Simple Chromatographic Method for Simultaneous Analyses of Phosphatidylcholine, Lysophosphatidylcholine, and Free Fatty Acids

This study describes a simple chromatographic method for the simultaneous analyses of phosphatidylcholine (PC) and its hydrolytic degradation products: lysophosphatidylcholine (LPC) and free fatty acids (FFA). Quantitative determination of PC, LPC, and FFA is essential in order to assure safety and to accurately assess the shelf life of phospholipid-containing products. A single-run normal-phase high-performance liquid chromatography (HPLC) with evaporative light scattering detector has been developed. The method utilizes an Allsphere silica analytical column and a gradient elution with mobile phases consisting of chloroform: chloroform–methanol (70:30%, v/v) and chloroform–methanol–water–ammonia (45:45:9.5:0.5%, v/v/v/v). The method adequately resolves PC, LPC, and FFA within a run time of 25 min. The quantitative analysis of PC and LPC has been achieved with external standard method. The free fatty acids were analyzed as a group using linoleic acid as representative standard. Linear calibration curves were obtained for PC (1.64–16.3 μg, r² = 0.9991) and LPC (0.6–5.0 μg, r² = 0.9966), while a logarithmic calibration curve was obtained for linoleic acid (1.1–5.8 μg, r² = 0.9967). The detection and quantification limits of LPC and FFA were 0.04 and 0.1 μg, respectively. As a means of validating the applicability of the assay to pharmaceutical products, PC liposome was subjected to alkaline hydrolytic degradation. Quantitative HPLC analysis showed that 97% of the total mass balance for PC could be accounted for in liposome formulation. The overall results show that the HPLC method could be a useful tool for chromatographic analysis, stability studies, and formulation characterization of phospholipid-based pharmaceuticals.KEY WORDS: evaporative light scattering detection, free fatty acid, lysophosphatidylcholine, phosphatidylcholine     

Fatty Acids of Mycobacterium kansasii

The cellular fatty acids of 35 strains of Mycobacterium kansasii isolated from clinical material were analyzed to establish properties by which we could identify and characterize these acid-fast microorganisms. The fatty acids were extracted from cells grown in liquid synthetic media, and they were analyzed as methyl esters by gas-liquid chromatography. The fatty acid profiles of all strains were similar. They differed from fatty acid profiles of other mycobacteria by their content of a saturated fatty acid with a methyl group at C2.     

Fatty Acids of Thiobacillus thiooxidans

Fatty acid spectra were made on Thiobacillus thiooxidans cultures both in the presence and absence of organic compounds. Small additions of glucose or acetate had no significant effect either on growth or fatty acid content. The addition of biotin had no stimulatory effect but did result in slight quantitative changes in the fatty acid spectrum. The predominant fatty acid was a C₁₉ cyclopropane acid.     

Fatty Acids of Myxococcus xanthus

Fatty acids were extracted from saponified vegetative cells and myxospores of Myxococcus xanthus and examined as the methyl esters by gas-liquid chromatography. The acids consisted mainly of C₁₄ to C₁₇ species. Branched acids predominated, and iso-pentadecanoic acid constituted half or more of the mixture. The other leading component (11–28%) was found to be 11-n-hexadecenoic acid. Among the unsaturated acids were two diunsaturated ones, an n-hexadecadienoic acid and an iso-heptadecadienoic acid. No significant differences between the fatty acid compositions of the vegetative cells and myxospores could be detected. The fatty acid composition of M. xanthus was found to be markedly similar to that of Stigmatella aurantiaca. It is suggested that a fatty acid pattern consisting of a large proportion of iso-branched C₁₅ and C₁₇ acids and a substantial amount of an n-16:1 acid is characteristic of myxobacteria.     

Polyunsaturated Fatty Acids: Biotechnology

ABSTRACT

Polyunsaturated fatty acids like EPA and DHA have attracted a great attention due to their beneficial effects on human health. At present, fish oil is the major source of EPA and DHA. Various alternative sources are being explored to get these essential fatty acids. Genes encoding enzymes involved in the biosyntheses of PUFAs have been identified, cloned and gene prospecting becomes a novel method for enhanced PUFA production. Desaturase and elongase genes have important biotechnological appeal from genetic engineering point of view. This review highlights the research and results on such enzymes.     

多不饱和脂肪酸的研究进展

多不饱和脂肪酸(PUFA)是人类的必需脂肪酸,对人体有重要的生理功能,能调节人体的脂质代谢、治疗和预防心脑血管疾病、抗癌、对抗肥胖,促进生长发育和提高幼体的成活率等．综述了PUFA的生理作用和来源的研究进展,特别是在对抗肥胖、促进生长发育、提高幼体的成活率等方面的研究情况进行了阐述．     

Bitter Taste of Oxidized Fatty Acids

A strong bitter taste was reported previously in highly autoxidized soybean oil, especially in the free fatty acid fraction. In the present study, for the elucidation of bitter substances in autoxidized oils, autoxidized linoleic acid was fractionated by silicic acid column chromatography, and the fraction with maximum peroxide value was treated with cysteine-ferric trichloride, reduced with sodium borohydride, oxidized with chromic acid and esterified with diazomethane. The results indicated that the bitter substances of autoxidized linoleic acid possessed a particular structure between the carboxyl group and other functional groups. To verify this fact, four oxidized fatty acids [ricinoleic(12-hydroxyoctadecenoic), 12-oxooctadecenoic, 9,12-dioxooctadecenoic, and dihydroxyoctadecenoic acids] were synthesized from castor oil, and their correlation to the bitter taste was investigated. The results showed that ricinoleic acid had a strong bitter taste, along with a strong stimulation, but this bitter taste was greatly reduced by methyl esterification. On the other hand, 12-hydroxy- and 9,10-dihydroxystearic acids were tasteless. From these results we conclude that the presence of an unsaturated bond, a carboxyl, a hydroxyl and/or peroxyl groups is a requisite for the bitterness of ricinoleic and autoxidized fatty acids.     

Cellular Fatty Acids of Pathogenic Neisseria

The cellular fatty acid composition of 20 isolates of Neisseria gonorrhoeae and 21 isolates of N. meningitidis was examined by gas-liquid chromatography. Each isolate of the two species possessed similar fatty acid profiles which were characterized by five major acids, accounting for 80 to 85% of the total. The three most abundant acids in each species were palmitic, palmitoleic, and beta-hydroxylauric acids; lauric and myristic acids were the next most abundant. The presence of large amounts of beta-hydroxylauric acid (20% or greater) and the relative concentrations of the other four major acids appear to be useful markers for distinguishing N. gonorrhoeae and N. meningitidis fatty acids from those of other bacteria.     

Separation and Identification of Fatty Acids

For the characterization of fatty acids, 2, 4-dinitrophenylhydrazones of p-bromophenacyl esters of even-numbered saturated fatty acids from C₂ to C₂₀ and several unsaturated fatty acids from monoethenoid to triethenoid were prepared. The derivatives of linoleic and linolenic acids as well as those of the other unsaturated and saturated acids, were successfully obtained in crystalline forms which showed sharp and high melting points, 72° and 69°, respectively. It was found that the derivatives of unsaturated acids were valuable for characterizing the parent acids, while those of saturated acids were unsuitable for this purpose owing to the similarity of their melting points.     

Inhibition of Topoisomerases by Fatty Acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure-activity relationships and mechanism of action were studied. Saturated fatty acids (C_6:0 to C_22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C_16:1 to C_22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C_18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Separation and Identification of Fatty Acids

An improved method is described for separating saturated fatty acids by reversed-phase paper chromatography. The 2,4-dinitrophenylhydrazides prepared from saturated fatty acids from C₂ to C₂₂ are run upward on paper impregnated with tetralin, using 90% methanol—acetic acid—tetralin, 80% ethanol—acetic acid—tetralin, or 90% methanol—tetralin as the moving solvent. The simultaneous separations of all even-carbon acids from C₆ to C₂₂, all odd-carbon acids from C₇ to C₁₉, and also all odd- and even-carbon acids from C₇ to C₁₉ can be successfully performed by means of this paper chromatography. The method is useful for the detection of component saturated fatty acids in natural fats.     

Detailed Analyses of Individual Neutral Lipid Classes of Pneumocystis

SUMMARY Individual neutral lipid classes of Pneumocystis carinii carinii were isolated and purifed by thin-layer chromatography. The fatty acids in steryl esters, triglycerides, free fatty acids, diglycerides, and monoglycerides were quantified by gas-liquid chromatography. The fatty acid compositions of these lipids were similar to those of the neutral lipid classes in whole rat lung controls, unlike comparable studies of phospholipid classes. The free fatty alcohols of rat lung controls and P. carinii were also identified and quantified; only saturated fatty alcohols were detected.     

Spin Label Motion in Fatty Acids

Spin labels dissolved in highly purified fatty acid systems exhibit nearly identical tumbling rates in liquid and solid phases. Even though the spin labels do not have the same molecular geometry as the lipid matrix the melting point of the matrix can be inferred by measurements of the temperature dependency of molecular motion.