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10 20 O 10

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Figure 9.82 (/I) HPLC chromatogram obtained with 10 /xL of a mixture of authentic steroid standards (1.0 fi% of each) on a Varian 0.22 mm X 25 cm MicroPak silica column with 16% tetrahydrofuran in water. The flow rate was 60 mL/h at 300 atm. (B) Typical chromatographic profile obtained with extracts of rat adrenal mitochondria incubated with 60 fiM 11-deoxycorticosterone. An aliquot (0.8 mL) was removed at 5 minutes of incubation and extracted with 10 mL of methylene chloride containing 10 fig of internal standard (11-deoxycortisol). The extract was evaporated to dryness and solubilized with 50 fiL of ethanol. Then 10 fiL was injected onto the column. (From Gallant et al„ 1978.)

and placed into methylene chloride containing 11-deoxycortisol as an internal standard and extracted. After further processing of the extracts, the samples were injected, and the amount of 11-/3- and 18-hydroxylation reaction products were determined from peak areas. Figure 9.82B shows a chromatogram of a sample taken after 5 minutes of incubation. The two products and the unreacted substrate as well as the internal standard are shown.

The hydroxylase activity was assayed using intact mitochondria obtained from the adrenal cortex of the rat.

9.8.3 25-Hydroxyvltamin D3-1 «-Hydroxylase (Tanaka and DeLuca, 1981)

This hydroxylase carries out the 1-hydroxylation of the compound 25-hydroxyvitamin D3 (25-OH-D3) to form the product 1,25-dihydroxy-vitamin D3 [l,25-(OH)2-D3]. The HPLC assay developed replaces those using radiolabeled substrates.

Separation was carried out by normal phase HPLC on a Zorbax Sil column with a solvent of 10% 2-propanol in hexane. The column was eluted isocrati-cally and monitored at 254 nm.

The reaction mixture contained 1 mL of tissue homogenate, sucrose, Tris-acetate (pH 7.4), magnesium acetate, and sodium succinate. The reaction was initiated by the addition of the substrate 25-OH-D3 and at intervals was stopped by the addition of a methanol-chloroform mixture (2:1). The sample was prepurified and analyzed by HPLC.

The hydroxylase was obtained from both chicken kidney and liver by homogenization.

9.8.4 Cholesterol 7a-Hydroxyiase (Hyiemon et ai., 1989)

Cholesterol 7a-hydroxylase, a cytochrome P450-dependent enzyme, catalyzes the first and rate-limiting step in the biosynthesis of bile acids from cholesterol. Potential mechanisms for regulation of the enzyme have been extensively studied.

In work published in 1989, Hyiemon et al. used cholesterol oxidase to convert 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one. Cholesterol that remained was converted to 4-cholesten-3-one. 7/3-Cholesterol, which was added as an internal steroid recovery standard, was oxidized to 7/3-hydroxy-4-cholesten-3-one. These steroid products were analyzed by Cis reversed-phase chromatography on an Altex Ultrasil-ODS column (4.6 mm X 25 cm) using 70: 30 (v/v) mixture of acetonitrile and methanol (Fig. 9.83). The eluate was monitored at 240 nm, and the amount of product determined from a calibration curve.

Figure 9.83 Separation of 7a-hydroxy-4-cholesten-3-one (A), 7/3-hydroxy-4-cholesten-3-one (B), and 4-cho!esten-3-one (C) by C-18 reversed-phase HPLC.

One milligram of microsomal protein was added to 0.1 M potassium phosphate buffer (pH 7.4), 50 m M NaF, 5 m M dithiothreitol, 1 m M EDTA, 20% glycerol, and 0.015% CHAPS. After a 5-minute incubation at 37°C, the reaction was initiated by adding a NADPH regenerating system consisting of (final concentrations) 5 m M isocitrate, 5 m Ai MgCl2, 0.5 mM NADPH, and 0.075 unit of isocitrate dehydrogenase. The final volume was 1.0 mL. The reaction was allowed to proceed for 20 minutes before being terminated by adding 30 /xL of 20% sodium cholate and 1 fig 7/3-hydroxycholesteroI as an internal recovery standard. Then, 44 /xL of 0.1% cholesterol oxidase in 10 m M potassium phosphate buffer containing 1 m M dithiothreitol and 20% glycerol was added. The mixture was incubated 10 minutes at 37°C and the reaction terminated by adding 2 mL of 95% ethanol. Cholesterol metabolites were extracted by adding 6 mL of petroleum ether, vortexing and centrifuging. The ether layer was collected and dried under nitrogen at 40°C. The extraction was repeated three times and the residues resuspended in 0.10 mL of the mobile phase; 20 fiL was injected. Cholesterol 7 a-hydroxylase activity was linear for 0.5 to 3 mg of microsomal protein and for up to 40 minutes using 1 mg of microsomal protein.

Microsomes from rat liver were prepared in 100 mM potassium phosphate buffer (pH 7.2) containing 100 mM sucrose, 50 mM KC1, 50 mM NaF,

5 mM EGTA, 3 mM dithiothreitol, 1 mM EDTA, 1 mM PMSF, and 100 /xM leupeptin.

9.8.5 3/3-Hydroxy A5-C27-sterold Oxldoreductase (Hylemon et al., 1991)

The second step in the synthesis of bile acids, according to Hylemon et al. (1991), is the conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one by NAD+-dependent 3/3-hydroxy-A5-C27-steroid oxidoreductase. This enzyme is located in the endoplasmic reticulum of liver, and its catalysis of the 3/3-hydroxy group also results in isomerization of the double bond from A5 to A4.

The steroid products are separated by reversed-phase chromatography on a Beckman Ultrasphere ODS (4.6 mm x 25 cm, 5 fxM) column equlibrated with 70:30 (v/v) acetonitrile-methanol. The absorbance is monitored at 240 nm and the amount of product is determined from a calibration curve.

One milligram of microsomal protein is added to 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaF, 10 mM dithiothreitol, 1 mM EDTA, 20% glycerol (v/v), 150 (xM 5-cholestene-3/3, 7a-diol, and 0.915% CHAPS. The reaction is initiated by 1 mM NAD+ to give a final reaction volume of 1.0 mL. After incubation at 37°C for 5 minutes, the reaction is terminated by adding 2 mL of 95% ethanol. An internal recovery standard, 4-cholesten-3-one (3 /xg in methanol) is also added. The steroid products are extracted into 5 mL of petroleum ether (repeated twice). After the ether has been removed at 40°C under a stream of nitrogen, the products are dissolved in 100 /xL of mobile phase and 20 ¡xL is injected into the column. The amount of product formed is linear with protein (to 1.5 mg) and with time (up to 10 min, 1 mg protein). The assay is much more sensitive than the direct spectrophotometric assay, and it avoids the use of thin-layer chromatography and radioisotopes described in other methods.

The source of enzyme is rat liver microsomes prepared by standard techniques.

Figure 9.84 shows a representative chromatogram.

9.8.6 Cytochrome P450acc (Sugano et al., 1989)

The side chain cleavage of cholesterol, producing pregnenolone, is catalyzed by cytochrome P450scc. This is the initial step in the biosynthesis of several steroid hormones. In this assay, the initial product, pregnenolone, is quantitatively converted to progesterone by treatment with cholesterol oxidase, which increases by about 10-fold the sensitivity of the assay.

Cholestenone (derived from unreacted cholesterol) and progesterone were separated from deoxycorticosterone acetate (internal standard) by normal phase chromatography on a TSK-gel silica 150 column (4 mm X 250 mm)

Figure 9.84 Separation of 7a-hydroxy-4-cholesten-3-one (A) from 4-cholesten-3-one (B) by Cis reversed-phase HPLC. Microsomes (1 mg) prepared from cholestyramine-fed rats. (From Hylemon et al., 1991.)

from Tosoh. The mobile phase of n-hexane-isopropanol (100:2, v/v) was run at a flow rate of 1.5 mL/min. The effluent was monitored at 240 nm.

The cholesterol side chain cleavage reaction was carried out in 0.9 mL containing 20 mM potassium phosphate buffer (pH 7.4), 0.3% (w/v) Tween 20, 100 nmol of cholesterol dispersed in 10 /¿L of ethanol, 70 pmol of cytochrome P450SCC, 10 nmol of adrenodoxin, 1 nmol of adrenodoxin reductase, 5 /¿mol G6P, 0.5 unit of G6P dehydrogenase, and 4 /¿mol of MgCl2. The reaction was initiated by adding 100 nmol of NADPH. After incubating at 37°C for 5 minutes, the reaction was terminated by heat treatment. Pregnenolone was converted to progesterone by adding 100 /¿L of cholesterol oxidase (0.4 U) dissolved in 20 mM potassium phosphate buffer (pH 7.4) containing 1% sodium cholate. After incubation at 37°C for 10 minutes, the steroids were extracted into dichloromethane. Deoxycorticosterone acetate (5 nmol) was added as an internal standard. The extracts were dried under nitrogen before reconstitution and analysis by normal phase HPLC. The amount of product was determined from a standard curve. Production of progesterone was linear with amounts of P450 up to 150 pmol.

Cytochrome P450scc was purified from mitochondria isolated from bovine adrenal cortex by a published protocol.

Figure 9.85 shows chromatograms.

9.8.7 Steroid 17a-Hydroxylase/C17_2o Lyase (Cytochrome P4502ikc) (Schatzman et al., 1988)

Cytochrome P4502iscc is involved in the syntheses of both glucocorticoids and androgens.

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