Abstract:

Changes in the ratio of reduced glutathione and oxidized glutathione and total antioxidant power of red drum fed a diet of pellets saturated with south Louisiana crude oil.

Suggested Citation:

DL Wetzel, TA Sherwood, CA Miller, RL Medvecky. 2019. Red drum diet exposure to Deepwater Horizon crude oil contaminated feed: Oxidative Stress Assays. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7Q23XRS

Purpose:

Controlled oil exposure study to evaluate the effects of south Louisiana crude oil, via contaminated food, on oxidative stress.

Data Parameters and Units:

Test 103 Sample Key worksheet: Date Collected (MM/DD/YYYY), Location Collected (MAP - Mote Aquaculture Park), Field ID, Matrix, MML, Species, Project, Tank ID. Food Preparation worksheet: Prep start and end date (MM/DD/YYYY), Est. total mass needed for daily feeding and samples (g), Feed Prep. Method, Feed mass (g), Mass of oil added (g), Mixing Speed, Mixing Time, Oil Source. Oxidative Stress Results worksheet: Treatment, Fish ID, Tank ID, Fish Tag Number, Wt (g), Length (cm), MML, T.A.P (Total Antioxidant Power) Rep 1 (uM), Rep 2 (uM), Average Trolox Concentration (uM), GSH (glutathione uM), GSSG (oxidized glutathione uM), GSH/GSSG (Ratio= (GSHt-2GSSG)/(GSSG)), MDA (malondialdehyde) Rep 1 (uM), Rep 2 (uM), Average (uM).

Methods:

Sample Collection: plasma 4 mL of whole blood was collected into a sodium citrate blood collection tube. The tube was inverted gently 5 times. The tube was centrifuged at 2000 x g for 10 minutes between 30- 60 minutes from collection time. The supernatant was removed and placed in a clean cryovial and stored in -80degC freezer. Samples were removed and thawed for the first time and are aliquotted out to prevent numerous freeze/thaw cycles. Sample Processing: Samples were run according to the manufacturer's protocol. Total Antioxidant Power Colorimetric Microplate Assay (TA02) by Oxford Biomedical Research, and 2-Thiobarbituric Acid Reactive Substances (FR40) by Oxford Biomedical Research. Sample Collection: whole blood 100ul of whole blood were collected into a microcentrifuge tube containing 30ul of Scavenger. 50ul of whole blood were collected into an empty microcentrifuge tube. Tubes were gently inverted and stored in -80degC freezer or in LN2. Sample Processing: Samples assayed according to the manufacturer's protocol. Total Antioxidant Power Colorimetric Microplate Assay (TA02) by Oxford Biomedical Research, GSH/GSSG Microplate Assay (GT40) by Oxford Biomedical Research.

Instruments:

DS2 Automated ELISA instrument by Dynex Technologies.

Error Analysis:

%CV for the standard curve less than 25% is acceptable, less than 15% for sample replicates.

Individual Files: