Mototeru Yamane

Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography

The quantitative determination of saturated and unsaturated fatty acids (ranging from acetic acid to lignoceric acid) in biological samples is presented. The secondary amine group of 5-(dimethylamino)-1-naphthalenesulponyl-semipiperazide (dansyl-semipiperazide) reacts with the carboxyl group of the fatty acids to form an amide linkage in order to obtain fluorescent derivatives of the acids. The fluorescent derivatives are analysed by high-performance liquid chromatography (HPLC) using an internal standard.

High-performance liquid chromatography—thermospray mass spectrometry of epoxy polyunsaturated fatty acids and epoxyhydroxy polyunsaturated fatty acids from an incubation mixture of rat tissue homogenate

A method for the analysis of epoxy polyunsaturated fatty acids (EpPUFAs) and epoxyhydroxy polyunsaturated fatty acids (EpHPUFAs) in rat tissue homogenate, with homo-gamma-linolenic acid (20:3, n - 6), arachidonic acid (20:4, n - 6), eicosapentaenoic acid (20:5, n - 3) or docosahexaenoic acid (22:6, n - 3) as a substrate, has been developed. Extraction with dichloromethane at pH 4-5 and concentration in the presence of pyridine were performed. Spectral analysis of chromatograms obtained with high-performance liquid chromatography-thermospray mass spectrometry showed the presence of EpPUFAs, EpHPUFAs and dihydroxy metabolites (DiHPUFAs) of EpPUFAs corresponding to each precursor fatty acid. On a selected-ion monitoring chromatogram, many EpPUFAs, EpHPUFAs and DiHPUFAs in an extract from an incubation mixture of each precursor fatty acid in aged rat tissue homogenate were detected simultaneously within 70 min. EpPUFAs and DiHPUFAs derived from 20:3 (n - 6) or 20:5 (n - 3) were detected in significant amounts. From these results, a highly active cytochrome P450 system or non-enzymic oxidative reactions in aged rat tissue homogenate were suggested.

Docosahexaenoic/arachidonic acid ω-hydroxylation system and differentiation in the human colonic adenocarcinoma cell line, Caco-2

The homogenate from Caco-2 cells of day 13 or 15 after subculturing had high omega-hydroxylation activity of docosahexaenoic acid (22:6(n-3)) or arachidonic acid (20:4(n-6)). Activity, maximal at pH 8.0, was inhibited in the presence of CO or metyrapone and in the absence of NADPH. Omega-hydroxylation activity of lauric acid in the homogenate was not detected. Apparent Michaelis constant (Km) values for 22:6(n-3) and 20:4(n-6) were found to be 4 and 7 microM. Omega-hydroxylation activity considerably increased with growth up to day 13 and then decreased until day 20 even though alkaline phosphatase (ALP) and leucine-aminopeptidase (LAP) activity increased with growth to day 20. Metyrapone in cultures caused omega-hydroxylation, ALP and LAP activity to decrease, while sodium butyrate dose-dependently increased that of omega-hydroxylation, ALP and an endogenous endonuclease and decreased lactate dehydrogenase (LDH) activity in culture medium. The omega-hydroxylation system thus appears quite likely to be associated with cytochrome P450, differentiation and/or apoptosis rather than cytotoxic cell death of Caco-2 cells. Substrate specificity, however, differed from that of human cytochrome P450 4A11.

Prevention of growth of human lung carcinoma cells and induction of apoptosis by a novel phenoxazinone, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one.

Anti-tumor effects of a novel phenoxazinone, 2-amino-4,4-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one (Phx), which was synthesized by the reaction of 2-amino-5-methylphenol with bovine hemoglobin, were studied in terms of suppression of the proliferation of human lung carcinoma cells and apoptosis induction. When Phx was added to cultures of the human lung carcinoma cell lines A549 (adenocarcinoma) and H226 (squamous carcinoma), it caused the growth inhibition and the death of these cells. Phx also fragmented the DNA of these cells to oligonucleosomal-sized fragments, which is characteristic of the apoptosis, dependent on the dose and exposure time. The cellular death caused by the administration of Phx was partially reversed by the addition of Z-VAD-fmk, a caspase family inhibitor. Present results suggest that Phx demonstrates anti-cancer activity against human lung carcinoma cell lines A549 and H226, by inhibiting growth and inducing apoptosis.

D,L-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) increases endoplasmic reticulum stress, autophagy and apoptosis accompanying ceramide accumulation via ceramide synthase 5 protein expression in A549 cells

In A549 cells, the addition of D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) led to marked autophagy with massive microtubule-associated protein 1 light chain 3B (LC3B)-II protein expression as an indication of autophagy and a steep decrease of p62 protein as a co-indication of autophagy. The addition of DL-PDMP caused massive autophagy with an increase of CAAT/enhancer binding protein homologous protein (CHOP) expression as the marker of endoplasmic reticulum (ER) stress, lactate dehydrogenase (LDH) release without caspase 3 activation and many autophagic vacuoles/devoid of a cell membrane on morphology. On the other hand, the addition of DL-PDMP caused an increase in cellular or subcellular ceramides (Cers), especially palmitoyl-Cer, based on de novo synthesis of Cer, and led to caspase-independent apoptosis. Marked increases of Cer levels in the nuclear envelope were observed 17 h after the addition. The elevations of Cer synthase activity and longevity-assurance homologue (LASS)5 protein expression were observed in subcellular fractions from 30 min until 2 h after the addition. However, the elevations of Cer synthase activity were independent of reactive oxygen species generation or cytochrome P450 4F2 activity. Since an increase in LASS5 protein expression in subcellular fraction occur in preference to the variation of LC3B-II protein expression via CHOP expression after the addition and Cer accumulation induced by the addition contributes to ER stress, it is thought that an elevation of Cer synthase activity via LASS5 protein expression associate to autophagy via CHOP expression (ER stress) with the addition.

ω-Hydroxylation of docosahexaenoic acid or arachidonic acid in human colonic well differentiated adenocarcinoma homogenate

Human colonic well differentiated adenocarcinoma homogenate was incubated with NADPH and docosahexaenoic acid (22:6(n-3)) or arachidonic acid (20:4(n-6)) as a substrate. On a selected ion monitoring chromatogram obtained with reversed phase-high-performance liquid chromatography thermospray mass spectrometry, omega-hydroxydocosahexaenoic acid (omega-HDHE) or omega-hydroxyeicosatetraenoic acid (omega-HETE) from an incubation mixture of the homogenate was detected in significant amount, compared to that from a colonic region remote from the carcinoma. In contrast, epoxydocosapentaenoic acids and the dihydroxy derivatives from 22:6(n-3) or epoxyeicosatrienoic acids and the dihydroxy derivatives from 20:4(n-6) were detected in low amounts, compared to that from a colonic region remote from the carcinoma. The results suggest that highly active NADPH-dependent omega-oxidations of polyunsaturated fatty acids occur in colonic adenocarcinoma homogenate.

High-performance liquid chromatography—thermospray mass spectrometry of hydroxy-polyunsaturated fatty acid acetyl derivatives

A method for the determination of hydroxy-polyenoic acids has been developed. Hydroxy groups of hydroxy-polyunsaturated fatty acid were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, fifteen kinds of hydroxy-polyunsaturated fatty acid derivative could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatograph without a gradient system. Generally, the base ion, [M + H - n(60)]+, is produced through elimination of acetic acid (n = number of the hydroxy group of hydroxy-polyunsaturated fatty acid). The detection limit for these derivatives was ca. 0.2 pmol at the levels of hydroxy-polyenoic acids prior to derivatization. They could be analysed in the range 0.5-25 pmol. The assay was successfully applied to hydroxy-polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acid was added.

High-performance liquid chromatography—thermospray mass spectrometry of ω-hydroxy polyunsaturated fatty acids from rat brain homogenate

A method for the analysis of omega-hydroxy polyunsaturated fatty acids (omega-HPUFAs) in rat tissue homogenate, supplemented with NADPH and homo-gamma-linolenic acid [20:3(n-6)], arachidonic acid [20:4(n-6)], eicosapentaenoic acid [20:5(n-3)] or docosahexaenoic acid [22:6(n-3)] as a substrate was developed. By ion analysis of chromatograms obtained with reversed-phase HPLC-thermospray MS, many omega-HPUFAs corresponding to each precursor fatty acid could be characterized by the high intensity of the molecular ion (MH+) and quasimolecular ion (MNH4+, MNa+), while other common HPUFAs were characterized by the high intensity of the base ion of MH+--H2O. On a selected-ion monitoring chromatogram of rat brain homogenate, significant amounts of omega-HPUFA from each precursor fatty acid, especially from 22:6(n-3), were detected compared with the amounts found in rat large intestine homogenate. Based on these results, a highly active NADPH-dependent omega-oxidation system is suggested for rat brain homogenate resulting in extensive oxidation of 22:6(n-3).

High-performance liquid chromatography-thermospray mass spectrometry of hydroperoxy polyunsaturated fatty acid acetyl derivatives

A method for the analysis of hydroperoxy polyunsaturated fatty acids was developed. The hydroperoxy groups were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography with thermospray mass spectrometry. Generally, the base ion, [M+H - n(60)]+ or [M+H - n(60) - n(H2O)]+, is produced through elimination of acetic acid or water (n = number of hydroperoxy groups). The detection limit for these derivatives was ca. 1 pmol at concentrations of hydroperoxy polyenoic acids prior to derivatization. Using this method, many hydroxy and hydroperoxy polyunsaturated fatty acid derivatives could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatogram without a gradient system. The assay was successfully applied to hydroxy and hydroperoxy polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acids had been added.

High-performance liquid chromatography–thermospray ionization-mass spectrometry of the oxidation products of polyunsaturated-fatty acids

Abstract The characteristic fragmentation of acetyl derivatives or underivatives in reverse-phase high-performance liquid chromatography–thermospray-mass spectrometry (RPLC–TSP-MS) analysis were investigated, and the utility of RPLC–TSP-MS analysis as a basal method for the detection of oxidation products of polyunsaturated-fatty acids (PUFAs) was reviewed. In polyhydroxy-PUFAs, the acetyl derivatives were suitable for RPLC–TSP-MS analysis, since high-stability in the sensitivity by a single mobile phase and high-sensitivity by strong base ion, [MH− n (60)] + ( n =number of the hydroxy groups), based on the elimination of acetic acid (60 mass units) from MH + ion without other-fragmentation were obtained. In hydroperoxy-PUFAs, the acetyl derivatives were suitable for RPLC–TSP-MS analysis, since strong base ions, [MH−60] + and [MH−60−H 2 O] + , were obtained, and the values of m/z differed from the value of m/z from the corresponding hydroxy-PUFA. For fractional determination of underived ω-hydroxy-PUFAs, the characteristics of the high-intensity of the adduct ion (MH + , MNH 4 + , MNa + ) in RPLC–TSP-MS analysis were utilized, since other common monohydroxy-PUFAs were characterized by the high-intensity of [MH−H 2 O] + ion. For fractional determination of underived epoxy-PUFAs, epoxyhydroxy-PUFAs and 5,6-dihydroxyeicosatrienoate-1,5-lactone (5,6-DiHETriE-δ-lactone), the characteristics of the high-intensity of the adduct ion (MH + , MNH 4 + , MNa + ) in RPLC–TSP-mass spectra were utilized. Then, the fragment ions formed by cleavage of two-types in the oxirane ring of epoxy-PUFA under electron-beam on conditions provided information as to the position of the oxirane ring.