Dimethylaminobenzaldehyde

The total hepatic hydroxyproline content was quantified as described previously with minor modifications.⁵² (link)^,53 (link) Briefly, 50 mg mouse liver tissue was hydrolyzed in 1 mL 6 N HCl at 110°C for 14 hours. Hydrolysates were filtered through 45-μm pore filters (Sartorius, Göttingen, Germany). A total of 15 μL of the hydrolysate was dried under nitrogen flow and subsequently redissolved in 50 μL 50% 2-propanol. A total of 100 μL of 0.6% chloramine-T (Merck, Darmstadt, Germany) solution was added to the samples and hydroxyproline standard probes (4-hydroxy-L-proline; Sigma-Aldrich, Taufkirchen, Germany) and incubated for 10 minutes at room temperature. Ehrlich’s solution (100 μL, 3 g dimethylamino-benzaldehyde [Sigma-Aldrich] in 26 mL 2-propanol + 8 mL 70% perchloric acid) was added and the samples again were incubated for 45 minutes at 50°C. Absorbance was measured at 570 nm using a microplate reader (Packard BioScience, Meriden, CT). Hydroxyproline levels were calculated against standard curves and expressed as milligrams of hydroxyproline per gram of liver tissue.

Total collagen was estimated by measuring the hydroxyproline, which is an amino acid characteristic of collagen. A portion of the sample was excised with a knife. The weight of the excised tissue was measured, the organ fragment was transferred to a biosmasher tube (Nippi), and four volumes of tissue weight 6 N HCl (Wako) was added and homogenized. The liver samples were hydrolyzed for 12–15 h in 5 mL of 6 N HCl at 96 °C. After removing from the heater and cooling at room temperature, these samples were centrifuged at 15,000 rpm for 5 min. Then, we collected an appropriate amount of the sample and added 0.5 volume of H₂O. After preparing 20 μL of the sample, we added 75 μL of 50% 2-opropanol in citrate-acetate-buffered chloramine T. The mixture was vortex and mixed. Subsequently, we added 75 μL of a mixture of Ehrlich’s reagent (2-propanol (Wako), Dimethylaminobenzaldehyde (Sigma) and perchloric acid (Sigma). Tubes were incubated for 10 min in a water bath at 60 °C. The samples were placed on the range of 560 nm absorbance band in Hitachi spectrophotometer (U-2000). Results were expressed as μmol hydroxyproline/g of liver tissue.

Kynurenine concentration was assessed by Ehrlich reaction. 100μL conditioned media was combined with 50μL 30wt% trichloroacetic acid (TCA) in water in a round-bottom 96 well plate. A standard curve of purified L-kynurenine (Sigma, St. Louis, MO) dissolved in R10 media was also included. This mixture was spun down at 2200RCF. 100μL supernatant was combined with fresh 2 wt% dimethylaminobenzaldehyde (Sigma, St. Louis, MO) in glacial acetic acid (Fisher, Waltham, MA). This mixture was immediately read at 490nm on a plate reader.

Sodium dodecyl sulfate (SDS), hydrochloric acid (HCl), sodium hydroxide (NaOH), phosphate-buffered saline (PBS), phenol/chloroform/isoamyl alcohol, chloramine T/oxidation buffer, dimethylaminobenzaldehyde, pepsin, urea, thiourea, dithiothreitol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fetal bovine serum (FBS) were obtained from Sigma-Aldrich, St. Louis, MO. Dulbecco’s Modified Eagle Medium (DMEM)/F12, penicillin and streptomycin were purchased from Life Technologies, Inc. (Carlsbad, CA).

Low molecular weight acid-soluble chitosan (manufacturer’s specification, M_w = 50000 g/mol; DDA = 80%), dimethylamino benzaldehyde (DMAB), and dibutyl phosphite were purchased from Sigma-Aldrich, USA and used without further purification unless otherwise stated.