Profiling cellular bioenergetics, glutathione levels, and caspase activities in stomach biopsies of patients with upper gastrointestinal symptoms

Figure 2 Representative measurements of gastric corpus cellular respiration, glutathione, and caspase activity. A: A run of cellular mitochondrial O₂ consumption by the gastric mucosa of Patient 16. The rate of respiration (k, μmo/L O₂ min^-1) was set as the negative of the slope of [O₂] vs t. The value of k_c (μmo/L O₂ min^-1) and the additions of 10 mmo/L cyanide (a specific inhibitor of cytochrome oxidase) and 50 μg/mL glucose oxidase (catalyzes the reaction of D-glucose + O₂ to D-glucono-δ-lactone + H₂O₂) are shown. O₂ consumption was inhibited by cyanide, confirming the oxidation occurred in the mitochondrial respiratory chain. O₂ was depleted by the addition of glucose oxidase, confirming the presence of dissolved O₂; B: A representative HPLC run of 150 pmol glutathione (GSH) standard (GSH retention time = 14.2 min); GSH standard curve is also shown [insert; GSH (pmol) = 0.00000117 x GSH peak area]; C: A representative HPLC run of cellular GSH in a stomach biopsy (23.9 mg mucosal fragment) from Patient 10. GSH peak area was 354508365 arbitrary units per 50 μL injection volume (reaction volume = 1.0 mL). Thus, cellular GSH content = 347 pmol mg^-1 [(354508365 × 0.00000117 × 20)/23.9]; D: Representative HPLC runs for the Ac-DEVD-AMC cleavage reaction by human active caspase-3 with and without the pan-caspase inhibitor zVAD-fmk. The caspase-3 substrate Ac-DEVD-AMC was detected by absorbance at 380 nm with a retention time of about 3 min (insert). The product AMC was detected by fluorescence (380 nm excitation and 460 nm emission) with a retention time of about 4.8 min; E: Representative HPLC runs of caspase activities in the presence (solid line; 22.3 mg mucosal fragment) and absence (dashed line; 21.2 mg mucosal fragment) of the pan-caspase inhibitor zVAD-fmk for Patient 12. The AMC peak area without zVAD-fmk was 16015 arbitrary units/mg and with zVAD-fmk 2810 arbitrary units/mg. Intracellular caspase activity was set as AMC peak area without zVAD-fmk minus with zVAD-fmk, or 13205 (rounded down to 13 × 10³) (Table 1); F: Representative HPLC runs of caspase activities with (solid line; 23.3 mg mucosal fragment) and without (dashed line; 19.1 mg mucosal fragment) zVAD-fmk for Patient 11. The AMC peak area without zVAD-fmk was 51207 arbitrary units/mg and with zVAD-fmk 37086 arbitrary unit mg^-1. Intracellular caspase activity, thus, was about 14 × 10³ (Table 1).