Oxidative stress in algae: Method development and effects of temperature on antioxidant nuclear signaling compounds

Non-enzymatic antioxidants neutralize the oxidative stress through scavenging reactive oxygen species. The structural complexity of non-enzymatic nuclear signaling antioxidants poses are more challenges for traditional quantitative analysis as these compounds have multiple forms and structurally diverse than proteins and therefore, challenge traditional analytical techniques. Current analytical methods cannot distinguish the carotenoids, and vitamin A and D metabolites within a single sample. To find an appropriate method to measure all the nuclear signaling compounds in algae, Isochrysis sp., under thermal stress, HPLC/ESI-MS and HPLC /APPI-MS was used. Standards of those compounds were run by QQQ HPLC/ ESI-MS and HPLC /APPI-MS methods by multiple reaction monitoring to optimize the most sensitive transition ion for each compound. Organic solvents (cold acetone and petroleum ether) were used for extraction. Neither APPI nor ESI method showed the sensitivity to detect all targeted non- enzymatic compounds. ESI method detects lower number of compounds than APPI method, but the sensitivity of this method was higher than APPI. Isochrysis sp., was kept at 25 °C, 30 °C and 35 °C for 96 hrs to observe the changes of non-enzymatic antioxidants (carotenoids and vitamins) oxidative stress. Algae were grown with continuous light and aeration in 70 ml test tube. Samples were collected from culture tubes (50 ml) at 0, 6, 12, 24, 48, 72 and 96 hrs Isochrysis showed best growth at 30 °C and lowest at 35 °C. Algae grown at 35 °C had significantly (P\u3c0.05) higher concentrations of MDA compared to algae grown at lower temperatures and the MDA concentrations decreased over time. Fucoxanthin was the dominating pigment followed by diadinoxanthin for all treatment groups. The quantity of carotenoids (fucoxanthin, diadinoxanthin, diatoxanthin and antheraxanthin) and pro-vitamin A (retinol, retinal, retinoic acid) was higher in algae grown at 35 °C and algae grown at 30 °C had lower quantity of these carotenoids, Algae grown at higher temperature had higher MDA. These data indicate that OS induced by thermal change alter carotenoids of antioxidants nuclear signaling compounds