Unresolved issues in the analysis of F₂-isoprostanes, F₄-neuroprostanes, isofurans, neurofurans, and F₂-dihomo-isoprostanes in body fluids and tissue using gas chromatography/negative-ion chemical-ionization mass spectrometry

Abstract

F₂-isoprostanes (F₂-IsoPs) generated from arachidonic acid (AA) have been recognized as the most reliable marker of nonenzymatic lipid peroxidation in vivo. F₂-IsoPs are initially produced in esterified form on phospholipids, and then released into body fluids in free form. The same mechanism can lead to generation of F₄-neuroprostanes (F₄-NPs) and F₂-dihomo-IsoPs from docosahexaenoic acid (DHA) and adrenic acid, respectively. In addition, isofurans (IsoFs) and neurofurans (NFs) may be preferentially produced from AA and DHA, respectively, under high oxygen tension. The detection of F₂-IsoPs using gas chromatography/negative-ion chemical-ionization mass spectrometry (GC/NICI-MS) has been widely employed, which is important for human body fluids containing low quantity of free-form F₂-IsoPs. F₄-NPs have also been detected using GC/NICI-MS, but multiple peaks need to be quantified. In this paper, we summarize the basic workflow of the GC/NICI-MS method for analyzing F₂-IsoPs and F₄-NPs, and various formats of assays conducted by different groups. We then discuss the feasibility of simultaneous analysis of IsoFs, NFs, and F₂-dihomo-IsoPs with F₂-IsoPs or F₄-NPs. Representative GC chromatograms for analyzing these markers in human body fluids and rat brain tissue are demonstrated. Furthermore, we discuss several factors that may affect the performance of the analysis, such as those related to the sample processing steps, interference from specimens, types of GC liners used, and the addition of electron multiplier voltage in the method setting for the MS detector. Finally, we question the appropriateness of measuring total (free plus esterified) levels of these markers in body fluids.

Keywords::

• free form
• esterified form
• human body fluid
• thin-layer chromatography
• triphenylphosphine

Acknowledgement

The methods of sample purification and GC/NICI-MS analysis for detecting F₂-IsoPs and F₄-NPs in body fluids and tissue, conducted in the laboratory of the corresponding author, were modified from those originally obtained from Professor Jason Morrow's laboratory at Vanderbilt University before the year 2008.

Declaration of interest

The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.

The work was supported by grants to H-C Yen from Ministry of Science and Technology (formerly known as National Science Council), Taiwan (NSC97-2320-B-182-012-MY3) and from the Ministry of Education, Taiwan (EMRPD180451). The grant funders play no role in the study design, data acquisition, or manuscript preparation.