Development of enzyme-linked immunosorbent assay for 8-iso-prostaglandin F_2α, a biomarker of oxidative stress in vivo, and its application to the quantification in aged rats

Abstract

8-iso-Prostaglandin F_2α (8-iso-PGF_2α) is one of the isoprostanes that are mainly generated nonenzymatically in vivo from arachidonic acid through free radical-induced lipid peroxidation. To assess oxidative stress in vivo, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) for 8-iso-PGF_2α. A sensitive calibration curve allowed the quantification of the amounts from 0.23 pg to 98.4 pg with 4.7 pg of 50% displacement in one assay. The ELISA method was applied to the measurement of the plasma levels of 8-iso-PGF_2α in young rats (4–8 weeks of age) and aged rats (106–123 weeks). The average level of esterified form in the plasma from aged rats was 30.6-fold higher than that in the plasma from young rats, reflecting the enhanced status of oxidative stress in aged animals. In addition, the aged rats exhibited higher levels of this F₂-isoprostane esterified to lipids from liver and kidney, suggesting local oxidative injury in specific organs. These results indicate the utility and accuracy of our ELISA for 8-iso-PGF_2α as a biomarker in vivo to assess systemic oxidative stress in animals or humans as well as oxidative injury at local sites.

Introduction

A series of prostaglandin F₂-like compounds termed F₂-isoprostanes are known to be produced in vivo nonenzymatically from arachidonic acid, an essential nutrient, in animals and humans predominantly by a free radical-induced lipid peroxidation [1], [2], [3]. A large body of evidence has suggested that the measurement of F₂-isoprostanes is a reliable and useful approach to assess lipid peroxidation and oxidative stress in vivo [4]. Of these, the levels of 8-iso-prostaglandin F_2α (8-iso-PGF_2α) have been studied more extensively as a biomarker of oxidative marker in vivo until now. The majority of arachidonic acid is esterified to the sn-2 position of phospholipids in cell membranes of various tissues or blood cells. Earlier studies have shown that F₂-isoprostanes including 8-iso-PGF_2α are initially generated in situ from arachidonic acid esterified in phospholipids under the oxidative stress and subsequently released as a free form by the action of phospholipases [2]. Thus, 8-iso-PGF_2α can be detectable as both free and esterified forms. The free form of 8-iso-PGF_2α can be quantified to evaluate total generation of it in vivo that reflects the enhanced systemic lipid peroxidation. By contrast, the measurement of 8-iso-PGF_2α esterified in phospholipids in tissues would provide the methods for assessing the oxidative injury in specific cells, tissues, or organs. For example, the administration of CCl₄ to animals has been shown to cause the marked elevation of circulating levels of F₂-isoprostanes [1], [2]. Alternatively, higher levels of F₂-isoprostanes have been recognized in plasma of smokers due to free radicals in cigarette smoke, whereas the intake of antioxidants such as vitamin C decreased the plasma levels of F₂-isoprostanes as biomarkers of lipid peroxidation [5]. Hence, the development of a sensitive and specific assay for 8-iso-PGF_2α should be highly useful as a noninvasive approach by analyzing the levels in plasma and urine to assess the involvement of oxidative stress and lipid peroxidation in human or animal disease from the point of prognostic diagnosis and the evaluation of antioxidants in pharmaceuticals and nutraceuticals.

Until now, most studies have employed gas chromatography–mass spectrometry (GC–MS) to quantify the levels of 8-iso-PGF_2α as a more sensitive and specific way [3], [6]. However, the mass spectrometric analysis requires high-cost apparatus and an analytical expert to operate the quantitative analysis. Additionally, the GC–MS analysis requires extensive sample preparations and derivatization of analytes. Moreover, the use of a deuterium-labeled internal standard required for the quantification with mass spectrometric quantification becomes more expensive for the analysis of multiple samples. As an alternative to GC–MS, liquid chromatography–mass spectrometry (LC–MS) is becoming useful for the analysis and quantification of analytes in clinical laboratories. However, many methods still require extensive sample purification prior to quantitative analysis. More recently, Saenger et al. has reported an improved assay for the measurement of urinary 8-iso-PGF_2α by LC–MS without sample pretreatment [7]. Recent advances have been reviewed regarding measurement of oxidative stress parameters using LC–MS [8]. Nevertheless, this mass spectrometric approach is also limited by smaller numbers of analytes assayed from biological sources due to the sequential operation of expensive instrumentation as compared with immunological methods. Therefore, we planed to develop a convenient and sensitive immobilized enzyme-linked immunosorbent assay (ELISA) specific for 8-iso-PGF_2α. This immunological method is applicable for many samples at the same time without high-cost facilities and an analytical expert. The present study was undertaken to prepare monoclonal antibodies specific for 8-iso-PGF_2α. Then, using the specific monoclonal antibody, we attempted to develop the immobilized ELISA for 8-iso-PGF_2α. Finally, we applied our method to the quantification of 8-iso-PGF_2α free in plasma and esterified in plasma lipids as well as the form esterified in tissues lipids from liver and kidney in young and old rats.