Spectrophotometric versus spectrofluorometric assessment in the study of the relationships between lipid peroxidation and metabolic dysregulation

• Published in: Chemical biology & drug design 2019-06, Vol.93 (6), p.1026-1035
• Main Authors: Ungurianu, Anca, Șeremet, Oana, Grădinaru, Daniela, Ionescu‐Tîrgoviște, Constantin, Margină, Denisa, Dănciulescu Miulescu, Rucsandra
• Format: Article
• Language: English
• Subjects: Aged; Animals; Biomarkers - metabolism; carbohydrate; chemical biology; Diabetes Mellitus, Type 2 - metabolism; Female; fluorescence spectroscopy; Humans; lipid; Lipid Peroxidation; Male; Middle Aged; Mitochondria - metabolism; nucleic acid; Oxidative Stress; Phenols - chemistry; Pilot Projects; Rats; Rats, Wistar; Reactive Oxygen Species - metabolism; Spectrometry, Fluorescence - methods; Spectrophotometry, Ultraviolet - methods; Sulfoxides - chemistry
• ISSN: 1747-0277; 1747-0285
• DOI: 10.1111/cbdd.13474

Reactive oxygen species are crucial to normal cell function, but are also part of the pathogenesis of multiple modern maladies. As such, sensitive, fast, and reliable methods of appreciating redox status are needed. We aimed to optimize the Amplex Red (AR) and ferric–xylenol orange (FOX) methods using human serum samples, rat tissue homogenates, and mitochondrial preparations. For AR, we intended to reduce probe concentration, maintaining method sensitivity, as well as extending its use from isolated lipoproteins samples, and readjust it for a high‐throughput application. Also, we evaluated the usefulness of a modified xylenol orange‐based spectrophotometric protocol, comparing and contrasting these methods in terms of clinical relevance and suitability for their further use in assessing redox status of various biological samples in different pathological conditions. Our results show that these optimized protocols are suitable for complex in vivo studies, as they require low quantities of sample and reagents, and are sensitive, rapid, and economical, with the option of adapting them for high‐throughput analysis. For a better assessment of oxidative status of serum‐derived samples, the two methods can be used concurrently, while for tissue‐derived ones, either can be employed for the measurement of a global redox status. This paper was focused on the optimization of two methods (based on Amplex Red and, respectively, xylenol orange) for the detection of redox imbalances in serum samples, rat tissue homogenates, and mitochondrial preparations. The developed protocols are suitable for studies involving various biological samples in different pathological conditions, as they require low quantities of sample and reagents, and are sensitive, rapid, and economical, with the possibility of adjusting them for a high‐throughput analysis.