H_2S concentrations in the heart after acute H_2S administration: methodological and physiological considerations

摘要

In this study, we have tried to characterize the limits of the approach typically used to determine H_2S concentrations in the heart based on the amount of H_2S evaporating from heart homogenates-spontaneously, after reaction with a strong reducing agent, or in a very acidic solution. Heart homogenates were prepared from male rats in control conditions or after H_2S infusion induced a transient cardiogenic shock (CS) or cardiac asystole (CA). Using a method of determination of gaseous H_2S with a detection limit of 0.2 nmol, we found that the process of homogenization could lead to a total disappearance of free H_2S unless performed in alkaline conditions. Yet, after restoration of neutral pH, free H_2S concentration from samples processed in alkaline and nonalkaline milieus were similar and averaged -0.2-0.4 nmol/g in both control and CS homogenate hearts and up to 100 nmol/g in the CA group. No additional H_2S was released from control, CS, or CA hearts by using the reducing agent tris(2-carboxyethyl)phosphine or a strong acidic solution (pH < 2) to "free" H_2S from combined pools. Of note, the reducing agent DTT produced a significant sulfide artifact and was not used. These data suggest that 1) free H_2S found in heart homogenates is not a reflection of H_2S present in a "living" heart and 2) the pool of combined sulfides, released in a strong reducing or acidic milieu, does not increase in the heart in a measurable manner even after toxic exposure to sulfide.