96, 201-207 (1979)

Separation of Acetanilide and Its Hydroxylated Metabolites and Quantitative Determination of “Acetanilide 4-Hydroxylase Activity” by High-Pressure Liquid Chromatography THOMAS Developmental

M. GUENTHNER, Pharmacology National

Branch, Institutes




National Institute of Child Health of Health. Bethesda, Maryland


and Human 20014


Received October 27, 1978 A simple and very sensitive method for the separation of 4-hydroxyacetanilide, 3-hydroxyacetanilide, 2-hydroxyacetanilide, and acetanilide was developed with the use of high-pressure liquid chromatography. Each of these phenolic derivatives can be separated completely from acetanilide and from one another. A simple assay for “acetanilide 4-hydroxylase activity” is thus described. The limit of sensitivity for cytochrome P-450mediated acetanilide 4-hydroxylase activity is estimated to be 1.O pmof/min/mg microsomal protein, thereby allowing this assay to be useful in detecting monooxygenase activity in “low level” nonhepatic tissues. Hepatic acetanilide 4-hydroxylase activity is induced about fourfold in C57BL/6N mice by 3-methylcholanthrene. Although acetanilide 2hydroxylase activity is about seven times lower than the 4-hydroxylase activity, the 2-hydroxylase is also induced about three- or fourfold in C57BL/6N mice by 3-methylcholanthrene. The “Zhydroxylase activity” cannot, however, be strictly quantitated under the conditions described herein. The K, values of both the 3-methylcholanthrene-induced and control 4-hydroxylase activity are about 0.55 mM; V,,, values for 3-methylcholanthrenetreated and control mice, respectively, are 4.9 2 1.1 and 1.1 + 0.31 nmoUmin/mg microsomal protein. The 4-hydroxylase in the liver of both 3-methylcholanthrene-treated and control mice appears to represent two or more catalytic activities, i.e., two or more forms of P-450 having widely differing affinities for the substrate acetanilide.

Aryl hydroxylation is an important route of metabolism for aromatic compounds. Cytochrome P-450-mediated microsomal monooxygenases catalyze the oxidation of a large number of aromatic substrates, and it has long been recognized that more than one form of “aryl hydroxylase” exists. Studies in our laboratory with the rabbit (1,2) and the rat and mouse (3) have demonstrated differences in the temporal control of two polycyclic aromatic compound-inducible microsomal monooxygenase activities: one is associated with 3-methylcholanthrene (MC)-induced’ PI450 and specifically catalyzes the oxidation r Abbreviations used: MC, 3-methylcholanthrene; hplc, high-pressure liquid chromatography.

of benzo[a]pyrene and other polycyclic hydrocarbons; the other is associated with MC-induced P-448 and catalyzes the 4hydroxylation of acetanilide and the N-hydroxylation of 2-acetylaminofluorene. Because 2-acetylaminofluorene is a potent hepatocarcinogen, we chose to develop as a marker for MC-inducible P-448 a sensitive assay for acetanilide 4-hydroxylase activity. The microsomal monooxygenase-catalyzed conversion of acetanilide to 4hydroxyacetanilide has been studied by calorimetric (4) and radiometric (5,6) assays. The calorimetric assay lacks sensitivity, and the radiometric assays have the disadvantage of being either tedious or incapable of measuring all products of the 201

0003-2697/79/090201-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproductmn in any form reserved.



reaction. We present here a method for assaying the hydroxylation of acetanilide that utilizes high-pressure liquid chromatography (hplc) for the separation of metabolites. With the use of double-label radioisotope counting techniques, moreover, each metabolite can be quantitated. The method is highly sensitive, rapid, and capable of measuring the formation of not only the main product of the reaction, 4&ydroxyacetanilide, but other minor products as well. MATERIALS


Materials. Acetanilide and 4-hydroxyacetanilide were purchased from Eastman Organic (Rochester, N. Y .). 3-Hydroxyand 2-hydroxyacetanilide were generously given to us by Dr. Jack Hinson (National Heart, Lung, and Blood Institute, Bethesda, Md.). These four chemicals were recrystallized from ethanol and water and were judged to be greater than 99% pure by inspection with hplc. [14C]Acetanilide (11 mCi/mmol) was bought from California Bionuclear (Sun Valley, Calif.). [‘“ClAcetanilide was mixed with nonlabeled acetanilide to achieve a final specific radioactivity of 0.5 mCi/mmol. Generally tritiated 4-hydroxy[3H]acetanilide (400 mCi/ mmol), purchased from New England Nuclear (Boston, Mass.) was mixed with nonlabeled 4-hydroxyacetanilide to achieve a final specific radioactivity of 5.0 mCi/ mmol. Water for hplc was processed through a Miflipore Mini-Q purification system (Millipore Corporation, Bedford, Mass.). “High-pressure liquid chromatography grade” methanol was obtained from Fisher Scientific Company (Silver Spring, Md.). Weanling male C57BL/6N inbred mice were supplied by the Veterinary Resources Branch, National Institutes of Health. methods . Liver microsomes from control or MC-treated mice were prepared as previously described (7), resuspended in 0.05

M Tris chloride-lo%

glycerol (10 mg protein/ml), and stored at -80°C until used. As described, assay conditions were optimized empirically and found to be the foIlowing. Each sample contained 0.25 ml of 0.2 M Tris chloride buffer, pH 7.6; 0.03 ml of 0.1 M MgC&, 0.62 ml water, 0.3 mg each of NADPH and NADH. To this mixture in a 25-ml Erienmeyer Aask was added 50 ~1 of a microsomal suspension (which was approximately 0.5 mg of protein). Blanks were incubated without microsomes. The reaction was initiated by the addition of 3.0 gmol of [‘4C]acetanilide (0.5 mCi/ mmol) in 25 ~1 of acetone. Following a lo-min incubation at 37”C, the reaction was terminated by adding 2.5 ml of ice-cold ethylacetate and placing the flask on ice. At this point, microsomes were added to the blanks. The incubation mixtures were transferred to extraction tubes containing approximately 300 mg NaCl and 0.1 pmol of 4-hydroxy[3H]a~etanilide (5.0 mC~mmo1) in 10 ~1 of acetone. After the stoppered tubes had been shaken for 10 min at 4°C the organic phase was removed and saved. The aqueous layer was again extracted with 2.5 ml of ethylacetate, and the two extracts were combined. The solvent was then removed under a nitrogen stream at room temperature; the use of high temperatures or air for evaporation promoted nonenzymic oxidation of acetanilide. The dried extracts were redissolved in 7.5 ~1 of methanol, and 2.5 ~1 of this solution was subjected to hplc analysis. Because 4-hydroxy[3H]acetanilide is the internal standard, there is no need for a quantitative recovery of the organic layer, although recovery was always found to be greater than 95%. The separation of hydroxylated products from the parent compound was achieved by using a reverse-phase microparticle hplc column. A Waters Associates (Milford, Mass.) liquid chromatograph with a U6K injector and a Waters ODS column (4.6 x 25 cm) was used. Similar results





were also obtained using a DuPont Model 830 chromatograph with a DuPont Zorbax (R) ODS column (Wilmington, Del.). The sample was eluted with methanol-water (33:67 by volume) at a flow rate of 2 ml/ min. The volume of the flow cell was 15.5 ~1, and the length of the light path was 10 mm. Optical density of the eluted material was examined at 254 nm. Each fraction (either a volume of about 0.33 ml 4 or the entire amount corresponding to the 0.10 0.0. UNITS 4-hydroxyacetanilide absorbance peak) was collected in a scintillation vial, dissolved in 10 ml of Aquasol (New England Nuclear, Boston, Mass.), and counted simulI i 6 8 taneously for 14C and DH radioactivity in a RETENTION TIME lminl Packard Tri-Carb scintillation counter FIG. 1. High-pressure liquid chromatogram of 4(Grove, Ill.). Disintegrations per minute of hydroxy-, 3-hydroxy-. 2-hydroxyacetanilide, and ‘*C, divided by the specific activity of the acetanilide. About 12.5 pg of each of the four substrate, equals the amount of product re- compounds in a total volume of 25 ~1 of methanol covered. Disintegrations per minute of 3H was injected. Approximately 0.33-m] fractions were recovered in the same sample, divided by collected. the number of disintegrations per minute equivalent to the amount of tritiated product somes had been incubated with the subadded to the original extraction, equals the strate [14C]acetanilide. Essentially the efficiency of recovery. With the use of these same retention time was found (right) for two values, the total amount of product the tritiated internal standard added after formed (nmo~mi~mg protein) is easily the incubation. calculated. RESULTS Known Standards A sample containing 4-hydroxy-, 3-hydroxy-, 2-hydroxyacetanilide, and the parent compound was injected into the liquid chromatograph (Fig. I), and an excellent separation of each of the three phenolic metabolites from the parent substrate and from one another can be seen. Approximate retention times were 2.3, 2.8, 3.3, and 4.8 min for 4-hydroxy-, 3-hydroxy-, 2hydroxyacetanilide, and acetanilide, respectively. An excellent correlation was found (Fig. 2) between the 4-hydroxy product detected optically (at left) and radiometrically (center) when control micro-

Dependence of Enzyme Activity on Protein, Incubation Time, and pH Acetanilide 4-hydroxylation required NADPH (data not illustrated) and was linear (Fig. 3) when 0.50 mg of microsomal protein or less was used. The formation of 4-hydroxy product remained linear for at least 40 min when the reaction mixture was shaken at 37°C. Substitution of a NADPH-regenerating system (glucose 6phosphate and glucose-6-phosphate dehydrogenase) for NADPH did not alter the linearity of enzyme activity as a function of time (data not shown). The pH profile for the enzyme in microsomes from both MCtreated and control mice showed very little difference between pH values of 7.2 and 8.5 (Fig. 3).










2.4 min

‘r I.10 0 D. UNITS



2.3 min 1 lO.Mx) ;pm ‘H INTERNAL,STANDARC


L -L 0

L 2 4 6 RETENTION TIME iminl

L 0





FIG. 2. Comparison of absorbance (left) and 14C scintillation spectrometry (center) for detecting biologically generated metabolites when [Ylacetanilide was added to control microsomes in the reaction mixture. Also shown (right) is the tritium peak from the internal standard 4-hydroxy[3H]acetanilide, which had been added to this same reaction mixture following the IO-min incubation at 37°C. The ratio of peak heights between the 4-hydroxy product and the parent compound cannot be directly compared between the absorbance experiment (left) and the 14C experiment (center), because the carrier 4-hydroxy[sH]acetanilide is measured by absorbance but not by “C scintillation spectrometry. Retention times, with arrows, are shown for all three chromatograms. Approximately 0.33-m] fractions were collected.

Differences between MC-Treated Control C57BLl6N Mice


Approximately fourfold greater acetanilide 4-hydroxylase activity was found (Fig. 4) in


MC-treated than in control mice. Of interest, 2-hydroxyacetanilide formation was also MC-inducible (depicted by arrow, Fig. 4, left). This assay clearly cannot be used to quantitate acetanilide 2-hydroxylase ac-



1.0 mg protein/FLASK



10 20 30 min INCUBATION




6.0 PH



FIG. 3. Formation of 4-hydroxyacetanilide: in nanomoles per minute as a function of microsomal protein concentration (left); in nanomoles per milligram protein as a function of incubation time (center); and in nanomoles per minute per milligram protein as a function of pH of the reaction mixture (right). MC microsomes only are shown at left and center; both MC and control microsomes are shown at right. For these studies the entire 4-hydroxyacetanilide peak (approximately 1.5 ml) was collected.











$ L 4









FIG. 4. High-pressure liquid chromatogram of acetanilide and its metabolites generated by hepatic microsomes from MC-induced (left) and control (right) mice. Approximately 0.33-m] fractions were collected.

tivity, however, because of another radioactive (unidentified) metabolite having a retention time of about 3.6 min (Fig. 2, center; Fig. 4, right). Both a-naphthoflavone and benzo[a]pyrene preferentially inhibited acetanilide 4- and 2-hydroxylase activities in MC microsomes, compared with those activities in control microsomes (data not shown). These results are consistent with other studies from this laboratory [Refs. (2,8); reviewed in Ref. (9)]: both PI-450and P-44%associated monooxygenase activities are extremely sensitive to inhibition by a-naphthoflavone in vitro. Very small unidentified peaks with approximate retention times of 1.3, 3.6, and 6.1 min with control microsomes and 6.1 min with MC microsomes were also observed (Fig. 4; also Fig. 2). These peaks may represent contamination of the starting acetanilide substrate by other unknown chemicals. N-Hydroxyacetanilide has a broad tailing peak with a retention time greater than that for acetanilide (unpublished data); the greater peak width and

longer retention time probably represent a pH equilibrium between ionized and nonionized N-hydroxyacetanilide. The small 6.1-min peak thus probably reflects the Nhydroxy derivative. None of these very minor peaks appeared to be MC inducible. Both the control and MC-induced acetanilide 4-hydroxylase activities appear to have the same K, value of approximately 0.55 mM (Fig. 5). A similar K, value was found on several repeated experiments with C57BL/6N mice and with several other inbred strains. V,,, values for MC-treated and control C57BL/6N mice, respectively, were 4.9 2 1.1 and 1.1 t 0.31 nmol/min/mg protein. At increased substrate concentrations (Fig. 5), the control enzyme appeared to become inhibited whereas the MC-induced activity did not. When the data are examined with the use of an Eadie-Hofstee plot, both the MC-induced and control 4-hydroxylase activities can each be seen to reflect most likely two or more distinct enzyme-active sites, having widely different affinities for the substrate acetanilide.




FIG. 5. Lineweaver-Burk (top) and Eadie-Hofstee (bottom) plots for liver acetanilide 4-hydroxylase activity from MC-treated and control C57BL/6N mice. For these studies, the entire 4-hydroxyacetanilide peak (about 1.5 ml) was collected.


The technique presented here provides a rapid and sensitive method for assaying the microsomal hydroxylation of acetanilide , Our method offers a great increase in sensitivity over the previously published colotimetric technique (4), and an improvement in rapidity and specificity over previously published radiometric methods (5~5). Four dozen samples can be assayed with ease by one person in the same day. We have also used this assay successfuily in determining acetanilide 4-hydroxylation in such “low activity” nonhepatic tissues as bone marrow, spleen, intestinal epithelium, and various fetal tissues. By increasing the amount of specific radioactivity in the assay, we estimate that the limit of sensitivity of 4-hydroxyacetanilide formation is 1.O pmol/ min/mg protein. Such low-level determina-


tions are not possible using previously published techniques. Daly has published (5) a sensitive technique which assays aryl hydroxylase activity by measuring the release of tritium into the aqueous medium. This assay, however, has drawbacks in that the 4-hydroxy-, 3-hydroxy-, and 2-hydroxyacetanilide products cannot be separately quantitated. Further, the “NIH shift” may prevent the quantitative release of tritium [discussed in Ref. (lo)] and therefore may complicate the interpretation of results. Selander and co-workers more recently reported (6) a method which separates the 4-hydroxy, 3-hydroxy, and 2-hydroxy metabolites from the substrate acetanilide. Their method, however, requires thin-layer chromatography clean-up, is less sensitive because double-labeling is not used, and the hplc takes about three times longer per sample than the method described in this report. MC-Induced acetanilide 4-hydroxylase activity has been known (8) to be associated with the murine Ah locus. Although 2-hydroxyacetanilide formation is about seven times lower than 4-hydroxyacetanilide formation, we suspect that acetanilide 2-hydroxylase induction by MC is also correlated with the Ah locus. This result may indicate that the same MC-induced enzymeactive site which oxygenates acetanilide at the 3,4-ring positions also oxygenates acetanilide-to a lesser extent-at the 2,3ring positions; the proposed (highly unstable) 3,4- and 2,3-arene oxides probably form nonenzymicaily the 4- and 2-hydroxy products, respectively. A second form of MC-induced P-450, however, may be principally responsible for the 2-hydroxylase activity. The fact that multiple forms of cytochrome P-450 handle the same substrate with varying degrees of specificity has been suspected for years (9,11-19). This concept is finally becoming more widely appreciated and accepted. The enzyme assay reported here has now become commonplace in our laboratory for determining catalytic activity of a certain inducible form



of P-450: MC-induced P-448, a microsomal membrane-bound structural gene product under temporal control and different from PI-450 but ultimately governed by the Ah regulatory gene (3,9). ACKNOWLEDGMENTS We thank Mr. Bruce M. Kisliuk for valuable technical assistance. The expert secretarial assistance of Ms. Ingrid E. Jordan is also greatly appreciated.

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4. Mitoma, C., and Udenfriend, S. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, p. 816, Academic Press, New York. 5. Daly, J. W. (1970) Anal. Biochem. 33, 286. 6. Selander, H. G., Jerina, D. M., and Daly, J. W. (1974) Arch. Biochem. Biophys. 164, 241. 7. Boobis, A. R., Nebert, D. W., and Felton, J. S. (1977) Mol. Pharmacol. 13, 259.




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Separation of acetanilide and its hydroxylated metabolites and quantitative determination of "acetanilide 4-hydroxylase activity" by high-pressure liquid chromatography.

ANALYTICAL BIOCHEMISTRY 96, 201-207 (1979) Separation of Acetanilide and Its Hydroxylated Metabolites and Quantitative Determination of “Acetanilid...
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