoxides are detected with 50% aqueous sulfuric acid as spray, with heating to color development (yellow to tan to brown) and to charring. In some cases these methods may not resolve the 5,6-epoxides from other common companion oxysterols such as 3/3-hydroxycholest-5-en-7one (7-ketocholesterol) or the cholesterol 7-hydroperoxides. The pres10 E. Chicoye, W. D. Powrie, and O. Fennema, Lipids 3, 335 (1968). it L. S. Tsai and C. A. Hudson, J. Food Sci. 49, 1245 (1984). 12j. Nourooz-Zadeh and L.-A. Appleqvist, J. Food Sci. 52, 57 (1987). 13 j. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 22,6, 497 (1957). 1, E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 31, 911 (1959). 1~ A. Hara and S. N. Radin, Anal. Biochem. 90, 420 (1978).
[45]
CHOLESTEROLEPOXIDES
441
ence of the 7-ketone is revealed by its UV-absorbing properties on chromatoplates containing an UV-fluorescing phosphor; hydroperoxide contamination is revealed by N,N-dimethyl-p-phenylenediamine spray applied before the sulfuric acid spray. ~6The 7-ketone and sterol hydroperoxide contaminations do not interfere in the subsequent HPLC analysis of 5,6-epoxides. As the 5,6-epoxides are subject to hydration, for some studies it is expedient to include their common hydration product, 5a-cholestane3/3,5,6/3-triol, among analytes examined. Moreover, the 5/3,6/3-epoxide (2) is less stable than the 5a,6a-epoxide (1) toward other decomposition, and care must be exercised to avoid alteration of the 5,6-epoxides during recovery and analysis.
Analysis of 5,6-Epoxides Three major means have been successfully used for analysis of mixtures of the 5,6-epoxides: high-performance liquid chromatography (HPLC), proton NMR spectroscopy, and chemical reduction to cholestanediols. Chromatography. The 5,6-epoxides are not resolved by thin-layer or packed-column gas chromatography, but their 3fl-acetates and 3/3-trimethylsilyl ethers may be. 3,4,6,1°Generally the 5t~,6a-epoxide (2) is more mobile. Preferred operations are conducted with HPLC and gas chromatography using fused silica capillary columns. Adsorption (silica) HPLC columns with binary solvents, e.g., hexane-2-propanol (24: l, 49: l, or 100:3, all v/v), used isocraticaily 3,~,~7,~8 or in a curvilinear gradient ~9 are very suitable, the 5a,6a-epoxide (1) being the more mobile. Resolution is also achieved with reversed-phase partition columns using acetonitrilewater (9: 1, v/v) or methanol-water (9: l, v/v), the 5fl,6/3-epoxide (2) being the more mobile. 17Detection of the 5,6-epoxides in column effluent by UV absorption is not suitable, but detection by differential refractive index or hydrogen flame ionization monitoring is effective. 5,6-Epoxide 3/3-benzoates and 3/3-p-nitrobenzoates are also resolved by HPLC using adsorption columns with isooctane-2-propanol (499: l, v/v) or reversed-phase columns with acetonitrile-water (19: 1, v/v) or methanol-water (9: 1, v/v) isocratically 17and with linear gradients of acetonitrile-2-propanol. 2° Esters of the 5/3,6/3-epoxide (2) are the more mobile and are readily detected by UV absorption. Gas chromatographic resolution of the isomeric 5,6-epoxides and their 16 L. i~ G. is L. 19 G. 20 K.
L. Smith and F. L. Hill, J. Chromatogr. 66, 101 (1972). A. S. Ansari and L. L. Smith, J. Chromatogr. 175, 307 (1979). S. Tsai, K. Ichi, C. A. Hudson, and J. J. Meehan, Lipids 15, 124 (1980). Maerker, E. H. Nungesser, and I. M. Zulak, J. Agric. Food Chem. 36, 61 (1988). Sugino, J. Terao, H. Murakami, and S. Matsushita, J. Agric Food Chem. 34, 36 (1986).
442
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[45]
3fl-trimethylsilyl ethers is conveniently achieved using fused silica capillary columns coated with silicone polymers such as SE-30, SE-54, DB-1, or DB-5, with gas effluent monitoring by the flame ionization method. The 5/3,6/3-epoxide (2) and its 3/3-trimethylsilyl ether are the more mobile. 3,2~-24 Detailed protocols using these various methods for recovery and analysis of the 5,6-epoxides from human body fluids 24,25and cholesterol-rich foods a r e a v a i l a b l e , llA2A9,2t,26,27
Proton NMR Spectroscopy. The proton NMR signals of the 6-proton of the isomeric 5,6-epoxides are significantly distinct from one another so as to constitute a ready means of analysis of mixtures. The 6/3-proton signal (in deuteriochloroform) of the 5c~,6a-epoxide (1) appears as a doublet at 8n 2.90 (J = 3.3-4.1 Hz); the 6a-proton of the isomeric 5/3,6/8epoxide (2) is also a doublet found at 6H 3.05 (J = 2.1-3.0 Hz). These key signals serve to identify the individual 5,6-epoxides, and integration of the signals affords ready estimation of relative amounts of each component. Except where the key signals are obscured by extraneous signals from other components of the sample, the proton NMR method is preferred for 5,6-epoxide estimation. Chemical Reduction Analysis. In the absence of an adequate HPLC or gas chromatography apparatus and NMR spectrometer, chemical reduction analysis may be used. The chemical reduction of mixtures of isomeric 5,6-epoxides yields mixtures of reduction products resolved by TLC with benzene-ethyl acetate (3 : 2, v/v). The 5a,6a-epoxide (1) yields 5a-cholestane-3/3,5-diol (4, Rf 0.30) as a single product; the isomeric 5/3,6/3-epoxide (2) yields 5et-cholestane-3/3,6/3-diol (5, Rf 0.19) and 5/3-cholestane-3/3,5diol (6, Rf 0.53). All three diols give a red color with 50% sulfuric acid, and
,o-.../I..1. OH
H I OH
4
5
ON
6
2t G. Maerker and J. Unruh, J. Am. Oil Chem. Soc. 63, 767 (1986). 22 S. R. Missler, B. A. Wasilchuk, and C. Merritt, J. Food Sci. 50, 595 (1985). 23 S. W. Park and P. B. Addis, J. Agric. Food Chem. 34, 653 (1986). 24 L. D. Gruenke, J. C. Craig, N. L. Petrakis, and M. B. Lyon, Biomed. Environ. Mass Spectrom. 14, 335 (1987). 25 I. Bj6rkhem, O. Breuer, B. Angelin, and S.-A Wikstr6m, J. LipidRes. 29, 1031 (1988). 26 L. S. Tsai and C. A. Hudson, J. Food Sci. 49, 1245 (1984). 27 S. W. Park and P. B. Addis, J. Food Sci. 42, 1500 (1987).
[46]
443
OXYSTAT TECHNIQUE
following heating to char the components the amounts of each may be estimated using densitometric scanning of the chromatoplate, eluted and weighed, 28 or estimated by gas chromatography. 5 28L. L. Smith and M. J. Kulig, Cancer Biochem. Biophys. 1, 79 (1975).
[46] O x y s t a t T e c h n i q u e in S t u d y o f R e a c t i v e Oxygen Species By
HERBERT
DE GROOT
Introduction There are two principal approaches to maintain steady-state oxygen partial pressures (Po:) in suspensions of respiring biological material. One approach is to deliver 02 via diffusion from a gas phase to the liquid phase. The other is to infuse O2-saturated aqueous medium into the suspension of respiring biological material. Based on the second principle we have developed an oxystat technique where 02 supply is maintained by injecting O2-saturated aqueous medium using a computer-supported feedback control system (Fig. 1). 1,2 Major advantages of this technique are (1) it is capable of maintaining steady-state Po: at very low levels, (2) it rapidly responds to alterations in the O2 uptake rate, and (3) it allows calculations of 02 uptake from the amounts of O2-saturated medium added. Oxystat Technique Apparatus
The oxystat system (Fig. 1) consists of an incubation chamber, an Oz sensor, a pump, and a computer. The water-jacketed incubation chamber is made of plexiglass. It is equipped with a magnetic stirrer and a port for the Oz sensor. At the top two stainless steel needles serve as entrances. The inlet for the O2-saturated infusion medium ends directly above the magnetic stirring bar. The second needle, which does not project into the 1T. Noll, H. de Groot, and P. Wissemann, Biochem. J. 236, 765 (1986). 2H. de Groot, A. Littauer, D. Hugo-Wissemann, P. Wissemann, and T. Noll, Arch. Biochem. Biophys. 264, 591 (1988). Copyright © 1990by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 186
All rights of reproduction in any form reserved.
ASSAY A N D REPAIR OF B I O L O G I C A L D A M A G E
[45]
ides are very poor activators of the cyclooxygenase in contradiction to a preliminary observation reported earlier. 7 For example, the esterified hydroperoxide in triglyceride and phosphatidylcholine were 1-4% as effective as the nonesterified 15-HPETE in activating cyclooxygenase. However, after nearly complete enzymatic hydrolysis (determined by TLC analysis), the preparations were 72-76% as effective as 15-HPETE.II Any sample likely to contain esterified material should thus be treated with esterases before assay with the cyclooxygenase method, so as to obtain a more valid assessment of the total hydroperoxide content. We find the assay described above useful in quantitating fatty acid hydroperoxide in amounts ranging from 50-200 pmol (-100-300 t~l sample per assay). However, materials other than hydroperoxide may activate cyclooxygenase (e.g., ethanol, acetone); added controls are valuable. Treating samples with glutathione peroxidase selectively reduces the activating fatty acid hydroperoxide and permits measurement of any nonhydroperoxide activator. Comparing the amounts of glutathione peroxidase-reducible and glutathione peroxidase-resistant activator is essential in determining how much of the activator in a sample is hydroperoxide. Acknowledgments This work was supported by grants GM30509 and HL34422 from the United States Public Health Service.
[45] C h o l e s t e r o l E p o x i d e s : F o r m a t i o n a n d M e a s u r e m e n t By G. A. S. ANSAm and LELAND L. SMITH
The isomeric cholesterol 5,6-epoxides 5,6a-epoxy-5a-cholestan-3fl-ol (cholesterol a-oxide, 1) and 5,6/3-epoxy-5/3-cholestan-3/3-ol (cholesterol /3-oxide, 2) are found in human tissues and foods and on bioassay exhibit diverse toxic effects in oitro.~,2 Both are formed together from cholesterol (cholest-5-en-3/3-ol, 3) by many defined oxidants, including air oxidation, 1,3 and by the actions of soybean lipoxygenases and of liver microsomal lipid peroxidation systems in oitro. 4 Both are formed by an un1 L. L. Smith, "Cholesterol Autoxidation." Plenum, New York, 1981. 2 L. L. Smith, Chem. Phys. Lipids 44, 87 (1987). s j. Gumulka, J. S. Pyrek, and L. L. Smith, Lipids 17, 197 (1982). 4 L. Aringer and P. Eneroth, J. Lipid Res. 15, 389 (1974).
METHODS IN ENZYMOLOGY, VOL 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[45]
CHOLESTEROLEPOXIDES
No C 7
.o~¢.+>/
: "" O
439
ceM7
.o-~x..--%/
O
1
2
3
characterized in vivo epoxidation system in mammals, 5 and the 5a,6c~epoxide (1) is formed by a liver microsomal enzyme in vitro. 6 Of all the recognized oxidation products of cholesterol formed by attack on cholesterol of defined active oxygen species, the 5,6-epoxides (1 and 2) are the most broadly formed, being formed by ground state dioxygen (302), electronically excited (sing,let) dioxygen (IO2), peroxide (O22+), ozone (O3), dioxygen cation (O2+), and hydroxyl radical (HO .). The ratio of product 5,6-epoxides (1:2) favors the 5/3,6/3-epoxide (2) in most cases, the 5a,6ot-epoxide (1) predominating only in oxidations involving the specific 5a,6ot-epoxidase, HO., and peracids. 3 In new experimental systems generating the 5,6-epoxides it is of importance that the products be properly identified and the 5,6-epoxide ratio (1:2) ascertained. The 5a,6aepoxide (1) alone has been posed as the 5,6-epoxide formed in biological systems and as the active agent in numerous bioassays but without adequate supporting evidence. Sources
The 5a,6a-epoxide (1) is commercially available from several sources, including Sigma Chemical Co. (St. Louis, MO), Research Plus Inc. (Bayonne, NJ), and Steraloids Inc. (Wilton, NH); the 5fl,6/3-epoxide (2) is also available from Research Plus Inc. The 5a,6a-epoxide (1) is readily prepared from cholesterol by peracid oxidation according to simple directions. 7 Synthesis of the 5fl,6fl-epoxide (2) from cholesterol8 and from 5acholestane-3fl,5,6fl-triol triacetate 9 also provides ready access. The limited commercial availability of the 5fl,6//-epoxide (2) is probably responsible for the lack of attention given this isomer in prior studies. Commercial and synthetic samples may contain significant amounts of the isomeric 5,6-epoxide, and the identity and purity of all reference samples 5 A. Sevanian, J. F. Mead, and R. A. Stein, Lipids 14, 634 (1979). 6 T. Watabe and T. Sawahata, J. Biol. Chem. 254, 3854 (1979). 7 L. F. Fieser and M. Fieser, "Reagents for Organic Synthesis," Vol. I, p. 136. Wiley, New York, 1967. s S. G. Levine and M. E. Wall, J. Am. Chem. Soc. 81, 2826 (1959). 9 A. T. Rowland and H. R. Nace, J. Am. Chem. Soc. 82, 2833 (1960).
440
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[45]
should be determined by appropriate chromatography and ready reliable means, generally including melting point, optical rotation, and proton nuclear magnetic resonance (NMR) spectra. The literature gives data in ranges: 5oL,6a-epoxide (1), mp 139-148 °, [OdD - 4 0 to -48.5 °, 8n 2.90 (d, J = 3.3-4. I Hz, 6fl-proton); 5fl,6fl-epoxide (2), mp 130-136 °, [a]D +8 to +11.5 °, 8rl 3.05 (d, J = 2.1-3.0 Hz, 6a-proton). ~ The isomers exhibit minor differences in infrared and mass spectra that are useful for confirming the identity of pure samples, but they are not useful for analysis of mixtures.l°-~z Characterization of the 5,6-epoxides as the 3/i-acetate, 3flbenzoate, or 3fl-trimethylsilyl ethers should also be considered in special cases. Recovery o f 5,6-Epoxides
Typically, total lipids from tissues, biological fluids, in vitro incubations, etc. may be extracted with chloroform-methanol (2: 1, v/v) using the classic methods of Folch et al. ~3or Bligh and Dyer ~4or with hexane-2propanol (3:2, v/v) according to Hara and Radin. 15 For foods and tissues containing phospholipids, preliminary removal of phospholipids from neutral lipids is necessary; for this purpose chromatography on silica gel using hexane-diethyl ether (9: 1, v/v) may be used to elute hydrocarbons, triacylglycerols, and other neutral lipid esters, followed by hexanediethyl ether (5:3, v/v) to elute sterols and oxysterols. Polar phospholipids are retained on the column. For oxysterols recovered from in vitro incubations in which massive amounts of triacylglycerols and phospholipids are not involved, direct thin-layer chromatography (TLC) of the recovered total lipid fraction may provide satisfactorily resolved 5,6epoxide preparations for analysis. The column fractionation should be monitored by TLC even though TLC does not resolve the isomeric 5,6-epoxides. Silica gel-coated chromatoplates irrigated with benzene-ethyl acetate (3:1, v/v) in three ascending irrigations or with cyclohexane-diethyl ether (9: 1, v/v) resolve the mixed 5,6-epoxides from other oxidized cholesterol derivatives. The 5,6-epoxides are detected with 50% aqueous sulfuric acid as spray, with heating to color development (yellow to tan to brown) and to charring. In some cases these methods may not resolve the 5,6-epoxides from other common companion oxysterols such as 3/3-hydroxycholest-5-en-7one (7-ketocholesterol) or the cholesterol 7-hydroperoxides. The pres10 E. Chicoye, W. D. Powrie, and O. Fennema, Lipids 3, 335 (1968). it L. S. Tsai and C. A. Hudson, J. Food Sci. 49, 1245 (1984). 12j. Nourooz-Zadeh and L.-A. Appleqvist, J. Food Sci. 52, 57 (1987). 13 j. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 22,6, 497 (1957). 1, E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 31, 911 (1959). 1~ A. Hara and S. N. Radin, Anal. Biochem. 90, 420 (1978).
[45]
CHOLESTEROLEPOXIDES
441
ence of the 7-ketone is revealed by its UV-absorbing properties on chromatoplates containing an UV-fluorescing phosphor; hydroperoxide contamination is revealed by N,N-dimethyl-p-phenylenediamine spray applied before the sulfuric acid spray. ~6The 7-ketone and sterol hydroperoxide contaminations do not interfere in the subsequent HPLC analysis of 5,6-epoxides. As the 5,6-epoxides are subject to hydration, for some studies it is expedient to include their common hydration product, 5a-cholestane3/3,5,6/3-triol, among analytes examined. Moreover, the 5/3,6/3-epoxide (2) is less stable than the 5a,6a-epoxide (1) toward other decomposition, and care must be exercised to avoid alteration of the 5,6-epoxides during recovery and analysis.
Analysis of 5,6-Epoxides Three major means have been successfully used for analysis of mixtures of the 5,6-epoxides: high-performance liquid chromatography (HPLC), proton NMR spectroscopy, and chemical reduction to cholestanediols. Chromatography. The 5,6-epoxides are not resolved by thin-layer or packed-column gas chromatography, but their 3fl-acetates and 3/3-trimethylsilyl ethers may be. 3,4,6,1°Generally the 5t~,6a-epoxide (2) is more mobile. Preferred operations are conducted with HPLC and gas chromatography using fused silica capillary columns. Adsorption (silica) HPLC columns with binary solvents, e.g., hexane-2-propanol (24: l, 49: l, or 100:3, all v/v), used isocraticaily 3,~,~7,~8 or in a curvilinear gradient ~9 are very suitable, the 5a,6a-epoxide (1) being the more mobile. Resolution is also achieved with reversed-phase partition columns using acetonitrilewater (9: 1, v/v) or methanol-water (9: l, v/v), the 5fl,6/3-epoxide (2) being the more mobile. 17Detection of the 5,6-epoxides in column effluent by UV absorption is not suitable, but detection by differential refractive index or hydrogen flame ionization monitoring is effective. 5,6-Epoxide 3/3-benzoates and 3/3-p-nitrobenzoates are also resolved by HPLC using adsorption columns with isooctane-2-propanol (499: l, v/v) or reversed-phase columns with acetonitrile-water (19: 1, v/v) or methanol-water (9: 1, v/v) isocratically 17and with linear gradients of acetonitrile-2-propanol. 2° Esters of the 5/3,6/3-epoxide (2) are the more mobile and are readily detected by UV absorption. Gas chromatographic resolution of the isomeric 5,6-epoxides and their 16 L. i~ G. is L. 19 G. 20 K.
L. Smith and F. L. Hill, J. Chromatogr. 66, 101 (1972). A. S. Ansari and L. L. Smith, J. Chromatogr. 175, 307 (1979). S. Tsai, K. Ichi, C. A. Hudson, and J. J. Meehan, Lipids 15, 124 (1980). Maerker, E. H. Nungesser, and I. M. Zulak, J. Agric. Food Chem. 36, 61 (1988). Sugino, J. Terao, H. Murakami, and S. Matsushita, J. Agric Food Chem. 34, 36 (1986).
442
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[45]
3fl-trimethylsilyl ethers is conveniently achieved using fused silica capillary columns coated with silicone polymers such as SE-30, SE-54, DB-1, or DB-5, with gas effluent monitoring by the flame ionization method. The 5/3,6/3-epoxide (2) and its 3/3-trimethylsilyl ether are the more mobile. 3,2~-24 Detailed protocols using these various methods for recovery and analysis of the 5,6-epoxides from human body fluids 24,25and cholesterol-rich foods a r e a v a i l a b l e , llA2A9,2t,26,27
Proton NMR Spectroscopy. The proton NMR signals of the 6-proton of the isomeric 5,6-epoxides are significantly distinct from one another so as to constitute a ready means of analysis of mixtures. The 6/3-proton signal (in deuteriochloroform) of the 5c~,6a-epoxide (1) appears as a doublet at 8n 2.90 (J = 3.3-4.1 Hz); the 6a-proton of the isomeric 5/3,6/8epoxide (2) is also a doublet found at 6H 3.05 (J = 2.1-3.0 Hz). These key signals serve to identify the individual 5,6-epoxides, and integration of the signals affords ready estimation of relative amounts of each component. Except where the key signals are obscured by extraneous signals from other components of the sample, the proton NMR method is preferred for 5,6-epoxide estimation. Chemical Reduction Analysis. In the absence of an adequate HPLC or gas chromatography apparatus and NMR spectrometer, chemical reduction analysis may be used. The chemical reduction of mixtures of isomeric 5,6-epoxides yields mixtures of reduction products resolved by TLC with benzene-ethyl acetate (3 : 2, v/v). The 5a,6a-epoxide (1) yields 5a-cholestane-3/3,5-diol (4, Rf 0.30) as a single product; the isomeric 5/3,6/3-epoxide (2) yields 5et-cholestane-3/3,6/3-diol (5, Rf 0.19) and 5/3-cholestane-3/3,5diol (6, Rf 0.53). All three diols give a red color with 50% sulfuric acid, and
,o-.../I..1. OH
H I OH
4
5
ON
6
2t G. Maerker and J. Unruh, J. Am. Oil Chem. Soc. 63, 767 (1986). 22 S. R. Missler, B. A. Wasilchuk, and C. Merritt, J. Food Sci. 50, 595 (1985). 23 S. W. Park and P. B. Addis, J. Agric. Food Chem. 34, 653 (1986). 24 L. D. Gruenke, J. C. Craig, N. L. Petrakis, and M. B. Lyon, Biomed. Environ. Mass Spectrom. 14, 335 (1987). 25 I. Bj6rkhem, O. Breuer, B. Angelin, and S.-A Wikstr6m, J. LipidRes. 29, 1031 (1988). 26 L. S. Tsai and C. A. Hudson, J. Food Sci. 49, 1245 (1984). 27 S. W. Park and P. B. Addis, J. Food Sci. 42, 1500 (1987).
[46]
443
OXYSTAT TECHNIQUE
following heating to char the components the amounts of each may be estimated using densitometric scanning of the chromatoplate, eluted and weighed, 28 or estimated by gas chromatography. 5 28L. L. Smith and M. J. Kulig, Cancer Biochem. Biophys. 1, 79 (1975).
[46] O x y s t a t T e c h n i q u e in S t u d y o f R e a c t i v e Oxygen Species By
HERBERT
DE GROOT
Introduction There are two principal approaches to maintain steady-state oxygen partial pressures (Po:) in suspensions of respiring biological material. One approach is to deliver 02 via diffusion from a gas phase to the liquid phase. The other is to infuse O2-saturated aqueous medium into the suspension of respiring biological material. Based on the second principle we have developed an oxystat technique where 02 supply is maintained by injecting O2-saturated aqueous medium using a computer-supported feedback control system (Fig. 1). 1,2 Major advantages of this technique are (1) it is capable of maintaining steady-state Po: at very low levels, (2) it rapidly responds to alterations in the O2 uptake rate, and (3) it allows calculations of 02 uptake from the amounts of O2-saturated medium added. Oxystat Technique Apparatus
The oxystat system (Fig. 1) consists of an incubation chamber, an Oz sensor, a pump, and a computer. The water-jacketed incubation chamber is made of plexiglass. It is equipped with a magnetic stirrer and a port for the Oz sensor. At the top two stainless steel needles serve as entrances. The inlet for the O2-saturated infusion medium ends directly above the magnetic stirring bar. The second needle, which does not project into the 1T. Noll, H. de Groot, and P. Wissemann, Biochem. J. 236, 765 (1986). 2H. de Groot, A. Littauer, D. Hugo-Wissemann, P. Wissemann, and T. Noll, Arch. Biochem. Biophys. 264, 591 (1988). Copyright © 1990by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 186
All rights of reproduction in any form reserved.
