Determination of Lipid Hydroperoxides in Marine Diatoms by the FOX2 Assay

Abstract

Ecologically-relevant marine diatoms produce a plethora of bioactive oxylipins deriving from fatty acid oxidation, including aldehydes, hydroxy-fatty acids, epoxy-hydroxy-fatty acids, and oxo-acids. These secondary metabolites have been related to the negative effect of diatoms on copepod reproduction, causing low hatching success and teratogenesis in the offspring during periods of intense diatom blooms. The common intermediates in the formation of oxylipins are fatty acid hydroperoxides. The quantitative measurement of these intermediates can fundamentally contribute to understanding the function and role of lipoxygenase metabolites in diatom-copepod interactions. Here, we describe the successful adaptation of the ferrous oxidation-xylenol orange 2 (FOX2) assay to diatom samples, which showed several advantages over other spectrophotometric and polarographic methods tested in the present work. Using this method we assessed fatty acid hydroperoxide levels in three diatom species: Skeletonema marinoi, Thalassiosira rotula, and Chaetoceros affinis, and discuss results in light of the literature data on their detrimental effects on copepod reproduction.

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

“…For the formation of the detectable complex, an incubation step is required, from 5 (Mehta et al., ) up to 30 min (Marasca, Greetham, Herring, & Fisk, ; Santas et al., ), being detectable at either 530 (Bilgili et al., ), 550 (Timabud et al., ), or 560 nm (Gay & Gebicki, ; Gay et al., ; Santas et al., ). Butylated hydroxytoluene (BHT) is often used in assay of lipid peroxides, at concentrations of 4 or 4.4 mM to prevent the process of chain oxidation (Gay & Gebicki, ; Nourooz‐Zadeh, ; Orefice et al., ). Usually, a standard curve of cumene hydroperoxide (Marasca et al., ), Fe 3+ (Mehta et al., ), or H 2 O 2 (Bilgili et al., ) is employed.…”

“…The reported concentrations of the FOX reagent components vary in literature for both the ferrous salt and xylenol orange (Dzoyem, Kuete, McGaw, & Eloff, 2014;Dzoyem, Nkuete, Ngameni, & Eloff, 2017;Gay & Gebicki, 2000;Orefice et al, 2015;Santas, Guzmán, Guardiola, Rafecas, & Bou, 2014;Timabud, Sanitchon, & Pongdontri, 2013). The acidic medium is crucial for the assay, with a value of 1.8 being reported as optimal (Gay, Collins, & Gebicki, 1999;Gay & Gebicki, 2000;Timabud et al, 2013), and usually, sulphuric acid 25 mM (Dzoyem et al, 2017;Gay et al, 1999;Nourooz-Zadeh, 1999;Santas et al, 2014) or perchloric acid 110 mM (Gay & Gebicki, 2003;Orefice et al, 2015;Timabud et al, 2013) is chosen as reagent solvent. For the formation of the detectable complex, an incubation step is required, from 5 (Mehta et al, 2015) up to 30 min (Marasca, Greetham, Herring, & Fisk, 2016;Santas et al, 2014), being detectable at either 530 (Bilgili et al, 2013), 550 (Timabud et al, 2013), or 560 nm (Gay & Gebicki, 2000;Gay et al, 1999;Santas et al, 2014).…”

“…The FOX method is based on the generation of a xylenol orange–Fe 3+ complex, as the reagent's Fe 2+ is oxidized upon interacting with the sample‐contained oxidants (Fukuzawa et al., ; Nourooz‐Zadeh, ; Reis et al., ). This is a sensitive and accurate method, with good stability to light/oxygen and with high reproducibility (Gay & Gebicki, ; Mehta, Darji, & Aparnathi, ; Orefice et al., ; Yu, Wang, Zhou, Zhao, & Qiu, ). The FOX method can be employed for determining H 2 O 2 (Nourooz‐Zadeh, ; Sturza et al., ; Yu et al., ), lipid (Ceylan et al., ; Gay & Gebicki, ; Nourooz‐Zadeh, ; Venturini, Simao, & Dichi, ), and protein (Gay & Gebicki, ) hydroperoxides in a wide range of biological samples, from serum (Takci et al., ) and lipoproteins (Erel, ) to cerebrospinal fluid, urine (Erel, ), and tissue homogenates (de Andrade et al., ).…”

Exploring Valuable Lipids in Diatoms

“…A large number of HBI isomers were produced in different diatoms that were cultivated under a series of growth conditions and determined by a combined spectroscopic and chromatographic analysis approach using NMR spectroscopy and chiral gas chromatography, respectively . Alkenes from the Haslea genus exhibit configurational but not geometric isomerism and the stereoisomeric centers (C-22) were identified (Orefice et al, 2015). In contrast, HBIs isolated from P. intermedium and R. setigera show evidence of homochirality, although they clearly exist as a mixture of geometric isomers .…”

“…Biosynthesis of oxylipins (Figure 1) is condition-dependent and immediately activated upon predation (D'ippolito et al, 2005;Cutignano et al, 2011;Nanjappa et al, 2014). Particularly, oxylipin production starts by the oxidation action of iron non-heme enzymes lipoxygenases (LOXs) on the precursor PUFAs and membrane phospholipids upon loss of cell integrity (Orefice et al, 2015). A LOX provides a specific and precise incorporation of a hydroperoxide group into the carbon chains and mediate the further transformations, resulting in generation of a group of important oxylipins (Wichard et al, 2005).…”