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

Research output: Contribution to journalArticle

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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 languageEnglish (US)
Pages (from-to)6-13
Number of pages8
JournalBiochimica et Biophysica Acta - General Subjects
Volume1622
Issue number1
DOIs
StatePublished - Jun 20 2003

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Arachidonate 12-Lipoxygenase
Metabolites
Metabolism
Aorta
Rabbits
Acids
Microsomes
Arachidonic Acid
High pressure liquid chromatography
Esters
High Pressure Liquid Chromatography
Lipoxygenase
Phenylephrine
Reverse-Phase Chromatography
Prostaglandin-Endoperoxide Synthases
Gas Chromatography
Ether
Derivatives
Ions
Bioactivity

Keywords

  • Arachidonic acid
  • Lipoxygenase
  • Rabbit aorta
  • Vasorelaxation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C3 by rabbit aorta. / Pfister, Sandra L.; Spitzbarth, Nancy; Nithipatikom, Kasem; Falck, J R; Campbell, William B.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1622, No. 1, 20.06.2003, p. 6-13.

Research output: Contribution to journalArticle

Pfister, Sandra L. ; Spitzbarth, Nancy ; Nithipatikom, Kasem ; Falck, J R ; Campbell, William B. / Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C3 by rabbit aorta. In: Biochimica et Biophysica Acta - General Subjects. 2003 ; Vol. 1622, No. 1. pp. 6-13.
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AU - Falck, J R

AU - Campbell, William B.

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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

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KW - Vasorelaxation

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