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1.
J Chromatogr A ; 1585: 113-120, 2019 Jan 25.
Article in English | MEDLINE | ID: mdl-30598291

ABSTRACT

Comprehensive separation of fatty acids, including various isomers, is very important for chemical analyses associated with detailed physiological function investigations. We found that a basic eluent is effective for realizing clear separation of double-bond regioisomers by reversed-phase high-performance liquid chromatography. We carried out a comprehensive analysis of fatty acids, including double-bond positional isomers, trans-fatty acids, and iso-fatty acids using reversed-phase high-performance liquid chromatography coupled with Fourier transform mass spectrometry (LC/FTMS). Clear separation of these fatty acids was achieved using a C18 column and a basic eluent. A three-pump gradient system using acetone provided rapid elution within 22 min. In a single run, 184 fatty acid molecular species contained in a dietary fish oil supplement were detected by the FTMS full scan. A previously unreported peak was also detected, which was assigned as tetratriacontadecaenoic acid by comparison with the MS2 profiles of fatty acids with known chemical structures. This result demonstrates that the developed method is useful for clarifying the composition of fatty acid molecular species in target samples, providing a promising approach to discover unreported fatty acids.


Subject(s)
Chemistry Techniques, Analytical/methods , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Fatty Acids/chemistry , Mass Spectrometry , Chemistry Techniques, Analytical/instrumentation , Fatty Acids/analysis , Fourier Analysis , Solvents/chemistry
2.
J Biochem ; 164(2): 127-140, 2018 Aug 01.
Article in English | MEDLINE | ID: mdl-29554278

ABSTRACT

It is commonly observed that freshwater fish contain lower amounts of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), than marine fish species. In this study, we performed a detailed comparative analysis of phospholipids (PLs) and triacylglycerols (TAGs) from Gymnogobius isaza, a freshwater goby endemic to Lake Biwa inhabiting the lake bottom, and Gymnogobius urotaenia, a related goby that inhabits the shore of Lake Biwa. We found that tissues from G. isaza contain remarkably high amounts of omega-3 LC-PUFAs in both PLs and TAGs. Mass spectrometry analysis of TAGs demonstrated that the most abundant TAG molecular species were TAG (16:0/18:1/20:5), followed by TAG (14:0/18:1/20:5), in which EPA is incorporated into TAG at either the sn-1 or sn-3 positions. We isolated cDNAs encoding acyl-CoA: diacylglycerol acyltransferase designated as GiDGAT1 and GiDGAT2, from G. isaza. Expression studies using a neutral lipid-deficient Saccharomyces cerevisiae mutant strain demonstrated that both GiDGAT1 and GiDGAT2 possessed diacylglycerol acyltransferase activity, and preferential incorporation of LC-PUFA into TAG was observed in the presence of GiDGAT1. This study revealed the novel lipid profiles of G. isaza and identified the enzymes that were involved in the production of PUFA-containing TAGs.


Subject(s)
Diacylglycerol O-Acyltransferase/metabolism , Fatty Acids, Omega-3/metabolism , Triglycerides/biosynthesis , Animals , Fatty Acids, Omega-3/chemistry , Fishes , Japan , Lakes , Triglycerides/chemistry
3.
Electrophoresis ; 37(23-24): 3196-3205, 2016 12.
Article in English | MEDLINE | ID: mdl-27649837

ABSTRACT

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl-γ-cyclodextrin (HP-γ-CD)-modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate-15 mM borate buffer (pH 9.0) containing 30 mM HP-γ-CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)- and (R)-enantiomers of regioisomeric 8-, 11-, 12-, and 15-HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8- and 12-HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8-HETE was found to be a mixture of 98% (R)-enantiomer and 2% (S)-enantiomer, while the 12-HETE was a mixture of 98% (S)-enantiomer and 2% (R)-enantiomer. The present study demonstrates that the HP-γ-CD-modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.


Subject(s)
Chromatography, Micellar Electrokinetic Capillary/methods , Hydroxyeicosatetraenoic Acids , gamma-Cyclodextrins/chemistry , Hydroxyeicosatetraenoic Acids/analysis , Hydroxyeicosatetraenoic Acids/chemistry , Hydroxyeicosatetraenoic Acids/isolation & purification , Linear Models , Reproducibility of Results , Sensitivity and Specificity , Stereoisomerism
4.
Environ Toxicol Chem ; 32(2): 406-9, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23147916

ABSTRACT

Existing standard bioconcentration tests (e.g., the Organization for Economic Cooperation and Development [OECD] test guideline 305) require large numbers of test animals and resources. The minimized aqueous exposure test is a new approach based on the standard bioconcentration test but allows estimation of bioconcentration factor (BCF) by minimized sampling of the test fish. The authors collected BCF data (298 curves from 155 chemicals, using common carp as test species) from Japan's Chemical Substances Control Law database and resampled the data to simulate the calculation of BCF that would be obtained if studies had been designed to obtain kinetic BCF derived from minimized aqueous exposure tests (BCF(km)). The correlation was high (r(2) = 0.967) between BCF derived from standard bioconcentration tests (BCF(full)) and BCF(km). The average value of the BCF(full) to BCF(km) ratio (BCF(full):BCF(km)) was 1.04 and ranged from 0.54 to 1.93, the 5th and 95th percentiles being 0.74 and 1.45, respectively. The results based on the 5th and 95th percentiles of the BCF(full):BCF(km) ratio suggest that BCF(full) 2,000 corresponds to BCF(km) 1,400 to 2,700, whereas BCF(full) 5,000 corresponds to BCF(km) 3,400 to 6,800. The authors also emphasize that the standard bioconcentration test should be performed when the resulting BCF(km) is in the region of regulatory concern.


Subject(s)
Environmental Monitoring/standards , Water Pollutants, Chemical/metabolism , Animals , Carps/metabolism , Computer Simulation , Databases, Factual , Environmental Monitoring/methods , Japan , Kinetics , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/standards , Water Pollution, Chemical/statistics & numerical data
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