Figure 2.

Urine BrTyr released after incubation with different enzymes or methanesulfonic acid (MSA). Each enzymatic reaction was carried out in 0.3 M ammonium acetate buffer at different pH, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4, at different temperatures, 37°C, 45°C, 55°C, and 60°C, for 2 and 17 h. The highest BrTyr released for each source of enzyme reacted among different pH values and temperatures at each time point was shown. The BrTyr in urine sample without enzymatic incubation (free) was carried out in 0.3 M ammonium acetate, pH 7.0 at 37°C. BrTyr released by MSA hydrolysis was carried out by adding MSA to a final concentration of 4 M and incubated at 110°C for 24 h. Stable isotope labeled internal standard mix was added before hydrolysis reaction either by enzyme or MSA. After reaction at the indicated time, the urine sample was diluted with 0.1% formic acid followed by solid phase extraction. BrTyr, 3-bromotyrosine.