A Simple and Sensitive Method for the Determination of Fibric Acids in the Liver by Liquid Chromatography

Abstract

Fibrates are used in biochemical and pharmacological studies as bioactive tools. Nevertheless, most studies have lacked information concerning the concentrations of fibric acids working inside tissues because a simple and sensitive method is not available for their quantitation. This study aimed to develop a simple and sensitive bioanalytical method for the quantitation of clofibric, bezafibric and fenofibric acids in samples of very small portions of tissues. Fibric acids were extracted into n-hexane-ethyl acetate from tissue homogenates (10 mg of liver, kidney or muscle) or serum (100 µL) and were derivatized with 4-bromomethyl-6,7-dimethoxycoumarin, followed by HPLC with fluorescence detection. These compounds were separated isocratically on a reversed phase with acetonitrile-water. Standard analytical curves were linear over the concentration range of 0.2-20 nmol/10 mg of liver. Precision and accuracy were within acceptable limits. Recovery from liver homogenates ranged from 93.03 to 112.29%. This method enabled the quantitation of fibric acids in 10 mg of liver from rats treated with clofibric acid, bezafibric acid or fenofibrate. From these analytical data, it became clear that there was no large difference in ratio of acyl-CoA oxidase 1 (Acox1) mRNA level to fibric acid content in the liver among the three fibric acids, suggesting that these three fibric acids have similar potency to increase expression of the Acox1 gene, which is a target of peroxisome proliferatoractivated receptor α. Thus, the proposed method is a simple, sensitive and reliable tool for the quantitation of fibric acids working in vivo inside livers.Key words clofibric acid; bezafibric acid; fenofibric acid; HPLC quantitation; rat liverFibrates are known to increase low-density lipoprotein size, high-density lipoprotein synthesis, reverse cholesterol transport and insulin sensitivity, and suppress plasma triacylglycerol, and vascular and systemic inflammation. [1][2][3][4][5] Owing to these beneficial effects, fibrates are utilized as drugs for therapies of diseases, such as primary dyslipidemia, metabolic syndrome, type 2 diabetes, nonalcoholic fatty liver disease and atherosclerosis. Most of these actions of fibrates are considered to be mediated through peroxisome proliferatoractivated receptor α (PPARα), which then forms a heterodimer with retinoid X receptor (RXR); once activated, the PPARα/ RXR heterodimer binds to peroxisome proliferator response element, causing increased or decreased transcription of many genes in various tissues.6,7) Tissues with the highest expression of PPARα are liver, kidney, heart and brown adipose. Fatty acids and eicosanoids are the endogenous ligands of PPARα [7][8][9] ; before starting to function, fibrates, which are ester forms, have to be converted by hepatic esterases to fibric acids, carboxylic acid forms that are active forms as PPARα ligands. 7,10) Because of their potent activities, fibrates or fibric acids are widely used in biochemical and pharmacological studies as tools. Nevertheless, most...

Fibrates Reduce Triacylglycerol Content by Upregulating Adipose Triglyceride Lipase in the Liver of Rats

“…Fibric acids were extracted and analyzed as described previously (20). In brief, fibric acids were extracted into n-hexane-ethyl acetate (85:15, by vol.)…”