Chem Biol Drug Des. 2019;1–10. wileyonlinelibrary.com/journal/cbdd | 1 © 2019 John Wiley & Sons A/S 1 | INTRODUCTION Reactive oxygen species (ROS) are part of normal cell life, being involved in a myriad of cellular processes, including cell signaling, proliferation and differentiation, senescence, and apoptosis. However, they are also part of the pathogene- sis of multiple modern maladies; therefore, maintaining redox balance is crucial for normal cell functioning (Kalyanaraman et al., 2012; Miwa et al., 2016; Zielonka et al., 2012). As such, to better appreciate the redox status of different biological samples or experimental models and different pathological conditions, rapid and reliable methods are needed. In this purpose, Amplex Red (AR) or 10-acetyl-3,7-dih ydroxyphenoxazine is a well-known probe used for spectro- fluorimetric assessments of hydrogen peroxide and other peroxides, as AR-based methods exhibit high sensitivity and stability, with measurements being conducted at physiological pH (Kalyanaraman et al., 2012; Krumschnabel et al., 2015; Miwa et al., 2016; Starkov, 2010; Zielonka et al., 2012). The oxidation of the non-fluorescent AR compound consists of Received: 2 August 2018 | Revised: 10 December 2018 | Accepted: 27 December 2018 DOI: 10.1111/cbdd.13474 SPECIAL ISSUE ARTICLE Spectrophotometric versus spectrofluorometric assessment in the study of the relationships between lipid peroxidation and metabolic dysregulation Anca Ungurianu 1 | Oana Șeremet 2 | Daniela Grădinaru 1 | Constantin Ionescu-Tîrgoviște 3 | Denisa Margină 1 | Rucsandra Dănciulescu Miulescu 3,4 1 Faculty of Pharmacy, Department of Biochemistry, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2 Faculty of Pharmacy, Department of Pharmacology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 3 “N. Paulescu” National Institute of Diabetes, Nutrition and Metabolic Diseases Bucharest, Bucharest, Romania 4 Faculty of Dentistry, Department of Endocrinology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Correspondence Denisa Margină, Faculty of Pharmacy, Department of Biochemistry, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania. Email: denisa.margina@gmail.com Abstract 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 spectrophoto- metric protocol, comparing and contrasting these methods in terms of clinical rele- vance 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 oxi- dative 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. KEYWORDS carbohydrate, chemical biology, fluorescence spectroscopy, lipid, nucleic acid