PC-12 Cells were cultured in 5% CO₂, – 95% atmosphere in high humidity at 37°C in Nutrient Mixture, F-12 Hams cell culture medium supplemented with 2.5% fetal bovine serum (FBS), 15% horse serum, 0.2% sodium bicarbonate and antibiotic cocktail from Gibco. For all the metabolism studies, passage 6–10 cells were used. Viability of the cells was measured by trypan blue dye exclusion.

All the specified chemicals, reagents, diagnostic kits, were purchased from Sigma Chemical Company Pvt. Ltd. St. Louis, MO, USA, unless otherwise stated. Nutrient mixture F-12 Hams culture medium, antibiotics, fetal bovine serum and horse serum were purchased from Gibco BRL, USA. Culture wares and other plastic consumables used in the study were procured commercially from Nunc, USA. Milli Q water (double distilled deionized water) was used in all the experiments. HNE was purchased from Caymen Chemicals, U.S.A.

The radio labeled [4-³H] HNE was synthesized from the dimethylacetal of HNE, which was oxidized to the 4-keto derivative using polymer-supported chromic acid as an oxidizing agent (¹). The resulting ketone was further reduced to the dimethylacetal of HNE by using tritiated NaBH₄ The [4-³H] HNE obtained by acid hydrolysis was purified on HPLC. Structural identity of HNE was established by gas chromatography-mass spectroscopy (GC-MS).

1,4-Dihydroxy-2-nonene (DHN) was synthesized enzymatically by incubating60 nmol of [4-³H]HNE with 300 milliunits of human placenta recombinant aldose reductase (AR) and 0.1 mM NADPH in 0.05 M potassium phosphate, pH 6.0, containing 0.4 M Li₂SO₄. The reaction was monitored by following the decrease in absorbance at 224 nm. The enzyme was removed by ultrafiltration using an Amicon Centriprep-10, and DHN in the filtrate was purified on HPLC as described above. Structural identity of DHN was established by gas chromatography-mass spectroscopy (GC-MS).

4-Hydroxy-trans-2-nonenoic acid (HNA) was synthesized by incubating 100 nmol of HNE (³H-HNE) with 1.0 unit of aldehyde dehydrogenase, and 1.5 mM NAD in 0.1 M potassium phosphate, pH 7.4, at 25 °C. The reaction was monitored by following the increase in absorbance at 340 nm. The enzyme was removed by ultrafiltration and HNA in the filtrate was purified by HPLC. Structural identity of HNA was established by gas chromatography-mass spectroscopy (GC-MS).

The conjugate of reduced glutathione (GSH) with HNE (GS-HNE) was prepared by incubating 1 μmol of [4-³H]HNE (55,000 cpm/nmol) with 5 μ of GSH in 0.1 M potassium phosphate, pH 7.0, for 1 h at room temperature. The reaction was monitored by following the consumption of HNE at 224 nm. The GS-HNE conjugate was purified by reverse phase HPLC as described below. For the synthesis of the reduced form of the glutathione-HNE conjugate (GS-DHN), 100 nmol of GS-HNE was incubated with 300 nmol of NADPH and 100 μg of aldose reductase in 0.1 M potassium phosphate, pH 6.0, for 3 h at 37°C. The reaction was monitored by following the consumption of NADPH at 340 nm. Structural identity of HNA was established by electrospray ionization-mass spectroscopy (GC-MS).

Synthesized standards and metabolites of GS-HNE and GS-DHN were separated by HPLC using a Varian reverse phase ODS C₁₈ column pre-equilibrated with 0.1% aqueous trifluoroacetic acid. The compounds were eluted using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (100% acetonitrile) at a flow rate of 1 ml/ min. The gradient was established such that solvent B reached 24% in 20 min, 26% in 30 min, and was held at this value for 10 min. Furthermore, in the next 10 min solvent B reached 60%, and in an additional 5 min it reached 100% and was held at this value for 10 min.

For GC-MS analyses of DHN and HNA, the samples were derivatized in 20 μl of acetonitrile with 20 μl of N, O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) for 1 h at 60°C. The mixture was cooled to room temperature and 2 μl aliquots were used for analysis. The GC-CI/MS analysis was performed using a HP5890/ HP5973 GC/MS system (Hewlett Packard; Palo Alto, CA, USA) under 70 eV electron ionization conditions. The compounds were separated on a bonded phase capillary column (DB-5MS, 30 m × 0.25 mm ID × 0.25μm film thickness from J7W Scientific Folsom, CA, USA. The GC injection port and interface temperature were set to 280°C, with helium gas (carrier) maintained at 14 psi. Injections were made in the split less mode with the inlet port purged for 1 min following injection. The GC oven temperature was held initially at 100°C for 1 min and then increased at a rate of 10°C min⁻¹ to 280°C, which was held for 5 min. Under these conditions, the retention time for DHN and HNA derivatives was 8.08 and 9.52 min respectively (^1,2,20).

For the GC-MS analysis of HNE, 0.4 ml of a 0.05 M solution of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) was added to the samples and the mixtures were vortexed for 1 min and incubated for 30 min at room temperature. After incubation the samples were extracted in 4 ml hexane containing (12 drops of concentrated sulfuric acid. The mixtures were centrifuged at 2000 rpm and the upper hexane layer was aspirated and dried under nitrogen. The dried samples were derivatized with BSTFA as described above and analyzed by GC-MS (^17,21).

Chemical identities of the GS-HNE and GS-DHN were established by electrospray ionization mass spectrometry (ESI/MS). The samples were analyzed on a single quadrapole Micromass LCZ instrument as described (²⁰). The ESI⁺/MS operating parameters were as follows: capillary voltage, 3.0 kV; cone voltage, 13 V; extractor voltage, 9 V; source block temperature, 100 °C; and dissolvation temperature, 200 °C. Nitrogen at 3 p.s.i. was used as nebulizer gas. Samples were reconstituted in 50 μl of acetonitrile/water/acetic acid (50/50/0.1) (v/v/v), and applied to the mass spectrophotometer using a Harvard syringe pump at a rate of 5 μl/min. Spectra were acquired at the rate of 200 atomic mass units/s over the range of 20–2000 atomic mass units.

Initial experiments described the time course of HNE metabolism. PC-12 Cells cultured in 10 cm dish were incubated in pre-warmed (37°C) modified Hank's Balanced Salt Solution (HBSS). Pre-warmed (37°C) HBSS had no observable effect on the viability of PC12 cells for the duration of the experiment. The incubation was started with the addition of 1–20 μM [³H]-HNE in 2.5 ml HBSS. Aliquots were withdrawn at indicated times, centrifuged at 10 000 × g for 10 min at 4°C, and the supernatant was ultra filtered and applied to Varian ODS C₁₈ reverse-phase column. The metabolites of [³H]-HNE were determined by quantifying the radioactivity in each fraction. Individual peaks were analyzed by ESI/MS or GC-CI/MS.

PC-12 cells were incubated in 2.5 ml HBSS without or with 0.05 mM sorbinil, 0.01 mM tolrestat, 0.5 mM miconazole or 5.0 μM benomyl for 30 min at 37°C. (³H)-HNE, 5 μM was then added in the incubation media of each sample and the incubation was carried out for an additional 30 min at 37°C. After the incubation, cells were separated from the incubation medium and the radio labeled metabolites extruded in the medium were resolved by reverse phase HPLC and analyzed by ESI/MS or GC/MS as described above.

In order to characterize and quantify radio labeled HNE and its putative metabolites, we established a reverse phase HPLC method as described in Materials and Methods. As shown in Figure 1, HNE and most of its putative metabolites nicely separated under our chromatographic conditions. However, glutathionyl conjugates of HNE (GS-HNE) and its reduced product (GS-DHN) co-eluted with the retention time of 23 min. DHN, the reduced metabolite of HNE eluted with the retention time of 31 min, whereas HNA, the oxidized metabolite of HNE eluted with the retention time of 37 min. HNE was eluted at 43 min. For the quantification and characterization of HNE and its metabolites, one ml fractions were collected and an aliquot of each fraction (100–250 μl) was used for quantification of radioactivity whereas the remaining aliquot was used for mass spectrometric analysis as described below.

Glutathionyl conjugate of HNE(GS-HNE) and reduced metabolite (GS-DHN) were characterized by ESI-MS. Samples corresponding to the glutathionyl conjugates (HPLC Peak I) were dried under nitrogen on speed vac and analyzed by ESI-MS as described under Materials and Methods. ESI-MS chromatogram of reagent GS-HNE and GS-DHN is illustrated in Fig. 2. The ESI⁺ mass spectrum of the reagent GS-HNE showed a molecular ion [M+H] ⁺ with a m/z of 464. An additional prominent ion with m/z 446 was identified and ascribed to daughter ion of GS-HNE arising from the loss of a single water molecule from the parent 464 ion, because the 446 species could be completely converted at lower cone voltages to the 464 species. The ESI⁺ mass spectrum of reagent GS-DHN displayed a single pseudo-molecular ion [M+H] ⁺ with a m/z value of 466. No daughter ions due to dehydration of the parent ion were observed.

For the characterization of DHN, HPLC Peak II, was subjected to GC-MS analysis. The samples were sililated as described under Materials and Methods and one micro liter of the derivatized sample was injected into the GC. As shown in Fig 3A, on GC, DHN eluted with the retention time of 8.08 min. Fig. 3B shows the fragmentation pattern of DHN. The signature ions with m/z values of 199,231 and 287 were due to M-CH2OTMS (cleavage between C1-C2), M-C5H11 (saturated carbon chain where D is labeled) and M-CH3 respectively. Structural identity of HNA, HPLC Peak III, was established by GC-MS analysis. The samples were sililated and analyzed as described for DHN. As shown in Fig 4A, on GC, HNA eluted with the retention time of 9.52 min. Fig. 4B shows the fragmentation pattern of HNA. The signature ions with m/z values of 245 and 301 were due to M-C5H11 and M-CH3 respectively. Structural identity of HNE (HPLC Peak IV) was established by GC-MS under chemical ionization conditions. Figure 5A shows the GC profile of HNE (21.99 min) and Fig. 5B shows its fragmentation pattern.

To examine HNE metabolism in PC-12 cells, cells were cultured in 10cm dishes. The culture medium was removed and the cells were incubated with 1–20 μM [³H]-HNE in 2.5 ml HBSS. Incubation of the cells with 1–20 μM HNE for 3 h did not cause a significant change in cell viability as determined by the MTT assay. For measuring the rate of HNE metabolism, aliquots were withdrawn at various times and the radioactivity in the medium was separated by HPLC. HNE remaining in the medium was determined by measuring radioactivity in the peak eluting with a retention time (T_R) of the HNE. As shown in Fig. 6, PC-12cells efficiently metabolized HNE in a time and concentration dependent manner. When the cells were incubated with 1 μM [³H]-HNE, 90% of HNE was metabolized in 5 min whereas incubation of the cells with 20 μM [³H]-HNE, resulted in the metabolism of ∼ 50% HNE in 5 min. Under these conditions, after 1h of incubation, only ∼ 50% unmetabolized HNE remained in the medium. The total radioactivity recovered from the incubation medium was 84±4%; = 1% radioactivity was recovered from the acid-insoluble fraction after 1h of incubation.

For the quantification and characterization of HNE-derived metabolites, PC-12 cells were incubated with 5 μM [3H]-HNE for 1h. Incubation medium was separated from the cells and the radio labeled metabolites extruded in the incubation medium were separated on HPLC. Upon HPLC separation of the medium, individual peaks were assigned to specific HNE metabolites (Fig. 7) on the basis of the retention time (T_R) of synthesized standards. As shown in Fig. 7, after 1h of incubation with 5 μM [³H]-HNE, 46% of the radioactivity in the medium was present as glutathione conjugates. Thus, conjugation with glutathione appears to be a rapid, high-affinity route of HNE elimination in PC-12 cells. Because the T_R of Peak I of the HNE-treated PC-12 cells was identical to reagent glutathionyl conjugates, fractions corresponding to this peak were subjected to ESI⁺/MS in order to characterize the metabolite(s) present in that peak. The ESI⁺ mass spectrum of HPLC Peak I showed a predominant peak at m/z 466.2, which was assigned to the + 1 charge state of GS-DHN (Fig. 8). Ions with m/z values of 464.2 and 446.2 respectively, represent GS-HNE and its daughter ion originating from the in source dehydration of GS-HNE. Quantification of the signals for the ions for GS-HNE and GS-DHN showed that 62% of the conjugate was in the reduced form (GS-DHN), whereas 38 % of the conjugate was present as GS-HNE.

The T_R of HPLC Peak II, which accounted for 4% of the total radioactivity, corresponded to the T_R of reagent DHN (Fig. 7). Structural identity of this peak was established by GC-MS. As shown in Fig. 9, the retention time and fragmentation pattern of this peak was identical to reagent DHN, indicating that peak II is due to DHN.

The T_R of HPLC Peak III, which represents 40% of the total radioactivity, was identical to reagent HNA (Fig. 7). To characterize this peak further, fractions corresponding to this peak were pooled, sililated and subjected to GC-CI/MS. The gas chromatograph (Fig. 10A) shows a prominent solvent-independent peak with a T_R value identical to reagent HNA. This peak was further subjected to MS analysis. As shown in Fig. 10B, a molecular ion with a m/z value of 301, corresponding to derivatized HNA, was observed. The fragmentation pattern of this ion was found to be identical to that of synthetic HNA, indicating that peak III is due to HNA.

The HPLC, Peak IV, representing 1% radioactivity, displayed high absorbance at 224 nm and appears to be due to the unmetabolized HNE, since it eluted with a T_R value identical to reagent HNE (43 min). GC-CIMS of this peak (Fig. 11) showed that the retention time and fragmentation pattern of this peak is identical to reagent HNE, suggesting this peak is due to HNE.

Two other peaks were observed on HPLC, eluting with the retention time of 53 (Peak V) and 57 min (Peak VI) respectively. These peaks represented 5 and 4 % radioactivity. Structural identity of these peaks has yet not been established.

Next we examined concentration dependent metabolism of HNE in PC12 cells. As described above when PC-12 cells were incubated with 1 or 5 μM [³H]-HNE for 1h, ∼ HNE was metabolized and glutathionyl conjugates and HNA were the major metabolites accounting for 90% of the metabolism. Incubation of the cells with 10 ìM [³H]-HNE for 1h, resulted in a concentration dependent increase in glutathione conjugates formation whereas no further increase was observed in the oxidation of HNE to HNA (Fig. 12). Under these conditions, a robust increase was observed in the reduction of HNE to DHN (Fig. 12). More significant increases were also observed in the conversion of HNE to Peak V and Peak VI. Incubation of the cells with 20 μM [³H]-HNE for 1h did not cause a significant change in the formation of glutathione conjugates, DHN, HNA and Peak V. However metabolism of HNE to Peak V was increased by three fold under these conditions. Moreover, 25% of HNE remained non metabolized under these conditions.

The HPLC and mass spectroscopic analyses already described clearly demonstrate that the major metabolic products of low concentrations of HNE in PC-12 cells are GS-HNE, GS-DHN and HNA. To identify the biochemical pathways involved in the formation of these metabolites, the PC-12 cells were incubated for 1 h at 37°C with inhibitors of AR (sorbinil, 50 μM or tolrestat, 10 μM), ALDH (benomyl, 5 μM) and Cytochrome P45 (miconazole 500 μM) in 2.5 ml HBSS, after which 5 ìM [³H]-HNE was added to the medium, and the incubation was continued for an additional 30 min. Cells incubated with HBSS under identical conditions served as control. Incubation of the PC-12 cells with 5.0 μM HNE, with or without these inhibitors for 3 h, did not significantly affect cell viability as determined by the MTT assay.

Radioactivity in the incubation medium of sorbinil or tolrestat -treated PC-12 cells separated on HPLC as described above. No change was observed in the amount of radioactivity recovered in Peaks I-VI. The ESI+ mass spectrum of peak I obtained from sorbinil or tolrestat -treated cells also did not show an difference in the abundance ration of GS-HNE:GS-DHN (Fig. 13).

The metabolism of HNE in PC-12 cells was, however, significantly inhibited by the ALDH inhibitor, benomyl. (Fig. 14) The radioactivity in Peak III corresponding to HNA decreased by >75% in cells pre-incubated with benomyl as compared with those treated with [³H]-HNE alone. In addition, the abundance of GS-HNE and Peak V was significantly increased in benomyl treated samples as compared to controls. Based on these observations, we infer that the formation of HNA is catalyzed by ALDH, and that inhibition of HNA formation leads to a corresponding increase in the glutathione-linked metabolism and abundance of Peak V.

Incubation of PC-12 cells with miconazole, decreased the rate of HNE metabolism. As shown in Fig. 15 there was a >6 fold increase in the abundance of unmetabolized HNE. Miconazole also slightly inhibited the glutathionyl conjugates formation. A significant decrease (>40 %) was evident in the formation of HNA in miconazole-treated cells as compared to control. A corresponding increase was also observed in Peak V formation in maconazole-treated cells.