Metabolism of 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone by Retinal Pigmented Epithelial Cells

Abstract

4-Hydroxy-7-oxo-5-heptenic acid (HOHA)-lactone is a biologically active oxidative truncation product released (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation from docosahexaenoate lipids. We now report that HOHA-lactone readily diffuses into retinal pigmented epithelial (RPE) cells where it is metabolized. A reduced glutathione (GSH) Michael adduct of HOHA-lactone is the most prominent metabolite detected by LC-MS in both the extracellular medium and cell lysates. This molecule appeared inside of ARPE-19 cells within seconds after exposure to HOHA-lactone. The intracellular level reached a maximum concentration at 30 min and then decreased with concomitant increases in its level in the extracellular medium, thus revealing a unidirectional export of the reduced GSH-HOHA-lactone adduct from the cytosol to extracellular medium. This metabolism is likely to modulate the involvement of HOHA-lactone in the pathogenesis of human diseases. HOHA-lactone is biologically active, e.g., low concentrations (0.1-1 μM) induce secretion of vascular endothelial growth factor (VEGF) from ARPE-19 cells. HOHA-lactone is also a precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives of primary amino groups in proteins and ethanolamine phospholipids that have significant pathological and physiological relevance to age-related macular degeneration (AMD), cancer, and wound healing. Both HOHA-lactone and the derived CEP can contribute to the angiogenesis that defines the neovascular "wet" form of AMD and that promotes the growth of tumors. While GSH depletion can increase the lethality of radiotherapy, because it will impair the metabolism of HOHA-lactone, the present study suggests that GSH depletion will also increase levels of HOHA-lactone and CEP that may promote recurrence of tumor growth.

Figure 1

HOHA-lactone production and protein adduction in rod photoreceptor cells.

Figure 2

HPLC purification of compound 4.

Figure 3

LC-MS analysis of extracellular media from ARPE-19 cells incubated with HOHA-lactone (0 or 10 μM). Top: Positive ion monitoring (m/z 400-500), TIC chromatograms of control cell ECM (Left) and HOHA-lactone treated cell ECM (Right). Inserts: Mass spectrum at retention time 4.41 min. Bottom: Two-dimensional plots (m/z values vs retention times) of control cell ECM (Left) and HOHA-lactone treated cell ECM (Right).

Figure 4

LC-MS and LC-MS/MS analysis of the reduced HOHA-lactone-GSH adduct 5. Left: Selected ion monitoring (SIM) chromatograms of m/z 450.4; Right: MS/MS spectrum of fragmentation ions from the CID of m/z 450.4. (A) Authentic standard 5. (B) ECM of ARPE-19 cells after incubation with 10 μM HOHA-lactone for 2 h. (C) Cell lysate from ARPE-19 cells after incubation with 10 μM HOHA-lactone for 2 h.

Figure 5

(A) LC-MS/MS analysis of metabolite 5 in cell lysates (Left) and ECM (Right) of APE-19 cells. Selected ion recording at m/z 321.0 from CID fragmentation of m/z 450.4 in the positive ion mode was used to specifically identify compound 5. (B) Time course of appearance of 5 (in % yield) in (closed square) cell lysates and (open square) ECM of ARPE-19 cells and reported as means ± S.D. of three independent experiments. (C) Calibration curve of metabolite 5 in LC-MS analysis.

Figure 6

LC-MS/MS analysis of metabolite 4 production in HOHA-lactone treated ARPE-19 cells. Left: LC chromatograms showing the MS2 transition m/z 448.4→301.0; Right: MS/MS spectrum of fragmentation ions from CID of m/z 448.4. (A) Authentic Standards 4. (B) cell lysate of ARPE-19 cells incubated with 10 μM HOHA-lactone for 5 min. (C) ECM of ARPE-19 cells incubated with 25 μM HOHA-lactone for 2 h. (D) Calibration curve of metabolite 4 in LC-MS analysis. (E) Time course of the intracellular appearance of 4 (% yield) in cell lysates from cells treated with 10 μM HOHA-lactone. (F) Proposed structures of fragmentation ions in the MS/MS spectrum of 4.

Figure 7

LC-MS/MS analysis of metabolites 4 and 5 formed in APRE-19 cells treated with HOHA-lactone in a concentration dependence study. (A) Total amount of 5 (in % yield) detected in the (open square) extracellular media and (closed square) cell lysates after 2 h incubation. (B) Total amount of 4 in (open circle) extracellular media and (closed circle) cell lysates after 2 h incubation. Results are reported as means ± S.D. of three independent experiments.

Figure 8

Time course and quantification of HOHA-lactone-derived GSH conjugates in hRPE cells after treatment with 10 μM HOHA-lactone. Quantitation of alcohol 5 in the extra-cellular medium (open square) and in cell lysates (closed circle). Values represent means ± S.D. of three independent experiments.

Figure 9

Time course of intracellular glutathione levels in (A) ARPE-19 cells and (B) hRPE cells after treatment with 10 μM HOHA-lactone. Values represent means ± S.D. of three independent experiments.

Figure 10

GC-EI⁺/MS analysis of TMS derivatives of authentic standard 2 and 3. (A) & (B) GC-MS spectrum of 2 standard; (C) & (D) GC-MS spectrum of 3 standard.

Figure 11

Appearance of metabolite 3 in the extracellular medium from ARPE-19 cells treated with 10 μM HOHA-lactone. ECM was extracted with chloroform, then the chloroform layer was dried, and the residue was derivatized by treatment with BSTFA. GC-MS was done in the SIM mode with m/z = 213 (TMS-M-CH₃)⁺. (A) GC-MS spectrum of ECM collected at 0 h. (B) GC-MS spectrum of ECM collected at 2 h. (C) Calibration curve of compound 3. (D) Evolution of metabolite 3 in ECM (n = 3).

Figure 12

Metabolic processing in RPE cells of HOHA-lactone released from oxidatively damaged rod photoreceptor disc membranes. ALDH = aldehyde dehydrogenase; GST = glutathione S-transferase; ADH = alcohol dehydrogenase.

Scheme 1

Putative metabolites of HOHA-lactone in ARPE-19 cells.

Scheme 2

Proposed structures of CID fragments of m/z 450 in mass spectrometry.

Scheme 3

Chemical synthesis and TMS derivatization of putative metabolites 2 and 3.