ACE/ACE2 Ratio and MMP-9 Activity as Potential Biomarkers in Tuberculous Pleural Effusions

Hsieh, Wen-Yeh; Kuan, Tang-Ching; Cheng, Kun-Shan; Liao, Yan-Chiou; Chen, Mu-Yuan; Lin, Pei-Heng; Hsu, Yuan-Chang; Huang, Chen-Yi; Hsu, Wei-Hua; Yu, Sheng-Yao; Lin, Chih-Sheng

Gelatin zymography for detecting MMP-2 and MMP-9 activity was performed with gelatin-containing gels as our previous report [4] . Briefly, 10 l pleural fluid for each subject was loaded to a 10% SDS-PAGE containing 0.1 mg·ml -1 gelatin (Sigma-Aldrich, St. Louis, MO, USA). The gelatinase activities were identified as clear zones against a blue background on the SDS-PAGE and quantified using Scion Image software (NIH, Bethesda, MD, USA), which quantifies the extent of lysis in bands containing gelatinase. Enzyme activity was expressed as ng·ml -1 . [13] . The assay was performed in a micro-quartz cuvette with 20 μl pleural fluid, 50 μM fluorogenic substrate and protease inhibitor cocktail (1:200; Sigma-Aldrich) in a final volume of 100 μl in ACE or ACE2 assay buffer. The reaction was followed kinetically for 1 hour using a fluorescence reader at an excitation wavelength of 330 nm and an emission wavelength of 390 nm. Data were fitted and plotted using Grafit v.4.0 (Sigma-Aldrich), and enzyme activity was expressed as RFU·h -1 ·ml -1 . Parallel samples were incubated with the above-mentioned reaction mixture in the presence of 1 μM captopril (Sigma-Aldrich), a specific ACE inhibitor for determining specific ACE activity, or 1 μM DX600 (AnaSpec), a specific ACE2 inhibitor for determining specific ACE2 activity.