Abstract
A method for the simultaneous quantification of urinary linoleic and arachidonic acid derived epoxides and diols, as well as the arachidonate omega hydroxylated product has been developed. The method employs negative mode electrospray ionization and HPLC with tandem mass spectroscopy for quantification. Odd chain length saturated epoxy and dihydroxy fatty acids are used as analytical surrogates resulting in linear calibrations (r (2) > or = 0.9995). Standard addition analyses showed that matrix effects do not prevent these surrogates from yielding reliable quantitative results. Using 4 ml urine aliquots at a final extract volume of 100 micro l and injecting 10 micro l, method detection limits and limits of quantification were < or =0.5 and 1.5 nM, respectively. The sensitivity for dihydroxy lipids was from 3- to 10-fold greater than the corresponding epoxy fatty acid. Shot to shot run times of 31 min were achieved. Rodent and human urine analyses indicated the method sensitivity is sufficient for general research applications. In addition, diurnal fluctuations in linoleate and arachidonate derived metabolites were observed in human subjects.
Highlights
A method for the simultaneous quantification of urinary linoleic and arachidonic acid derived epoxides and diols, as well as the arachidonate omega hydroxylated product has been developed
Epoxy fatty acids lack a strong chromophore, limiting the sensitivity of detection based on UV absorption
The molar extinction coefficient () is defined as the amount of radiant energy absorbed on a path through a solution of known molar concentration expressed in Abs moleϪ1cmϪ1
Summary
A method for the simultaneous quantification of urinary linoleic and arachidonic acid derived epoxides and diols, as well as the arachidonate omega hydroxylated product has been developed. The simultaneous quantification of cytochrome P450 dependent linoleate and arachidonate metabolites in urine by HPLC-MS/MS. The 20-HETE inhibits large and medium conductance calcium dependent potassium (KϩCa) channels (19), preventing cellular hyperpolarization This action of 20-HETE is opposed by the epoxides of arachidonic acid, i.e. epoxy eicosatrienoic acids (EETs), which increase the open state probability of KϩCa channels (3, 20, 21). These octadecanoid diols or dihydroxyoctadecenoic acids (DiHOMEs) inhibit mitochondrial respiration (8, 29, 30), increase vascular permeability (31), and have been identified as their glucuronide conjugates in the urine of children with generalized peroxisomal disorders (10). The simultaneous determination of both linoleate and arachidonate derived oxidation products in urine may provide unique insights into the associations between these compounds under various physiological conditions, including disease
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