Metabolism of the lipid peroxidation product, 4-hydroxy-trans-2-nonenal, in isolated perfused rat heart

Abstract

The metabolism of 4-hydroxy-trans-2-nonenal (HNE), an alpha, beta-unsaturated aldehyde generated during lipid peroxidation, was studied in isolated perfused rat hearts. High performance liquid chromatography separation of radioactive metabolites recovered from [3H]HNE-treated hearts revealed four major peaks. Based on the retention times of synthesized standards, peak I, which accounted for 20% radioactivity administered to the heart, was identified to be due to glutathione conjugates of HNE. Peaks II and III, containing 2 and 37% radioactivity, were assigned to 1, 4-dihydroxy-2-nonene (DHN) and 4-hydroxy-2-nonenoic acid, respectively. Peak IV was due to unmetabolized HNE. The electrospray ionization mass spectrum of peak I revealed two prominent metabolites with m/z values corresponding to [M + H]+ of HNE and DHN conjugates with glutathione. The presence of 4-hydroxy-2-nonenoic acid in peak III was substantiated using gas chromatography-chemical ionization mass spectroscopy. When exposed to sorbinil, an inhibitor of aldose reductase, no GS-DHN was recovered in the coronary effluent, and treatment with cyanamide, an inhibitor of aldehyde dehydrogenase, attenuated 4-hydroxy-2-nonenoic acid formation. These results show that the major metabolic transformations of HNE in rat heart involve conjugation with glutathione and oxidation to 4-hydroxy-2-nonenoic acid. Further metabolism of the GS-HNE conjugate involves aldose reductase-mediated reduction, a reaction catalyzed in vitro by homogenous cardiac aldose reductase.

Figure 1. Typical HPLC analysis of tritated metabolites collected from isolated hearts perfused with [³H]HNE

Isolated rat hearts were perfused with Krebs-Henseleit (KH) buffer, and a bolus of [³H]HNE (55 nmol in 1 ml) was injected into the aorta. The coronary effluent (perfusate) was collected every 30 s. After 3 min, the heart was removed and homogenized, and the supernatant was collected. Pooled fractions of the perfusate (A) and the supernatant (B) were separated by reverse phase HPLC using an ODS C₁₈ column. Radioactivity in the HPLC eluate was measured in 1-ml fractions. The peaks I–IV are marked. The arrows indicate the retention time of the synthetic indicated reagents. Further details of HPLC separation are described under “Experimental Procedures.”

Figure 2. GC/CIMS analysis of peak III obtained from HPLC separation of radioactive metabolites recovered in the coronary effluents of isolated buffer-perfused rat heart exposed to a bolus of [³H]HNE

Collected coronary effluents were separated by HPLC as described in the text, and the fractions corresponding to peak III (see Fig. 1) were pooled, derivatized, and subjected to GC/CIMS. Panel A shows a typical gas chromatogram of derivatized peak III. Panel B shows the negative-ionization mass spectrum of the sililated metabolite in peak III. Molecular ions of derivatized HNA are located at m/z 269.1, 343.2, 401.3, and 418.2. Panel C shows the gas chromatograph of reagent DHN, prepared and derivatized as described under “Experimental Procedures.”

Figure 3. Electrospray mass spectra of reagent GS-HNE (A) and GS-DHN (B)

The glutathione adducts of HNE and DHN were prepared as described under “Experimental Procedures” and injected into electrospray. Peaks at m/z464.0 and 466.3 were assigned to [M + H]⁺ of GS-HNE and GS-DHN, respectively. Peaks at m/z 308.2 and 446.2 in the spectrum of GS-HNE correspond to [M + H]⁺ of GS-HNE fragmentation due to loss of HNE and one water molecule, respectively. Insets show peaks due to GS-HNE and GS-DHN at a higher resolution. Block temperature = 225 °C; Repeller voltage = 4 V.

Figure 4. Electrospray mass spectrum of metabolites of [³H]HNE

Isolated perfused rat hearts were exposed to [³H]HNE, and the metabolites recovered in the coronary effluent were collected and separated on HPLC as described in the text. Pooled fractions corresponding to peak I (see Fig. 1) were collected and injected into electrospray. Inset shows separation of peaks at m/z 464 and 466.1 at a greater resolution. Other conditions identical to Fig. 3.

Figure 5. Effect of inhibiting aldose reductase and aldehyde dehydrogenase on the metabolism of HNE

Isolated perfused hearts were exposed to [³H]HNE (55 nmol in 1 ml) in the presence of 0.2 mm sorbinil (A), 2 mm cyanamide (B), or 0.2 mm sorbinil + 2.0 mm cyanamide (C), and the coronary effluents of the hearts were collected, pooled, and separated on HPLC as described under “Experimental Procedures.” The peaks recovered are marked according to their position in Fig. 1. The arrows indicate the retention time of the synthetic indicated reagents.

Figure 6. Electrospray-mass spectra of metabolites of [³H]HNE collected in coronary effluents of hearts perfused with either 0.2 mm sorbinil (A), 2 mm cyanamide (B), or 0.2 mm sorbinil and 2 mm cyanamide (C)

Coronary effluents were collected, pooled, and separated on HPLC, and fractions corresponding to peak I (see Fig. 5) were analyzed by ESI-MS. Peaks atm/z 464 and 466 were ascribed to GS-HNE and GS-DHN, respectively.