Oxidation of polyunsaturated (PUFA) lipids, catalyzed by free radicals either by non-enzymatic or enzymatic mechanisms, is highly critical in living cells during toxicity, pathophysiological events and disease states, radiation exposure, xenobiotic and drug metabolism, and aging. However, the methods of analysis to characterize the structures of different peroxidized lipids in cells (including PUFA, either free or esterified in the cellular membrane PLs) and the oxidative lipid metabolites, and determination of the extent of lipid peroxidation in the biological systems, are challenging and tedious due to the choice/availability of a suitable analytical method and also the complexity of different peroxidation products of lipids formed in a temporal scale which is system-dependent. Therefore, the current chapter discusses various available analytical methods/techniques to analyze the oxidized lipids and determine lipid peroxidation in the biological systems with an emphasis on health and disease. Finally, the importance of oxidative lipidomics has been discussed as a rapidly emerging discipline of measuring oxidative stress.
Cholesterol is an important plasma membrane component, precursor for hormones and vitamins, and is a regulator of metabolism. However, the oxidized forms of cholesterol (oxysterols) can cause toxicity and induce pro-inflammatory responses and are implicated in chronic degenerative diseases. In general, oxysterols with a modified sidechain serve in various physiological and/or pathophysiological functions. The source of these oxysterols may be exogenous, from the food we ingest, or endogenous, as the by-product of normal cholesterol metabolism, free radical-mediated oxidation, or autoxidation of cholesterol. This chapter discusses the nature of oxysterols as oxidized cholesterol species, oxysterol signaling and pathophysiology, oxysterols and neurodegenerative diseases, ozone-oxidized cholesterol as a new class of oxysterols, detection of 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (cholesterol secoaldehyde, ChSeco or atheronal-A) at sites of inflammation and evidence for in vivo existence, cytotoxicity of ChSeco and pro-inflammatory actions in the cells of mammalian systems, and ChSeco signaling in neuronal cells and implications in Alzheimer’s pathology.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules, with significant effects in human diseases including cancer and cardiovascular disease. The ability to accurately and precisely detect the formation of free radicals within cells and tissues is crucial to developing proper treatments for the problems caused by ROS/RNS. Fluorescent probes have become widely available reagents of detecting ROS/RNS within cells. Several commercially available kits have shown their specificity toward detecting the formation of ROS and RNS. In this chapter, we discuss the principle behind each kit and the benefits and shortcomings of these kits, namely dihydroethidium (DHE), dichlorohydrofluoresin diacetate (DCF-DA), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate), and 10-acetyl-3,7-dihyrdroxyphenoxazine (Amplex red). DHE is used to specifically detect superoxide, while DCF-DA readily detects hydroxyl radicals. Amplex red is used to detect hydrogen peroxide, and DAF-FM is used for measuring nitric oxide. However, due to the nature of their reactivity, the probes are not absolutely specific for the noted ROS/RNS species, and will react with others. ROS measurement may need to be made in real time, and they are short-lived within the cell, especially superoxide and nitric oxide. This chapter explains the mechanism behind each chemical kit, the protocols used with the kit, and show typical results after imaging. Additionally, an assessment is made on the use of the kit, identifying the advantages and disadvantages of each probe.
In pulsed electron paramagnetic resonance (EPR), free-induction decay (FID) or spin echo (SE) signals of unpaired electrons are recorded in the time-domain. In both methods, electromagnetic waves play an important role in the detection of unpaired electrons in EPR spectroscopy. The resonator generates and senses electromagnetic waves and therefore serves as a critical interface between unpaired electrons and the transmit/receive systems of an EPR spectrometer. Since a resonator is a sensitive electrical circuit that can amplify voltages and currents when the electrical circuit of the resonator is on resonance, the resonator is an essential component for EPR detection in continuous wave and pulsed EPR. Without the resonator, EPR signals cannot be detected with sufficient sensitivity. In this chapter, the basics of resonators and some examples of resonators used in preclinical studies with small animals and human subjects are explained.
