Studies on Moss Spores. IV. Mass Spectrometric Identification of Saturated and Monoenoic Long Chain Fatty Acids in the Triglyceride Fraction of Polytrichum commune Spores

The saturated long chain fatty acid methyl esters of the triglyceride fraction of Polytrichum commune spores were separated by silver nitrate TLC and identified by a combination of gas chromatographic-mass spectrometric technique. The saturated fatty acid methyl esters were straight-chained, and even-numbered with carbon numbers ranging from 12 to 26 or odd-numbered with carbon numbers ranging from 13 to 25. The major components of the fraction containing saturated fatty acid methyl esters were methyl palmitate and methyl stearate. The fatty acid methyl esters of the monoenoic fraction isolated by silver nitrate TLC were converted to TMSO derivates which were analysed by gas chromatography-mass spectrometry. The analysis gave evidence of positional isomers. The fraction contained the following straight chain monoenoic fatty acid methyl ester isomers: methyl 7-cis-hexadecenoate, methyl 9-cis-hexadecenoate, methyl 9-cis-heptadecenoate, methyl 9-cis-octadecenoate, methyl 11-cis-octadecenoate, and methyl 11-cis-eicosenoate. The major components were methyl 9-cis-octadecenoate and methyl 7-cis-hexadecenoate.     

MODULATION OF FATTY ACIDS IN THE MEMBRANES OF ANACYSTIS NIDULANS (CYANOBACTERIA): INCORPORATION OF ODD-NUMBERED CARBON FATTY ACIDS1

Anacystis nidulans Richt., a unicellular cyanobacterium, can incorporate exogenously supplied fatty acids, including odd-numbered carbon fatty acid (OFAs), into the acylglycerols of cell membranes. Data are presented for the uptake of undecanoic acid (11:0) into cells of A. nidulans, the subsequent elongation up to C17, and incorporation of OFA into the four major membrane acylglycerols. The incorporation of OFAs was followed by desaturation of part of the saturated fatty acid to monoenoic fatty acid. Positional analyses of the double bonds of these manoenoic fatty acids suggest that there is one desaturase that inserts a Δ⁹ bond in both odd- and even-numbered fatty acids of varying chain length. Our data also suggest that there is no positional specificity for chain length on the glycerol backbone by the acyltransferases.     

The use of clustering techniques in the elucidation or confirmation of metabolic pathways. Application to the branched-chain fatty acids present in the milk fat of lactating goats.

The aim of this paper is 2-fold. (1) To propose the use of a group of mathematical techniques, called clustering, in the elucidation of complex metabolic relationships. (2) To apply clustering for the identification of related groups of saturated fatty acids having a common metabolic pathway for their biosynthesis in the milk fat of lactating goats. In this way, four groups of branched-chain fatty acids and two groups of straight-chain fatty acids are identified; the odd-numbered iso-, the even-numbered iso-, the anteiso-acids and the branched-chain fatty acids with methyl substitution in the chain, the odd-numbered straight-chain and the even-numbered straight-chain fatty acids. The long-chain fatty acids are not part of any group. The different metabolic pathways for their biosynthesis are discussed. From the results, it is concluded that clustering is indeed a potentially useful tool in the study of complex metabolic relationships.     

Feeding stimulants eliciting the probing behavior for Peregrinator blannulipes Montrouzier et Signore (Hemiptera: Ruduviidae) from Tribolium confusum (Jacquelin du Val)

Four fatty acid methyl esters identified in the solvent extract of Tribolium confusum (Jacquelin du Val) larvae as kairomones were individually and collectively tested for probing behavior of Peregrinator biannulipes Montrouzier et Signoret. All identified fatty acid methyl eaters, methyl palmitate, methyl linolate, methyl oleate and methyl stearate, exhibited characterisitic kairomonal probing behavior of P. biannulipe toward the lure. These fatty acid methyl ester were active at 0.2 microg/lure but a synergistic effect was not observed among them. Commercially available C8-C14 even-numbered fatty acid methyl esters that were not detected in the extract of T. confusum larvae also elicited a probing behavior but their activities were weaker than those of four fatty acid methyl ester (C16:0, C18:0, C18:1 and C18:2) identified in the extract. On the other hand, C17 and C19 odd-numbered fatty acid methyl esters did not show any activity at all.     

Novel wax esters and hydrocarbons in the cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus

The cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus, were found to contain minor amounts of novel wax esters, in addition to the major components, hydrocarbons. The wax esters ranged in carbon number from C19 to C31 and consisted of esters of both odd- and even-numbered alcohols and acids. Each wax ester with a given carbon number eluted at several different retention times indicating possible methyl branching in either the fatty acid or alcohol moiety, or in both moieties. Each eluting peak of wax esters consisted of a mixture of wax esters of the same carbon number in which the fatty acid moiety ranged from C8 to C18, and the alcohol moiety ranged from C8 to C17. Some wax esters were largely found on the head indicating they may be of a glandular origin. The hydrocarbons consisted of: n-alkanes, C23 to C33; odd-numbered n-alkenes, C27 to C35; and the major components, methyl-branched alkanes, C26 to over C49. Notable components of the methyl-branched alkanes were 2-methyltriacontane, and the novel trimethylalkanes with a single methylene between the first and second branch points, 13,15,19-trimethylhentriacontane and 13,15,21-trimethyltritriacontane.     

The effect of sodium propionate and sodium caprylate on the fatty acid content of Trichophyton rubrum

Summary From a metabolic point of view, sodium propionate and sodium caprylate in sub-fungistatic concentrations, manifest some interesting effects upon the metabolism, fatty acid synthesis, and pigmentogenesis ofTrichophyton rubrum in its earlier growth phases.Propionate at the higher concentration and caprylate inhibit growth as measured by mycelial dry weight.The higher concentration of propionate suppressed pigment production but a yellow pigment, apparently differing from others previously described, was observed consistently in cultures with the lower concentration of the salt.Caprylate in the concentration tested resulted in a culture in which fatty acid concentration was significantly decreased, however, there appears to be no correlation between fatty acid content depression and mycelial dry weight.Data on fatty acid composition suggest that propionate, an odd-numbered carbon chain fatty acid, diverts synthesis of even-numbered carbon chain fatty acids to the odd-numbered ones.     

Availability of Energy in Esters of Aliphatic Acids and Alcohols by Growing Chicks

Biological availability of 106 esters of alcohols and aliphatic mono-, di- and tri-carboxylic acids and diethylene glycol succinate was compared by the mini-test with chicks. Chicks can utilize methyl esters of saturated fatty acids of carbon chain from 10 to 14, ethyl esters of those from 9 to 12, propyl caprate, n-butyl esters of those from 8 to 12, n-amyl esters of those from 6 to 12, n-hexyl n-butyrate and i-vaterate, and n-octyl and n-decyl acetates. Only 3 dicarboxylates, i.e. di-octyl and di-lauryl succinates and di-methyl cis-cyclopropane-l,2-dicarboxylate, were available among the dicarboxylates tested. Availability of ethyl esters of succinic, fumaric and citric, acid was unexpectedly low.     

Incorporation of Specific Fatty Acid Precursors During Spore Germination and Outgrowth in Bacillus thuringiensis

The selective incorporation of precursors specific for individual fatty acids in germinating and outgrowing spores of Bacillus thuringiensis is described. The specific precursors utilized were [¹⁴C]butyrate, -isobutyrate, -valerate, and -isovalerate, which were incorporated into even-numbered normal-chain isomers, even-numbered iso-isomers, odd-numbered normal-chain acids, and odd-numbered isohomologs, respectively. This preferential incorporation by B. thuringiensis allows the terminal carbons of specific normal and branched-chain fatty acids, contained within the cytoplasmic membrane, to be labeled with ¹⁴C and, potentially, ¹³C.     

Fatty acid ethyl esters in the liverwort Conocephalum conicum

A series of fatty acid ethyl esters ranging from C₁₄ to C₂₄ was isolated from a hexane extract of the liverwort Conocephalum conicum, these esters accounted for 77% of the extract. The ethyl esters consisting of even-numbered fatty acids were predominant and ethyl palmitate was the major constituent.     

Composition of suberin-associated waxes from the subterranean storage organs of seven plants

The waxes associated with the suberin in the periderm of the underground storage organs of parsnip (Pastinaca sativa L.), carrot (Daucus carota L.), rutabaga (Brassica napobrassica Mill.), turnip (Brassica rapa L.), red beet (Beta vulgaris L.), sweet potato (Ipomoea batatas L.) and potato (Solanum tuberosum L.) were isolated, fractionated into hydrocarbon, wax ester, free fatty alcohol and free fatty acid fractions, and analyzed by combined gas chromatography and mass spectrometry. The amount of wax extracted from the periderm of the storage organs ranged from 2 to 32 μg/cm². The hydrocarbons from the suberin layer have a broader chain-length distribution, a predominance of shorter carbon chains, and a higher proportion of even-numbered carbon chains than the leaf alkanes from the same plants. The major components of the free and esterified fatty alcohols and fatty acids have an even number of carbon atoms, and are similar in chain-length distribution to their counterparts found covalently attached to the suberin polymers; however, these suberin components are shorter in chain length than their cuticular analogues from the leaves. Also extracted from the storage organs were polar components which included fatty alcohols and fatty acids in a conjugated form, and ω-hydroxy acids and dicarboxylic acids. Evidence is presented that removal of the wax from the periderm of whole storage organs results in a decrease in diffusion resistance to moisture. Scientific Paper No. 5516, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164, USA     

Waxmonoester fermentation in Euglena gracilis T. Factors favouring the synthesis of odd-numbered fatty acids and alcohols

Waxmonoester fermentation at the expense of endogenous paramylon was followed in the dark in autotrophically grown Euglena gracilis. With reduced oxygen tension and decreasing O₂-consumption rates the proportion of odd-numbered fatty acids and alcohols increased up to a molar ratio of nearly 1:1 under strictly anaerobic conditions. Labelled ¹⁴CO₂, succinate and propionate were incorporated into odd-numbered fatty acids and alcohols 11 to 33 times faster than in even-numbered chains. The electron-flow inhibitor rotenone diminished waxester formation in total, but especially CO₂ fixation and the synthesis of odd-numbered chains, without impeding anaerobic carbohydrate breakdown. These findings are indicative for propionyl-CoA as an intermediate in the synthesis of odd-numbered chains. Its probable synthesis in the methylmalonyl-CoA pathway is discussed with regard to energetics.Abbreviation CCCP carbonylcyanide m-chlorophenyl hydrazone     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

Mycolic acids from Rhodococcus, Gordonia, and Dietzia

The mycolic acids from 11 species of Rhodococcus, seven species of Gordonia, and one species of Dietzia were analyzed using capillary gas chromatography and mass spectrometry (GLC/MS). All strains tested in this study were divided into three groups according to the degree of double bonds and the average carbon number (Av.Nc.) of their mycolic acids. The genus Gordonia belongs to the first group possessing an Av.Nc. in the upper 50s and 60s with 0 to 5 double bonds. Some Rhodococcus species possessed Av.Nc. in the 40s with a variety of distributions of polyunsaturated fatty acids from 0 to 4. The rest of the Rhodococcus species and the genus Dietzia possessed Av.Nc. in the 30s with saturated fatty acids. We previously reported on Nocardia strains whose Av.Nc. were in the 50s. Considering the identification of mycolic acid-containing Actinomycetales at the generic level, the Av.Nc. proved to be useful as a means of differentiating the genera Rhodococcus, Gordonia and Nocardia. The genus Dietzia was found to have its own characteristic constitution of mycolic acid molecular species. The mycolic acids from D. maris 58001T were characterized by an almost equal amount of constituents of even- and odd-numbered carbon chains, whereas the major components of mycolic acids in all other strains had even-numbered carbon chains. Another characteristic of Dietzia was some even-numbered mycolic acids which contained odd-numbered straight chains with odd-numbered alpha-branches. These characteristics indicated that Dietzia might possess a novel fatty acid biosynthesis system.     

Distribution of C22-, C24- and C26-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄-α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary The yeast Candida maltosa precultivated on liquid n-alkanes utilized different solid n-alkanes (especially C₂₀–C₂₅) in the presence of pristane as an organic phase with rates comparable to, or somewhat larger than, those of liquid n-alkanes. Analysis of cellular fatty acids indicated an assimilation of solid n-alkanes via monoterminal oxidation. The resulting fatty acids with substrate chain length were chain-shortened by C₂ units down to an optimal range of chain length from C₁₆ to C₁₈ and incorporated into cellular, lipids directly or after desaturation. The intermediates of chain-shortening with numbers of carbon atoms higher than C₁₈, as well as the unusually long-chain fatty acids of substrate chain length, were detected in trace amounts only. Even-carbon-numbered and odd-numbered fatty acids predominated in experiments with evenchain and odd-chain n-alkanes, respectively. Studies with cerulenin indicated that de novo synthesis of fatty acids was negligible. Oxidation of solid n-alkanes by the yeast C. maltosa yielded fatty acid patterns similar to those of cells grown on liquid n-alkanes.     

Methylmalonic and propionic acidemias: lipid profiles of normal and affected human skin fibroblasts incubated with [1-14C]propionate

Normal human skin fibroblasts and those from methylmalonic acidemia and propionic acidemia patients were grown in culture. Following incubation with [1-14C]propionate, the major lipid classes in the cells were separated by thin layer chromatography and isolated fractions analyzed by radio gas chromatography for the presence of odd-numbered long-chain fatty acids; the pattern of even-numbered long-chain fatty acids was obtained also. Normal fibroblasts incorporated a small percentage of propionate into odd-numbered fatty acids which were present in all lipids studied. The abnormal cells incorporated a larger amount while maintaining the characteristic ratios of odd-numbered fatty acids found in the normal line. Most of the radioactivity was associated with phospholipids which are the predominant constituents of cell membranes. A characteristic C15/C17 ratio was found for different phospholipids and the triglyceride fraction; pentadecanoic acid was the principal odd-numbered fatty acid utilized in the assembly of complex lipids. Compared to even-numbered long-chain fatty acids the absolute amount of odd-numbered fatty acids was low (1-2%), even in affected cells. An unusual polar lipid fraction was isolated in the course of the study. In the normal cell it contained several unlabeled eicosanoids which were missing from the same fraction of both affected cell lines.     

Microautoradiographic studies on host-parasite interactions II. The exchange of 3H-lysine between Uromyces phaseoli and Phaseolus vulgaris

A new species of anaerobic bacterium that degrades the even-numbered carbon fatty acids, butyrate, caproate and caprylate, to acetate and H₂ and the odd-numbered carbon fatty acids, valerate and heptanoate, to acetate, propionate and H₂ was obtained in coculture with either an H₂-utilizing methanogen or H₂-utilizing desulfovibrio. The organism could be grown only in syntrophic association with the H₂-utilizer and no other energy sources or combination of electron donor and acceptors were utilized. It was a Gram-negative helical rod with 2 to 8 flagella, about 20 nm in diameter, inserted in a linear fashion about 130 nm or more apart along the concave side of the cell. It grew with a generation time of 84 h in co-culture with Methanospirillum hungatii and was present in numbers of at least 4.5×10^-6 per g of anaerobic digestor sludge.     

Formation of Volatile Esters in Strawberries

Aliphatic alcohols including methyl, ethyl, isopropyl, npropyl, isobutyl, nbutyl, isoamyl, namyl and hexyl alcohol were converted to their acetate, propionate, nbutyrate, isovalerate and caproate esters during the incubation with strawberry fruit tissue. Formate, isobutyrate and n-valerate esters were formed when alcohols were incubated together with these fatty acids and strawberry.

Seventy esters were formed from various combinations of alcohols and acids by means of incubation with strawberry.

No ester formation was observed when strawberry was homogenized.     

Microbial Incorporation of Fatty Acids Derived From n-Alkanes Into Glycerides and Waxes

When n-alkanes with 13 to 20 carbon atoms were fed to a Nocardia closely related to N. salmonicolor, the produced cellular triglycerides and aliphatic waxes invariably contained fatty acids with an even or an odd number of carbon atoms subject to this feature of the n-alkane substrate. Beta-oxidation and C₂ addition are both operative, as evidenced by the spectra of fatty acids incorporated into the cellular lipid components. There is no distinction in the rate of microbial incorporation of the odd-or even-numbered carbon chains. The fatty acids are apparently directly derived from the long chain n-alkanes, rather than synthesized via the classic C₂-condensation route. The alcohol component of waxes produced by the Nocardia is invariably of the same chain length as the n-alkane substrate.     

Fatty acid composition of Simonsiella strains

Gas-liquid chromatography of methyl esters of bound fatty acids extracted from the cells of 48 Simonsiella strains showed that these aerobic, gliding, multicellular-filamentous bacteria have fatty acid profiles of the pattern considered typical of Gramnegative eubacteria. All strains contained predominantly tetradecanoic acid (29.5%), 9-hexadecenoic acid (22.2%), an unidentified acid with an equivalent chain length of approximately 20 carbon atoms (15.8%), and dodecanoic acid (11.4%).Discriminant analysis of the mean relative percentages of 12 fatty acids correctly assigned 94% of the strains to groups based on their source of origin (i.e., the oral cavities of sheep, cat, human or dog); the relative amounts of only 3 of the fatty acids (9-octadecenoic acid, hexadecanoic acid, and tetradecanoic acid) provided most of this discrimination.