Sulfatase from helix pomatia type h 2

We determined the concentration of urinary CAs, such as noradrenaline (NA), adrenaline (AD) and their metabolites, by treatment with the enzyme (sulfatase from Helix pomatia Type H-2, Sigma–Aldrich, St. Louis, MO, USA) according to the previous report [43 (link)]. Briefly, the urine was heated for 10 min following incubation with 500 U/mL of enzyme at 37 °C for one hour. After addition of isoprenaline (Sigma-Aldrich, St. Louis, MO, USA) as the internal standard, CAs were purified using a Monospin PBA (GL sciences, Tokyo Japan). The HPLC system (Prominance HPLC System Shimazu Corporation, Kyoto, Japan) consisted of a quaternary pump with a vacuum degasser, thermostatted column compartment, autosampler, and equipped with an electrochemical detector (ECD 700 S, Eicom Corporation, Kyoto, Japan). A reverse-phase column (Inertsil ODS-4; 250 × 3.0 mm ID, 5 µm, GL Sciences, Tokyo, Japan) was used, and the column temperature was maintained at 35 °C. The HPLC mobile phase was 24 mM acetate–citrate buffer (pH 3.5)-CH₃CN (100/14.1, v/v). The mobile phase flow rate was 0.3 mL/min, and the injection volume was 20 μL. The eluents were detected and analyzed at 500 mV. Excretion of CAs were expressed as a ratio, with urinary creatinine concentration measured using a LabAssay Creatinine (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan).