Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo

Biochimica et Biophy.~ica Acta. 111146( I t)t) I } i 15- i 24

115

i '~ it)91 Elsevier Science Publishers B.V. All rights rest:fred 1)1)05-276{I/t)1/$113.50 ADONIS I)(1(15276(191(111284V

BBALIP 53729

Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo Fumie Hashimo,o and Hidenori Hayashi Department of Physiok~.~,ical Cltemistr}'. l:acuhy oJ"Ph~,,~ ,aceutical Sciem'e~. Josai Unirersity. Sakado. Saitama (Japan) {Received IS Maruh It~91)

Key words: Aminotriazole: Cholesterol: 4,-Methyl-5c~-cholest-7-en-3/J-ol: 4.4-Dimethyl-5~-cholest-g-en-3/:l-ol: Intermediale: Peroxisome

Cholesterol synthesis from mevalonate is inhibited by aminotriazole treatment in vivo. We tried to identify intermediates accumulated in liver of aminotriazole-treated rats. At 6 h after the aminotriazole treatment, the liver was e:~cised. $terols were extracted from it, and subjected to capillary gas-liquid chromatography, high-performance liquid chromatography, gas-liquid chromatography linked to mass spectrometry and gas-liquid chromatography linked to Fomqer-transform infrared spectrometry. It was found that 4a~methyl-Sa-cholest-7-en-3~l-ol and 4,4-dimethyl-Sa-cholest-8-en-3[I-ol were accumulated in the liver, mainly as the free forms. The contents of the former and the latter were increased to 25- and 64.times the control values, respectively. In another experiment, [2-1~C]mevalonate was i~ected at 2 h after aminotriazole treatment, and 4 h later the liver was excised. The sterols extracted from the liver were subjected to gas-liquid chromatography linked to mass spectrometry. Specific fragment ions reflecting the incorporation of [ t~C] mevalonate were detected in the mass spectra of the intermediate sterols. Accumulation of 4a~-methyl-Sa-cholest-7-en-3g-ol and 4,4-dimethyl-Sa-cholest-8-en-3g-ol after aminotriazole treatment suggests that elimination of the 4a-methyl group from 4-methyl intermediate sterols is inhibited by aminotriazole.

Introduction

Peroxisomes have many physiological roles. They participate in g-oxidation of very-long-chain fatty acids [1-5] and dicarboxylic acids [6-8] and in the syntheses of plasmalogens [9-11], prostaglandines [12,13] and bile acids [14-19]. Recently, it was also found that peroxisoraes take part in the synthesis of cholesterol. 3-Hydroxy-3-methylglutaryl-CoA reductase, the ratelimiting enzyme of cholesterol synthesis, is present in peroxisomes in addition to microsomes [20,21]. Sterol carrier protein-2 is required in the enzymatic conversion of sterol intermediate to cholesterol during choles-

Abbreviations: TLC, thin-layer chromatography; GLC, gas-liquid chromatography: GC-MS, gas-liquid chromatography linked to mass spectrometry; GC-FTIR, gas-liquid chromatography linked lo Fourier-transform infrared spectrometry; HPLC. high-performance liquid chromatography: TMS. trimethylsilyl. Correspondence: F. ttashimoto, Department of Physiological Chemistry, Faculty of Phar:. aceutical Sciences, Josai University, Sakado, Saitama 350-02. Japan.

terol biosynthesis, and had been thought to be exclusively localized in cytoplasm. However, it was recently found that this nonenzymatic protein is localized almost entirely in peroxisomes [22-25]. Further, cholesterol is synthesized from mevalonate in peroxisomes in addition to microsomes in the presence of cytosolic fraction in vitro [26]. It was also reported that peroxisomes catalyze the initial step in cholesterol synthesis (the condensation of acetyi-CoA units into acetoacetylCoA) [27]. Many oxidases, such as D-amino acid oxidase, urate oxidase and fatty acyI-CoA oxidase, are present in peroxisomes and produce H 2 0 2 during oxidase reaction. Hydrogen peroxide is degraded by catalase, which is localized in peroxisomes. Catalase is irreversibly inactivated by aminotriazole. We previously studied cholesterol synthesis after inhibition of catalase activity by aminotriazole and found that cholesterol synthesis from mevalonate was inhibited by aminotriazole treatment and unidentified intermediate(s)were accumulated in the liver, serum and bile [28]. In the present experiment, we established that 4a-methyl-Sa-cholest7-en-3/3-ol and 4,4-dimethyl-5a-cholest-8-en-3/3-ol were

i16 accumulated in the liver. These results suggest th~:t the elimination of the 4or-methyl group iron, 4-methyl sterol intermediates is inhibited by aminotriazole. Materials and Methods

Materials Aminotriazole was purchased from Tokyo Chemical Industry (Japan). 4a-Methyl-5a-cholesl-7-en-3/3-yl acetate and 4a-methyl-3a-cholest-8-en-3t3-yl acetate were kindly provided by Dr. Toshihiro Akihisa (Nihon University), and the free forms of these compounds were obtained by hydrolysis. 4,4-Dimethyl-5o~-cholesta-8,14dien-313-ol was kindly provided by Dr. James M. Trzaskos (E.I. du Pont de Nemours and Company). Other sterols, cholestyramine, NADPH, isocitrate and isocitrate dehydrogenase were obtained from Sigma Chemicals (U.S.A.). [2-~C]D,L-Mevalonolactone was purchased from M.S.D. Isotopes (Canada). Triton WRI339 was purchased from Rugal Chemical (U.S,A.). All other reagents were of analytical grade from Wako Pure Chemicals (Japan).

Treatment of the rats Male Wistar rats (250-300 g) were fed ad libitum on Clea laboratory chow pellets (Japan) in a 12-h light-dark cycle. Aminotriazole was intraperitoneally administered to the rats at a dose of 1 mg per 1 g body weight in physiological saline 3 h into their light cycle. Control rats were injected with the same volume of physiological saline instead of the aminotriazole solution. At 2 h after the injection, bile duct cannulation was carried out in order to perform the experiment under similar conditions to those in the previous paper [28]. The liver was perfused with cold saline and excised immediately after exsanguinatien through the abdominal aorta under anesthesia at 4 h after the operation. In the stable isotope experiment, [2-13C]mevalono lactone was administered to the rats at a dose of 15 ttg per 1 g body weight in saline through the portal vein immediately after the operation. The liver was excised at 4 h after the operation.

20% H:SO4 was sprayed on the positions ol marker steroids and the plate was heated. The areas corresponding to esterified sterol (Fr. 1), the region between esterified sterol and free cholesterol (Fr. II) and free cholesterol (Fr, I!1) (Fig. I) were scraped from the plate into centrifuge tubes. Lipids were extracted with Folch's solution and the solvent was evaporated off. After hydrolysis, sterols were extracted with petroleum ether. The extract was washed with water and evaporated to dryness. For gas-liquid chromatography (GLC), 5a-cholestane (100/zg) was added to the extract as an internal standard. The mixture was allowed to react with n-trimethylsilylimidazole at 100 °C for 2 h. The derivatives thus obtained were subjected to capillary GLC, gasliquid chromatography linked to mass spectrometry (GC-MS) and gas-liquid chromatography ',ir~ked to Fourier-transform infrared spectrometry (GC-FTIR). For high-performance liquid chromatography (HPLC), the extract was dissolved in ethanol.

Capillary gas.liquid chromatography Gas chromatographic analysis was performed using a Shimadzu GC-7A gas chromatograph equipped with an OV-1701 bonded column (0.25 mm × 25 m, i.d. 0.3 /.tin, GL Sciences, Japan). The separation conditions were: oven temperature, 270 ° C; injection port temperature, 290 ° C; carrier gas, N2; flow rate, 1 ml/min.

Gas-liquid chromatography linked to mass spectrometry Mass spectrometric analysis was performed using an JMS DX-300 instrument (JEOL Ltd., Japan) equipped with an OV-1701 bonded column (0.25 mm x 25 m, GL Science, Japan).

Gas-liquM chromatography linked to Fourier-transform h~frared spectrometry Fourier-transform infrared spectrometric analysis was performed using a JIR 5500 instrument (JEOL Ltd., Japan) equipped with an OV-1701 bonded column (0.25 mm × 25 m, GL Science, Japan).

High-performance liquM chromatography Extraction of sterols fi'om lit'er and preparation for analysis Liver was homogenized with 20 volumes of Foleh's solution (chloroform/methanol, 2: 1, v/v) and lipids were extracted. The extract was washed with 1/5 volume of water. The chloroform layer was concentrated and subjected to thin-layer chromatography (TLC) using reversed-phase KC isF plate (20 × 20 cm, thickness 0.2 mm, Whatman, U.S.A.). A mixture of cholesteryl palmitate and cholesterol (2(1 /.tg each) was loaded on the side of the origin as markers of esterified sterol and free sterol, respectively. After development with n-hexane/diethyi ether/formic acid (80 : 20 : 2, v/v),

The extracted samples were injected on an lnertsil C , column (4.6 × 250 ram, 5 ~ m particles, GL Science, Japan), and eluted with acetonitrile/ methanol/ water (47:47:6, v/v) at 45 °C, at a flow rate of l ml/min. The ultraviolet absorption was monitored at 214 rim.

Preparation of microsomes for biosynthesis of 4,4.dimethyl-Sa-cholest-8-en-3~-ol Microsomes were prepared as follows [29]. Wistar rats (v, eighing about 250 g) were maintained on a powdered diet (Clea, Japan) containing 3% (w/w) cholestyramine on a 12-h light-dark cycle for 10 days [30,31]. The rats were killed 6 h into the dark period

and the liver was perfuscd in situ with cold 0.25 M sucrose solution. Liver homogenate was prepared with 0.1 M potassium phosphate buffer (also containing 2 mM glutathione, l mM EDTA, 4 mM MgCl 2, 0.25 M sucrose, pH 7.4). Microsomcs were obtained by centrifugation at 105000 × g for 1 h. The microsomal pellet thus obtained was washed once by suspension in fresh 0.1 M potassium phosphate buffer (also containing l mM glutathione, 0.1 mM EDTA, pH 7.4) and centrifugation. Isolated microsomes were suspended in the latter buffer for incubation experiments, and stored at -80°C.

Biosynthesis of 4, 4-dimethyb 5 a-cholest-8-en-3 fl-ol from 4,4-dimethyl-5t~-cholesta-8,14-dien-3~-ol b~ rat licer mi£rosomes 4,4-Dimethyl-5a-cholest-8-en-3/3-ol was biosynthesized from 4,4-dimethyl-5a-eholesta-8,14-dien-3fl-ol by using microsomes according to the method of Shaficc et al. [32]. In the presence of 0.5 mM CN-, the final product of the reaction of 4,4-dimethyi-5a-cholesta8,14-dien-3/3-ol and microsomes is 4,4-dimethyl-Saeholest-8.en-3t~-ol [29]. The reaction mixture for synthesis of 4,4-dimethyi5a-cholest-8-en-3a-ol contained, in a final volume of 20 ml: 100 mg of microsomal protein, 0.5 mM NaCN, 2 mM NADPH, 50 mg of isocitrate, 0.4 mM MgCl2, 3,5 units of isocitrate dehydrogenase, 0.1 M potassium phosphate buffer (also containing 1 mM glutathione, 0,1 mM EDTA and 20% glycerol, pH 7.4). The reaction was initiated by the addition of 250 ~ M 4,4-dimethyl-5a-cholesta-8,14-dien-3/3-ol (suspended in Triton WR-1339, 75:1 (w/w) detergent-sterol at a sterol concentration of 1000 nmol/ml); incubation was continued for 2 h at 37 ° C. The reaction was terminated by the addition of 20 ml of 15% KOH in 95% methanol. After saponification and extraction with petroleum ether, the extraction solvent was evaporated off. The residue was dissolved in 0.5 ml of ethanol for purification by reverse-phase HPLC (see above). Repeated 50-p,I injections of extracted incubation mixture were made and 1 ml fractions were collected. Fractions containing 4,4-dimethyl-5a-cholest-8-en-3/~-ol were pooled. Results

Analysis of sterols in licer after aminotriazole treatment Thin-layer chromatography of standard steroids was performed. The R F values of esterified sterols were 0.73-0.83; that of free cholesterol was 0.35. 4-Methyl sterols were developed to the area between esterified sterols and free cholesterol, and their RF values were 0.42-0.44 (Fig. 1). After TLC of the liver extract, the zone was separated into 3 fractiohs: Fr. I (esterified

117

Front-~I 0

o

FF. I

o

Fr. II 0

0

o

!

2

3

~

5

Fr. III

6

Fig. i, Thin-la~er chromatography profiles of ester and free forms of sterols. Standard steroids ( 2 0 / z g each) were loaded on reverse-phase KCt~F plates ( 2 0 × 2 0 cm. thickness 0.2 turn, Whatman, U.S.A.). After development with n - h e x a n e / d i e t h y l e t h e r / f o r m i c acid (S0: 2(|: 2. v/v). the plate was sprayed with 2(1~ H . S O ~ and heated,

The plate was separated to three zones: Ft. l (esterified sterol area), Fr, II (free 4-methyl sterol area) and Fr 111 (free cholesterol area). ). 4(~-methyl-5t~-cholest-7-en-3/3-yl-acetate; 2. 4a-methyl-5a-cholest-~

en-3/3-yl-acelate: 3, choleslerylpalmitale; 4, 4a-methyl-5a-chotest-Yen-3/3-oh 5, 4a-methyl-5a-cholest-S-en-3/3-oh6, choleslerol.

sterolst Fr. !I (free 4-methyl sterols) and Fr. Ill (free cholesterol) as shown in Fig. I. Fig. 2 shows capillary gas chromatograms of Fr. i - I l l of liver sterols from control and aminotriazoletreated rats, In Fr. ! of control rats, the main peak (P- l) was detected at the retention time of 20 rain, with minor peaks at 25.1 and 30.5 rain. Aminotriazole did not affect the heights of these peaks. However, another minor peak (1:'-2) was detected at 27.4 rain in the treated rats. In Fr, II of both rats, peaks were detected at retention times of 20 (P-l), 25.1, 27.4 (P-2), 28.3 (P-3) and 30.5 min. Peak heights of P-2 and P-3 were greatly increased by aminotriazole treatment. In Fr Ill of both rats, the main peak (P-l) was detected at 20 min, with minor peaks at 25.1 and 30.5 min. No effect of aminotriazole treatment was observed. Table I shows the -elative retention times of standard and sample sterols on capillary GLC. The retention times of P-l, P-2 and P-3 coincided with those of standard cholesterol, 4t~-methyl-5~t-cholest-7-en-3fl-ol and 4,4-dimethyl-5a-cholest-8-en-3B-ol, respectively. On capillary GLC, the relative retention time of 4,4-dimcthyl-5u-chole~t-8-en-3/3-ol was close to that of 4,4-d;methyl-5a-eholesta-8,14-dien-3/3-ol. These sterois can 0e clearly separated on HPLC [32], and we confirmed that the retention times of P-l, P-2 and P-3 coincided with those of standard cholesterol, 4amethyl-5a-cholest-7-en-3/3-ol and 4,4-dimethyl-5acholest-8-en-3/~-ol, respectively, on HPLC. 4,4-Dimethyl-Sa-cholesta-8,14-dien-3fl-ol was hardly detected (Table iI). Fig. 3 shows the GC-MS profiles. Mass spectra of P-I, P-2 and P-3 were identical with those of standard

118 cholesterol, 4a-methyi-5a-cholest-7-en-313-ol and 4,4dimethyI-5 a-cholest-8-en-313-ol, respectively.

TABLE I

T h e i n f r a r e d s p e c t r u m o f P-1 s h o w e d m a x i m a at 2947, 1466, 1381, 1265, I099, 895 a n d 845 c m - I . T h e s p e c t r u m o f P-2 s h o w e d m a x i m a at 2962, 1458, 1381, 1265, 1(199, 903 a n d 845 c m - J ; t h a t o f P-3 g a v e m a x i m a at 2958, 1458, 1370, 126I, 1107, 1076, 937, 895 a n d 849 c m - ~ . T h e s e s p e c t r a o f P - l , P-2 a n d P-3 w e r e i d e n t i c a l with those of standard cholesterol, 4a-methyl-5acholest-7-cn-3fl-ol and 4,4-dimethyl-5a-choIest-8-en3/3-oi, r e s p e c t i v e l y ( d a t a n o t s h o w n ) . F r o m t h e s e r e s u l t s , P - l , P-2 a n d P-3 w e r e i d e n t i f i e d

Trimethylsilyl dericatives of stcrols were analyzed by GLC employing, a capillary column of OV-I70I (0.25 ram×25 m, i.d. 1).3 /.Lm) as described in the text. Relative retention is with respect to 5a-choleslane (its retention time was 10.69 rain). Data are means_+ S.D. of 10 samples. P-l-P-3 indicate the sample sterols as shown in Fig. 2.

Control

Amtnotrlozole

Fr, I P-1

P-I I.S

CaFilla,'T gas chromatographic :malysL~ of standard and samph" sterols

Relative retention time

5a-Cholestane Cholesterol Desmosterol 7-Dchydrocholesterol Lathosterol 4a-Methyl-5a-cholest-8-en-3/3-ol 4a-Methyl-5a-cholest-7-en-3/]-ol 4,4-Dimethyl-5a-cholest-8-en-3¢l-ol 4,4-Dimethyl-5o~-cholesta-8,14-dien-3fl-ol Lanosterol

standard

sample

1 1.88 2.08 2.11 2.14 2.51 2.57 2.65 2.67 2.80

1 1.87+0.01 (P-i) 2.56:1:0.02(P-2) 2,65 + 0.01 (P-3) -

i

I

P-2

Ft.

II i S

as cholesterol, 4a-methyl-5a-cholest-7-en-3/3-ol and 4,4-dimethyl-5a-cholest-8-en-3/3-ol, respectively. Table III shows the effects of aminotriazole treatment on the contents of cholesterol, 4a-methyl-5acholest-7-en-3/3-ol and 4,4-dimethyl-5a-cholest-8-en3 ~ - o l in t h e liver. C o n t e n t s o f c h o l e s t e r o l in e s t e r i f i e d a n d f r e e f o r m s w e r e n o t a f f e c t e d by a m i n o t r i a z o l e treatment. 4a-Methyl-5a-cholest-7-en-3g-ol was mainly

I.S.

present as the free form, and was increased to 25-times the control by aminotriazole treatment. 4,4-Dimethyl5a-cholest-8-en-3/3-ol was also mainly present as the free form, a n d w a s i n c r e a s e d t o 64 t i m e s t h e c o n t r o l .

Fr, I l l P- i

However, total contents (free and ester) of 4a-methyl5a-cholest-7-en-3g-ol and 4,4-dimethyl-5a-cholest-gen-3g-ol were very small compared with that of cholest e r o l , amounting to only 0.8 and 1.6%, respectively, of

P-1 r.s.

1.:5.

I

cholesterol content.

o

lb

2o

i0

do

2o

10

Time (mln) Fig. 2, Effects of aminotriazole treatment on the capillary gas chromatography profiles of liver sterols. 2 h after the injection of aminotriazole into rats. bile duct cannulation was performed. 4 h later, the liver was excised and lipids were extracted. Total lipid extracts were subjected to TLC. At-ler the development, the plate was separated into hree zones (Fr. I-liD as shown in Fig. t. After hydrolysis of the extract from the plate, sterols were extracted with petroleum ether. They were derivatized with n-trimethylsilylimidazole and analyzed by GLC employing a capillary colurnn of OV-170I (0.25 mm×25 m, i.d. 0.3 ~.m). A nitrogen carrier gas flow rate of 1 ml/min was employed wilh an oven temperature of 270°C and an injection port temperature of 2911°C. 5a-Cholestane was added to the samples as an internal standard (I.S.). The injected samples of Fr. 1, Fr. il and Fr. Iii correspond to 30, 60 and 3 mg of liver, respectively. P-I, P-2 and P-3 indicate the sterols at retention times of 20, 27.4 and 28.3 min, respectively.

TABLE I! Retention times of standard and sample sterols on high-performance liquid chromatography Sterols were analyzed by HPLC employing an Inertsil Ca column (4,6 × 250 ram) with a mobile phase of acetonitrile/methanol/water (47:47:6) at 45°C, at a flow rate of I ml/min. The sterols were dissolved in etha~:ol for injection and detection was done by measuring ultraviolet absorbance at 214 nm. Data are means+S.D, of 10 samples. P-I-P-3 indicate the sample sterols as shown in Fig. 2. Retention time (min) stan- sample dard Cholesterol 4,4-Dimethyl-5ec-cholesta-8,14-dien-3fl-ol 4a-Methyl-5a-cholest-7-en-3g-ol 4,4-Dimethyl-5a-cholest-8-en-3fl-ol

35.26 37.82 41.99 52.34

35.47 ± 0.73 (P-l) 41.75_+0.68 (P-2) 51.90 + 0.60 (P-3)

119 T A B L E !!!

Confirmation that 4-methyl sterols are intermediates #z the synthetic pathway from met'alonate to cholesterol

Effects of aminotriazoh' treatment on contents of choh,sterol and 4-methyl sterols m rat liter

In o r d e r to p r o v e that P-1 a n d P - 2 are i n t e r m e d i a t e s

Sterols were extracted from the livers of control and amlno~riazoleIreated rats as described in the text. The derivatives of slerols were analyzed by GLC employing a capillary column of O V - i 7 0 1 . D a t a are means + S.D. of five animals. ~' Represents a significance change (P < 0.005). Contents ( / ~ g / g liver) control

aminolriazole

Cholesterol Ester 151 _+46 Free 1510± 54

in c h o l e s t e r o l s y n t h e s i s f r o m m e v a l o n a t e , w e t o o k t h e ma~.¢ 'Lneetra o f s t e r o l s e x t r a c t e d f l o r a t h e liver a f t e r

injection of [ L~C]mevalonate. Sterols is synthesized from six molecules of mevalonate through squalene and therefore sterol synthesized from [2-'3C]mevalonate contains '3C at positions C1, C7, ClS, C2z, C2~ and C3~ of the steroid structure. Scheme 1 shows characteristic fragment ions of cholesterol, 4a-methyl-5a-cholest-7en-3/3-oi and 4,4-dimethyl-5a-cholest-8-en-3/3-ol derived from endogenous mevalonate and [13C]mevalonate [33,34]. Fig. 4 shows mass spectra of P-l, P-2 and P-3 of aminotriazolc-treated rats which had been injected with ['3C]mcva|onate. The mass spectrum of P-1 was hardly changed compared with that of P-I of Fig. 3. The

Aminotriazole / C o n t r o l ratio

175±61 1468± 76

1.16 I).g7

4 ~- M e t h y l - 5 a - c hoIest-7-e n-3~-ol Ester trace 2.18 4:_I).52 Free 0.46_+0.15 11.5+_3.11

25.1) *

4,4-Dimethyl-5~-eholest-8-en-3/~-ol

Ester

0

trace

Free

0.40 + 0. I 0

25.5 _+ ! .2

63.8 *

spectrum

o f P-2 i n d i c a t e d

specific

ions of m/e

477

Cholesterol i00 r--~

P-I

1°°i '['I "........

i

J~'9 L !

4"J ~1

0 200

400

•

600

600

i00

c

i 50

5O

>

llllIii l ILi I"3

,

l.ml

.,.-1%.

r

400

4~-Methyl-5~-cholest-7-en-39-ol

P-2 i00

.q,-..l

;

200

d

~*I

|"i~'

lid 200

P-3 i00

. .~:3;

IIll~o ~.~

~ 7

Ii

J

400

600

0

l llllIIl, Lg..,=. ,,,lJ 200

]'

400

600

4,4-Dimethyl-5(~-cholest-8-en-3~-ol

'r

i00

Its

;)

50

50

4U~.

3 1

0

p

i '°' 'I': T 200

400

~ 600

0

,~

"

I

1:11

" I~]Lrb'i'n~? P , 200

'ti

"16

r-

400

"? J

600

role Fig. 3. Mass spectra of standard and sample sterols. Sterols were extracted lrom rat liver as described in the text, and then the trimethylsilyl derivatives were subjected to G C - M S . P - t - p - 3 indicate the sample sterols as shown in Fig. 2.

120 (M + 5), 462 (a + 51, 387 (b + 5), 372 (c + 5), 272 (e + 3), 246 (i + 3) and 229 (g + 2) of 4a-methyl-5a-cholcst-

c r e a s e d by a m i n o t r i a z o l e t r e a t m e n t . T h e ( M + 5 1 / M ratio was 0,26. T h e ion p e a k of m / e 492 ( M + 6) of 4,4-dimethyl-5ot-cholest-8-en-3~-oI (P-31 s y n t h e s i z e d from [ 13C]mevatonate was greatly i n c r e a s e d . T h e ( M +

7-en-3/3-ol d e r i v e d from [B3C]mevalonate. T h e spect r u m of P-3 r e v e a l e d specific ions of m / e 492 ( M + 61, 477 (a + 61, 402 (b + 6), 387 (c + 6) 358 (j + 5), 287 (e + 41, 261 (i + 4) and 244 (g + 3) of 4,4-dimethyl-5o~-

6)/M ratio was (I.79. 4,4-Dimethyi-5ot-cholest-8-en-3/3ol seems to be derived from exogenou:, :nevalenate to a much greater extent than 4a-methyl-5a-cholest-7-cn3/3-ol, under these conditions. In the mass fragmen-

cholest-8-en-3/3-ol labeled with ~~C. These results confirmed that P-2 and P-3 arc 4a-methyl-5a-choIest-7cn-3~-ol and 4,4-dimcthyl-5a-cholest-8-cn-3/3-ol, respectively, and arc intermediates of cholesterol synthesis from mevalonatc. Fig. 5 shows the effects of aminotriazole treatment

t o g r a m of Fr. III (free c h o l e s t e r o l ) , the ion p e a k of m / e 333 (k + 4) of c h o l e s t e r o l (P-31 d e r i v e d from [I3C]mevalonate was d e c r e a s e d to a b o u t one-fifth of

the control by aminotriazole treatment. Other fragment peaks were hardly affected. As shown in Table !!I, the intermediates were accumulated in the liver after aminotriazole treatment, but cholesterol content was apparently unaffected. How-

on mass f r a g m e n t o g r a m s of sterols e x t r a c t e d from the liver after injection of [13C]mevalonate, In "he frag-

mentogram of Fr. I (esterified sterol), the ion peak of m / e 333 (k + 4 ) of cholesterol (P-l) derived from [~3C]mcvalonate was not detected in aminotriazoletreated rats, but was in control rats. The specific ion

ever, Fig. 5 d e m o n s t r a t e s t h a t de novo synthesis o f c h o l e s t e r o l from m e v a l o n a t e was i n h i b i t e d by a m i n o t r i azole t r e a t m e n t , a n d 4 a - m e t h y l - S a - c h o l e s t - 7 - e n - 3 / 3 - o l and 4,4-dimethyl-5a-cholest-8en-3/3.ol were accumul a t e d in t h e liver.

p e a k of m / e 477 ( M + 5) of 4 a - m e t h y l - 5 a - c h o l e s t - 7 en-3/3-ol (P-2) s y n t h e s i z e d from [13C]mevalonate was i n c r e a s e d by a m i n o t r i a z o l c t r e a t m e n t . T h e ( M + 5 ) / M ratio of 4 a - m c t h y l - 5 a - c h o l e s t - 7 - e n - 3 / 3 - o l of a m i n o t r i a zole t r e a t e d rats was O 22. In the f r a g m e n t o g r a m of Fr. II (free form of 4-methyl stcrol), thc specific ion p e a k of m / e 477 ( M + 5) of 4 a - m e t h y l - 5 a - c h o l e s t - 7 - e n - 3 / 3 ol (P-21 d e r i v e d from [13C]mevalonate was clearly in-

Discussion We previously reported that cholesterol synthesis from mevalonate is inhibited after injection of amino-

Scheme 1

Expected fragment ions of trimethyl.~ilyl (TMS) tk'riratires of sterols .~ynthesized from endogenous meralonate and [t'~C]meralonate on mass .~pectrmneto' Fragment ions

M. M.Iccular ion a. M-CI |.~ b. M - T M S O H c. M-(C'tI.~ + T M S O I 1 )

d. M-side chain e. M-(sidc chain +TMSOII) I. M-(.sidc chain+42)

g. M-(side chai1~. +TMSOII+ 421 h, M-(sidc chain+27) i. M-(side chain + T M S O 4- 27)

13C

4a-Methyl-Sa-eholest-7-en-3/~-ol 4,4-Dimethyl-5a-cholest-8-en-3/~-ol

increased number

(m / e )

increased number

(m / e)

increased number

( m / e)

+ + -

0 5 0 5 ()

(4581 (403) (443) (448) (368)

0 5 0 5 i)

(472) (477) (457) (462) (382)

11 6 (I 6 0

(486) (492) (471) (477) (396)

+

5

(373)

5

(387)

6

-

I)

(353)

0

(367)

0

+ + + + +

5 0 3 0 3

(358) (345) (348) (255) (258)

5 {} 3 11 3

(372) (3591 (362) (269) (272)

6 l) 4 0 4

(402) (3811 (387) (373) (377) (283) (287)

0

(3(131

0

(3171

0

(3311

2 {l 2

(305) (213t (215)

2 0 2

3 0 3

(334) (241) (244)

+

() 3

(318) (321)

0 3

(3191 (227) (229) (3321

() 4

(346) (350)

-

()

(229)

0

(243)

0

(257)

+

3

(232)

3

(246)

4

(261)

0 5

(3531 (358) 0

(353)

5

(358)

0 5

(357) (362)

j. 4~-mcthyl stcrol M-(TMSOIt + 291 4.4-dimcthyl stcrol

+ -

M - I T M S O l t + 43)

+

k. M-(C~~ ('-~ +TMSO)

Cholesterol

+

() 4

(32~)) (333)

11 4

(335)

(333) (337)

1"~! triazole in rats [28]. In the present work, we sought to identify the i n t e r m e d i a t e ( s ) that accumulated in the liver after aminotriazole treatment. Steroids extracted from the liver were separated into Fr. I-I11 by TLC and t h e n subjected to c:,pillary GC. In Ft. ! (ester form of sterol), only 4 a - m e t h y l - 5 a cholest-7-en-3~-ol (P-2) was clearly increased by aminotriazole t r e a t m e n t (Fig. 2). in the previous paper, we showed that the radioactivity in Ft. 1 or sterol synthesized from [l'~C]mevalonate was increased about twice by aminotriazole t r e a t m e n t [28]. T h e r e f o r e , the increased radioactivity in Fr. I may be d u e to ester form of 4a-methyl-5a-cholest-7-en-3H-ol. in Fr, II (free form of 4-methyl sterol), 4a-methyl-5cPcholest-7-cn3/3-oi (P-2) and 4,4-dimethyl-5c~-cholcst-8-en-3~-ol (P3) were increased (Fig. 2). T h e radioactivity of Fr. ll

Control

Amlnotriazole

IS. P-1

l~S,

P;1

e;2

mle Fr,

I

__.L

1

_

k

._1

n

--

,i

-' L

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Fr, II

S

P-I

P-2

P-3

____Lrl

p-[

!.$.

1

~_

p-2

p-~.

I

372

~JL______ L

333 472

II |

F~ ,It

.

!

~77

492 !

1

I

•

I

r

'.r.-

~72 486

a77 '"

= :;86

L ?.q

P-1

9

t

P-I . . . .

329 333

~ -

_

1 O0

372

L ~

492

11

___1

372

Ik__._~

------------ 329

J

333

~L

i

' ,....

50 4 ~,,

i

51

II~l->l

qt

e'~ 1 iLL~

i"'1

o 5~i411iiiL-'-"~" I00 200

'~ ,-'A- -

. ..... 400

300

--

100

~92 / L,k

,

m

,

. . . .

500

:,~,

e~"~

600

-

_

_

,

,~I

o

8 ==

ll!lQt

',t <,' II

1 >,+,

<-+¢:..

,-',;q~l--~+~

~i

,,+g!'r

' _~ •

tO0

200

•

~

b+5

~

300

400

500

600

P-3 100 " r . - - - - ~'-. . . . . . . . . . . . . . . . . . . . . . . '

~'1 I.

I

' I

I

5

lO

!5

20

0

S

tO

15

20

Time (min) Fig. 5. [:.ffcch of amint)tri:tzole on mass fragmcnlograms of sterols from ra! liver after injection of [It(')muvtih)nate, The sterols v,'erc ¢xlra¢lcd from lh¢ livers of control and aminolriazole-trealed rats

(k + 4} of cholestcrok m / e 472 (31) and 477 ( M + 5) of 4 ~ - m e i h y b 5,-chl)lcsl-7-cn-3~-olz m / c 486 ( M ) and 492 ( M + 6 ) of 4.4-di-

,llllll.~liiil_; ~ 2 ~ ....~ ," :

0

• m

~,ftcr the injccti(m off ~~(']mevalonateas described in the text. M~,ss fn|gme||togr,|phy of Fr, I (cslerified sterolK Fr. II (free 4-methyl stcrois) and Ft. I11 {free cholesterol)was perfl)rmed in the presence of 5.-choleqane as an intern:it standard (I.S.). Specific Rms were monitored: m / c 372 (M) of 5.-cholestane: m / e 329 (k) and 333

50

11i{t'?

_

r,~

P-2

8

t~72 477

t ,II 7! til;

-

,nethyl-5.-cholcst-X-en-3/~qfl.The Io:|ded samples of Ft. I. Fr. I! and Fr. I!! correspond Io fill, 60 and ~ mg of liver, respectively. P-I-P-3 indicalc the slcmls :is sho~vnin Fig. 2.

,

50 ,,:!;7 I, ! il

o

.~-

~,=., I

.

x

~6 c

"~

~

.....

,~I

"...filllllflia,,,a~_._!:Th__i'~.......... 100

200

300

400

500

600

m/e Fig. 4. Mass spectra of cholesterol (P-I) and 4-methyl slerols (P-2

and P-3) from rat liver after injection of [I;(']nwvahmi,tc. 2 h i, ftcr the injection of amim)lrilizolc, Ihc rats were subjected to bile duct

cannulation. [t3(']Mevalonate was administered to Ih¢ rats through the portal vein immediately after the op:ration. 4 h laler, the liver was excised, Sterols were extracted from the liver, and then the derivatives were subjcct~-d to GC-MS as described in the text. M, a, b. c. c. g, i. j and k indicate fragment ions as '~hownin Scheme !.

was increased about 5(}-times by ;lminotriazole treatment [28]. The large increase of radioactivity in Ft. II seems to be due to the free forms of 4 a - m e t h y l - 5 a cholest-7-cn-3fl-ol and 4.4-dimethyl-Sa-cholest-8-en3~-ol. As shown in Fig. 2, P-1 was also d e t e c t e d in Fr. I! and corresponds to about I% of cholesterol in Ft. Ill. 4-Methyl sterols w e r e d e v e l o p e d to nearly the same position as cholesterol on T L C (Fig. 1). T h e r e f o r e , P-I of Fr. ll may represent cholesterol contamination. The mass spectrum of 4a-methyl-5ce-cholest-7-en3/3-ol t?-2) is shown in Fig. 3. T h e molecular weight of 4a-methyl-Sa-cholest-7-en-3~-ol is identical to that of 4a-methyl-5a-cholest-8-en-3~B-ol, and the mass spectra of the two arc very similar to each other, though the relative intensity of the ion at m / e 269 of the former is

122 Souc!ene

l

->Dlhvdrolnnosterol

Lonosterol

1

I I

@ 4,4-DImethyl-5~-cholest-8-en-3~.-O1

~A-Dimethvl-5~-cholesto-8,24-dten-3&ol I 4N-~thvl-5~-cholesto-8,2~-0ien-58-ol

....

I I I I

-> a~-Methv]-Sm-cholest-8-en-3g-ol /

4~-~ethv I-Sa-ch01es t-7-en-SB-01

/

I

1

/ /

Cholest-8-en-SB-ot . . . . . @ Cho!es ta-5,2t~-dlen- 3~-oI

4

1

I

¢

+ Cholest-7-en-3g-ol

1

--> Cholestcrnl

Fig. (). Cholesterol .synthesis from squalene. Major p a t h w a y s are illustrated with c o n t i n u o u s arrows: m i n o r and hypothetical p.'lthways a r e shown hy broken-line arrows. Thick arrows ( ~ ) indicate the i n t e r m e d i a t e sterols which are a c c u m u l a t e d after aminotriazole treatment in vivo.

larger than that of the latter [35]. We obtained the same result, though the mass spectrum of 4a-methyl5a-cholest-8-en-3/3-ol is not shown. The different relative retention times of the two on capillary GLC also indicate that P-I is not 4a-r, ethyl-5ot-cholest-8-en-3/3ol, but 4a-methyl-5o~-cholest-7-en-3/3-ol (Table I). The mass spectrum of cholesterol (P-I) from rats administered with [13C]mevalonate (Fig. 4)was almost the same as that in Fig. 3. Because the content of cholesterol synthesized de novo from [L3C]mevalonate is much smaller than that of cholesterol already presen,' in the liver, the ratio of the former to the latter is small. Therefore, the spectrum was apparently unchanged when the relative abundance was displayed on the vertical axis. However, as shown in Fig. 5, the fragment peak of m/e 333 (k + 4) of cholesterol was reduced by aminotriazole treatment. These results clearly indicated that cholesterol synthesis was inhibited by aminotriazole treatment. These results suggest that the intermediates accumulated in the liver after the inhibition of cholesterol synthesis by aminotriazole treatment are 4a-methyl5o~-cholest-7-en-3/3-ol and 4,4-dimethyl-5ot-cholest-8en-3/~-ol. In the process of cholesterol synthesis, the 14amethyl group is eliminated from lanosterol, producing 4,4-dim ethyl-5 a-cholesta-8,24-dien-3/3-ol. 4,4-Dimethyl-5a-cholesta-8,24-dien-3~-ol is oxidized to 4,4dimethyl-5~v-chnlest-8-en-3/3-ol. The 4a-methyl group is eliminated from 4,4-dimethyl-5a-cholest-8-en-3/3-ol, and then the 4/3-methyl group is converted to a 4amethyl group, forming 4a-methyl-5o~-cholest-8-en-313ol. The double bond is transferred from C~ to C7, producing 4c~-methyl-5a-cholest-7-en-3/3-ol. The 4amethyl group is eliminated from 4a-methyl-5a-cholesl7-en-313-ol, and the double bond is transferred from C 7

to C.~, finally producing cholesterol (Fig. 6) [36,37]. That is, the 4or-methyl group is eliminated from 4,4-dimethyl-5ot-cholest-8-en-3/~-ol and 4a-methyl-5a-cholest-7-en-3~-oi. These 4a-methyl sterols were accumulated in the liver after aminotriazole treatment (Fig. 2 and Table liD. These results suggest that the elimination of the 4a-methyl group was inhibited by aminotriazole treatment. There are a number of reports about inhibitors of cholesterol synthesis, but most of them are reports about 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors [38]. Inhibitor of demethylation of steroids is rarely studied. Some ianosterol derivatives suppress C~4 demethylation process in vitro [39]. Ketokonazole impairs demethylation process at Ct4 and to some extent at C~ in vivo [40,41]. in the present study, 14a-methyl sterol was not detected, and so the elimination of 4a-methyl group may be specifically inhibited by aminotriazole. Non-mercurial sufhydryl 1eagents (sodium arsenite, /~-mereaptoethanol, dithiothreitol and ethanethiol), cholesterol, several oxygenated sterols and cholesterol esters impair C 4 demethylation process in vitro [42,43]. Cytochrome c, C N - and 4-hydroxymethylene-5a-cholest-7-en-3-one also suppress demethylation process at C~ in vitro [44]. Therefore, aminotriazole is the first reported inhibitor of the elimination of 4a-methyl group in vivo. Many pathways of cholesterol synthesis are reported [36,37,45], In the present study, we could not detect 4(~-methyl-5(~-cholesta-7,24-dien-3/3-ol, but detected 4o~-methyl-Sc~-cholest-7-en-38-ol and 4,4-dimethyl-5acholest-8-en-3/3-ol in the liver after aminotriazole treatment. These results indicate that pathway including the latter two 4-methyl sterols is main route of cholesterol synthesis in rat liver. 4-Methyl sterois are useful as indicators for elucidation of cholcsterol-syn-

123 thetic pathway in vivo. Using aminotriazole, the investigation of biosynthetic route of cholesterol may be further advanced. The elimination of the 4a-methyl group occurs as follows. The 4a-methyl group is converted to a carboxyl group by microsomal 4a-methyl sterol oxidase in the presence of NAD(P) and O2, and then CO, is eliminated by 4a-carboxylic acid decarboxylase, producing a 3-ketosterol. Steroid-3-ketoreductase regenerates the 3/3-hydroxy sterol from 3-ketosteroid [2%31,45]. Thompson et al. reported that peroxisomcs in addition to microsomes synthesize cholesterol from mevalonatc in the presence of cytosol in vitro [26]. Recently, Appelkvist et al. reported that lanosterol, 4,4-dimcthyl5a-cholesta-8,14-dien-3g-oi, 4,4-dimethyl-5a-cholest-8en-3/3-ol and desmethylsterols were generated after incubation of [3HI mevalonate and peroxisomcs in the presence of cytosol, and furthermore dihydrolanosterol oxidase, steroid-14-reductase, steroid-8-en-isomerase and steroid-3-ketoreductase were present not only in microsomes, but also in peroxisomes [46]. Dihydrolanosterol oxidase eliminates 14a-methyl group from dihydrolanosterol, forming 4,4-dimethyl-5acholesta-8,14-dien-3#-ol. Steroid-14-reductase produces 4,4-dimethyl-5a-cholest-8-en-3/3-ol from 4,4-dimethyl-5a-cholesta-8,14-(hen-3/J-ol. ~iteroid-3-ketoreductase takes place in the final step of demethylation at C 4 as stated above. Noland et ai. reported that sterol carrrier protein-2 activates the microsomal conversion of 4,4-dimethyl-5a-cholest-8-en-3B-ol to C_,7 steroid [47]. Recently sterol carrier protein-2 is reported to be localized almost entirely in peroxisomes [23,24]. These suggest that the 4a-methyl sterol oxidase reaction is also taken place in peroxisomes. When considered with these reports relating to peroxisomes, it is interesting that 4,4-dimethyl-Sa-cholest-8-en-3,8-ol and 4a-methyl-5a-cholest-7-en-3/3-ol were accumulated in the liver after aminotriazole treatment, in the present study, The 4a-methyl sterol oxidase reaction may be inhibited by aminotriazole treatment in vivo. it is not yet clear whether the 4a-methyl sterol oxidase reaction of peroxisomes a n d / o r microsomes is directly inhibited by aminotriazole, or whether the elimination of the 4a-methyl group is suppressed by HzO 2 accumulated owing to the inhibition of catalase activity of peroxisomes by aminotriazole treatment. If subcellular localization of 4a-methyl sterols as indicators is clarified, synthetic site of cholesterol in liver may be elucidated. That is, aminotriazole may be useful in helping to elucicate the participation of peroxisomes in cholesterol synthesis in vivo. In conclusion, it was clarified that cholesterol synthesis was inhibited by aminotriazole treatment, resulting in accumulation of 4a-methyl-5a-cholest-7-en-3B-ol and 4,4-dimethyl-5a-eholest-8-en-3g-ol in the liver. These results suggest that the elimination reaction of

the 4a-methyl group from these intermediate sterols may be inhibited by aminotriazole.

Acknowledgements We are grateful to Dr. James M. Trzaskos (E.I. du Pont de Nemours and Company) for providing authentic 4,4-dimethyl-5 a-cholesta-8,14-dien-3/3-ol, and to Dr. Toshihiro Akihisa (Nihon University) for 4a-methyl5a-cholest-7-en-3B-yl acetate and 4a-methyl-5acholest-8-en-3/3-yl acetate. Our thanks are also due to Dr, Yuzo Yoshida (Mukogawa University) for valuable advice on the determination of sterol structure. References I Singh. I., Moser, E., Goldfischer, S. and Motor, II. ~.~t. 119,'441 Proc. Natl. Acad. Sci. USA 81. 4203-42(|7. 2 Wanders, RJ.A., Van Roermund, C.W,T., Van Wijland, M.J.A., Schutgcns. R.B.H., Schram, A.W., Van den Bosch, H. and Tager, J.M. (1987) Biochim. Biophys, Acta 919, 21-25. 3 Wanders, R.J.A,, Van Roermund, C.W,T., Van Wijland, M.J,A., Sch:ttgens, R.B.H., Heikoop, J., Van den Bosch, I,!., Schram, A,W. and Tager, J.M, (1987)J. Clin. invest. 80, 1778-1783. 4 Singh, !-i., Derwas, N. and Poulos, A. 119871 Arch. Biochem. Flit~phys. 250, 382-3~). 5 Singh, !t. and Poulos, A. (1988),',rch. Biochem. Biophys. 266, 486-495. 6 Bj/hkhem, I., Blomstrand, S., Haga, P., Kase, B.F., Plonek. E., Pedersen, J.l., Strandvik, B. and Wikstrom, S.-A, 119841 Biochim. Biophys. Acta 795, 15-19. 7 Kolvraa, S. and Gregersen, N, 11986) Biochim. Biophys. Acta 876, 515-525. 8 Suzuki, H.. Yamada, J,, Watanabe, T, and Suga, T. 11989) Bitvchim. Biophys. Acta 900, 25-30. t) Jones, C.L. and Hajra, A.K. 119771 Biochem, Biophys, Res. Commun. 76. 1138-1143, 10 Hajra, A.K., Burke, C.L. and Jones, C.L. (19791 J. Biol. Chem. 254, 10896-111900. i I Patel, B.N.. Mackne~, M.l. and Connock, MJ. 119871 Biochem. J, 244, 443-448. 12 Diczfalusy, U., Alexson, S.E.H. and Pedersen, J.F. (1987) Biochem. Biophys. Res. Commun. 144, 1206-1213. 13 Diczfalusy, U. and Alexson. S.E.H. (191/01 J. Lipid Res. 31. 307-314, 14 Pedersen, J.I. and Gustaf,~on, J. 1198111FEBS Lett, 121,345-348. 15 Hagey. L.R., Krisans, S.K. (19821 Biochem. Biophys, Res. Commun. 107, 834-841. 16 Haya~hi, H.. Fukuyama, K. and Yamasaki, F. (19831 Chem. Pharm. Bull. 31,653-658, 17 Hayashi, H., Fukui, K. and Yamasaki, F. 119841J. Biochem. 96, 1713-1719. 18 Farrants, A.-K.O., Bj6rkhem, !. and Pedersen, J.I. 11989) Biochim. Biorhys, Acta 1002, 198-202. 10 Farr:v,ts. A.-K.O,, Bibrkhem, I, and Pedersen, J.I. (19001 Biochim. Bioph3~s. Acta 1046, 173-177. 211 Keller, G.A., Barton, M.C., Shapiro, D.J. and Singer, S,L, 11985) Proc. Natl. Acad. Sci. USA 82, 7711-774. 21 Keller. G.A,, Parirandeh. M. and Krisans, S,K. 119861J. Cell Biol. IG3, 875-886. 22 Van det Krift, T.P., Leunissen, J., Teerlink, T., Van Heusden, G,P.H., Verkleij, A.J. and Wirts, K,W.A. (1985) Biochim. Biophys. Acla 812, 387-392.

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36 Schmepfer. Jr. O.J. 1!9821 Annm Rev. Biochem. 51,555-585. 37 Strandverg, T.E, "[ilvis. R.S. and Miellinen, T.A. ( 10891 Biochim. Biophys. Acta !1101, 15{I-156. 38 Schroepfer, Jr. G.J. (19811 Annu. Rev. Biochem. 5{I, 585-621. 39 Frye, L.L. and Robinson. C.H. 119881 J. Chem. Sot., Chem. Commun. 129-131. 411 Strandberg, T.E., Tiivis, R.S. and Mieltinen. T.A. (1987~ Lipids 22. 10211-11124. 41 Mieltinen, T.A. (1988) J. Lipid Res. 29, 43-51. 42 Abcrnelhy, D., Hignite, C. and Azarnoff. D.L. (|976) Steroids 27, 297-307. 43 Gaylor. J,L. and Delwiche, C.V. (I976) J. Biol. Chem, 251, 6638-6645. 44 Brady, D.R. and Crowder. R.D. 110781 J. Biol. Chem. 253, 31111-3105. 45 H.C. Rilling and L.T. Chayet 119851 in Sterols and Bile Acids (Danielsson, H. and Sjovall, J., eds.), pp. 1-39, Elsevier. Amsterdam. 46 Appelkvist, E.-L,, Reinhart, M,, Fischer, R., Billheimer, J. and Dallner, G, 1199111 Arch. Biochem. Biophys. 282, 318-325. 47 Noland, B.J., Arevalo, R.E., Hansbury, E. and Seallen, T.J. 119801 J. Biol. Chem. 255, 4282-4289.