Figure 4.

Deuterated (d₈-) and non-labeled (H-) eicosanoid production in the presence of exogenous d₈-arachidonic acid in non-failing control and failing human heart mitochondria. Isolated mitochondria (1 mg) were placed in 10 mm HEPES buffer (pH 7.4) containing 10% glycerol and 1 mm DTT, and homogenized by sonication on ice. Mitochondrial phospholipase activity was activated by addition of either 2 mm EGTA or 0.2 mm free Ca²⁺ in the presence of 8 nmol of d₈-AA at 35 °C. After 20 min of incubation, the reaction was terminated by addition of methanol to 20% (v/v) final concentration at 4 °C. Eicosanoids were immediately extracted by solid phase extraction with internal standards, derivatized with AMPP, and analyzed by high-resolution high mass accuracy mass spectrometry. The quantities of deuterated (D₈-) and non-labeled (H-) eicosanoids, including HETEs (A), EETs (B), and prostaglandins (C), were determined by calculations based on either internal standards (250 pg each of TXB₂-d₄ and PGE₂-d₄) or by calibration utilizing an external standard (250 pg of 12-HETE-d₈) and are presented as the average ± S.E. (n = 4). §, p < 0.05, and §§, p < 0.01. *, p < 0.05, and **, p < 0.005 when compared with EGTA incubations.