APAP-ALF patient plasma analysis

APAP-overdose Patient Plasma Analysis

The following were performed with the APAP-ALF patient plasma samples:

Syndecan-1 ELISA (Human Syndecan-1 ELISA, CellSciences)

1. Perform according to manufacturer's instructions.

HMGB1 ELISA (HMGB1 ELISA, IBL International, Tecan)

1. Perform according to manufacturer's instructions.

ALT assay (ALT Infinity Reagent, Thermo Fisher)

1. Perform according to manufacturer's instructions.

LC/MS disaccharide analysis

1. Pool individual plasma samples of healthy control subjects and APAP-ALF patients and digest using 10 mU each of recombinant heparin lyase I, II, and III (pH 7.0 – 7.5) and recombinant chondroitin lyase ABC (pH optimum 7.4) in 300 μl digestion buffer (50 mM ammonium acetate containing 2 mM calcium chloride adjusted to pH 7.0). Mix samples well.
2. Place samples in a water bath at 37 °C for 12 hours,
3. Terminate enzymatic digestion by removing enzymes by centrifugation using 3000 Da molecular weight cut-off spin filter units (AmiconÒ Ultra centrifugal unit). Wash the units twice with 250 μl of distilled water. Dry the filtrates, containing the disaccharide products, by vacuum centrifuge. 
4. Label samples by adding 10 μl of 0.1 M 2-aminoacridone (AMAC) in DMSO/acetic acid (17/3, vol/vol). Incubate at room temperature for 10 min, followed by adding 10 μl of 1 M aqueous sodium cyanoborohydride and incubating for 1 hour at 45 °C.
5. Prepare 0.5 ng/μl disaccharide standard mixture (Iduron) and AMAC-label following step 4. 
6. After the AMAC-labeling reaction, centrifuge samples at 13500 x g for 10 minutes and collect supernatant. 
7. LC was performed on an Agilent 1200 LC system at 45 °C using an Agilent Poroshell 120 ECC18 (2.7 μm, 3.0 × 50 mm) column. Mobile phase A is 50 mM ammonium acetate aqueous solution, and the mobile phase B is methanol. The mobile phase passed through the column at a flow rate of 300 μl/min. The gradient was 0-10 min, 5-45 % B; 10-10.2 min, 45-100 % B; 10.2-14 min, 100 % B; 14-22 min, 100-5% B. Injection volume was 5 μl. 
8. A triple quadrupole mass spectrometry system equipped with an ESI source (Thermo Fisher Scientific) was used a detector. The online MS analysis was at the Multiple Reaction Monitoring (MRM) mode. MS parameters: negative ionization mode with a spray voltage of 3000 V, a vaporizer temperature of 300 °C, and a capillary temperature of 270 °C. 

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