Linoleic Acid-Induced Ultra-Weak Photon Emission from Chlamydomonas reinhardtii as a Tool for Monitoring of Lipid Peroxidation in the Cell Membranes

Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the detection of lipid peroxidation in the cell membranes

The oxidation of dehydroascorbic acid and 2,3-diketogulonate by distinct reactive oxygen species

l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), 'superoxide' (a ∼50 : 50 mixture of [Formula: see text] and [Formula: see text]), hydroxyl radicals (•OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1O2). Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate (OTP; '2-keto-l-xylonate'). Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2 The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product 'fingerprints' are analytically useful, indicating which ROS are acting in vivo

Determination of glycolic acid level in higher plants during photorespiration by stable isotope dilution mass spectrometry with double-labelling experiments

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