Malondialdehyde tetrabutylammonium salt

The MDA levels as an index of oxidative stress and the efficacy of antioxidant administration were determined at the end of the WBE or IRB sessions, respectively. The MDA-HPLC method was mainly based on the procedure by Nielsen et al.21 (link) The reagents potassium phosphate monobasic, potassium hydroxide, phosphoric acid (H₃PO₄), sodium hydroxide (NaOH), HPLC-grade methanol, and thiobarbituric acid (TBA) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Malondialdehyde tetrabutylammonium salt (96% purity) was also purchased from Sigma-Aldrich Co. and used for standards’ preparation.
For sample preparation, 100 µL of plasma was added to 700 µL of 1% H₃PO₄ and 200 µL of 42 mmol/L TBA. The mixture was heated for 30 minutes at 100°C and then ice-cooled for 2 minutes. A 250 µL of the aliquot was separated and added to 250 µL of 1 M NaOH in methanol (1:6). After centrifugation (5 minutes at 10,000 g) and filtration, 20 µL of the supernatant was injected in the HPLC apparatus at a flow rate of 1 mL/min, column temperature and pressure at 26°C and 2,200 psi, respectively. The absorbance was read at 532 nm.
For the MDA standard curve preparation, a stock solution of 100 µM of MDA was prepared in 0.01 mmol/L HCl. Dilutions from stock solution of 10–0.5 µmol/L were then performed. The same treatment described for plasma was used for the standard curve reaction.

Formaldehyde was purchased from Merck (Darmstadt, Germany). Catechin hydrate, malondialdehyde tetrabutylammonium salt, thiobarbituric acid (TBA), 5, 5′-dithiobis 2-nitrobenzoic acid (dTNB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ascorbic acid and vitamin E were obtained as the generous gift samples from Square Pharmaceuticals Ltd., Dhaka, Bangladesh. SGOT and SGPT assay kits were purchased from Human (Germany). Pyrogallol, gallic acid, Folin-Ciocalteu reagent (FCR), 2, 2-diphenyl-1-picrylhydrazyl, KOH, xylene, hematoxylin, and eosin were purchased from Loba Chemie (India). All other chemicals were of analytical grade and procured from reliable commercial suppliers.

Ultrapure water was obtained from a PURELAB flex 2 water purification system (ELGA LabWater, Paris, France). Ethanol (99.7%) and acetic acid were purchased from VWR (Mannheim, Germany). Ethanol (HPLC grade) and citric acid monohydrate (≥95.5%) were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). MEthanol (HPLC grade) and sulphuric acid (95%) were from Th. Geyer (Renningen, Germany). Formic acid (99.9%), malondialdehyde tetrabutylammonium salt (≥97%), potassium sodium tartrate tetrahydrate (≥98%), and 3,5-dinitrosalicylic acid (≥98%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). n-Propanol, propionic acid, 2-thiobarbituric acid and sodium azide were purchased from Merk (Darmstadt, Germany); sodium hydroxide was from Honeywell (Morris Plains, NJ, USA). D-(+)-GalAc monohydrate (99%) was obtained from Fluka (Munich, Germany), sodium nitrate (99%) was from Acros Organics (Geel, Belgium), and trisodium citrate dihydrate (≥98%) was from Alpha Aesar (Ward Hill, MA, USA). Sugar beet pectin Betapec RU 301, apple pectin Classic AU-L 036/18 (low methylated, DM 38%) and apple pectin Classic AU-L 022/17 (high methylated, DM 71%) were kindly provided by Herbstreith & Fox GmbH & Co. KG (Neuenbürg, Germany).