Colorimetric assay kit

A caspase-3 assay kit evaluated the apoptosis (Beyotime, Shanghai, China). Caspase 3 belongs to the CED-3 subfamily of the Caspase family and is a protease that plays an important role in the process of apoptosis (17 (link)). Caspase 3 Assay Kit is based on Caspase 3 which can catalyze the substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) to produce yellow p-nitroaniline (pNA), which has strong absorption around 405 nm The activity of Caspase 3 can be detected by measuring absorbance (yellow product is proportional to the activity of Caspase 3 enzyme). The MG-63 cells were treated with paraffin in 96-well plates for 90 minutes and passed through 80 µL of reaction buffer (caspase-3 substrate, 2 mmol/L, 10 µL). Ten µL of cell lysate protein was incubated in each sample in an incubator (37 °C, 5% CO₂) for 2 h. Used R&D Systems’ colorimetric assay kit (Thermo Scientific, USA) to measure samples at 405 nm.

Fasting serum lipids (total cholesterol, high‐density lipoprotein [HDL] cholesterol, and triglycerides) and hsCRP (Dade Behring, Deerfield, IL) were measured at the San Francisco General Hospital clinical lab. Blood was drawn in the fasting state at the San Francisco General Hospital clinical laboratories and aliquots of serum and plasma were stored at −80°C until analysis. Total cholesterol, HDL‐cholesterol, and triglycerides were measured by colorimetric assay kits (Thermo Fisher Scientific, Middletown, VA). ApoA1 and apoB were measured by nephelometric immunoassays (BNII; Siemens Healthcare Diagnostics, Deerfield, IL). Platelet‐activating factors acetylhydrolase activity (PAH) and secretory phospholipase A₂ levels were measured by kits from Cayman Chemical (Ann Arbor, MI). Lecithin‐cholesterol acyltransferase, and oxidized low‐density lipoprotein levels were measured with commercially available ELISA kits from ALPCO (Salem, NH), and Mercodia (Winston Salem, NC), respectively. Paraoxonase arylesterase activity was determined by using phenyl acetate as substrate as described by Furlong et al.^{20 (link)}Percentage of CD19 B‐cells was assessed using quantitative flow cytometry performed at the San Francisco General Hospital reference laboratory (ARUP Laboratories, test code 0095920).

Hepatic lipids were determined by using a modified Hara and Radin protocol (Hara and Radin, 1978 (link)). Briefly, 75 mg of hepatic tissue was homogenized in 1 ml of hexane:isopropanol (3:2) with 0.01% (w/v) of butylated hydroxytoluene to prevent lipid peroxidation. Following the addition of 300 μl of 0.47 M Na₂SO₄, samples were centrifuged for 20 min at 7500g and the upper organic phase was transferred to a clean tube and evaporated under nitrogen. To solubilize the lipid, 500 μl hexane containing 0.1% Triton-X was added and mixed gently for 20 min. The hexane was evaporated under nitrogen and 500 μl of dH₂O was added to the tube. Prior to assaying for triglyceride and cholesterol levels, samples were incubated at 37 °C for 15 min. Triglyceride and cholesterol concentrations in lipid extracts were determined enzymatically with colorimetric assay kits (ThermoFisher Scientific, Middletown, VA).

Plasma total cholesterol, triglyceride, and glucose concentrations were measured enzymatically using colorimetric assay kits (ThermoFisher, Middletown, VA). Plasma insulin (Mercodia, Winston Salem, NC) and adiponectin (ALPCO, Salem, NH) concentrations were determined using ELISA kits. Plasma concentrations of inflammatory markers IL-1β, IL-6, and TNFα were determined by ELISA (eBioscience, ThermoFisher, Middletown, VA). HOMA-IR was calculated from fasting glucose and insulin levels (glucose, mg/dL x insulin, mU/L ÷ 450).