A1887

All chemicals and reagents were of analytical grade, including fatty acid-free HSA (A1887, lyophilized powder, Sigma-Aldrich, St. Louis, MO, USA), perfluorobutanoic acid (PFBA, 98%, Sigma-Aldrich, St. Louis, MO, USA), perfluorohexanoic acid (PFHxA, 98%, Sigma-Aldrich, St. Louis, MO, USA), PFOA (98%, Chemical Industry, Tokyo, Japan), perfluorodecanoic acid (PFDA, 98%, Sigma-Aldrich, St. Louis, MO, USA). The stock solution of each PFCA was prepared in phosphate-buffered saline (PBS, pH 7.4 ± 0.1). HSA solution was freshly prepared in PBS (pH 7.4 ± 0.1) 15 min before use. In order to prevent PFCAs from adsorbing to glass surface, polypropylene containers were used.

Routine markers were measured in serum using ABX Pentra 400 (HORIBA, Kyoto, Japan). Capillary blood glucose concentrations were measured using a glucose oxidase method on a Dr. Müller Super GL (Dr. Müller Glucose Analyzer, Freital, Germany). WISP-1/CCN4 levels were measured by human direct sandwich WISP-1/CCN4 DuoSet ELISA kit (DY1627; R&D Systems, Germany) in combination with bovine serum albumin (A7030, Sigma, Munich, Germany) or human serum albumin (A1887, Sigma) and performed on 96-well high-binding assay plates (82.1581, Sarstedt, Nümbrecht, Germany) as described in [17 (link)]. Commercially available ELISA kits were used for the measurements of serum/plasma insulin (Insulin ELISA, Mercodia AB, Uppsala, Sweden) and TIMP1 (all from R&D Systems, Minneapolis, MN, USA), and the U-Plex assay was used to measure IL6, tumor necrosis factor alpha (TNFα), and MCP1 (MSD, Rockville, MD, USA).
Analysis of hepatic triglyceride content was performed according to the Triglyceride Determination Kit (Sigma Aldrich Chemie, Steinheim, Germany), and the absorbance changes were detected at 540 nm by spectrophotometry.

To further compare the UP with commercial human albumin (A1887, Sigma) and commercial glycated human albumin (A8301, Sigma), the iso-electro point of the three proteins was determined (Figure 1C). Equal amounts (6.9 μg) of the three proteins were run along with standards on a precast Bio-Rad IEF gel pH 3–10 (Bio-Rad, Hercules, CA, United States). The IEF standards were soybean trypsin inhibitor, 4.6 pI (10109886001, Sigma), and bovine milk β-lactoglobulin A, 5.1 pI (L3908, Sigma). The protein bands of interest were flanked on both sides by the standards in the gel. The gel was run for 3 h at various voltages; 1 h at 100 V, 1 h at 250 V, and 30 min at 500 V. The gel was fixed in 10% trichloroacetic acid (TCA) for 10 min. Excess ampholytes were removed by an additional overnight 1% TCA soak. To detect the protein bands, the gel was washed in water three times and stained with GELCODE Blue Stain Reagent (Pierce, Rockville, IL, United States).