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A practical radiochromatographic assay for cholesterol epoxide hydrase
ANALYTICAL
BIOCHEMISTRY
94,
383-385
(1979)
A Practical Radiochromatographic Assay For Cholesterol Epoxide Hydrasel HOMER S. BLACK Photobiology
A. LENGER
AND WANDA
Laboratory, Veterans Administration Hospital, Biochemistry, Baylor College of Medicine,
and Departments Houston. Texas
of Dermatology
and
77030
Received July 12, 1978 A method for the assay of cholesterol epoxide hydrase activity is described. The assay involves the thin-layer chromatographic separation and quantitation of radiolabeled cholestan-3P,%,6a-epoxide and its major hydration product, cholestan-3/3,5ru,6@riol. Radiochromatographic scanning is employed to quantitate the reaction. The procedure is sensitive, rapid, and nondestructive.
MATERIALS
Cholestan-3&5a,6a-epoxide (CAE)2 is formed in both human and mouse skin upon exposure to ultraviolet (uv) radiation (1 - 3). This sterol has been reported to be carcinogenic (4). More recent studies, however, suggest that if this sterol is involved in the etiology of uv carcinogenesis, it is not as an ultimate carcinogen and further metabolism of the compound must be involved (5). Thus a practical means of studying the metabolism of CAE is required. Although several methods for assay of hydratases of polycyclic hydrocarbon-derived expoxides have been described (6- 1 l), one of the most rapid methods involves partitioning of substrate and major reaction product by cross-extraction into a lipophilic- hydrophilic biphasic system (12). However, because of the polar characteristics of both CAE and its major metabolite, the assay method described here avoids the need for solvent searches to find suitable crossextraction systems and enjoys a further advantage in that it is nondestructive to either substrate or product.
AND METHODS
Chemicals. Radiolabeled cholestan3/3,5a,6a-epoxide was prepared from 4[‘4C]cholesterol (New England Nuclear, Boston, Massachusetts) by the method described previously (13). Cholestan-3&5a,6ptrio1 (triol) was prepared by reacting [“C]cholesterol with formic acid, treating with hydrogen peroxide, followed by hydrolysis (14). Both radioactive compounds were purified, after recrystallization, by thinlayer chromatography (tic). Radiopurity was at least 98%. Preparation of liver homogenates. Hairless mice (SKH-HR-1) were sacrificed by cervical dislocation and the livers excised. The livers were weighed and homogenized in 4 vol of Krebs-Ringer phosphate buffer (pH 7.4) with a Polytron 10 equipped with saw-toothed generator. The homogenate was centrifuged at 1OOOgfor 15 min at 5°C and the supernatant (enzyme) used in the assay. Protein was determined according to the method of Lowry et al. (15) and the supernatant fraction, prepared as described, usually contained about 25 mg protein/ml. Assay. Ten microliters of radiolabeled CAE (95 PCi; 4.24 mCi/mmol specific activity) was transferred to 16 x loo-mm tubes
1 This investigation was supported in part by Grant CA-1346446awarded by the National Cancer Institute, DHEW. ’ Abbreviations used: CAE, cholestan-3/3,5a,6cepoxide; triol, cholestan3P,5~,6/Mriol. 383
0003-2697/79/060383-03$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
BLACK
AND
LENGER 25 -
-2
FIG. 1. Linearity of detector response for epoxide hydrase quantitation. Ordinate represents detector response in counts per minute of disk integrator. Abscissa represents quantity of cholesterol epoxide. The radiolabeled epoxide was spotted on tic plates, developed, and scanned as in the described assay.
containing 10 vg of nonlabeled substrate. The sterol, a total of 19 pg, was dried under Nz and solubilized in 100 ~1 of Tween 80 solution (86.6 mg of Tween 80 in 1% ethanol). One milliliter of enzyme was added and the reaction carried-out at 37°C for 30 min. The reaction was halted by addition of 5 ml of chloroform:methanol(2:1, v/v). Total lipids were extracted twice with this solvent and
0
2
4 s
6
8
, 10
FIG. 3. Relation of substrate concentration to reaction rate. The assays were performed as described under Materials and Methods. Values represent the mean of duplicate samples from at least two experiments. S is expressed as micromolar CAE and ranges from 0.22 to 8.41 FM. Values for V are expressed as microgram of product formed/per milligram protein/ per hour.
finally with chloroform. Nonlipid contaminants were removed by chromatography on a Sephadex G-25 (100-300 pm) column (16). The pooled lipid extract was dried in vacua and made to 0.5 ml with chloroform: methanol. One-fifth of the sample was spotted on high performance tic plates (Silica gel 60, EM Laboratories Incorporated, Elmsford, N. Y .) and developed in chloroforrnacetone (8:2, v/v). Quantitation was accomplished with a Berthold Model LB 2760 tic scanner to which was attached a recorder with disk integrator. Thin-layer chromatography plates were scanned under the following conditions: detector bandwidth 2 mm; 10-s time constant; propane gas flow 7-8 ml/mitt; scanner drive speed 600 mm/h; and recorder speed 1 cm/min.
Minutes
FIG. 2. Enzymatic hydration as a function of time. Values two experiments.
of cholesterol epoxide represent the mean of
FIG. 4. Enzymatic as a function of pH.
hydration
of cholesterol
epoxide
CHOLESTEROL
EPOXIDE
RESULTS AND DISCUSSION
The cholesterol epoxide hydrase assay method described here measures the hydration reaction of cholestan-3/3,5a,6aepoxide to cholestan-3/3,5a,6@triol. Although there are other intermediates resulting from metabolism of CAE, about 90% of the initial radioactivity in the reaction mixture can be recovered as the epoxide or trio1 in the 30-min incubation period. A requisite for quantitative tic is linearity between amount of compound present and the respective detector response. Stevenson (17) has discussed several reasons for departure from linearity in tic quantitation. Notably, differences in sample mass, resulting from uneven spot size, can attenuate weak /3 emitters such as 14C and 3H. In addition, the tic absorbent itself can markedly attenuate these isotopes and result in erroneous counting efficiencies for different sample sizes. Whereas the latter can be mathematically corrected, this was not necessary in the present case where ratio of peak areas were used to calculate percentage conversion of substrate. Further, linearity of detector response, over the range of radioactivities used in the assay, was shown to hold (Fig. 1). It can also be seen that quantities of substrate, in the range of 1 to 2 nmol could be accurately measured, although sensitivity could be greatly enhanced using a substrate of higher specific activity. Using this assay procedure, the timecourse for epoxide hydration was examined. As can be seen in Fig. 2, the reaction approached linearity through 30 min and thereafter reached a plateau. This time was chosen for the standard assay. The validity of the assay procedure is further demonstrated in Fig. 3 where effect of substrate concentration on reaction rate is represented. An apparent K, of 1.49 PM and V,,, of 0.452 Fg/mg protein/h. were calculated from the plot (18). The substrate concentration used in our assay, 4.70 PM, approaches saturation levels and yet pro-
HYDRASE
ASSAY
385
vides sufficient conversion of substrate to be accurately quantitated. To achieve the latter, at least 5% of the substrate must be converted. The reaction conditions were further characterized when the effect of pH on reaction rate was determined. Using a series of Tris and Krebs phosphate buffers the pH optimum was shown to be 7.4 (Fig. 4). In conclusion, a sensitive radiochromatographic sterol epoxide hydrase assay procedure is described which is more rapid than previous radiometric assays and is nondestructive. REFERENCES Black, H. S., and Lo, W. B. (1971) Nature (London)
234, 306-308.
Lo, W. B., and Black, H. S. (1972) .Z. Invest. Dermatol.
58, 278-283.
Black, H. S., and Douglas, D. R. (1973) Cancer Res. 33, 2094-20%.
Bischoff, F. (1%9) In Advances in Lipid Research Vol. 7, pp. 165-244, Academic Press, New York. Black, H. S., and Chan, J. T. (1976) Oncology 33, 119-122. Oesch, F., Jerina, D. M., and Daly, J. (1971)Arch. Biochem. Biophys. 144, 253-261. Leutz, J. D., and Gelboin, H. V. (197.5) Arch. Biochem. Biophys. 168, 722-725. Leibman, K. C., and Ortiz, E. (1969) Biochem. Pharmacol. 18, 552-554. ,. Stoming, T. A., and Bresnick, E. (1973) Science 181, 951-952. 10. Dansette, P. M., Yagi, H., Jerina, D. M., Daly, J. W., Levin, W., Lu, A. Y. H., Kuntzman, R., and Conney, A. H. (1974) Arch. Biochem. Biophys. 164, 511-517. 11. Nesnow, S., and Heidelberger, C. (1975) Anal. Biochem.
67, 525-530.
12. Schmassmann, H. U., Glatt, H. R., and Oesch, F. (1976) Anal. Biochem. 74, 94- 104. 13. Chan, J. T., and Black, H. S. (1976) J. Invest. Dermatol. 66, 112- 116. 14. Fieser, L. F., and Rajagopalan, S. (1949)Z. Amer. Chem. Sot. 71, 3938-3944. 15. Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 16. Christie, W. W. (1973) Lipid Analysis, Pergamon, Long Island City, N. Y. 17. Stevenson, R. (1970) Amer. Lab., May. 18. Dixon, M., and Webb, E. C. (1964) Enzymes, 2nd ed, p. 69, Longmans Green, London.
BIOCHEMISTRY
94,
383-385
(1979)
A Practical Radiochromatographic Assay For Cholesterol Epoxide Hydrasel HOMER S. BLACK Photobiology
A. LENGER
AND WANDA
Laboratory, Veterans Administration Hospital, Biochemistry, Baylor College of Medicine,
and Departments Houston. Texas
of Dermatology
and
77030
Received July 12, 1978 A method for the assay of cholesterol epoxide hydrase activity is described. The assay involves the thin-layer chromatographic separation and quantitation of radiolabeled cholestan-3P,%,6a-epoxide and its major hydration product, cholestan-3/3,5ru,6@riol. Radiochromatographic scanning is employed to quantitate the reaction. The procedure is sensitive, rapid, and nondestructive.
MATERIALS
Cholestan-3&5a,6a-epoxide (CAE)2 is formed in both human and mouse skin upon exposure to ultraviolet (uv) radiation (1 - 3). This sterol has been reported to be carcinogenic (4). More recent studies, however, suggest that if this sterol is involved in the etiology of uv carcinogenesis, it is not as an ultimate carcinogen and further metabolism of the compound must be involved (5). Thus a practical means of studying the metabolism of CAE is required. Although several methods for assay of hydratases of polycyclic hydrocarbon-derived expoxides have been described (6- 1 l), one of the most rapid methods involves partitioning of substrate and major reaction product by cross-extraction into a lipophilic- hydrophilic biphasic system (12). However, because of the polar characteristics of both CAE and its major metabolite, the assay method described here avoids the need for solvent searches to find suitable crossextraction systems and enjoys a further advantage in that it is nondestructive to either substrate or product.
AND METHODS
Chemicals. Radiolabeled cholestan3/3,5a,6a-epoxide was prepared from 4[‘4C]cholesterol (New England Nuclear, Boston, Massachusetts) by the method described previously (13). Cholestan-3&5a,6ptrio1 (triol) was prepared by reacting [“C]cholesterol with formic acid, treating with hydrogen peroxide, followed by hydrolysis (14). Both radioactive compounds were purified, after recrystallization, by thinlayer chromatography (tic). Radiopurity was at least 98%. Preparation of liver homogenates. Hairless mice (SKH-HR-1) were sacrificed by cervical dislocation and the livers excised. The livers were weighed and homogenized in 4 vol of Krebs-Ringer phosphate buffer (pH 7.4) with a Polytron 10 equipped with saw-toothed generator. The homogenate was centrifuged at 1OOOgfor 15 min at 5°C and the supernatant (enzyme) used in the assay. Protein was determined according to the method of Lowry et al. (15) and the supernatant fraction, prepared as described, usually contained about 25 mg protein/ml. Assay. Ten microliters of radiolabeled CAE (95 PCi; 4.24 mCi/mmol specific activity) was transferred to 16 x loo-mm tubes
1 This investigation was supported in part by Grant CA-1346446awarded by the National Cancer Institute, DHEW. ’ Abbreviations used: CAE, cholestan-3/3,5a,6cepoxide; triol, cholestan3P,5~,6/Mriol. 383
0003-2697/79/060383-03$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
BLACK
AND
LENGER 25 -
-2
FIG. 1. Linearity of detector response for epoxide hydrase quantitation. Ordinate represents detector response in counts per minute of disk integrator. Abscissa represents quantity of cholesterol epoxide. The radiolabeled epoxide was spotted on tic plates, developed, and scanned as in the described assay.
containing 10 vg of nonlabeled substrate. The sterol, a total of 19 pg, was dried under Nz and solubilized in 100 ~1 of Tween 80 solution (86.6 mg of Tween 80 in 1% ethanol). One milliliter of enzyme was added and the reaction carried-out at 37°C for 30 min. The reaction was halted by addition of 5 ml of chloroform:methanol(2:1, v/v). Total lipids were extracted twice with this solvent and
0
2
4 s
6
8
, 10
FIG. 3. Relation of substrate concentration to reaction rate. The assays were performed as described under Materials and Methods. Values represent the mean of duplicate samples from at least two experiments. S is expressed as micromolar CAE and ranges from 0.22 to 8.41 FM. Values for V are expressed as microgram of product formed/per milligram protein/ per hour.
finally with chloroform. Nonlipid contaminants were removed by chromatography on a Sephadex G-25 (100-300 pm) column (16). The pooled lipid extract was dried in vacua and made to 0.5 ml with chloroform: methanol. One-fifth of the sample was spotted on high performance tic plates (Silica gel 60, EM Laboratories Incorporated, Elmsford, N. Y .) and developed in chloroforrnacetone (8:2, v/v). Quantitation was accomplished with a Berthold Model LB 2760 tic scanner to which was attached a recorder with disk integrator. Thin-layer chromatography plates were scanned under the following conditions: detector bandwidth 2 mm; 10-s time constant; propane gas flow 7-8 ml/mitt; scanner drive speed 600 mm/h; and recorder speed 1 cm/min.
Minutes
FIG. 2. Enzymatic hydration as a function of time. Values two experiments.
of cholesterol epoxide represent the mean of
FIG. 4. Enzymatic as a function of pH.
hydration
of cholesterol
epoxide
CHOLESTEROL
EPOXIDE
RESULTS AND DISCUSSION
The cholesterol epoxide hydrase assay method described here measures the hydration reaction of cholestan-3/3,5a,6aepoxide to cholestan-3/3,5a,6@triol. Although there are other intermediates resulting from metabolism of CAE, about 90% of the initial radioactivity in the reaction mixture can be recovered as the epoxide or trio1 in the 30-min incubation period. A requisite for quantitative tic is linearity between amount of compound present and the respective detector response. Stevenson (17) has discussed several reasons for departure from linearity in tic quantitation. Notably, differences in sample mass, resulting from uneven spot size, can attenuate weak /3 emitters such as 14C and 3H. In addition, the tic absorbent itself can markedly attenuate these isotopes and result in erroneous counting efficiencies for different sample sizes. Whereas the latter can be mathematically corrected, this was not necessary in the present case where ratio of peak areas were used to calculate percentage conversion of substrate. Further, linearity of detector response, over the range of radioactivities used in the assay, was shown to hold (Fig. 1). It can also be seen that quantities of substrate, in the range of 1 to 2 nmol could be accurately measured, although sensitivity could be greatly enhanced using a substrate of higher specific activity. Using this assay procedure, the timecourse for epoxide hydration was examined. As can be seen in Fig. 2, the reaction approached linearity through 30 min and thereafter reached a plateau. This time was chosen for the standard assay. The validity of the assay procedure is further demonstrated in Fig. 3 where effect of substrate concentration on reaction rate is represented. An apparent K, of 1.49 PM and V,,, of 0.452 Fg/mg protein/h. were calculated from the plot (18). The substrate concentration used in our assay, 4.70 PM, approaches saturation levels and yet pro-
HYDRASE
ASSAY
385
vides sufficient conversion of substrate to be accurately quantitated. To achieve the latter, at least 5% of the substrate must be converted. The reaction conditions were further characterized when the effect of pH on reaction rate was determined. Using a series of Tris and Krebs phosphate buffers the pH optimum was shown to be 7.4 (Fig. 4). In conclusion, a sensitive radiochromatographic sterol epoxide hydrase assay procedure is described which is more rapid than previous radiometric assays and is nondestructive. REFERENCES Black, H. S., and Lo, W. B. (1971) Nature (London)
234, 306-308.
Lo, W. B., and Black, H. S. (1972) .Z. Invest. Dermatol.
58, 278-283.
Black, H. S., and Douglas, D. R. (1973) Cancer Res. 33, 2094-20%.
Bischoff, F. (1%9) In Advances in Lipid Research Vol. 7, pp. 165-244, Academic Press, New York. Black, H. S., and Chan, J. T. (1976) Oncology 33, 119-122. Oesch, F., Jerina, D. M., and Daly, J. (1971)Arch. Biochem. Biophys. 144, 253-261. Leutz, J. D., and Gelboin, H. V. (197.5) Arch. Biochem. Biophys. 168, 722-725. Leibman, K. C., and Ortiz, E. (1969) Biochem. Pharmacol. 18, 552-554. ,. Stoming, T. A., and Bresnick, E. (1973) Science 181, 951-952. 10. Dansette, P. M., Yagi, H., Jerina, D. M., Daly, J. W., Levin, W., Lu, A. Y. H., Kuntzman, R., and Conney, A. H. (1974) Arch. Biochem. Biophys. 164, 511-517. 11. Nesnow, S., and Heidelberger, C. (1975) Anal. Biochem.
67, 525-530.
12. Schmassmann, H. U., Glatt, H. R., and Oesch, F. (1976) Anal. Biochem. 74, 94- 104. 13. Chan, J. T., and Black, H. S. (1976) J. Invest. Dermatol. 66, 112- 116. 14. Fieser, L. F., and Rajagopalan, S. (1949)Z. Amer. Chem. Sot. 71, 3938-3944. 15. Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 16. Christie, W. W. (1973) Lipid Analysis, Pergamon, Long Island City, N. Y. 17. Stevenson, R. (1970) Amer. Lab., May. 18. Dixon, M., and Webb, E. C. (1964) Enzymes, 2nd ed, p. 69, Longmans Green, London.