Oxiselect tac assay kit

TAC, in terms of copper reducing equivalent (CRE) of the sample, was evaluated using OxiSelect™ TAC Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA; Catalog Number: STA- 360) [25 (link)]. The components of the kit were uric acid standard (Part No. 236001), reaction buffer, 100X (Part No. 236002), copper ion reagent 100X (Part No. 236003) and stop solution, 10X (Part No. 236004). The assay protocol was as per the manufacturer’s product manual. Briefly, 20 μL of the sample in various concentrations and 180 μL of 1X reaction buffer were transferred to each well in a 96 well plate and mixed thoroughly. An initial absorbance was taken at 490 nm. The reaction was started by adding 50 μL of 1X copper ion reagent into each well and incubated on an orbital shaker for 5 min. Then the reaction was stopped by adding 50 μL of 1X stop solution to each well and absorbance was measured again. The net absorbance was calculated by subtracting the initial reading from the final reading and the mM uric acid equivalent (UAE) was determined from the uric acid standard curve. Finally, the CRE was determined by multiplying UAE by 2189.

Plasma total antioxidant capacity (TAC) level was evaluated using the OxiSelect™ TAC Assay Kit (Cell Biolabs, Inc.), following the manufacturer’s instructions. In brief, TAC assay is based on a copper reduction reaction from copper (II) to copper (I) in the presence of an antioxidant such as uric acid. The absorbance values were read at 490 nm against the uric acid serial dilution standard curve (0–1 mM) and were directly proportional to the plasma TAC expressed as μM of uric acid equivalents.

Oleoresins obtained from dichloromethane extraction of lyophilized spirulina (AquaSolar™) and maqui (Isla Natura™) were prepared as described above for Gracilex^® production. The oleoresins were resuspended in DMSO, and 50 µL of sample was taken to measure the antioxidant capacity using an OxiSelect™ TAC Assay Kit according to the manufacturer’s instructions (Cell Biolabs, Inc., San Diego, CA, USA), which is based on the reduction of copper (II) to copper (I), also known as a cupric ion reduction antioxidant capacity (CUPRAC) assay [47 (link)]. Uric acid was used as a standard to calculate the milli-equivalents of antioxidant.

The following high grade chemicals were obtained commercially or as a generous gift: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) (Sigma-Aldrich, St Louis, MO, USA); raloxifene (Cipla Ltd., Goa, India); phosphorous, calcium, alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), hydroxyproline (HP), total cholesterol (TC) and triglyceride (TG) kits (Span Diagnostic Pvt. Ltd.); dimethyl sulfoxide (DMSO) and phosphate buffer saline (PBS) (Mediatech Inc., Manassas, VA, USA); xylazine (Indian Immunologicals Ltd., Hyderabad, India); ketamine (Neon Laboratories Limited, Thane, India); diclofenac (Troikaa Pharmaceuticals Ltd., Ahmedabad, India); gentamicin (Abbott, Pitampur, India); DPPH (1,1-diphenyl-2-picrylhydrazyl) (HIMEDIA Co. Ltd., India); ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay kit (Sigma-Aldrich, MO, USA, Catalog Number MAK187); OxiSelect™ TAC Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA; Catalog Number: STA- 360); fetal bovine serum (Mediatech, Manassas, VA); penicillin streptomycin solution 100X (10,000 IU/mL penicillin and 10,000 μg/mL streptomycin) (Mediatech, Manassas, VA) were procured.

Serum total cholesterol, high-density lipoprotein-cholesterol, and triglycerides were measured in CORE Laboratory (Hospital Clínic de Barcelona) by standard enzymatic methods. We assessed the serum ratio cholesteryl ester to free cholesterol by the Cholesterol/Cholesteryl Ester Quantitation Kit (Cat# 428901, Calbiochem, Merck, Darmstadt, Germany), and the serum total antioxidant capacity by the OxiSelect TAC Assay Kit (Cat# STA-360, Cell Biolabs Inc., San Diego CA, United States).

The cardiotoxicity indices including brain natriuretic peptide (BNP) and cardiac troponin I (cTnI) were determined in serum using a rat ELISA kit (Cloud-clone corp., USA). The markers of oxidative stress, malondialdehyde (MDA) and total antioxidant capacity (TAC), were determined in cardiac tissue homogenates using MDA ELISA kit (Cloud-clone corp. USA), and OxiSelect™ TAC assay kit (Cell Biolabs, USA), respectively. The antioxidant capacity is determined relative to uric acid standards and therefore results were expressed as mM uric acid. Cardiac tissue homogenate was further used to assess inflammatory markers (TNF-α and IL-1β) and the apoptotic marker caspase-3 using their respective ELISA kits (Cloud-clone corp., USA).