Analysis Of Total Fatty Acids Research Articles

Gamma-irradiation of liposomes composed of saturated phospholipids. Effect of bilayer composition, size, concentration and absorbed dose on chemical degradation and physical destabilization of liposomes

Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), or mixtures of these two phospholipids were exposed to gamma-irradiation in an air environment. Disappearance of the mother compounds was monitored by HPLC analysis. Plotting of the logarithmic values of residual DPPC or DPPG concentration versus irradiation dose resulted in straight lines. The slopes of these lines (overall degradation constants) depended on the type of phospholipids, concentration of the liposomes and the size of the liposomes. Under the chosen conditions, addition of DPPG in DPPC-liposomes did not affect the degradation rate constant of DPPC and visa versa. The presence of phosphate buffer (pH 7.4), pH or presence of sodium chloride did not affect the irradiation damage either. Minor changes were found upon analysis of total fatty acids by GLC and upon measurement of water soluble phosphate compounds. These changes were less pronounced than the changes monitored by HPLC of phospholipids, because the HPLC analysis monitored the overall degradation of the liposomal phospholipids. Thin-layer chromatography/fast atom bombardment mass spectrometry (TLC/FAB-MS) analysis of irradiated and non-irradiated DPPC or DPPG provided information on the structure of several degradation products. Degradation routes which include these degradation products are proposed. Gamma-irradiation neither affected the size of the liposomes nor the bilayer rigidity as determined by dynamic light scattering and fluorescence anisotropy of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. However, upon gamma-irradiation, changes in the melting characteristics of the liposomes were found by differential scanning calorimetry (DSC) measurements. The pre-transition melting enthalpy of the liposomal bilayer decreased or disappeared and the main-transition broadened. The changes found in DSC scans correlated qualitatively well with the changes recorded after HPLC analysis of phospholipids.

A mutant of Nannochloropsis deficient in eicosapentaenoic acid production

The most abundant fatty acids found in Nannochloropsis are 16:0, 16:1 ( n-7) and 20:5 ( n-3). By analysing the fatty acid composition of mutagenized cells by gas chromatography, we discovered a mutant strain of Nannochloropsis that is completely devoid of 5,8,11,14,17- cis-eicosapentaenoic acid [20:5 ( n-3)]. An analysis of total fatty acids from whole cells showed that the loss of 20:5 ( n-3) in mutant cells was accompanied by a concomitant increase in the relative amount of 20:4 ( n-6). However, the increase in 20:4 ( n-6) was not proportional to the loss of 20:5 ( n-3) within individual lipid classes. The percentage of 20:4 ( n-6) in monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol and phosphatidylglycerol (PG) in the mutant strain was lower than the wild type levels of 20:5 ( n-3) in these lipids. Conversely, the percentage of 20:4 ( n-6) in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the mutant cells was higher than the wild type levels of 20:5 ( n-3). Molecular species analysis of MGDG showed that, compared to the wild type strain, the mutant strain contained more 16:1 16:0 MGDG, and less of the MGDG species containing C 20 fatty acids. Relatively low levels of 20:4 20:4 MGDG were found in the mutant, although 20:5 20:5 MGDG was abundant in wild type cells. Relative membrane lipid levels were altered in the mutant compared to the wild type strain, particularly PG, which showed a fourfold decrease. The mutant cells grow more slowly than the wild type cells on both solid and in liquid media. Electron micrographs showed that the mutant strain had fewer thylakoid stacks per chloroplast, and fewer thylakoids per stack than wild type cells. We concluded that the mutation affects an extrachloroplastic ( n-3) desaturase that is required for the synthesis of 20:5 ( n-3), which is found in both chloroplast and extrachloroplast lipids.

Microbial community structure and biogeochemistry of Miocene subsurface sediments: implications for long‐term microbial survival

AbstractThirty closely spaced cores were obtained from Miocene‐aged fluvial, lacustrine and palaeosol subsurface sediments ranging in depth from 173 to 197 m at a site in south‐central Washington to investigate the size and composition of the microbial community in relation to sediment geochemical and geophysical properties. Total phospholipid fatty acid (PLFA) analysis indicated that the greatest concentrations of microbial bio‐mass were in low‐permeability lacustrine sediments that also contained high concentrations of organic carbon. Community structure, based on lipid analyses and on in situ hybridization of bacterial cells with 16S RNA‐directed DNA probes, also revealed the presence of metabolically active bacteria that respire sulphate and/or Fe(III) in the lacustrine sediments. Concentrations of pore water sulphate were low (4–8 mg/L) and HCI‐extractable Fe was predominantly Fe(II) in the same samples where total biomass and organic carbon were highest. The low hydraulic conductivity (10‐6 to < 10‐9 cm/s) of these sediments has likely contributed to the long term maintenance of both bacteria and organic carbon by limiting the supply of soluble electron acceptors for microbial respiration. These results suggest that the current subsurface microbial population was derived from organisms that were present during lake sedimentation = 6–8 million years ago.

Characterization of the cellulose-degrading bacterium NCIMB 10462

The gram-negative cellulase-producing bacterium NCIMB 10462 has been previously namedPseudomonas fluorescens subsp. or var.cellulosa. Because of renewed interest in cellulose-degrading bacteria for use in the bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its true metabolic potential. Metabolic and physical characterization of NCIMB 10462 revealed that this is an alkalophilic, nonfermentative, gram-negative, oxidase-positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium has few characteristics consistent with a classification ofP. fluorescens and a very low probability match with the genusSphingomonas. However, total lipid analysis did not reveal that any sphingo-lipid bases are produced by this bacterium. NCIMB 10462 grows best aerobically, but also grows well in complex media under reducing conditions. NCIMB 10462 grows slowly under anaerobic conditions on complex media, but growth on cellulosic media occurred only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIMB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37°C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is its ability to degrade cellulose, we suggest that it be calledPseudomonas cellulosa.

Bile formation and hepatic plasma membrane composition in guinea-pigs and rats

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl −, phosphate and Ca 2+ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na +,K + ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs. Total fatty acid analysis of the membrane revealed that palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2, n-6) were the predominant fatty acids in guinea-pigs, while palmitic acid (C16:0), stearic acid (C18:0) and arachidonic acid (C20:4, n-6) were the most important in rats. Thus, high bile flow rate and BAIF in the guinea-pig may be attributed to the low bile acid concentration (below the critical micellar concentration), secretion of hypercholeretic bile acids (e.g. ketolithocholate) and high bicarbonate output.

Phospholipid molecular species composition of developing fetal guinea pig brain.

Adequate accumulation of polyunsaturated essential fatty acids, in particular docosahexaenoic acid (22:6n-3), into membrane phospholipids is critical for optimal fetal brain development. This process is maximal during the period of rapid neurite outgrowth, neuritogenesis, which precedes the major growth phase, myelination. There is no information about differential changes during gestation to individual brain phospholipid molecular species which contain 22:6n-3. Such details of brain development would be concealed by total fatty acid analysis of isolated phospholipid classes. We have detailed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species compositions in developing fetal guinea pig brain. Total brain PC concentration increased substantially between 40 and 68 (term) d of gestation, corresponding to myelination, while PE increased in a biphasic manner between 25-35 d, which was coincident with onset of neuritogenesis, and 40-68 d. Fetal brain development was accompanied by complex changes in the concentration of individual phospholipid molecular species. During early gestation (25-40 d) 22:6n-3 was enriched in both PC and PE sn-1 16:0 molecular species. However, between 40 d and term there was no further increase in brain PC 22:6n-3 content, while brain PE was significantly enriched in both PE18:1/22:6 and PE18:0/22:6. We hypothesize that accumulation of 22:6n-3 into sn-1 18:1 and 18:0 species represents establishment of a 22:6n-3-containing membrane PE pool which may be turned over more slowly than sn-1 16:0 species. Identification of specific changes in membrane phospholipids which are associated with defined events in brain development may provide a basis for assigning functional roles to individual molecular species.

Limits and Possibilities of Total Fatty Acid Analysis for Classification and Identification of Bacillus Species

A numerical study of fatty acid patterns of 313 Bacillus strains was done based on standardized cultural and chemical techniques. All strains were characterized by the predominance of branched fatty acids (mainly il5:0, al5:0, often i17 :0, a17 :0, rarely i13 :0, i14 :0, i16 :0) ranging from 40% to more than 90% of total fatty acids. The ratios and proportions of the major fatty acids were found to be largely responsible for grouping strains into seven clusters (I to VII) by the UPGMA method using the correlation coefficient (S C ). Two main groups of Bacillus species can be defined on the basis of the ratio of the quantitatively predominant fatty acids i15:0 and a15:0. The species B. amyloliquefaciens , B. firmus , B. firmus-lentus intermediates, B. lentus, ‘B. freudenreichii’, B. laterosporus, B. licheniformis, B. megaterium, B. pasteurii, B. subtilis, B. alvei, B. circulans, B. coagulans, B. globisporus, B. lautus, B. macerans, B. pabuli, B. pantothenticus, B. polymyxa, B. psychrophilus and some strains of B. sphaericus (grouped into clusters I, II, and VII) are characterized by a ratio of i15 :0/a15 :0 < 2. The species B. thermoglucosidasius , B. badius , B. pumilus, the majority of B. sphaericus strains , B. stearothermophilus , B. cereus and B. thuringiensis grouped into clusters III to VI and exhibited a ratio of i15 :0/al5 :0 > 2. Significant quantitative variations were observed for particular fatty acids in different strains belonging to the species B. firmus , B. firmus-lentus intermediates, B. macquariensis , B. megaterium , B. subtilis , B. circulans , B. coagulans , B. macerans , and B. cereus . The species could be clustered into subgroups at a similarity level of 97.5% (S c ). With fatty acid analysis, Bacillus strains can be assigned to species groups, however, the enormous heterogeneity of fatty acid profiles within several species reflects the unsatisfactory taxonomic situation within the genus, which can be improved by further DNA-DNA hybridization studies.

Differentiation between fermenting and spoilage yeasts in wine by total free fatty acid analysis

AbstractDifferentiation between fermenting and spoilage yeasts in wine was estimated by cellular fatty acid profiles. Forty‐two strains of yeasts representing 17 genera were grown on a defined liquid medium for 48 h at 25°C in a rotary shaker. After saponification of yeast cells, free fatty acid extracts were analysed by gas chromatography. Multivariate analysis was performed by Principal Components Analysis (PCA) to define clusters of fatty acids and yeasts.Strains were characterised especially by long‐chain fatty acids, palmitic (C16) to linolenic (C18:3) acid under aerobic culture. Nevertheless, most of Saccharomyces cerevisiae and also Dekkera bruxellensis (former names D intermedia and Brettanomyces lumbicus) synthesized medium‐chain fatty acids, octanoic (C8) acid to dodecanoic (C12) acid. With this method it was possible to differentiate fermenting grape yeasts such as S cerevisiae from spoilage yeasts on the basis of the absence of linoleic (C18:2) and linolenic (C18:3) acids. However, the method seemed unreliable for the identification of strains, more particularly those of species of S cerevisiae.

Identification and characterization of a soil bacterium with extracellular emulsifying activity

Abstract A gram‐negative bacterial strain (designated UG2) isolated from oil‐contaminated soil was identified as Pseudomonas aeruginosa based on biochemical characterization, total cellular fatty acid analysis, and serotyping. The strain produced high extracellular emulsifying activity when grown on various aromatic and straight chain hydrocarbons such as naphthalene, stearic acid, hexadecane and sunflower oil. The emulsifying activity in crude culture filtrates of UG2 exhibited stability at 80°C. In addition, the presence of the emulsifying agent increased partitioning of 14C‐PCB into the aqueous phase in soil slurries by a factor of 30‐fold over that of the water control. The results suggest that UG2 emulsifying agent may have a potential application in remediation of PCB‐contaminated soil that has also been inoculated with PCB‐degrading organism(s).

Antimicrobial effect of human milk on Bordetella pertussis

It has been demonstrated that human milk, unlike bovine milk, can reduce the viability of Bordetella pertussis. This antibacterial activity was not due to the presence of antibiotics or antibodies in the human milk. Reducing the level of available iron or increasing the concentration of lysozyme in bovine milk did not induce anti-B. pertussis activity. Analysis of total fatty acids revealed that human milk contained significantly more linoleic acid than bovine milk. However, the addition of linoleic acid to bovine milk did not inhibit the growth of B. pertussis.

Method for the Quantitative Analysis of Volatile Free and Total Branched-Chain Fatty Acids in Cheese and Milk Fat

Volatile free fatty acids (<C12) in Parmesan cheese and milk fat were extracted with diethyl ether:hexane, adsorbed on neutral alumina, eluted from alumina with formic acid in diisopropyl ether, and separated from long-chain fatty acids by simultaneous distillation-extraction. Volatile branched-chain fatty acids in total lipid extracts were isolated similarly after alkaline hydrolysis. Butyl esters of isolated fatty acids were prepared, extracted into pentane, washed with methanol:water, and analyzed on a bonded polyethylene glycol (Supelcowax10) capillary column. Esters of fatty acids were identified by gas chromatographymass spectroscopy and were quantified by gas chromatography using 2-ethylnonanoic acid as an internal standard. In addition to the usual even carbon-numbered FFA, 2-methylbutanoic, 3-methylbutanoic, 2ethylbutanoic, pentanoic, 3-methylpentanoic, 4methylpentanoic. 2-ethylhexanoic, 4-methylhexanoic, heptanoic, a methylheptanoic, an ethylheptenoic, 4-ethylheptanoic (or 3-methyloctanoic), 4-methyloctanoic, a methyloctanoic, nonanoic, 4-methylnonanoic, 8-methylnonanoic, 2-ethyldecanoic, and 9-decenoic acids were identified and quantified. Substantial reservoirs of volatile branched-chain. odd carbon-numbered, and unsaturated fatty acids occur in cow's milk fat.

Direct transesterification of all classes of lipids in a one-step reaction.

Conventional techniques for the determination of fatty acid composition of lipids require solvent extraction, purification, hydrolysis, and derivatization procedures that are both lengthy and cumbersome. A 1-hr direct transesterification procedure carried out in methanol-benzene 4:1 with acetyl chloride circumvented all these steps and was applicable for analysis of both simple (triglycerides) and complex lipids (cholesteryl esters, phospholipids, and sphingomyelin). Recoveries (greater than 95%) of standards unaffected by the presence of 5% water and 200 mg of silica suggested that the technique could be used for the quantitative analysis of total fatty acids as well as of fatty acids in classes of lipids separated on silica from biological samples. When compared to the Folch procedure, the technique led to a 20.1% increase in total fatty acids for plasma, 3.9% for feces, 7.4% for bile, and 9.7% for rat liver. We therefore conclude that this one-step direct transesterification procedure is superior to currently used methods, not only because of its simplicity and speed, but also because of its added precision.

Gas chromatographic analysis of total fatty acids extracted from Schinus terebenthifolius berries

Gas chromatographic analysis of total fatty acids extracted from Schinus terebenthifolius berries

Blood and Milk Lipid Responses Induced by Growth Hormone Administration in Lactating Cows

Nine multiparous Holstein cows (average 5.8 yr, 63 to 132 days postpartum) were in a 28-day single reversal experiment in the Beltsville open circuit respiration chambers with two 14-day injection periods (placebo versus bovine growth hormone, 51.5 IU/day). With growth hormone treatment, milk fat percentage increased 25 to 29%, milk fat yield increased 41%, and cows averaged −13.7 Mcal/day energy balance. There were small increases of triglycerides and 1,2-diglycerides, core lipids, and small decreases of cholesterol and phospholipids, the membrane lipids. Fat from growth hormone treatment had 6% less short (6:0, 8:0, 10:0) and medium (12:0, 14:0, 14:1, 15:0, 16:0) chain fatty acids and 6% more long chain 18:1 fatty acids. Plasma of cows treated with growth hormone had an increase of concentrations of free fatty acids, but no shifts were apparent among other lipid classes. Analysis of total plasma fatty acids did not show any net changes, but within individual plasma lipid classes, 18:1 increased and 18:2 decreased. Overall changes of blood and milk lipids are consistent with the concept that adipose tissue reserves were mobilized in response to hormone treatment and that these mobilized lipids were the major carbon source for the 41% increase of milk fat secretion. Increases of de novo synthesis of short and medium chain fatty acids also occurred but much less. Cows treated with growth hormone were in negative energy balance, and the mechanism may differ when cows are in positive energy balance.

A Comparison of Comedonal and Skin Surface Lipids from Hairless Dogs Showing Clinical Signs of Acne

Certain animals in a colony of hairless dogs (Mexican Hairless x Beagle) display the clinical features of acne vulgaris, including both open and "closed" comedones, milia, pustules, and abscesses. In order to compare the canine disease with human acne, lipids from the various types of noninflamed lesions and from the skin surface were analyzed by thin-layer (TLC) and gas-liquid (GLC) chromatography. TLC of total nonhydrolyzed lipid from comedones and milia gave similar patterns, with free sterol, ceramides, and free fatty acids comprising the bulk of the material and sterol esters and wax diesters as minor components. Skin surface samples contained mainly sterol esters and wax diesters, with smaller amounts of free sterol and only trace amounts of ceramides. GLC analysis of total hydrolyzed fatty acids from comedones showed similar patterns for the various lesions types, with 22 to 26 carbon acids comprising 55-65% of the total; these acids comprised only 19% of the total hydrolyzed skin surface samples. Analyses by argentation TLC and GLC/mass spectrometry showed the lesional lipids contained significant amounts of alpha-hydroxy-16:0, 16:1, 18:1 and 18:2, but only trace amounts of these appeared in the surface lipid. Fatty acid analysis of individual lipid classes show that the long chain, saturated fatty acids occur predominantly in the free fatty acid and polar lipid fractions and not in the wax diester of sterol ester fractions. Since wax diesters and sterol esters are products of sebaceous gland lipid synthesis, whereas free sterols, fatty acids, and ceramides are characteristic epidermal lipids, the data indicate that the lipids obtained from the acne lesions of dogs are primarily epidermal in origin and that sebaceous gland contribution is minimal in plugged follicles.

Fractionation of fish oil fatty acid methyl esters by means of argentation and reversed-phase high-performance liquid chromatography, and its utility in total fatty acid analysis

The utility of reversed-phase and argentation high-performance liquid chromatography (HPLC) as pre-fractionation methods in fatty acid analysis is discussed. Both HPLC modes were applied to cod liver oil fatty acid methyl esters. Apart from positional isomers, the fractions obtained by reversed-phase HPLC were analysed by gas-liquid chromatography and appear to be free of the usually occurring “critical pairs”. The mechanism of retention of the fatty acid methyl esters on low-loaded silver nitrate-impregnated silicas is discussed. It is shown that argentation HPLC is a rapid semi-preparative pre-fractionation method for highly unsaturated fatty esters with 3–6 double bonds.

The Chloroplast Envelope ofPhaseolus vulgarisL.

An isolation procedure for the chioroplast envelope of Phaseolus vulgaris L. is described. A carotenoid-enriched membrane fraction was obtained from purified chioroplasts using hypotonic shock followed by sucrose density gradient centrifugation. Chlorophyll was almost absent from the isolated membranes. Total lipid, fatty acid, and protein analyses of the fraction suggested that it possessed properties closely analogous to some of those previously reported for chloroplast envelopes of other species.

Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF(3)-CH(3)OH, BCl(3)-CH(3)OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described.

Influence of dietary fish meal on egg fatty acid composition.

AbstractChanges in the fatty acid composition of egg‐yolk fat of hens fed diets with increasing fish meal content are studied by total fatty acid analysis. The fatty acid composition of the major lipid fractions in egg‐yolk fat after feeding a high‐level fish meal diet was determined by a combination of thin layer chromatography (t.l.c.) and gas‐liquid chromatography (g.l.c.) The changes produced show a fatty acid pattern similar to those of the diets themselves. Long‐chain polyunsaturated fatty acids are deposited preferentially in the phospholipids, reaching the highest concentration in cephalin and lecithin.

The Limitations of Whole Tissue Analysis to Define Linolenic Acid Deficiency

It has been claimed on the basis of a deficient diet and on whole head, liver and heart analysis, that linolenic acid is not essential to the rat (J. Nutr. 101: 937, 1971). The present studies show that the methodology was inadequate to support this conclusion. Analytical data are presented which illus trate the principle that whole tissue analysis need not define the content of lino lenic acid metabolites in cell membrane phosphoglycerides. In particular, rat brain ethanolamine phosphoglycerides (EPG) contained 22% of their fatty acids as C22:6,n-3 but the contribution of brain EPG to the total head lip id was only 1.5%. It was estimated that analysis of total head fatty acids could not be used as evidence of deficiency of linolenate metabolites in brain EPG. J. Nutr. 102: 1315-1322, 1972.