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Formation of 8,11,12-trihydroxyeicosatrienoic acid by a rat lung high-speed supernatant fraction
352
Biochirnica et Biophysics Acta, 665 (1981) 352-354 Elsevier/North-Holland
Biomedical
Press
BBA Report HBA 51312
FORMATION OF 8,11,12-TRIHYDROXYEICOSATRIENOIC SUPERNATANT FRACTION C.K. PACE-ASCIAK
a,* , K. MIZUNO
b and S. YAMAMOTO
ACID BY A RAT LUNG HIGH-SPEED
”
a RcsearL.k Institute, Hospital for Sick Chi!dretl, 555 Utriversity Aver~ur, Tormto MSG I X8 (Canada) and b Department of Biochemistry, Tokrtshima Ut?iversit>’School ofMedicine, Tokushirna [Japatz)
(Received
March 20th,
1981)
E;ey bvords: Arachidotlic acid; Trihydrosy fbtt!’ acid; (Rat lung)
enzyme was found in the high-speed (100 000 Xg) supematant fraction of a rat lung homogenate which catalysed the conversion of arachidonic acid into SJlJ24rihydroxyeicosatrienoic acid. The isomeric 8,9,12-trio1 was not detected. The structure of the isolated product was confirmed by mass spectrometric analysis of the methyl ester t-butyldimethylsilyl ether derivative. These results indicate that formation of both positional isomers is carried out by separate enzymes, the distributions of which are not restricted to platelets. An
Platelets from several species, including man, have been shown to convert essential fatty acids not only into prostaglandins and thromboxanes but also into open chain trihydroxyeicosenoic acids [l-3]. Thus, 8,11,14eicosatrienoic acid is converted into a mixture of the positional isomers 8,9,12- and 8,11,12trihydroxyeicosadienoic acids [ 1] while arachidonic acid is converted into the corresponding trihydroxyeicosatrienoic acids [2,3]. While platelets appear to form measurable amounts of these two open triols, it is not known whether other tissues also contain this activity and whether this activity is due to a particulate or a soluble enzyme. In this report we demonstrate the conversion of (1 -‘4C)-labelled arachidonic acid into only 8,ll ,12-trihydroxyeicosatrienoic acid by a soluble enzyme present in rat lung. Lungs were removed from 20 male Wistar rats (250-300 g), weighed and homogenized in 2 vol. 0.05 M KH2P04/NaOH buffer (pH 7.4). In order to reduce platelet contamination lungs were perfused in * On sabbatical
leave at Tokushima University. Recipient of a Josiah Macy Jr. Foundation Faculty Scholarship award. To whom reprint requests should be addressed.
vivo by injecting 25 ml of buffer through the heart immediately prior to their removal. The homogenate was centrifuged at 600 X g for 15 min and the resulting supernatant was centrifuged at 100 000 Xg for 60 min. The high-speed supernatant was used as enzyme source. It was incubated with 1 . lo7 cpm in (I-14C)labelled arachidonic acid (Amersham, spec. act. 50 mCi/mmol) mixed with 300 M unlabelled product for 40 min at 37°C in air. 5 vol. ethanol was added to terminate the incubation and the resulting precipitated protein was sedimented by centrifugation. The ethanol solution was evaporated to complete dryness and the residue was fractionated by silicic acid column chromatography. The fraction eluted with ethyl acetate was converted to the methyl ester with fresh ethereal diazomethane and to the t-butyldimethylsilyl ether with t-butyldimethylchlorosilane (Applied Science Labs.). This derivative was analysed by GC-MS using a Jeol - D300 equipped with a JMA - 2000 mass data analybis system. The column (1 m, 1.5% SE-30 on Chromosorb W) was operated at 250°C. Fig. 1 shows a mass spectrum of the major polar product isolated from incubation of rat lung high
353
315 (447-132) M-(131+132)
c z z
O100
150
200
250
450
500
550
660
3ot35
650
350
400
450
M/Z
700
750
800
M/Z
Fig. 1. Mass spectrum of the main polar product (methyl ester t-butyldimethylsily1 arachidonic acid by a rat high-speed supernatant fraction.
supernatant fraction with (l-14C)-labelled arachidonic acid. A small fragment ion at m/z 653, characteristic of a loss of t-butyl group (57), was indicative of the molecular weight of the product although the molecular ion was not present. Other fragment ions involving loss of a r-butyldimethylsilanol (131 or 132) include m/z 521 (M- (57 t 132)), 323 (455 - 132), 315 (447 - 132). Structurally characteristic fragments at m/z 255 (cleavage between CI1 and CIz -base peak) and 285 (Cl-Cs fragment) are indicative of vicinal hydroxyl groups at C1 1 and C12 and an isolated hydroxyl group at Ca, respectively. This mass spectrum is similar to that of 8,ll ,12-trihydroxyeicosatrienoic acid methyl ester trimethylsilyl. ether [2] except that major fragments at m/z 213, 243 and 281 in that spectrum are shifted upward by 42 units in our mass spectrum due to the presence of t-butyl groups in our derivative. Our isolated product is, therefore, 8,ll ,I 2-trihydroxyeicosatrienoic acid. Several experiments were carried out to quantitate the amount of 8,ll ,l L-trihydroxyeicosatrienoic acid formed from arachidonic acid. In these experiments (1 -14C)-labelled arachidonic acid (2 nmol) was incubated for 20 min at 37°C with 0.5 ml of highspeed supernatant fraction. The crude ethanol extract from the incubation was analysed on thin-layer speed
ether derivative, C value 30.2) formed from
chromatography after development with either chloroform/methanol/acetic acid/water (90 : 9 : 1 : 0.65, v/v) or the upper phase of ethyl acetate/2,2,4trimethyl pentane/acetic acid/water (90 : 50 : 20 : 100, v/v). 8,11,12-Trihydroxyeicosatrienoic acid (RF, 0.19 and 0.36, respectively) was formed in 8-12% yield (n = 8) and 12-hydroxyeicosatetranaenoic acid (RF, 0.51 and 0.81, respectively) in 40-60% yield (n = 8). As expected, negligible amounts of prostaglandins Ez, F,, and 6-keto F,, and thromboxane B2 were present (less than 1%). Further studies are under way to purify the enzyme responsible for this transformation. This study was supported by grants from the Medical Research Council of Canaca (C.P.-A. MT4181), the Josiah Macy Jr. Foundation (C.P.-A.) and by a grant-in-aid from the Ministry of Education, Science and Culture of Japan, by a research grant from the Intractible Diseases Division, Public Health Bureau, Ministry of Health and Welfare of Japan, and by grants from the Japanese Foundation on Metabolism and Diseases, the Naito Foundation, the Asahi Scholarship Promotion Fund and the Japan Research Foundation for Clinical Pharmacology (S.Y.). We thank Mrs. Yoshida, Tokushima University School of Pharmacy for recording the mass spectra.
354
References 1 Falardeau, P., Hamberg, M. and Samuelsson, Biochim. Biophys. Acta, 441, 193-200
2 Jones, R.L., Kerry, P.J., Poyser, N.L., Walker, PC. and B. (1976)
Wilson, N.H. (1978) Prostaglandins, 16,583-589 3 Bryant, R.W. and Bailey, J.M. (1979) Prostaglandins, 9-18
17,
Biochirnica et Biophysics Acta, 665 (1981) 352-354 Elsevier/North-Holland
Biomedical
Press
BBA Report HBA 51312
FORMATION OF 8,11,12-TRIHYDROXYEICOSATRIENOIC SUPERNATANT FRACTION C.K. PACE-ASCIAK
a,* , K. MIZUNO
b and S. YAMAMOTO
ACID BY A RAT LUNG HIGH-SPEED
”
a RcsearL.k Institute, Hospital for Sick Chi!dretl, 555 Utriversity Aver~ur, Tormto MSG I X8 (Canada) and b Department of Biochemistry, Tokrtshima Ut?iversit>’School ofMedicine, Tokushirna [Japatz)
(Received
March 20th,
1981)
E;ey bvords: Arachidotlic acid; Trihydrosy fbtt!’ acid; (Rat lung)
enzyme was found in the high-speed (100 000 Xg) supematant fraction of a rat lung homogenate which catalysed the conversion of arachidonic acid into SJlJ24rihydroxyeicosatrienoic acid. The isomeric 8,9,12-trio1 was not detected. The structure of the isolated product was confirmed by mass spectrometric analysis of the methyl ester t-butyldimethylsilyl ether derivative. These results indicate that formation of both positional isomers is carried out by separate enzymes, the distributions of which are not restricted to platelets. An
Platelets from several species, including man, have been shown to convert essential fatty acids not only into prostaglandins and thromboxanes but also into open chain trihydroxyeicosenoic acids [l-3]. Thus, 8,11,14eicosatrienoic acid is converted into a mixture of the positional isomers 8,9,12- and 8,11,12trihydroxyeicosadienoic acids [ 1] while arachidonic acid is converted into the corresponding trihydroxyeicosatrienoic acids [2,3]. While platelets appear to form measurable amounts of these two open triols, it is not known whether other tissues also contain this activity and whether this activity is due to a particulate or a soluble enzyme. In this report we demonstrate the conversion of (1 -‘4C)-labelled arachidonic acid into only 8,ll ,12-trihydroxyeicosatrienoic acid by a soluble enzyme present in rat lung. Lungs were removed from 20 male Wistar rats (250-300 g), weighed and homogenized in 2 vol. 0.05 M KH2P04/NaOH buffer (pH 7.4). In order to reduce platelet contamination lungs were perfused in * On sabbatical
leave at Tokushima University. Recipient of a Josiah Macy Jr. Foundation Faculty Scholarship award. To whom reprint requests should be addressed.
vivo by injecting 25 ml of buffer through the heart immediately prior to their removal. The homogenate was centrifuged at 600 X g for 15 min and the resulting supernatant was centrifuged at 100 000 Xg for 60 min. The high-speed supernatant was used as enzyme source. It was incubated with 1 . lo7 cpm in (I-14C)labelled arachidonic acid (Amersham, spec. act. 50 mCi/mmol) mixed with 300 M unlabelled product for 40 min at 37°C in air. 5 vol. ethanol was added to terminate the incubation and the resulting precipitated protein was sedimented by centrifugation. The ethanol solution was evaporated to complete dryness and the residue was fractionated by silicic acid column chromatography. The fraction eluted with ethyl acetate was converted to the methyl ester with fresh ethereal diazomethane and to the t-butyldimethylsilyl ether with t-butyldimethylchlorosilane (Applied Science Labs.). This derivative was analysed by GC-MS using a Jeol - D300 equipped with a JMA - 2000 mass data analybis system. The column (1 m, 1.5% SE-30 on Chromosorb W) was operated at 250°C. Fig. 1 shows a mass spectrum of the major polar product isolated from incubation of rat lung high
353
315 (447-132) M-(131+132)
c z z
O100
150
200
250
450
500
550
660
3ot35
650
350
400
450
M/Z
700
750
800
M/Z
Fig. 1. Mass spectrum of the main polar product (methyl ester t-butyldimethylsily1 arachidonic acid by a rat high-speed supernatant fraction.
supernatant fraction with (l-14C)-labelled arachidonic acid. A small fragment ion at m/z 653, characteristic of a loss of t-butyl group (57), was indicative of the molecular weight of the product although the molecular ion was not present. Other fragment ions involving loss of a r-butyldimethylsilanol (131 or 132) include m/z 521 (M- (57 t 132)), 323 (455 - 132), 315 (447 - 132). Structurally characteristic fragments at m/z 255 (cleavage between CI1 and CIz -base peak) and 285 (Cl-Cs fragment) are indicative of vicinal hydroxyl groups at C1 1 and C12 and an isolated hydroxyl group at Ca, respectively. This mass spectrum is similar to that of 8,ll ,12-trihydroxyeicosatrienoic acid methyl ester trimethylsilyl. ether [2] except that major fragments at m/z 213, 243 and 281 in that spectrum are shifted upward by 42 units in our mass spectrum due to the presence of t-butyl groups in our derivative. Our isolated product is, therefore, 8,ll ,I 2-trihydroxyeicosatrienoic acid. Several experiments were carried out to quantitate the amount of 8,ll ,l L-trihydroxyeicosatrienoic acid formed from arachidonic acid. In these experiments (1 -14C)-labelled arachidonic acid (2 nmol) was incubated for 20 min at 37°C with 0.5 ml of highspeed supernatant fraction. The crude ethanol extract from the incubation was analysed on thin-layer speed
ether derivative, C value 30.2) formed from
chromatography after development with either chloroform/methanol/acetic acid/water (90 : 9 : 1 : 0.65, v/v) or the upper phase of ethyl acetate/2,2,4trimethyl pentane/acetic acid/water (90 : 50 : 20 : 100, v/v). 8,11,12-Trihydroxyeicosatrienoic acid (RF, 0.19 and 0.36, respectively) was formed in 8-12% yield (n = 8) and 12-hydroxyeicosatetranaenoic acid (RF, 0.51 and 0.81, respectively) in 40-60% yield (n = 8). As expected, negligible amounts of prostaglandins Ez, F,, and 6-keto F,, and thromboxane B2 were present (less than 1%). Further studies are under way to purify the enzyme responsible for this transformation. This study was supported by grants from the Medical Research Council of Canaca (C.P.-A. MT4181), the Josiah Macy Jr. Foundation (C.P.-A.) and by a grant-in-aid from the Ministry of Education, Science and Culture of Japan, by a research grant from the Intractible Diseases Division, Public Health Bureau, Ministry of Health and Welfare of Japan, and by grants from the Japanese Foundation on Metabolism and Diseases, the Naito Foundation, the Asahi Scholarship Promotion Fund and the Japan Research Foundation for Clinical Pharmacology (S.Y.). We thank Mrs. Yoshida, Tokushima University School of Pharmacy for recording the mass spectra.
354
References 1 Falardeau, P., Hamberg, M. and Samuelsson, Biochim. Biophys. Acta, 441, 193-200
2 Jones, R.L., Kerry, P.J., Poyser, N.L., Walker, PC. and B. (1976)
Wilson, N.H. (1978) Prostaglandins, 16,583-589 3 Bryant, R.W. and Bailey, J.M. (1979) Prostaglandins, 9-18
17,