Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C3 by rabbit aorta

Sandra L. Pfister; Nancy Spitzbarth; Kasem Nithipatikom; J R Falck; William B. Campbell

doi:10.1016/S0304-4165(03)00097-7

Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C₃ by rabbit aorta

Sandra L. Pfister, Nancy Spitzbarth, Kasem Nithipatikom, J R Falck, William B. Campbell

Biochemistry

Research output: Contribution to journal › Article › peer-review

16 Scopus citations

Abstract

Arachidonic acid is metabolized by both the cyclooxygenase and lipoxygenase pathways by rabbit aorta. We investigated the metabolism of 12-hydroperoxyeicosatetraenoic acid by aortic homogenates and microsomes. Rabbit aortic homogenates were incubated in the presence of ¹⁴C-arachidonic acid plus 12-lipoxygenase and analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Under these experimental conditions, there was a ¹⁴C-metabolite that migrated at 17.6 min. This ¹⁴C-metabolite was not observed when aortic homogenates were incubated in the absence of 12-lipoxygenase. Similar results were obtained with aortic microsomes. Further analysis using a different HPLC solvent system resolved the ¹⁴C-metabolite into a number of products. Gas chromatography/mass spectrometric (GC-MS) analysis of the major product (labeled peak 3) after conversion to the methyl ester-trimethylsilyl derivative showed two major compounds (compounds A and B) eluting at 13.99 and 14.14 min. The two compounds differed in the intensities of the 213 and 243 m/z ions with 243 being greater than 213 in compound A and the opposite in compound B (relative abundance 213 vs. 243; 100% vs. 43% for compound A and 5% vs. 100% for compound B). Based on the mass spectra, peak 3 contained two metabolites identified as the methyl ester-trimethylsilyl ether derivatives of 8,11,12-trihydroxyeicosatrienoic acid (trioxilin A₃) and 8,9,12-trihydroxyeicosatrienoic acid (trioxilin C₃). Biological activity of the mixture of two trioxilins isolated from aortic homogenates was tested in phenylephrine-precontracted aortas and found to produce concentration-dependent relaxations (maximal relaxation: 20.1±7.6%). Further testing with authentic trioxilin A₃ and C₃ revealed that trioxilin C₃ was the active metabolite (maximal relaxation: 16.6±1.3%). In conclusion, trioxilin C₃ acid was isolated and identified as a novel biologically active arachidonic acid metabolite formed by rabbit aorta when 12-lipoxygenase is supplied exogenously.

Original language	English (US)
Pages (from-to)	6-13
Number of pages	8
Journal	Biochimica et Biophysica Acta - General Subjects
Volume	1622
Issue number	1
DOIs	https://doi.org/10.1016/S0304-4165(03)00097-7
State	Published - Jun 20 2003

Keywords

Arachidonic acid
Lipoxygenase
Rabbit aorta
Vasorelaxation

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology

Access to Document

10.1016/S0304-4165(03)00097-7

Cite this

@article{132f704a93574fd083040fe5752a1267,

title = "Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C3 by rabbit aorta",

abstract = "Arachidonic acid is metabolized by both the cyclooxygenase and lipoxygenase pathways by rabbit aorta. We investigated the metabolism of 12-hydroperoxyeicosatetraenoic acid by aortic homogenates and microsomes. Rabbit aortic homogenates were incubated in the presence of 14C-arachidonic acid plus 12-lipoxygenase and analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Under these experimental conditions, there was a 14C-metabolite that migrated at 17.6 min. This 14C-metabolite was not observed when aortic homogenates were incubated in the absence of 12-lipoxygenase. Similar results were obtained with aortic microsomes. Further analysis using a different HPLC solvent system resolved the 14C-metabolite into a number of products. Gas chromatography/mass spectrometric (GC-MS) analysis of the major product (labeled peak 3) after conversion to the methyl ester-trimethylsilyl derivative showed two major compounds (compounds A and B) eluting at 13.99 and 14.14 min. The two compounds differed in the intensities of the 213 and 243 m/z ions with 243 being greater than 213 in compound A and the opposite in compound B (relative abundance 213 vs. 243; 100% vs. 43% for compound A and 5% vs. 100% for compound B). Based on the mass spectra, peak 3 contained two metabolites identified as the methyl ester-trimethylsilyl ether derivatives of 8,11,12-trihydroxyeicosatrienoic acid (trioxilin A3) and 8,9,12-trihydroxyeicosatrienoic acid (trioxilin C3). Biological activity of the mixture of two trioxilins isolated from aortic homogenates was tested in phenylephrine-precontracted aortas and found to produce concentration-dependent relaxations (maximal relaxation: 20.1±7.6%). Further testing with authentic trioxilin A3 and C3 revealed that trioxilin C3 was the active metabolite (maximal relaxation: 16.6±1.3%). In conclusion, trioxilin C3 acid was isolated and identified as a novel biologically active arachidonic acid metabolite formed by rabbit aorta when 12-lipoxygenase is supplied exogenously.",

keywords = "Arachidonic acid, Lipoxygenase, Rabbit aorta, Vasorelaxation",

author = "Pfister, {Sandra L.} and Nancy Spitzbarth and Kasem Nithipatikom and Falck, {J R} and Campbell, {William B.}",

note = "Funding Information: We thank Mrs. Gretchen Barg for secretarial assistance. These studies were supported by grants from the National Heart, Lung, Blood Institute (HL-37981 and HL-57895), General Medicine (GM31278), and the Robert A. Welch Foundation.",

year = "2003",

month = jun,

day = "20",

doi = "10.1016/S0304-4165(03)00097-7",

language = "English (US)",

volume = "1622",

pages = "6--13",

journal = "Biochimica et Biophysica Acta - General Subjects",

issn = "0304-4165",

publisher = "Elsevier",

number = "1",

}

TY - JOUR

T1 - Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C3 by rabbit aorta

AU - Pfister, Sandra L.

AU - Spitzbarth, Nancy

AU - Nithipatikom, Kasem

AU - Falck, J R

AU - Campbell, William B.

N1 - Funding Information: We thank Mrs. Gretchen Barg for secretarial assistance. These studies were supported by grants from the National Heart, Lung, Blood Institute (HL-37981 and HL-57895), General Medicine (GM31278), and the Robert A. Welch Foundation.

PY - 2003/6/20

Y1 - 2003/6/20

N2 - Arachidonic acid is metabolized by both the cyclooxygenase and lipoxygenase pathways by rabbit aorta. We investigated the metabolism of 12-hydroperoxyeicosatetraenoic acid by aortic homogenates and microsomes. Rabbit aortic homogenates were incubated in the presence of 14C-arachidonic acid plus 12-lipoxygenase and analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Under these experimental conditions, there was a 14C-metabolite that migrated at 17.6 min. This 14C-metabolite was not observed when aortic homogenates were incubated in the absence of 12-lipoxygenase. Similar results were obtained with aortic microsomes. Further analysis using a different HPLC solvent system resolved the 14C-metabolite into a number of products. Gas chromatography/mass spectrometric (GC-MS) analysis of the major product (labeled peak 3) after conversion to the methyl ester-trimethylsilyl derivative showed two major compounds (compounds A and B) eluting at 13.99 and 14.14 min. The two compounds differed in the intensities of the 213 and 243 m/z ions with 243 being greater than 213 in compound A and the opposite in compound B (relative abundance 213 vs. 243; 100% vs. 43% for compound A and 5% vs. 100% for compound B). Based on the mass spectra, peak 3 contained two metabolites identified as the methyl ester-trimethylsilyl ether derivatives of 8,11,12-trihydroxyeicosatrienoic acid (trioxilin A3) and 8,9,12-trihydroxyeicosatrienoic acid (trioxilin C3). Biological activity of the mixture of two trioxilins isolated from aortic homogenates was tested in phenylephrine-precontracted aortas and found to produce concentration-dependent relaxations (maximal relaxation: 20.1±7.6%). Further testing with authentic trioxilin A3 and C3 revealed that trioxilin C3 was the active metabolite (maximal relaxation: 16.6±1.3%). In conclusion, trioxilin C3 acid was isolated and identified as a novel biologically active arachidonic acid metabolite formed by rabbit aorta when 12-lipoxygenase is supplied exogenously.

AB - Arachidonic acid is metabolized by both the cyclooxygenase and lipoxygenase pathways by rabbit aorta. We investigated the metabolism of 12-hydroperoxyeicosatetraenoic acid by aortic homogenates and microsomes. Rabbit aortic homogenates were incubated in the presence of 14C-arachidonic acid plus 12-lipoxygenase and analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Under these experimental conditions, there was a 14C-metabolite that migrated at 17.6 min. This 14C-metabolite was not observed when aortic homogenates were incubated in the absence of 12-lipoxygenase. Similar results were obtained with aortic microsomes. Further analysis using a different HPLC solvent system resolved the 14C-metabolite into a number of products. Gas chromatography/mass spectrometric (GC-MS) analysis of the major product (labeled peak 3) after conversion to the methyl ester-trimethylsilyl derivative showed two major compounds (compounds A and B) eluting at 13.99 and 14.14 min. The two compounds differed in the intensities of the 213 and 243 m/z ions with 243 being greater than 213 in compound A and the opposite in compound B (relative abundance 213 vs. 243; 100% vs. 43% for compound A and 5% vs. 100% for compound B). Based on the mass spectra, peak 3 contained two metabolites identified as the methyl ester-trimethylsilyl ether derivatives of 8,11,12-trihydroxyeicosatrienoic acid (trioxilin A3) and 8,9,12-trihydroxyeicosatrienoic acid (trioxilin C3). Biological activity of the mixture of two trioxilins isolated from aortic homogenates was tested in phenylephrine-precontracted aortas and found to produce concentration-dependent relaxations (maximal relaxation: 20.1±7.6%). Further testing with authentic trioxilin A3 and C3 revealed that trioxilin C3 was the active metabolite (maximal relaxation: 16.6±1.3%). In conclusion, trioxilin C3 acid was isolated and identified as a novel biologically active arachidonic acid metabolite formed by rabbit aorta when 12-lipoxygenase is supplied exogenously.

KW - Arachidonic acid

KW - Lipoxygenase

KW - Rabbit aorta

KW - Vasorelaxation

UR - http://www.scopus.com/inward/record.url?scp=0037757519&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037757519&partnerID=8YFLogxK

U2 - 10.1016/S0304-4165(03)00097-7

DO - 10.1016/S0304-4165(03)00097-7

M3 - Article

C2 - 12829255

AN - SCOPUS:0037757519

SN - 0304-4165

VL - 1622

SP - 6

EP - 13

JO - Biochimica et Biophysica Acta - General Subjects

JF - Biochimica et Biophysica Acta - General Subjects

IS - 1

ER -