RESUMEN
A method was developed for determination of proteinaceous free amino acids by gas chromatography-mass spectrometry (GC-MS). The guanidino group of arginine in amino acid samples was modified with 1,2-cyclohexanedione at room temperature under basic conditions, and all amino acids were directly derivatized with isobutyl chloroformate. The amino acid derivatives formed were analyzed by GC-MS. The method developed was applied successfully for the determination of amino acids in the Japanese alcoholic beverage, sake.
Asunto(s)
Aminoácidos/análisis , Vino/análisis , Etanol/química , Formiatos/química , Cromatografía de Gases y Espectrometría de Masas , JapónRESUMEN
A method was developed for determination of free fatty acids (FFAs) in plasma by gas chromatography. Plasma was extracted with 3 vol of methanol. Most cholesterol esters and triacylglycerols did not dissolve in the aqueous methanol. FFAs in the crude lipid solution were directly and selectively methylated with (trimethylsilyl)diazomethane at room temperature. Fatty acid methyl esters (FAMEs) formed were extracted with hexane, and nonreactive phospholipids were washed out with 95% methanol. The partially purified FAME preparation was analyzed by gas chromatography. The composition and amount of plasma FFAs closely approximated those obtained using two different methods.
Asunto(s)
Cromatografía de Gases/métodos , Ácidos Grasos no Esterificados/sangre , Ésteres/química , Hexanos , Metanol/química , Metilación , TemperaturaRESUMEN
A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH3ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.
Asunto(s)
Cromatografía de Gases , Ácidos Grasos/análisis , Triglicéridos/química , Ésteres/química , Hexanos/química , Metanol/química , Ácidos Oléicos/análisis , Ácidos Oléicos/metabolismo , TemperaturaRESUMEN
Rapid, convenient methods have been developed for fatty acid analysis of membrane glycerophospholipids in microorganisms. Fatty acid methyl esters derived from glycerophospholipids have been prepared directly from wet pellets of Escherichia coli cells or Saccharomyces cerevisiae spheroplasts without lipid extraction and fractionation in high yields under mild temperature conditions for analysis by gas chromatography.
Asunto(s)
Cromatografía de Gases/métodos , Escherichia coli/química , Ácidos Grasos/análisis , Glicerofosfolípidos/química , Saccharomyces cerevisiae/química , Fraccionamiento Químico , Esferoplastos/química , TemperaturaRESUMEN
Two improved methods have been developed for preparation of fatty acid methyl esters (FAME) from major O-ester lipid classes in blood, i.e., cholesterol ester, triacylglycerol, and glycerophospholipids. The methods involve simple operations, and use neither harmful solvents such as chloroform or benzene nor highly reactive volatile reagents such as acetyl chloride. The FAME synthesis reaction proceeds under mild temperature conditions. The methods include (1) extraction of lipids from 0.2 ml of blood with 0.2 ml of tert-butyl methyl ether and 0.1 ml of methanol, (2) separation of the total lipids into lipid classes using a solid-phase extraction column or thin-layer chromatography, and (3) methanolysis of each lipid class at room temperature or at 45 °C. In all the operations, solvent concentration is performed only once prior to gas-liquid chromatography (GC). No noticeable differences in composition determined by GC have been found between FAME prepared by the present methods and those prepared by a conventional method involving lipid extraction with chloroform/methanol. The mild reaction and simplified procedures of the present methods enabled safe and reproducible analysis of the fatty acid compositions of the major ester-lipid classes in blood.
Asunto(s)
Análisis Químico de la Sangre/métodos , Ácidos Grasos/análisis , Lípidos/análisis , Lípidos/clasificación , Algoritmos , Análisis Químico de la Sangre/normas , Fraccionamiento Químico/métodos , Cromatografía de Gases , Cromatografía en Capa Delgada , Ácidos Grasos/sangre , Ácidos Grasos/química , Ácidos Grasos/clasificación , Humanos , Lípidos/sangre , Lípidos/química , Metanol/química , Metanol/farmacología , Éteres Metílicos/química , Éteres Metílicos/farmacología , Extracción en Fase Sólida/métodos , Solventes/química , Solventes/farmacologíaRESUMEN
KOH in aqueous methanol catalyzes selective methanolysis of polar glycerolipids with O-ester-linked acyl residues, while triacylglycerols and sterol esters are inert in the solution. Based on these findings, a convenient and reliable method was developed for the preparation of fatty acid methyl esters (FAMEs) from polar glycerolipids in lipid mixtures without prior isolation. Methanolysis of polar glycerolipids was completed within 2.5 min by vortexing or 20 min by shaking with 0.7 M KOH/70% (v/v) methanol in the presence of hexane at 30 degrees C. The yields of FAMEs obtained by the present method were greater than 95%. The method was applied successfully to gas chromatographic analysis of the fatty acid compositions of polar glycerolipids in seed oil and blood. No obvious differences were found between the fatty acid compositions determined by the present method and those determined by conventional methods, including lipid extraction with chloroform/methanol followed by isolation of polar lipids by chromatography. The fatty acid composition of polar glycerolipids, including phospholipids, can be determined readily in many crude samples.
Asunto(s)
Ácidos Grasos/síntesis química , Glicéridos/metabolismo , Metanol/metabolismo , Metanol/farmacología , Sangre/metabolismo , Análisis Químico de la Sangre/métodos , Catálisis , Cromatografía de Gases , Ésteres , Ácidos Grasos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/sangre , Lípidos/aislamiento & purificación , Metano/análogos & derivados , Metano/química , Aceites de Plantas/química , Especificidad por Sustrato , Agua/farmacologíaRESUMEN
A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45 degrees C overnight or heated at 100 degrees C for 1-1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.
Asunto(s)
Ésteres/química , Ácidos Grasos/química , Cromatografía Liquida , Aceites de Pescado/química , Ácido Clorhídrico/química , Indicadores y Reactivos/química , Cinética , Lípidos/sangre , Lípidos/química , Metanol/química , Metilación , Aceites de Plantas/química , Solubilidad , Temperatura , Tolueno/química , Agua/químicaRESUMEN
Polyamines with diamine structures of chain length longer than 3C were essential for the synthesis of phosphatidic acid (PA) from ricinoleoyl-CoA and lysophosphatidic acid (LPA) by the castor LPA acyltransferase reaction, suggesting that polyamines modulate enzyme affinity for the acyl-CoA substrate in vivo.
Asunto(s)
Aceite de Ricino/biosíntesis , Lisofosfolípidos/metabolismo , Poliaminas/farmacología , Acilcoenzima A/metabolismo , Acilación/efectos de los fármacos , Ácidos Fosfatidicos/biosíntesis , Ácidos Ricinoleicos/metabolismoRESUMEN
The major fatty acid component of castor (Ricinus communis L.) oil is ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid), and unsaturated hydroxy acid accounts for >85% of the total fatty acids in triacylglycerol (TAG). TAG had a higher ricinoleate content at position 2 than at positions 1 and 3. Although lysophosphatidic acid (LPA) acyltransferase (EC 2.3.1.51), which catalyzes acylation of LPA at position 2, was expected to utilize ricinoleoyl-CoA preferentially over other fatty acyl-CoAs, no activity was found for ricinoleoyl-CoA in vitro at concentrations at which other unsaturated acyl-CoAs were incorporated rapidly. However, activity for ricinoleoyl-CoA appeared with addition of polyamines (putrescine, spermidine, and spermine), while polyamines decreased the rates of incorporation of other acyl-CoAs into position 2. The order of effect of polyamines on LPA acyltransferase activity was spermine > spermidine >> putrescine. At concentrations of spermine and spermidine of >0.1 mM, ricinoleoyl-CoA served as an effective substrate for LPA acyltransferase reaction. The concentrations of spermine and spermidine in the developing seeds were estimated at approximately 0.09 and approximately 0.63 mM, respectively. These stimulatory effects for incorporation of ricinoleate were specific to polyamines, but basic amino acids were ineffective as cations. In contrast, in microsomes from safflower seeds that do not contain ricinoleic acid, spermine and spermidine stimulated the LPA acyltransferase reaction for all acyl-CoAs tested, including ricinoleoyl-CoA. Although the fatty acid composition of TAG depends on both acyl-CoA composition in the cell and substrate specificity of acyltransferases, castor bean polyamines are crucial for incorporation of ricinoleate into position 2 of LPA. Polyamines are essential for synthesis of 2-ricinoleoyl phosphatidic acid in developing castor seeds.
Asunto(s)
Ácidos Fosfatidicos/biosíntesis , Poliaminas/metabolismo , Ácidos Ricinoleicos/metabolismo , Ricinus communis/metabolismo , Acetilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Carthamus tinctorius/embriología , Carthamus tinctorius/enzimología , Carthamus tinctorius/metabolismo , Ricinus communis/embriología , Ricinus communis/enzimología , Cationes Bivalentes , Metabolismo de los Lípidos , Lisofosfolípidos/metabolismo , Metales/química , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Ácidos Fosfatidicos/química , Semillas/enzimología , Semillas/crecimiento & desarrollo , Espermidina/farmacología , Especificidad por SustratoRESUMEN
An improved safe method that does not contaminate the environment with cadmium chloride, a toxic heavy metal salt, was developed for the synthesis of phosphatidylcholine (PC). PC was synthesized from sn-glycero-3-phosphocholine (GPC) and fatty acid in one step under mild conditions without the use of cadmium chloride. GPC was prepared from egg yolk PC and adsorbed by kieselguhr in a Teflon vessel. The GPC on kieselguhr was acylated with fatty acid in the presence of two reagents, dicyclohexylcarbodiimide for synthesis of fatty acid anhydride and 4-dimethylaminopyridine as an acylating catalyst, at 30 degrees C overnight. The PC thus produced was purified by silica gel column chromatography. The yield of dioleoyl PC was 90% based on the starting material, GPC.
Asunto(s)
Ácidos Grasos/química , Glicerilfosforilcolina/química , Fosfatidilcolinas/síntesis química , Fosforilcolina/química , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/química , Acilación , Animales , Cloruro de Cadmio , Catálisis , Cromatografía , Tierra de Diatomeas , Yema de Huevo/química , Espectrometría de MasasRESUMEN
Developing rice seeds rapidly accumulated storage lipids between 5 and 12 d after flowering. The contents of palmitic, oleic, and linoleic acids increased throughout seed development, while the alpha-linolenic acid content remained low. The activity of acyl-CoA synthetase varied coincidentally during the period of lipid accumulation, and rice seeds had a sufficient capacity to supply acyl-CoA substrates for TAG synthesis. Acyl-CoA synthetase showed a broad specificity for native FA of rice seeds except for stearic acid, and pi electrons of a delta9-delta11 double bond in the C16-C18 acyl chains were required for its maximal activity.
Asunto(s)
Coenzima A Ligasas/metabolismo , Lípidos/biosíntesis , Oryza/crecimiento & desarrollo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Semillas/metabolismo , Membrana Celular/enzimología , Ácidos Grasos/análisis , Concentración de Iones de Hidrógeno , Cinética , Metabolismo de los Lípidos , Lípidos/química , Oryza/enzimología , Oryza/metabolismo , Semillas/enzimología , Especificidad por SustratoRESUMEN
In developing seeds of Perilla frutescens var. crispa, the triacylglycerol fraction was found to accumulate between 15 and 19 days after flowering. Of this, 65% of the total fatty acids was alpha-linolenic acid in the mature seeds, with the latter being esterified in comparable amounts at all positions (sn-1, 2 and 3) of the glycerol residue. It was also demonstrated that, 1-acylglycerol-3-phosphate acyltransferase, which catalyzes esterification at the sn-2 position of the glycerol backbone, showed low activities for alpha-linolenoyl-CoA as substrate. These findings suggest that the diacylglycerol precursor for triacylglycerol synthesis is not directly derived from phosphatidic acid through the glycerol phosphate pathway.