A new method for determining liver microsomal cholesterol 7α-hydroxylase

663

A NEW METHOD FOR DETERMINING LIVER MICROSOMAL CHOLESTEROL 7=HYDROKYLASE M. R. Malinow, Phyllis McLaughlin,

Lynne Papworth and G. W. Kittinger

Oregon Regional Primate Research Center, Beaverton, Oregon 97005 and University of Oregon Medical School, Portland, Oregon 97201

ABSTRACT A method is described to determine the mass of 7o-hydroxy cholesterol synthetized in vitro by liver microsomes without the use of a radioactive substrate. Manuscript received on:

2126175 INTRODUCTION

Current methods for determining microsomal cholesterol 7a-hydroxylase are based on the incorporation of radioactive cholesterol into labeled 7U-OH cholesterol

[l-6].

the existing assay procedures: nonradioactive

There are three major criticisms of (1) they fail to quantify the endogenous

substrate pool that dilutes the radioactive substrate;

(2) the physical state of the added radioactive cholesterol may affect the labeling of the substrate [7]; (3) a double labeling procedure is required to determine the mass of the product formed [5, 8, 91.

To

determine the activity of microsomal cholesterol 7o-hydroxylase in the liver of rats, we have designed a method that measures the mass of 7ffOH cholesterol synthesized in vitro without the addition of a radioactive substrate. MATERIALS AND METHODS Principle of the method The activity of cholesterol 7o-hydroxylase is determined by the maximum rate at which 7o-OH cholesterol can be synthetized from endogenous cholesterol in incubated liver microsomes under conditions of excess substrate oncentration. After the reaction has been stopped with methanol, [7$H] 7o-OH cholesterol is added to the incubated microsomes as an internal standard. The product is then isolated by thin-

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layer chromatography matography (GLC).

(TLC) and its mass measured by gas liquid chro-

Reagents Chemicals were obtained from the following commercial sources: g-mercaptoethylamine and glucose-6-phosphate dehydrogenase (Sigma Chemical Co., St. Louis, MO.); cholesterol, D-glucose-6 phosphate, and NADP+ (Calbiochem, San Diego, Ca.); 7-oxocholesterol, cholesteryl igobutyrate, 7U-, and 78-OH cholesterol (Steraloids, Pawling, N.Y.); [ H] NaBH4 (New England Nuclear, Boston, Mass.) (S.A. ( 185 mCi/mmole). All other reagents and solvents were of analytical grade. Synthesis of [73H] 7o-OH cholesterol 7-oxocholesterol (0.07 mmoles) was dissolved in 9 ml of methanol and placed in a bath maintained at 4" C. Twenty-five mCi of tritium labeled NaBH4 (0.14 mmoles) were added to the methanolic solution in small portions. After standing overnight, 8 ml of water were added and the steroids extracted three times with 30 ml of petroleum ether (b.p. 60" C). The extracts were c?bined and evaporated to dryness under N2. To ensure that all remaining H label was stably linked to the 7-C position the steroid residue was dissolved in 5 ml of methanol. '$he solvent was distilled, collected by condensatio 3 and assayed for H in a Negligible H activity was present liquid scintillation spectrometer. The 7o- and 7$-OH cholesterol isomers in the residue in the distillate. were separated by TLC on 250 b precoated silica gel LQFD Quanta/Gram chromatography plates (Quantum Industries, Fairfield, N.J.) with authentic standards in adjacent lanes. Diethyl ether was used as the developing solvent and the Rf's of the 7o- and 7$-isomers were 0.36 and 0.47, respectively after four ascents (S.A. of 7o-OH cholesterol was theoretWhen scanned with a Packard Model 7201 scanner ical, - 40 mCi/mmole). the R 's of the radioactive peaks corresponded with those of the authentic sgandards. Preparation of microsomal fractions Sprague-Dawley rats of either sex, weighing around 350 g, were fed on Purina (R) rat chow and water ad libitum up to the time of the experiment and anesthetized with sodium pentobarbital (4 mg/lOO g) i.p. between 8:30 and 9:30 a.m. The liver was excised and imediately minced in an iced solution containing sucrose, 0.25 M; nicotinamide, 0.075 M; and potassium ethylene diamine tetra-acetate (EDTA), 0.002 M. An aliquot of 2.5 g of liver was further minced in a metal tube, delivered into 8 ml of the above media, and homogenized with 4 passes in a teflon glass homogenizing tube. After the homogenate had been centrifuged at 600 g for 10 min, the supernatant was poured off and centrifuged at 20,000 g for 15 min. The upper part of the supernatant was sliced off with a tube slicer and the rest was carefully aspirated to leave the lower mitochondrial rich fraction. The supernatant was then centrifuged for 60 min at 100,000 g. The pellet obtained was suspended in 2.5 ml of 0.154 M KC1 and centrifuged at 800 g for 5 min. The supernatant

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containing the microsomes was used on the same day for the in vitro assay. During all these steps, the preparation was kept at 4" C. Protein determination Before incubation, the protein concentration in the microsome preparation was estimated spectrometrically at 280 mlJ (Zeiss PMQ II); later assays were performed by the procedure of Lowry --et al. (10). Determination

of endogenous microsomal

cholesterol.

To 0.25 ml of microsomal preparation were added 0.25 ml of ethanol. Extraction was performed with 2 ml of petroleum ether, vortexing, and the addition of 2 ml of dichloromethane. The microsomes were extracted two more times with petroleum ether. The extracts were then combined, dried under N2, and dissolved in 100 cl1 of benzene. Adequate amounts-generally between 1 and 2 M--were used for GLC. Incubation of microsomal fraction The following medium was incubated in a covered Dubnoff shaker at 37" c: Phosphate buffer 0.1 M, pH 7.4, 2.0 ml;$-mercaptoethylamine, final concentration 10 mM, 1.0 ml; 1 ml of a NADPH generating system containing NADP+, 5 Moles; glucose-6-phosphate, 50 Nmoles; glucose-6phosphate dehydrogenase, 2 IU; microsomes, 3.0 ml (containing around 5 mg of protein). The inculpation was stopped by adding 6 ml of methanol and a known amount of [7- H] 7o-OH cholesterol was added to estimate recovery. Lipid extraction Extraction of the methanol solution of the incubate was performed with 12 ml of chloroform, vortexing and the further addition of 6 ml of ethylacetate with subsequent vortexing; 10 ml of water were then added. The tubes were vortexed, centrifuged and the water layer reduced by aspiration. This procedure was repeated and the solvent layer was aspirated, dried under N2 and dissolved in 100 c11 of benzene. Thin layer chromatography Approximately equal portions of the benzene extract were applied on three lanes of Quanta/Gram LQDF chromatography plates and authentic 7oand 78-OH cholesterol were applied on adjacent lanes. The plate was developed in a saturated tank of diethyl ether by ascending chromatography. The standards were visualized by immersing the lateral lane with the standards in a charring solution of 20 ml 80% phosphoric acid, 80 ml water, and 3 g cupric acetate. The Rf's of 7GOH cholesterol and of 78The plate was heated OH cholesterol were 0.13 and 0.21, respectively. in an oven at 70" for 5 minutes and the portions corresponding to 7~ OH cholesterol were scraped into glass stoppered tubes. Five ml of me hanol were added, vortexed for 30 set, and centrifuged. To determine f [7 H] 7o-OH cholesterol recovery, a 500 M aliquot of the supernatant was taken to dryness and the radioactivity assayed in a Packard Liquid Scintillation Spectrometer (Model 3003). Typical recoveries were

68.44 5 2.44% (mean t S-D.. N=lO). Determination

of ICY-OH cholesterol

Four ml of the supernatant from the TLC elution were dried under and 40 Wl of a pyridine solution of hexamethyldisilazene and triN2, methylchlorosilane (Tri-sil, Pierce Chemical Co., Rockford, Ill.) were added and vortexed. Adequate amounts, generally 2 to 6 ~1 containing approximately 0.1 pg of 7ar-OH cholesterol, were assayed by gas liquid chromatography in a Barber Coleman Model 5000 chromatograph with an 8 ft column of 1% SE31 on lOOf mesh Gas Chrom Q (Applied Science Laboratories, State College, Pa.). Typical conditions were: column temperature, 240" C; detector temperature, 240" C; injector temperature, 250' C; and N2 flow, 35 ccfmin. The recorded areas of peaks corresponding to 7~OH cholesterol were weighed and the mass of the sample was calculated by comparison with adequate standards of authentic 7o-OH cholesterol. The 7Cr-OH cholesterol of control samples whose reaction was stopped at 0 min was subtracted to obtain the net synthesized ICY-OH cholesterol. Identification of product Evidence that the product was identical with authentic ICY-OH cholesterol was obtained by TLC with 3 different solvent systems and by GLC of the trimethylsilyl ether. In addition, it was identified by mass spectrometry of the trimethylsilyl ether isolated by GLC on a DuPont 21491 B Gas Chromatograph Mass Spectrometer System. The mass spectrum was typical for a trimethylsilyl ether of 7-hydroxylated cholesterol, with a main peak corresponding to m/e 456. Precautions during chromatography Tubes used in the procedure were freed of organic material by being heated at 500" C in an oven muffler. Preparation of the trimethylsilyl ether as well as aspiration of the solution into the syringe for GLC, was best accomplished under N to prevent a precipitate from forming. The presence of any air bu g ble in the syringe used for injecting the sylated material into the gas chromatograph resulted in the appearance of two peaks, one with an Rf similar to ICY-OH cholesterol and an earlier peak with a mass spectrum with a main peak at m/e M-2. RESULTS Gas liquid chromatography The trimethylsilyl ether derivatives of authentic 7@-OH and 78OH cholesterol assayed by GLC under the conditions described above, had an R f of 0.57 and 0.80, respectively 1.0).

(eholesteryl isobutyrate Rf =

Figure 1 shows a calibration curve for different amounts of

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'7cu-OH cholesterol determined by GL,C.

I

20

I

I

I

40 60 80 7a-Hydroxycholesterol (ng)

Fig. 1 - Quantification of ICY-OH cholesterol by GLC. sents duplicate determinations.

I

100

Each point repre-

Recovery of authentic 7c+OH cholesterol Different amounts of 7o'-OH cholesterol together with known amounts of [7-3H] ICY-OH cholesterol

(S.A. - 40 mCi/mMole) were dissolved in

methanol and added to the incubating medium, extracted, chromatographed on thin layer plates and eluted; the mass of the trimethylsilyl ether derivative was determined by GLC as described above.

Table 1 shows the

correlation between the expected and the recovered compound. Synthesis of 7%-OH cholesterol by liver microsomes (a)

Effect of protein concentration.

sentative observations

Figure 2 shows two repre-

in which the synthesis of 7@-OH cholesterol was

assayed with different amounts of microsomal protein.

The amount of

7o'-OH cholesterol synthesized was proportional to the protein concen-

tration, 240 and 170 nanograms130 min/mg protein, respectively, within the limits studied. TABLE 1 Recovery of added X+hydroxycholesterol following extraction, TLC and GLC. Each value is the average of 2 determinations. 7a-hydroxycholesterol added (l.tg)

7a-hydroxycholesterol recovered (ng)

5.0 10.0 20.0

5.37 10.54 19.70

/c

Recovery (%> 107 105 98

240ng

7a-OH Cholesterol/mg

Protein

lsoo16CO-

0

2-

CholesteroVmg Protein

2

4

6 Protein (mg)

8

IO

Representative observations showing the synthesis of 7cr-OH cholesterol by different amounts of microsomal protein. Each point is the average of two determinations performed in duplicate. Abscissa, mg protein per incubating flask.

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Time course of the enzymic reaction.

Figure 3 shows a rep-

resentative observation where the synthesis of 7o-OH cholesterol in the microsomal preparations was studied for 45 minutes. the product was proportional

The appearance of

to the length of incubation within the time

limits of the study with a slight decrease in rate at 45 minutes.

I

4 20

30 40 Time (min)

I

50

Fig. 3 - A representative observation on the effect of varying the times of incubation on the synthesis of 7~OH cholesterol by liver Each point is the average of two determinations microsomes. performed in duplicate. (c)

Effect of added cholesterol.

The endogenous cholesterol

concentration of rat liver microsomal preparations was approximately 15 Pg/mg protein.

An increase ranging from 0 to 30% was observed in the

synthesis of 7o-OH cholesterol when 750 Pg of cholesterol dissolved in

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75 IJ.~of acetone were added to microsome incubations containing 4 to 5 mg of protein (exogenous substrate concentration of 270 mM). (d)

Enzyme activity in rats.

The rate of synthesis of 7o'-OR

cholesterol was 184.68 + 35.53 (mean + S.D., N=5) ngj20 min/mg protein and results were similar in 30 minute incubations

(see Fig. 2).

These

values are within the range reported by other authors using a double labeling procedure

(Table 2). TABLE

2

Synthesis of 7ct'-OHcholesterol by rat liver microsomes Mass in authors' units

d of animals

(Mean + S.D.).

Reduced units Reference (ng/min/mg prot)

1.33 + O.ll* n moll30 minlmg prot

4

0.5 pg/l5 min/12--18 mg prot

2

0.56 2 0.18"" n mo1/20 min/mg prot

9

11.3 5 3.6

184.68 + 35.53 ng/20 min/mg prot

5

9.2 +- 1.7

prot, protein;

17.8 t 0.5" 2.2

5 8, Fig. 3 11 present paper

*deviation not defined; **l.O p.m. DISCUSSION AND CONCLUSIONS

The present method assays the activity of liver microsomal cholesterol 7a-hydroxylase without the use of a radioactive precursor.

It

thus surmounts two important limitations of previous methods: the unknown contribution of the endogenous substrate and the possibility that enzymatic activity could be modified by agents used to disperse the exogenous substrate in the incubating medium

[5].

Moreover, the method

quantifies the mass of product formed without resorting to a double labeling procedure

[5,8,9] which necessitates careful consideration of

extraction recoveries

[9].

There are conflicting reports about the effect of exogenous substrate concentration on the activity of liver microsomal cholesterol 7o-hydroxylase.

Shefer et al. [l] demonstrated that the enzyme system

is saturated when added cholesterol attains a concentration of 208 mM in the incubating medium.

However, Bjb;khem and Danielsson

[8] reported

that the addition of cholesterol to the incubating medium did not increase the yield of product formed; thus the endogenous substrate seems to saturate the enzyme system.

Only occasionally did we observe

a minimal increase in the synthesis of 7o-OH cholesterol when the concentration of exogenous substrate rose from 0 to 270 mM.

Thus, our

results also suggest that the endogenous substrate saturates the hydroxylase

system under the present assay conditions. ACKNOWLEDGEMENTS

Publication 6781 of the Oregon Regional Primate Research Center. Aided by grants FR-00163, HL-16587 and HD-02715, National Institutes of Health. Mass spectrometry was performed at the Oregon Graduate Center with the cooperation of Dr. Doyle Daves. ADDENDUM Systematic names of the sterols are referred to in the text by their trivial name: cholesterol, cholest-5-en-38-01; 7o-OH cholesterol, cholest-5-ene-38,7Wdiol; 78-OH cholesterol, cholest-5-ene-38,7&diol; 7-oxocholesterol, cholest-5-en-3$-ol-7-one; cholesteryl isobutyrate, cholest-5-en-3$-yl isobutyrate. REFERENCES 1.

Shefer, S., S. Hauser and E. H. Mosbach, J. Lipid Res., 2, (1968).

2.

Johansson, G., Eur. J. Biochem., 2l, 68 (1971).

3.

Mitton, J. R., N. A. Scholan and G. S. Boyd, Eur. J. Biochem., 0, 569 (1971).

4.

Aringer, L., and P. heroth,

J. Lipid Res., l-4,

563 (1973).

328

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K. A., and S. Balasubramaniam,

Biochem. J., 128, 1

5.

Mitropoulos, 0972).

6.

Hulcher, F. H., W. H. Oleson and H. B. Lofland, Arch. Biochem. Biophys., I&, 313 (1974).

7.

Balasubramaniam, S., K. A. Mitropoulos and N. B. Myant, Eur. Biochem. J., 2, 77 (1973).

8.

Bjb'rkhem, I., and H. Danielsson, Anal. Biochem., 59, 508 (1974).

9.

Shefer, S., G. Nicolau and E. H. Mosbach, Circulation, 49 and 50 (Suppl.), 270 (1974).

10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 11. Mitropoulos, K. A., S. Balasubramaniam, Biophys. Acta, 326, 428 (1973).

and N. B. Myant, Biochim.