Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid

2000 ◽  
Vol 279 (2) ◽  
pp. H863-H871 ◽  
Author(s):  
Kristopher G. Maier ◽  
Lisa Henderson ◽  
Jayashree Narayanan ◽  
Magdalena Alonso-Galicia ◽  
John R. Falck ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Interstitial Fluid ◽  
Internal Standard ◽  
Dienoic Acid ◽  
Renal Tissue ◽  
Gas Chromatography Mass Spectrometry ◽  
Epoxyeicosatrienoic Acids ◽  
Hplc Assay ◽  
Standard Curve ◽  
Cytochrome P 450

This study describes a fluorescent HPLC assay for measuring 20-hydroxyeicosatetraenoic acid (20-HETE) and other cytochrome P-450 metabolites of arachidonic acid in urine, tissue, and interstitial fluid. An internal standard, 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, was added to samples, and the lipids were extracted and labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. P-450 metabolites were separated on a C18 reverse-phase HPLC column. Coelution and gas chromatography-mass spectrometry studies confirmed the identity of the 20-HETE peak. The 20-HETE peak can be separated from those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatrienoic acids. Known amounts of 20-HETE were used to generate a standard curve (range 1–10 ng, r 2 = 0.98). Recovery of 20-HETE from urine averaged 95%, and the intra-assay variation was <5%. Levels of 20-HETE were measured in 100 μl of urine and renal interstitial fluid or 0.1 mg of renal tissue. The assay was evaluated by studying the effects of 1-aminobenzotriazole (ABT) on the excretion of 20-HETE in rats. ABT reduced excretion of 20-HETE by >65% and inhibited the formation of 20-HETE by renal microsomes. The availability of this assay should facilitate work in this field.

Enhanced synthesis of epoxyeicosatrienoic acids by cholesterol-fed rabbit aorta

1991 ◽  
Vol 261 (3) ◽  
pp. H843-H852 ◽  
Author(s):  
S. L. Pfister ◽  
J. R. Falck ◽  
W. B. Campbell
Keyword(s):  
Arachidonic Acid ◽  
Mass Spectra ◽  
Acid Metabolism ◽  
Rabbit Aorta ◽  
Arachidonic Acid Metabolism ◽  
Gas Chromatography Mass Spectrometry ◽  
Epoxyeicosatrienoic Acids ◽  
Aortic Tissue ◽  
Cytochrome P 450 ◽  
Hydroxyeicosatetraenoic Acids

Arachidonic acid metabolism via cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase was investigated in thoracic aortic tissue obtained from rabbits fed either standard rabbit chow or chow containing 2% cholesterol. Aortic strips were incubated with [14C]arachidonic acid and A23187. Metabolites from extracted media were resolved by high-pressure liquid chromatography (HPLC). Normal and cholesterol-fed rabbit aortas synthesized prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs). The major cyclooxygenase products were 6-keto-PGF1 alpha and PGE2. Basal aortic 6-keto-PGF1 alpha production was slightly reduced in cholesterol-fed compared with normal rabbits. 12(S)- and 15(S)-HETE were the major aortic lipoxygenase products from both normal and cholesterol-fed rabbits. The structures were confirmed by gas chromatography-mass spectrometry (GC-MS). Only cholesterol-fed rabbit aortas metabolized arachidonic acid via cytochrome P-450 epoxygenase to the epoxyeicosatrienoic acids (EETs). 14,15-, 11,12-, 8,9-, and 5,6-EET were identified based on comigration on HPLC with known 14C-labeled standards and typical mass spectra. Incubation of normal aorta with 14,15-EET decreased the basal synthesis of 6-keto-PGF1 alpha. The other EETs were without effect. The four EET regioisomers relaxed the norepinephrine-precontracted normal and cholesterol-fed rabbit aorta. The relaxation response to 14,15-EET was greater in aortas from cholesterol-fed rabbits. These studies demonstrate that hypercholesterolemia, before the development of atherosclerosis, alters arachidonic acid metabolism via both the cyclooxygenase and epoxygenase pathways.

scholarly journals Investigations on the Key Odorants Contributing to the Aroma of Children Soy Sauce by Molecular Sensory Science Approaches

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1492
Author(s):  
Jia Huang ◽  
Haitao Chen ◽  
Zhiming Zhang ◽  
Yuping Liu ◽  
Binshan Liu ◽  
...  
Keyword(s):  
Gas Chromatography ◽  
Solid Phase ◽  
Internal Standard ◽  
Retention Indices ◽  
Soy Sauce ◽  
Gas Chromatography Mass Spectrometry ◽  
Volatile Components ◽  
Active Compounds ◽  
Standard Curve ◽  
Key Odorants

To investigate the key odor-active compounds in children’s soy sauce (CSS), volatile components were extracted by means of solvent extraction coupled with solvent-assisted flavor evaporation (SE-SAFE) and solid-phase microextraction (SPME). Using gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS), we identified a total of 55 odor-active compounds in six CSSs by comparing the odor characteristics, MS data, and retention indices with those of authentic compounds. Applying aroma extract dilution analysis (AEDA), we measured flavor dilution (FD) factors in SE-SAFE isolates, ranging from 1 to 4096, and in SPME isolates, ranging from 1 to 800. Twenty-eight odorants with higher FD factors and GC-MS responses were quantitated using the internal standard curve method. According to their quantitated results and thresholds in water, their odor activity values (OAVs) were calculated. On the basis of the OAV results, 27 odorants with OAVs ≥ 1 were determined as key odorants in six CSSs. These had previously been reported as key odorants in general soy sauce (GSS), so it was concluded that the key odorants in CSS are the same as those in GSS.

Role of PGI2 and epoxyeicosatrienoic acids in relaxation of bovine coronary arteries to arachidonic acid

1993 ◽  
Vol 264 (2) ◽  
pp. H327-H335 ◽  
Author(s):  
M. Rosolowsky ◽  
W. B. Campbell
Keyword(s):  
Arachidonic Acid ◽  
Coronary Arteries ◽  
Vascular Tone ◽  
Epoxyeicosatrienoic Acids ◽  
Lipoxygenase Inhibitor ◽  
Physiological Processes ◽  
Coronary Vascular Tone ◽  
Cytochrome P 450 ◽  
Endothelium Dependent Relaxation

Metabolites of arachidonic acid regulate several physiological processes, including vascular tone. The purpose of this study was to determine which metabolites of arachidonic acid are produced by bovine coronary arteries and which may regulate coronary vascular tone. Arachidonic acid induced a concentration-related, endothelium-dependent relaxation [one-half maximum effective concentration (EC50) of 2 x 10(-7) M and a maximal relaxation of 91 +/- 2% at 10(-5) M] of bovine coronary arteries that were contracted with U-46619, a thromboxane mimetic. The concentration of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), a metabolite of prostaglandin I2 (PGI2), increased from 82 +/- 6 to 328 +/- 24 pg/ml with arachidonic acid (10(-5) M). Treatment with the cyclooxygenase inhibitor indomethacin attenuated arachidonic acid-induced relaxations by approximately 50% and blocked the synthesis of 6-keto-PGF1 alpha. PGI2 caused a concentration-related relaxation (EC50 of 10(-8) M and a maximal relaxation of 125 +/- 11% at 10(-7) M). BW755C, a cyclooxygenase and lipoxygenase inhibitor, inhibited arachidonic acid-induced relaxation to the same extent as indomethacin. When vessels were treated with both indomethacin and BW755C, the inhibition of relaxation was the same as either inhibitor alone. SKF 525a, a cytochrome P-450 inhibitor, reduced arachidonic acid-induced relaxation by approximately 50%. When SKF 525a was given in combination with indomethacin, the relaxation by arachidonic acid was almost completely inhibited. SKF 525a inhibited the synthesis of epoxyeicosatrienoic acids (EETs).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of Volatile Organic Contaminants in Bulk Oils (Edible, Injectable, and Other Internal Medicinal) by Purge-and-Trap Gas Chromatography/Mass Spectrometry

1994 ◽  
Vol 77 (3) ◽  
pp. 647-654 ◽  
Author(s):  
Don W Thompson
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Vegetable Oils ◽  
Organic Contaminants ◽  
Internal Standard ◽  
Gas Chromatography Mass Spectrometry ◽  
Purge And Trap ◽  
Standard Curve ◽  
Volatile Organic ◽  
Volatile Organic Contaminants

Abstract Purge-and-trap gas chromatography/mass spectrometry is evaluated for the quantitation of part-per-billion levels of volatile organic contaminants in bulk vegetable oils. Results using 2 purge techniques (direct purging of the heated oil and purging after dispersing the oil on an aluminum oxide powder) and 2 quantitative methods (standard curve and deuterium-labeled internal standard addition) are reported. Twenty volatile compounds and 8 vegetable oils were investigated. Recovery data and estimated detection limits for each compound are reported for each purge technique. Generally acceptable recoveries (70-130% for more than 90% of the analyte spikes) and acceptable detection levels (approximately 4-10 ppb) were obtained for all compounds using either the external standard curve or the deuterium-isotope-labeled internal standard. The use of a dispersant (such as alumina) for sample purging resulted in poor recoveries of the highly volatile contaminants.

scholarly journals Role of endogenous CYP450 metabolites of arachidonic acid in maintaining the glomerular protein permeability barrier

2007 ◽  
Vol 293 (2) ◽  
pp. F501-F505 ◽  
Author(s):  
Jan Michael Williams ◽  
Mukut Sharma ◽  
Siddam Anjaiahh ◽  
John R. Falck ◽  
Richard J. Roman
Keyword(s):  
Arachidonic Acid ◽  
Essential Role ◽  
Dienoic Acid ◽  
Permeability Barrier ◽  
Selective Inhibitors ◽  
Glomerular Permeability ◽  
Isolated Glomeruli ◽  
Cytochrome P 450 ◽  
Protein Permeability

This study examined the metabolism of arachidonic acid (AA) by cytochrome P-450 enzymes in isolated glomeruli and the effects of selective inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatetraenoic acids (EETs) on glomerular permeability to albumin ( Palb). Glomeruli avidly produced 20-HETE, EETs, dihydroxyeicosatetraenoic acids (diHETEs), and HETEs when incubated with exogenous AA. N-hydroxy- N′-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 μM) selectively inhibited the formation of 20-HETE by 95% and increased Palb from 0.00 ± 0.08 to 0.73 ± 0.10 ( n = 43 glomeruli, 4 rats). Addition of a 20-HETE mimetic, 20-hydroxyeicosa-5( Z),14( Z)-dienoic acid (20-5,14-HEDE; 1 μM) opposed the effects of HET0016 (10 μM) to increase Palb (0.21 ± 0.10, n = 36 glomeruli, 4 rats). Preincubation of glomeruli with exogenous AA to increase basal production of 20-HETE had a similar effect. We also examined the effect of an epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MSPPOH; 5 μM), on Palb. MSPPOH (5 μM) significantly increased Palb but had no effect on the synthesis of EETs in glomeruli incubated with AA. However, MSPPOH (5 μM) selectively reduced epoxygenase activity by 50% in glomeruli incubated without added AA. Pretreatment with 8,9-EET (100 nM) attenuated the effects of MSPPOH (5 μM) on Palb. These results indicate that glomeruli produce 20-HETE, EETs, diHETEs, and HETEs and that endogenously formed 20-HETE and EETs play an essential role in the maintenance of the glomerular permeability barrier to albumin.

EET homologs potently dilate coronary microvessels and activate BKCa channels

2001 ◽  
Vol 280 (6) ◽  
pp. H2430-H2440 ◽  
Author(s):  
Yongde Zhang ◽  
Christine L. Oltman ◽  
Tong Lu ◽  
Hon-Chi Lee ◽  
Kevin C. Dellsperger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Eicosapentaenoic Acid ◽  
Epoxyeicosatrienoic Acids ◽  
Bkca Channels ◽  
Small Arteries ◽  
Structural Specificity ◽  
Cytochrome P 450 ◽  
Vascular Myocytes

Epoxyeicosatrienoic acids (EETs) are released from endothelial cells and potently dilate small arteries by hyperpolarizing vascular myocytes. In the present study, we investigated the structural specificity of EETs in dilating canine and porcine coronary microvessels (50–140 μm ID) and activating large-conductance Ca2+-activated K+(BKCa) channels. The potencies and efficacies of EET regioisomers and enantiomers were compared with those of two EET homologs: epoxyeicosaquatraenoic acids (EEQs), which are made from eicosapentaenoic acid by the same cytochrome P-450 epoxygenase that generates EETs from arachidonic acid, and epoxydocosatetraenoic acids (EDTs), which are EETs that are two carbons longer. With EC50 values of 3–120 pM but without regio- or stereoselectivity, EETs potently dilated canine and porcine microvessels. Surprisingly, the EEQs and EDTs had comparable potencies and efficacies in dilating microvessels. Moreover, 50 nM 13,14-EDT activated the BKCa channels with the same efficacy as either 11,12-EET enantiomer at 50 nM. We conclude that coronary microvessels and BKCa channels possess low structural specificity for EETs and suggest that EEQs and EDTs may thereby also be endothelium-derived hyperpolarizing factors.

scholarly journals 20-HETE modulates myogenic response of skeletal muscle resistance arteries from hypertensive Dahl-SS rats

2001 ◽  
Vol 280 (3) ◽  
pp. H1066-H1074 ◽  
Author(s):  
Jefferson C. Frisbee ◽  
Richard J. Roman ◽  
U. Murali Krishna ◽  
John R. Falck ◽  
Julian H. Lombard
Keyword(s):  
Skeletal Muscle ◽  
Arachidonic Acid ◽  
Dienoic Acid ◽  
Vessel Diameter ◽  
Transmural Pressure ◽  
Myogenic Response ◽  
Hydroxyeicosatetraenoic Acid ◽  
Resistance Arteries ◽  
Cytochrome P 450

The present study determined the role of 20-hydroxyeicosatetraenoic acid [20-HETE; produced by ω-hydroxylation of arachidonic acid via cytochrome P-450 (CP450) 4A enzymes] in regulating myogenic activation of skeletal muscle resistance arteries from normotensive (NT) and hypertensive (HT) Dahl salt-sensitive (SS) rats. Gracilis arteries (GA) were isolated from each rat and viewed via television microscopy, and changes in vessel diameter with altered transmural pressure were measured with a video micrometer. Under control conditions, GA from both groups exhibited strong, endothelium-independent myogenic activation. Treatment of GA with 17-octadecynoic acid (17-ODYA; inhibitor of CP450 4A enzymes) did not alter myogenic activation in NT rats, but impaired this response in HT animals. Treatment of GA from HT rats with dibromo-dodecynyl-methylsulfimide (DDMS; inhibitor of 20-HETE production) impaired myogenic activation, as did application of 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, an antagonist for 20-HETE receptors. Application of iberiotoxin, a Ca2+-activated potassium (KCa) channel inhibitor, restored myogenic activation from HT rats treated with DDMS. These results suggest that myogenic activation of skeletal muscle resistance arteries from NT Dahl-SS rats does not depend on CP450, whereas myogenic activation of these vessels in HT Dahl-SS rats is partly a function of 20-HETE production, inhibiting KCachannels through a receptor-mediated process.

Exaggerated response to adenosine in kidneys from high salt-fed rats: role of epoxyeicosatrienoic acids

2005 ◽  
Vol 289 (2) ◽  
pp. F386-F392 ◽  
Author(s):  
Elvira L. Liclican ◽  
John C. McGiff ◽  
Paulina L. Pedraza ◽  
Nicholas R. Ferreri ◽  
John R. Falck ◽  
...  
Keyword(s):  
High Salt ◽  
Gas Chromatography Mass Spectrometry ◽  
Epoxyeicosatrienoic Acids ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Dose Dependent ◽  
Cytochrome P 450 ◽  
Adenosine Analog ◽  
Krebs Buffer

Cytochrome P-450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels when adenosine2A receptors (A2AR) are stimulated. As high salt (HS) intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in HS-fed rats. Male Sprague-Dawley rats were fed either HS (4.0% NaCl) or normal salt (NS; 0.4% NaCl) diet. On day 8, isolated kidneys were perfused with Krebs' buffer containing indomethacin (10 μM) and l-NAME (200 μM) and preconstricted to ∼150 mmHg with infusion of phenylephrine (10−7 M). Renal effluents were extracted for analysis of eicosanoids by gas chromatography-mass spectrometry. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1–10 μg) resulted in dose-dependent dilation; at 10 μg, perfusion pressure (PP) was lowered to a greater extent in the kidneys of HS rats compared with NS rats (−60 ± 4 vs. −31 ± 8 mmHg; P < 0.05) and the area of response was increased (27 ± 6 vs. 9 ± 4 mm2; P < 0.05), as was EET release (132 ± 23 vs. 38 ± 18 ng; P < 0.05). HS treatment increased A2AR and CYP2C23 protein expression. A selective epoxygenase inhibitor, MS-PPOH (12 μM), significantly reduced the response to 2-CA in HS rats; PP, area of response, and EET release decreased by 40, 70, and 81%, respectively, whereas lesser changes were evident in NS kidneys. Thus the greater vasodilator response to 2-CA seen in kidneys obtained from HS-fed rats was mediated by increased EET release. As EETs are renal vasodilator and natriuretic eicosanoids, interactions between adenosine and EETs may contribute to the adaptive response to HS intake.

Cytochrome P-450-dependent HETEs: profile of biological activity and stimulation by vasoactive peptides

1996 ◽  
Vol 271 (4) ◽  
pp. R863-R869 ◽  
Author(s):  
M. A. Carroll ◽  
M. Balazy ◽  
P. Margiotta ◽  
D. D. Huang ◽  
J. R. Falck ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Perfusion Pressure ◽  
Internal Standard ◽  
Gas Chromatography Mass Spectrometry ◽  
Ang Ii ◽  
Biological Profile ◽  
Hormonal Stimulation ◽  
Dose Dependent ◽  
Cytochrome P 450 ◽  
Hydroxyeicosatetraenoic Acids

The cytochrome P-450 pathway is capable of metabolizing arachidonic acid to omega- and subterminal hydroxylase metabolites, 16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acids (P-450 HETEs). We have quantitated, by gas chromatography-mass spectrometry (GC/MS), endogenous HETEs exiting the rabbit isolated perfused kidney elicited by hormonal stimulation. Kidneys were perfused with Krebs-Henseleit solution containing indomethacin (2.8 microM) to prevent further metabolism of HETEs by cyclooxygenase. Phenylephrine (2-3 microM) was added to the perfusate to raise perfusion pressure to approximately 80 mmHg. Angiotensin II (ANG II), arginine vasopressin (AVP), and bradykinin (BK) were injected into the renal artery and perfusates collected throughout the vasoactive response. After addition of an internal standard, deuterated 19-HETE, perfusates were extracted and purified and P-450 HETEs were derivatized for GC/MS analysis. Under basal conditions, 16-, 18-, 19-, and 20-HETEs were released (range: 50-270 pg/ml), 19-HETE being the highest and fivefold greater than 16-HETE, the lowest. Injection of 50 ng ANG II increased by two- to sixfold P-450 HETE release associated with an increase of 40 +/- 11 mmHg in perfusion pressure. An equipressor dose of AVP (50 ng) did not release P-450 HETEs nor did a 5-micrograms dose of the vasodilator peptide BK, which decreased perfusion pressure by 22 +/- 6 mmHg. Authentic 19- and 20-HETE isomers resulted in dose-dependent dilation, as did 18(R)- and 16(R)-HETEs, whereas their enantiomers and 17-HETE isomers were without effect on perfusion pressure. The vasodilator effects of 18(R)- and 16(R)-HETEs, like 20- and 19-HETEs, were inhibited by indomethacin. Furthermore, P-450 HETEs exhibited both regio- and stereoselective inhibition of proximal tubule adenosine triphosphatase (ATPase) activity. The (S) enantiomers of 16- and 17-HETE potently inhibited activity, whereas their (R) isomers and other P-450 HETEs had negligible effects on ATPase activity. The quantity of HETEs released from the kidney, either under basal conditions or when stimulated by ANG II, and their biological profile suggest that subterminal HETEs may participate in renal mechanisms affecting vasomotion and tubular transport.

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