Chiral Phase High-performance Liquid Chromatography Research Articles

An sn-2 regioselective lipase with cis-fatty acid preference from Cordyceps militaris: Biochemical characterization and insights into its regioselective mechanism

Lipase with unique regioselectivity is an attractive biocatalyst for elaborate lipid modification. However, the excavation of novel sn-2 regioselective lipases is difficult due to their scarcity in nature, with Candida antarctica lipase A (CALA) being the pronouncedly reported one. Here, we identified a novel CALA-like lipase from Cordyceps militaris (CACML7) via in silico mining. Through chiral-phase high-performance liquid chromatography, we determined that CACML7 displays sn-2 regioselectivity (>68 %) as does CALA, but exhibits distinctive chain length selectivity and bias against unsaturated fats. Notably, the curvature of the acyl-binding tunnel was expected to contribute to the 2.2-fold higher preference for cis-fatty acid (C18:1, cis-Δ9) over trans-fatty acid (C18:1, trans-Δ9) unlike trans-active CALA. Random pose docking of trioleoylglycerol (TOG) into the active site of a lid-truncated mutant of CACML7 revealed that TOG accepts a tuning fork conformation, of which the precise positioning of the reactive ester group towards the catalytic center was only favorable via sn-2 binding mode. The unique active site morphology, which we refer to as an “acyl-binding tunnel with a narrow entrance,” may contribute to the sn-2 regioselectivity of CACML7. Our data provide an attractive model to better understand the mechanism underlying sn-2 regioselectivity.

Phenolic Constituents with Glucose Uptake and GLUT4 Translocation Bioactivities from the Fruits of Cordia dichotoma.

From the fruits of Cordia dichotoma, 11 new phenolic compounds, dichotomins A-K, were isolated, together with 19 known compounds. Through the analysis of detailed NMR data and HRESIMS data, the planar structures of all compounds were confirmed. Using NMR calculations, the absolute configuration of dichotomins A-K was elucidated by comparing their observed and computed electronic circular dichroism (ECD) spectra. Dichotomin H (8) and dichotomin I (9) were determined as two pairs of enantiomers. The enantiomers of compounds 8 and 9 were separated using chiral-phase high-performance liquid chromatography (HPLC), and the stereostructure of each enantiomer was determined by similarly calculating the ECD. Compounds 3, 5, 7, 17, 18, 23-25, and 27-30 increased glucose uptake by 1.04- to 2.85-folds at concentrations of 30 μg/mL. Further studies revealed that compounds 3 and 5 had a moderate effect on glucose transporter 4 (GLUT4) translocation activity in L6 cells. At 30 μg/mL, compound 3 significantly enhanced AMPK phosphorylation and GLUT4 expression. As a whole, compound 3 has the potential to be a drug candidate for the treatment of type 2 diabetes mellitus (T2DM).

Undescribed isoquinolines from Zanthoxylum nitidum and their antiproliferative effects against human cancer cell lines

Eleven previously undescribed alkaloids, including three pairs of enantiomers nitidumalkaloids A–C, a pair of scalemic mixtures nitidumalkaloid D and three optically pure or achiral alkaloids, nitidumalkaloids E–G, along with 20 known alkaloids, were isolated from an ethanolic extract of the whole Zanthoxylum nitidum (Roxb.) DC plant. The chemical structures of the alkaloids were elucidated using a combination of comprehensive nuclear magnetic resonance (NMR) and high-resolution electro-spray ionization mass spectrometry (HR-ESI-MS) analyses. The configuration of the stereogenic centers of all undescribed compounds was precisely established based on single-crystal X-ray diffraction and electronic circular dichroism (ECD) calculations. Racemic mixtures of nitidumalkaloids A–D were purified, and their enantiomers were analyzed via chiral-phase high-performance liquid chromatography with electrochemical detection measurements (HPLC-ECD). Twelve compounds exhibited significant antiproliferative activities against a panel of cancer cell lines. Further studies were designed to investigate the underlying molecular mechanism of (1′S, 6R)-nitidumalkaloid B, which was the most active antiproliferative agent against human cancer A549 cells. G2/M cell cycle arrest, induction of apoptosis, and suppression of the Wnt/β-catenin signaling pathway were in part associated with the antiproliferative activity of (1′S, 6R)-nitidumalkaloid B. Moreover, (1′S, 6R)-nitidumalkaloid B inhibited cell migration by downregulating the epithelial-mesenchymal transition process in A549 cells. These data suggest that the antiproliferation activity of (1′S, 6R)-nitidumalkaloid B was correlated with the stereoselectivity of the stereoisomers, and (1′S, 6R)-nitidumalkaloid B was prioritized as a potential leading compound for the management of aggressive human non-small-cell lung cancer (NSCLC) from natural products.

Integral Stereoselectivity of Lipase Based on the Chromatographic Resolution of Enantiomeric/Regioisomeric Diacylglycerols.

Stereoselectivity, a distinctive characteristic of lipase (EC 3.1.1.3), refers to the ability to differentiate between enantiomeric positions (sn-1 and sn-3) in triacylglycerol (TAG). This property has been determined based on the time course of enantiomeric excess of diacylglycerol (DAG) considering several consecutive steps of lipase-catalyzed hydrolysis of TAG; however, this concept is insufficient to represent the true nature of lipases which are capable of hydrolyzing the sn-2 position of TAG under the condition acyl migration occurs. Here, we suggest "integral stereoselectivity" to capture the preference of lipases for all ester groups of both TAG and DAG, as a novel index of the stereochemistry of lipase. To determine integral stereoselectivity, we established an analytical system based on the chromatographic resolution of dioleoylglycerol (DO) enantiomers and regioisomers. DO enantiomers were derivatized with 4-nitrophenyl isocyanate, and subsequently, resolved by chiral-phase high-performance liquid chromatography-ultraviolet. Regioisomers of monooleoylglycerol and DO were analyzed by HPLC with an evaporative light-scattering detector. Time-course analysis of three model lipases involved in the hydrolysis of trioleoylglycerol validated the analytical system designed to determine the integral stereoselectivity. As an accurate indicator of lipase stereochemistry reflecting all hydrolysis steps, integral stereoselectivity can expedite the development of lipases with unique stereochemistry from agricultural sources and their application to the food industry.

Tri- and Tetrameric Proanthocyanidins with Dentin Bioactivities from Pinus massoniana.

Guided by dentin biomechanical bioactivity, this phytochemical study led to the elucidation of an extended set of structurally demanding proanthocyanidins (PACs). Unambiguous structure determination involved detailed spectroscopic and chemical characterization of four A-type dimers (2 and 4-6), seven trimers (10-16), and six tetramers (17-22). New outcomes confirm the feasibility of determining the absolute configuration of the catechol monomers in oligomeric PACs by one-dimensional (1D) and two-dimensional (2D) NMR. Electronic circular dichroism as well as phloroglucinolysis followed by mass spectrometry and chiral phase high-performance liquid chromatography (HPLC) analysis generated the necessary chiral reference data. In the context of previously reported dentin-bioactive PACs, accurately and precisely assigned 13C NMR resonances enabled absolute stereochemical assignments of PAC monomers via (i) inclusion of the 13C NMR γ-gauche effect and (ii) determination of differential 13C chemical shift values (ΔδC) in comparison with those of the terminal monomer (unit II) in the dimers 2 and 4-6. Among the 13 fully elucidated PACs, eight were identified as new, and one structure (11) was revised based on new knowledge gained regarding the subtle, stereospecific spectroscopic properties of PACs.

A New Optical Biflavonoid, (2″R)-2″,3″-Dihydrorobustaflavone 7,4′-Dimethyl Ether, and Other Constituents from Selaginella trichoclada Alsto

A new optical biflavonoid, (2” R)-2″,3″-dihydrorobustaflavone 7,4′-dimethyl ether (1), and 6 known compounds (2-7) were isolated for the first time from the 70% ethanol extract of Selaginella trichoclada Alsto. The structures of the compounds were confirmed by extensive spectroscopic data analyses. Racemic compound 1 was separated by chiral-phase high-performance liquid chromatography, and the absolute configurations of (±)-1 were defined by circular dichroism spectroscopic data. Compound 1 exhibited moderate cytotoxicity against MCF-7, A549, and HepG2 human cancer cell lines.

Resolution of Atropisomeric Cyclic Catechol Monoether O-Sulfate Esters by a Molluscan Sulfatase.

Atropisomeric cyclic catechol ethers are notoriously difficult to resolve by classical chiral phase high-performance liquid chromatography. Here, we show the first application of sulfatase enzymes for the kinetic resolution of O-sulfato-catechol ethers with enantioselectivities ranging from 30 to 65% ee, as determined by preparation of their Marfey’s ether derivatives. Substrate-structure dependence was briefly explored.

Lipid Classes, Fatty Acid Composition, and Glycerolipid Molecular Species of the Red Alga <i>Gracilaria vermiculophylla</i>, a Prostaglandin-Producing Seaweed

The red alga Gracilaria vermiculophylla is a well-known producer of prostaglandins, such as PGE2 and PGF2α. In this study, the characteristics of glycerolipids as substrates of prostaglandin production were clarified, and the lipid classes, fatty acid composition, and glycerolipid molecular species were investigated in detail. The major lipid classes were monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), as well as phosphatidylcholine (PC), which accounted for 43.0% of the total lipid profile. Arachidonic acid (20:4n-6), a prostaglandin precursor, and palmitic acid (16:0) were the predominant fatty acids in the total lipid profile. The 20:4n-6 content was significantly high in MGDG and PC (more than 60%), and the 16:0 content was significantly high in DGDG and SQDG (more than 50%). Chiral-phase high-performance liquid chromatography determined that fatty acids were esterified at the sn-1 and sn-2 positions of those lipids. The main glycerolipid molecular species were 20:4n-6/20:4n-6 (sn-1/sn-2) for MGDG (56.5%) and PC (40.0%), and 20:4n-6/16:0 for DGDG (75.4%) and SQDG (58.4%). Thus, it was considered that the glycerolipid molecular species containing one or two 20:4n-6 were the major substrates for prostaglandin production in G. vermiculophylla.

New prenylated aeruginosin, microphycin, anabaenopeptin and micropeptin analogues from a Microcystis bloom material collected in Kibbutz Kfar Blum, Israel.

Thirteen new and eighteen known natural products were isolated from a bloom material of an assembly of various Microcystis spp. collected in November, 2008, from a commercial fishpond near Kibbutz Kfar Blum, the Jordan Valley, Israel. The new natural products included the prenylated aeruginosin KB676 (1), microphycin KB921 (2), anabaenopeptins KB906 (3) and KB899 (4) and micropeptins KB928 (5), KB956 (6), KB970A (7), KB970B (8), KB984 (9), KB970C (10), KB1048 (11), KB992 (12) and KB1046 (13). Their structures were elucidated primarily by interpretation of their 1D and 2D nuclear magnetic resonance spectra and high-resolution mass spectrometry. Marfey’s and chiral-phase high performance liquid chromatography methods were used to determine the absolute configurations of their chiral centers. Aeruginosin KB676 (1) contains the rare (2S,3aS,6S,7aS)-Choi and is the first prenylated aeruginosin derivative described in the literature. Compounds 1 and 5–11 inhibited trypsin with sub-μM IC50s, while Compounds 11–13 inhibited chymotrypsin with sub-μM IC50s. The structures and biological activities of the new natural products and our procedures of dereplication are described.

Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8 R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases

8-Hydroxyoctadeca-9 Z,12 Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8 E,12 Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8 R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10 S-HODE by Lentinula edodes, and 10 R-HODE by Epichloe typhina. Racemic [8- 2H]8-HODE and [10- 2H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess–Martin periodinane followed by reduction to hydroxy fatty acids with NaB 2H 4. The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) with 8 R-HODE and 10 S-HODE as standards. 8 R-HODE eluted after 8 S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10 S-HODE eluted before 10 R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5 S,8 R-Dihydroxyoctadeca-9 Z,12 Z-dienoic acid (5 S,8 R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8 R,11 S-dihydroxyoctadeca-9 Z,12 Z-dienoic acid (8 R,11 S-DiHODE) by Agaricus bisporus. 8 R-Hydroperoxylinoleic acid (8 R-HPODE) can be transformed to 5 S,8 R-DiHODE and 8 R,11-DiHODE by Aspergillus spp., and 8 R,13-dihydroxy-9 Z,11 E-dienoic acid (8 R,13-DiHODE) can also be detected. We prepared racemic [5,8- 2H 2]5,8- and [8,11- 2H 2]8,11-DiHODE by oxidation and reduction as above and 8 R,13 S- and 8 R,13 R-DiHODE by oxidation of 8 R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC–MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8 R-HODE (>95% R), 10 R-HODE (>70% R), and 5 S,8 R- and 8 R,11 S-DiHODE with high stereoselectivity (>95%), whereas 8 R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8 R,11 S-DiHODE.

The upstream oxylipin profile of Arabidopsis thaliana: a tool to scan for oxidative stresses.

Various physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.

Chiral Phase High-Performance Liquid Chromatographic Separation of Enantiomeric 1,2- and 2,3-O-Isopropylidene-sn-glycerols as 3,5-Dinitrophenylurethanes

High-performance liquid chromatographic separation of enantiomeric 1,2- and 2,3-O-isopropylidene-sn-glycerols was performed on two different stationary phases of opposite configurations: N-(S)-1-(α-naphthyl)ethylaminocarbonyl-(S)-tert-leucine and N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(R)-tert-leucine. This caused a reversal in the order of enantiomer elution. The isopropylideneglycerols were chromatographed as 3,5-dinitrophenylurethane derivatives. Complete enantiomer resolution, which permitted an accurate estimation of the optical purity of each enantiomer, was easily achieved on both columns (each 25 cm × 4.6 mm i.d.) within 15 min after an injection by an isocratic elution with hexane/dichloromethane/ethanol (40:12:3, by vol) as the mobile phase. The formations of diastereomeric π-π donor-accepter interaction, hydrogen bonding association, and dipole-dipole stacking between the solutes and the chiral stationary phases were considered as contributing factors to enantiomer resolution. Under optimal conditions, the optical purities of several commercially available 1,2-O-isopropylideneglycerol enantiomers were successfully determined in this study. The new method used in this study demonstrates that chiral phase high-performance liquid chromatography provides effective resolution, identification, and quantitation of synthetic 1,2- and 2,3-O-isopropylidene-sn-glycerol enantiomers.

Capillary electrophoresis of cytochrome P-450 epoxygenase metabolites of arachidonic acid. 2. Resolution of stereoisomers.

Each of the four regioisomers of epoxyeicosatrienoic acids (EETs) is a candidate for being an endothelial-dependent hyperpolarizing factor (EDHF). One regioisomer, 14,15-EET, stereospecifically blocks cyclooxygenases from converting arachidonic acid to prostaglandins and stereospecifically binds to cellular receptors. Both stereospecific actions emphasize the need to establish the tissue availability of the 14,15-EET enantiomers. The present work describes a method to quantitate picogram amounts of 14,15-EET enantiomers by capillary electrophoresis. The 14,15-EET enantiomers were baseline resolved (R = 1.3) using unsubstituted beta-cyclodextrin and 32% acetonitrile (v/v). When absorption at 194 nm was monitored using a photodiode array detector, 8 and 1 pg of underivatized 14,15-EET were readily quantitated and detected, respectively. Capillary electrophoresis accurately assessed chiral excesses up to 97:3 for either 14,15-EET enantiomer. Moreover, capillary electrophoresis with a photodiode array detector was sufficiently sensitive to detect and measure 14,15-EET enantiomers from murine liver. Thus, unlike chiral-phase high-performance liquid chromatography, capillary electrophoresis can be used to directly assess the chirality of trace amounts of underivatized eicosanoids.

Determination of stereochemical configuration of the glycerol moieties in glycoglycerolipids by chiral phase high‐performance liquid chromatography

This study reports a simple and sensitive method for determining the absolute configuration of the glycerol moieties in glycoglycerolipids. The method is based on chiral phase high-performance liquid chromatography (HPLC) separations of enantiomeric di- and monoacylglycerols released from glycosyldi- and monoacylglycerols, respectively, by periodate oxidation followed by hydrazinolysis. The released di- and monoacylglycerols were chromatographed as their 3,5-dinitrophenylurethane (3,5-DNPU) and bis(3,5-DNPU) derivatives, respectively. The derivatives were separated on two chiral phases of opposite configuration, (R)- and (S)-1-(1-naphthyl)ethylamine polymers for diacylglycerols and N-(R)- -(1-naphthyl)ethylaminocarbonyl-(S)-valine and N-(S)-1 -(1-naphthyl)ethylamino-carbonyl-(R)-valine for monoacylglycerols. Clear enantiomer separations, which permit the assignment of the glycerol configuration, were achieved for sn-1,2(2,3)-diacyl- and sn-1(3)-monoacylglycerols generated from linseed oil triacylglycerols by partial Grignard degradation on all the chiral stationary phases employed. Using the method, we have determined the glycerol configuration in the glycosyldiacylglycerols (monogalactosyl-, digalactosyl-, and sulfquinovosyldiacylglycerols) and glycosylmonoacylglycerols (monogalactosyl-, digalactosyl-, and sulfoquinovosylmonoacylglycerols) isolated from spinach leaves and the coralline red alga Corallina pilulifera. The results clearly showed that the glycerol moieties in all the glycoglycerolipids examined have S-configuration (sn-1,2-diacyl- and sn-1-monoacylglycerols). The new method demonstrates that chiral phase HPLC provides unambiguous information on the configuration of the glycerol backbone in natural glycosyldi- and monoacylglycerols, and that the two-step liberation of the free acylglycerols does not compromise glycerol chirality.

Optical resolution of asymmetric triacylglycerols by chiral-phase high-performance liquid chromatography

A simple method for direct optical resolution of some asymmetric triacylglycerols (TGs) has been established. The method employs chiral-phase high-performance liquid chromatography (HPLC). An enantiomeric pair of TGs comprising 1-eicosapentaenoyl-2,3-dicapryroyl- sn-glycerol (ECC) and 1,2-dicapryroyl-3-eicosapentaenoyl- sn-glycerol (CCE) was resolved on a CHIRALCEL OF™ or on a CHIRALCEL OD™ column. The separation of another pair of asymmetric TGs, 1-docosahexaenoyl-2,3-dicapryroyl- sn-glycerol (DCC) and 1,2-dicapryroyl-3-docosahexaenoyl- sn-glycerol (CCD), was achieved with the CHIRALCEL OD column. The chiral-phase HPLC method in combination with silver-ion HPLC and high-temperature gas chromatography was used for monitoring two interesterification reactions, whose products were chiral TGs. Interesterification of tricapryloylglycerol with ethyleicosapentaenoate or with ethyldocosahexaenoate was performed using Rhizomucor miehei lipase as the catalyst. The products targeted were the asymmetric pair of TGs, ECC and CCE or DCC and CCD. The amounts of sn-1-substituted products (ECC or DCC) were greater than their sn-3-substituted counterparts (CCE or CCD) throughout the reaction period, suggesting that R. miehei lipase had a stereopreference towards the sn-1 position over the sn-3 position.

High-performance liquid chromatographic resolution of reverse isomers of 1,2-diacyl- rac-glycerols as 3,5-dinitrophenylurethanes

The resolution of reverse isomers remains a major unsolved problem in glycerolipid chromatography. We have investigated the separation of the reverse isomers of 1,2-diacyl- rac-glycerols under a variety of high-performance liquid chromatography (HPLC) conditions. The reverse isomers of diacylglycerols having various pairs of acyl groups including short and highly unsaturated chains, which were prepared by partial Grignard degradation of the corresponding triacylglycerols, were chromatographed as 3,5-dinitrophenylurethanes. Excellent resolution was achieved for the reverse isomers of very different pairs of acyl groups, such as acetate–palmitate and docosahexaenoate–palmitate, by chiral-phase HPLC on columns containing ( R)- and ( S)-1-(1-naphthyl)ethylamine polymeric phases, reversed-phase HPLC on a highly efficient C 18 column (4 μm particle size) and silver ion HPLC on a silver loaded cation-exchange column. The chiral-phase HPLC also permitted complete enantiomer resolution for all the reverse isomers examined. No satisfactory resolution by any of the HPLC methods, however, was obtained for the reverse isomers possessing minor differences in chain lengths and degree of unsaturation, such as laurate–palmitate and oleate–linoleate. The limitations of resolution and characteristics of elution are described.

A Sensitive Gas Chromatography-Mass Spectrometry Assay Reveals Increased Levels of Monohydroxyeicosatetraenoic Acid Isomers in Human Plasma After Extracorporeal Photoimmunotherapy and Under In Vitro Ultraviolet A Exposure

Extracorporeal photoimmunotherapy (photopheresis) is a highly effective therapy in the treatment of various disorders. Although extracorporeal photoimmunotherapy has been successfully used for more than 10 y, its mechanism of action is still unclear. The formation of reactive oxygen species have been implicated in extracorporeal photoimmunotherapy, but malonyl dialdehyde as a marker of systemic lipid peroxidation did not increase significantly during treatment. To investigate further the involvement of reactive oxygen species in extracorporeal photoimmunotherapy, we have introduced a highly sensitive negative ion gas chromatography-mass spectrometry based method for quantitating oxygenated arachidonic acid isomers (hydroxyeicosatetraenoic acids) in plasma samples of patients treated with extracorporeal photoimmunotherapy. In the plasma of healthy volunteers pmole amounts of 2-, 3-, 5-, 8-12-, and 15-hydroxyeicosatetraenoic acid were detected and we observed a dose-dependent augmentation in these metabolites when the blood was irradiated with increasing doses of ultraviolet A in the presence of the photosensitizer 8-methoxypsoralen. Analysis of plasma samples obtained from patients before and after extracorporeal photoimmunotherapy revealed a characteristic increase in total hydroxyeicosatetraenoic acid levels, particularly of 5-hydroxyeicosatetraenoic acid which contributed 80% to the sum of all hydroxyeicosatetraenoic acid isomers. Chiral phase high-performance liquid chromatography indicated almost equal amounts of 5S- and 5R-hydroxyeicosatetraenoic acid suggesting that the majority of lipid peroxidation products are formed via nonenzymatic oxidation reactions.

Identification of 12-lipoxygenase products in the gills of carp, Cyprinus carpio

SUMMARY: A lipoxygenase was found in the crude enzyme solution from the gills of carp, Cyprinus carpio. It oxidized arachidonic acid (AA) more efficiently than linoleic acid, eicosapentaenoic acid and docosahexaenoic acid. Lipoxygenase activity was constantly detected in the gill microsomes and was found to be optimum at pH 7.2. It was not stimulated by reduced nicotinamide adenine dinucleotide phosphate and was not inhibited by SKF525A, a cytochrome P450 inhibitor. The oxygenated products extracted from the reaction mixtures of crude enzyme solution and AA were purified by reverse-phase and straight-phase high-performance liquid chromatography (HPLC). The major metabolite was identified as 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) by ultraviolet spectrophotometry, gas chromatography-mass spectrometry and chiral phase HPLC. In addition, 12-hydroxyeicosapentaenoic acid (12-HEPE) and 13-hydroxyoctadecadienoic acid (13-HODD) were also found in the reaction products as minor components. Similar results were obtained by the analysis of the reaction products of AA and carp gill microsomes. These results confirm the presence of 12-lipoxygenase in carp gill microsomes.

Simultaneous determination of RRR- and SRR-alpha-tocopherols and their quinones in rat plasma and tissues by using chiral high-performance liquid chromatography.

We established a method to simultaneously determine RRR- and SRR-alpha-tocopherol (alpha-Toc) and their quinones in biological samples by chiral-phase high-performance liquid chromatography (HPLC). Alpha-Toc had a shorter retention time than alpha-tocopherylquinones (alpha-TQ), and 2-ambo-alpha-Toc was completely separated into two peaks; the first peak was RRR-alpha-Toc and the second SRR-isomer by chiral HPLC connected Chiralcel OD-H column and Sumichiral OA4100 column. In contrast, of the two peaks of alpha-TQ, the first was the SRR-isomer. We also investigated differences in the distribution of RRR- and SRR-alpha-TQ in rat tissues after oral administration of 2-ambo-alpha-Toc by the above HPLC method. Rats deficient in vitamin E were divided into two groups, control and experimental, and tissues were collected at 3, 6, and 24 h after oral 2-ambo-alpha-Toc administration. The concentrations of RRR- and SRR-alpha-Toc and their quinones in plasma and each tissue were determined. The concentration of SRR-alpha-TQ in plasma and adrenal glands was not significantly different from RRR-alpha-TQ. However, the concentration of SRR-alpha-TQ in liver up to 6 h after oral administration was higher than that of RRR-alpha-TQ, and SRR- and RRR-alpha-TQ levels were similar at 24 h after oral administration. Therefore, we may assume that the formation of alpha-TQ in vivo was not different between RRR- and SRR-isomer and that it was not affected by the presence of alpha-Toc stereoisomers.

Human 12(R)-Lipoxygenase and the Mouse Ortholog: MOLECULAR CLONING, EXPRESSION, AND GENE CHROMOSOMAL ASSIGNMENT

Expressed sequence tag information was used to clone the full-length sequence for a new human lipoxygenase from the B cell line CCL-156. A related mouse sequence with 83% nucleotide identity to the human sequence was also cloned. The human lipoxygenase, when expressed via the baculovirus/insect cell system produced an approximately 80-kDa protein capable of metabolizing arachidonic acid to a product identified as 12-hydroxyeicosatetraenoic acid by mass spectrometry. Using chiral phase-high performance liquid chromatography, the product was identified as >98% 12(R)-hydroxyeicosatetraenoic acid as opposed to the S-stereoisomer formed by all other known mammalian lipoxygenases. The single copy human 12(R)-lipoxygenase gene was localized to the chromosome 17p13 region, the locus where most other lipoxygenase genes are known to reside. By reverse transcription-polymerase chain reaction, but not by Northern blot, analysis the 12(R)-lipoxygenase mRNA was detected in B cells and adult skin. However, the related mouse lipoxygenase mRNA was highly expressed in epidermis of newborn mice and to a lesser extent in adult brain cortex. By in situ hybridization the mouse lipoxygenase gene was demonstrated to be temporally and spatially regulated during embryogenesis. Expression was induced at embryonic day 15.5 in epidermis, nasal epithelium, and surface of the tongue. These results broaden the mammalian lipoxygenase family to include a 12(R)-lipoxygenase whose biological function remains to be determined.