The Structure of a Novel Neutral Lipid A from the Lipopolysaccharide of Bradyrhizobium elkanii Containing Three Mannose Units in the Backbone

The chemical structure of the lipid A of the lipopolysaccharide (LPS) from Bradyrhizobium elkanii USDA 76 (a member of the group of slow‐growing rhizobia) has been established. It differed considerably from lipids A of other Gram‐negative bacteria, in that it completely lacks negatively charged groups (phosphate or uronic acid residues); the glucosamine (GlcpN) disaccharide backbone is replaced by one consisting of 2,3‐dideoxy‐2,3‐diamino‐D ‐glucopyranose (GlcpN3N) and it contains two long‐chain fatty acids, which is unusual among rhizobia. The GlcpN3N disaccharide was further substituted by three D ‐mannopyranose (D ‐Manp) residues, together forming a pentasaccharide. To establish the structural details of this molecule, 1D and 2D NMR spectroscopy, chemical composition analyses and high‐resolution mass spectrometry methods (electrospray ionisation Fourier‐transform ion cyclotron resonance mass spectrometry (ESI FT‐ICR MS) and tandem mass spectrometry (MS/MS)) were applied. By using 1D and 2D NMR spectroscopy experiments, it was confirmed that one D ‐Manp was linked to C‐1 of the reducing GlcpN3N and an α‐(1→6)‐linked D ‐Manp disaccharide was located at C‐4′ of the non‐reducing GlcpN3N (α‐linkage). Fatty acid analysis identified 12:0(3‐OH) and 14:0(3‐OH), which were amide‐linked to GlcpN3N. Other lipid A constituents were long (ω‐1)‐hydroxylated fatty acids with 26–33 carbon atoms, as well as their oxo forms (28:0(27‐oxo) and 30:0(29‐oxo)). The 28:0(27‐OH) was the most abundant acyl residue. As confirmed by high‐resolution mass spectrometry techniques, these long‐chain fatty acids created two acyloxyacyl residues with the 3‐hydroxy fatty acids. Thus, lipid A from B. elkanii comprised six acyl residues. It was also shown that one of the acyloxyacyl residues could be further acylated by 3‐hydroxybutyric acid (linked to the (ω‐1)‐hydroxy group).     

The structure of lipid A of the lipopolysaccharide from Burkholderia caryophylli with a 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residue exclusively in glycosidic linkage

From the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli, the complete structure of lipid A has been characterized. For the first time, a 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residue was proven to be exclusively linked to the reducing end of lipid A from a wild-type LPS. The LPSs of B. caryophylli were degraded by mild acetate buffer hydrolysis at pH 4.4. The obtained lipid A was analyzed as such, and also after de-O-acylation or dephosphorylation. The structure of lipid A was identified mainly by means of matrix-assisted laser desorption/ionisation mass spectrometry, and by various 1D and 2D (1)H and (13)C NMR spectroscopic measurements.     

Structural variability of endotoxins from R-type isogenic mutants of Shigella sonnei

The structural variations in the rough-type endotoxins [lipopolysaccharides (LPSs)] of Shigella sonnei mutant strains (S. sonnei phase II-4303, R41, 562H and 4350) were investigated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS. A series of S. sonnei mutants had previously been the subject of analytical studies on the biosynthesis of heptose components in the core oligosaccharide region of LPSs. This study gives a complete overview on the structures of the full core and lipid A of S. sonnei mutant strains by MS. We found that the LPSs of the isogenic rough mutants were formed in a step-like manner containing 0:1:2:3 heptose in the deep core region of 4350, 562H, R41 and 4303, respectively, and the longest LPS from the mutant S. sonnei 4303 contained also five hexoses. The structural variations in the lipid A moiety and in the oligosaccharide part of the intact LPS were followed by MALDI-TOF-MS/MS. For the dissolution and the ionization of the samples, 2,5-dihydroxybenzoic acid in citric acid solution was applied as matrix. The detailed evaluation of the mass spectra indicates heterogeneity in the lipid part due to the differences in the phosphate and fatty acid composition.     

Structural characterization of lipopeptide biomarkers isolated from Bacillus globigii

Spectra obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of Bacillus globigii (Bacillus subtilis niger) spores, vegetative cells and the culture supernatant show a cluster of biomarkers centered at a molecular mass of 1478 Da. Three biomarkers were isolated from the cell-free culture supernatant by solid-phase extraction and reversed-phase high-performance liquid chromatography, and characterized using various kinds of mass spectrometry. A Fourier transform mass spectrometer with a MALDI source was used to determine the monoisotopic protonated masses at 1463.8, 1477.8, and 1505.8 Da in order of elution. The mass differences of 14 and 28 Da suggest that they are homologous molecules. Alkaline hydrolysis of each species showed that it contained a lactone linkage. Strong acid hydrolysis released a fatty acid from an amide bond, consistent with a lipopeptide. A quadrupole time-of-flight instrument with a nanospray source was used to sequence the hydrolyzed forms of the three biomarkers. The cyclic lipopeptides were found to have amino acid sequences identical with those in fengycins and plipastatins, antimicrobial compounds with phospholipase inhibitor activity, previously identified in related species of Bacillus subtilis and Bacillus cereus.     

Changes of phospholipid composition within the dystrophic muscle by matrix-assisted laser desorption/ionization mass spectrometry and mass spectrometry imaging

Duchenne muscular dystrophy (DMD) is a neuromuscular disease linked to the lack of the dystrophin, a submembrane protein, leading to muscle weakness and associated with a defect of the lipid metabolism. A study of the fatty acid composition of glycerophosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) and tandem mass spectrometry (MS/MS) enabled us to characterize a change of the lipid composition of dystrophic cells at the time of the differentiation. This modification has been used as a marker to identify with profiling and imaging MALDI-ToF MS regenerating areas in sections of an mdx mouse leg muscle. It is the first time that such a slight change in fatty acid composition has been observed directly on tissue slices using mass spectrometry. This approach will be useful in monitoring the treatment of muscular regeneration.     

252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.     

New conditions for matrix-assisted laser desorption/ionization mass spectrometry of native bacterial R-type lipopolysaccharides

A new sample preparation method for matrix-assisted laser desorption/ionization (MALDI) analysis of native rough-type lipopolysaccharides (R-type LPSs) is presented. In our MALDI mass spectra, besides the [M--H](-) ions, abundant ions originating from the cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) unit and the lipid A moiety are always present, giving important pieces of information about the structure of the molecules analyzed. Remarkably, in most cases, the comparison of the MALDI mass spectra of the intact R-type LPS with the O-deacylated one allowed us to obtain the structure of the lipid A moiety.     

A matrix-assisted laser desorption/ionization mass spectrometry approach to the lipid A from Mesorhizobium loti

The isolation, purification and analysis of the lipid A obtained from Mesorhizobium loti Ayac 1 BII strain is presented. Analysis of the carbohydrate moiety after acid hydrolysis by high-pH anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD) showed the presence of glucosamine and galacturonic acid as the only sugar components. Gas chromatographic (GC) and GC/mass spectrometric (MS) analysis of the fatty acids revealed the presence of 3-OH-C12:0; 3-OH-C13:0; 3-OH-C20:0 and 27-OH-C28:0 among the major hydroxylated species. In addition, C16:0, C17:0, C18:0 and C 20:0 were shown as main saturated fatty acids. Different polyacylated species were evidenced by thin layer chromatography of lipid A, allowing the purification of two fractions. Ultraviolet matrix-assisted laser desorption/ionization time-of-flight (UV-MALDI-TOF) MS analysis with different matrices, in the positive- and negative-ion mode, was performed. The fast moving component revealed the presence of hexa-acylated species, varying in the fatty acid composition. Species containing three 3-OH fatty acids and a 27-OH-C28:0 fatty acid were observed. Individual ions within this family differ by +/-14 mass units. The slow moving component was enriched mainly in penta-acylated species. Among them, three subgroups were detected: the major one compatible with the sugar core bearing two 3-OH 20:0 fatty acids, a 3-OH 13:0 or a 3-OH 12:0 fatty acid, a 27-OH 28:0 fatty acid and one saturated fatty acid. Each signal differs in a C18:0 acyl unit from the corresponding hexa-acylated family. On the other hand, a subgroup bearing one 3-OH 20:0 fatty acid, one 27-OH 28:0 fatty acid and two non-polar fatty acids was shown. A minor subgroup compatible with structures containing two hydroxylated and three non-polar fatty acids was also detected. The results obtained showed that nor-harmane was an excellent matrix for charged lipid A structural studies in both, positive and negative ion modes.     

Improved fatty acid detection in micro-algae and aquatic meiofauna species using a direct thermal desorption interface combined with comprehensive gas chromatography-time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography (GC x GC) with time-of-flight mass spectrometry detection is used to profile the fatty acid composition of whole/intact aquatic microorganisms such as the common fresh water green algae Scenedesmus acutus and the filamentous cyanobacterium Limnothrix sp. strain MRI without any sample preparation steps. It is shown that the technique can be useful in the identification of lipid markers in food-web as well as environmental studies. For instance, new mono- and diunsaturated fatty acids were found in the C(16) and C(18) regions of the green algae S. acutus and the filamentous cyanobacterium Limnothrix sp. strain MRI samples. These fatty acids have not, to our knowledge, been detected in the conventional one-dimensional (1D) GC analysis of these species due to either co-elution and/or their presence in low amounts in the sample matrix. In GC x GC, all congeners of the fatty acids in these microorganisms could be detected and identified due to the increased analyte detectability and ordered structures in the two-dimensional separation space. The combination of direct thermal desorption (DTD)-GC x GC-time-of-flight mass spectrometry (ToF-MS) promises to be an excellent tool for a more accurate profiling of biological samples and can therefore be very useful in lipid biomarker research as well as food-web and ecological studies.     

气相色谱/质谱法分析孔石莼中的脂肪酸

建立了孔石莼脂肪酸的气相色谱/质谱(GC/MS)测定方法。使用Folch法提取了孔石莼中的总脂,经过2 mol/L HCl-甲醇溶液的甲酯化处理后,采用GC/MS法对其脂肪酸组成进行了分离分析,同时结合有机质谱学规律,分别对饱和脂肪酸甲酯、单不饱和脂肪酸甲酯和多不饱和脂肪酸甲酯的裂解规律和质谱特征进行了分析归纳。通过质谱数据库检索和标准品对照,鉴定出孔石莼中的24种脂肪酸,其中9,12,15-十八碳三烯酸、4,7,10,13-十六碳四烯酸和6,9,2,15-十八碳四烯酸3种主要多不饱和脂肪酸占总脂肪酸含量的45.14%。通过对孔石莼中脂肪酸的分析,表明特征离子在脂肪酸甲酯尤其是多不饱和脂肪酸甲酯的定性方面具有很好的应用价值。     

Structural analysis of lipid A from Escherichia coli O157:H7:K- using thin-layer chromatography and ion-trap mass spectrometry

Rapid separation and structural identification of lipid A from Escherichia coli were performed using thin-layer chromatography (TLC) and mass spectrometry (MS). After the resolved spot of the lipid A had been scraped from TLC plate, the sample was re-extracted from the removed powder with chloroform-methanol (2 : 1, v/v) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) ion-trap MS. For detailed structural characterization, multiple-stage mass analysis (MS(4)) of the major species in ESI-MS/MS provided important information about the series of fragment ions. The dominant fragment ions in each MS stage were produced from the loss of fatty acyl groups mainly driven by charge-remote processes, and this information about the fragment ions can be used to deduce the composition or the position of the fatty acid substituent in the lipid A. In contrast, MALDI-TOFMS indicated that fragmentation resulted from charge-driven processes. Molecular mass profiling and fragmentation analysis provides essential information for clarifying the detailed structure of the lipid A from E. coli O157:H7:K(-).     

Analysis of amino acid mixtures by plasma desorption mass spectrometry

Plasma desorption mass spectrometry (PDMS) was investigated as a means of analysing mixtures of three, four and five amino acids in both positive- and negative-ion modes. Fifteen mixtures were tested; each mixture contained equimolar amounts of selected amino acids. The PD mass spectra exhibited MH⁺ and [M – H]^? molecular ions for all the aminoacids with different desorption–ionization yields. The spectra were more easily interpreted in the negative- than the positive-ion mode. The desorption order of the amino acids was progressively established by comparing the molecular ion desorption–ionization yields for each mixture. This desorption order was well correlated in both the positive- and negation-ion modes with the acid–base thermodynamic data for the amino acids in the gas phase. This observation gives some insight into the desorption–ionization mechanisms under PDMS conditions.     

Structural heterogeneity and environmentally regulated remodeling of <Emphasis Type="Italic">Francisella tularensis</Emphasis> subspecies <Emphasis Type="Italic">novicida</Emphasis> lipid a characterized by tandem mass spectrometry

The structural characterization of environmentally-regulated lipid A derived from Francisella tularensis subspecies novicida (Fn) U112 is described using negative electrospray ionization with a linear ion trap Fourier transform ion cyclotron resonance (IT-FT-ICR) hybrid mass spectrometer. The results indicate that a unique profile of lipid A molecular structures are synthesized in response to Fn growth at 25 degrees C versus 37 degrees C. Molecular species were found to be tetra-acylated, sharing a conserved glucosamine disaccharide backbone, a galactosamine-1-phosphate linked to the reducing glucosamine, and multiple O- and N-linked fatty acyl groups. Deprotonated molecules were interrogated by MS(n) scanning techniques at both high and nominal mass resolution and were found to be complex heterogeneous mixtures where structures differed based on the positions and identities of the O- and N-linked fatty acyl substituents. For the dominant ion series, which consisted of five peaks, 30 unique lipid A structures were identified. Estimates for the relative abundance of each structure were derived from MS relative abundance ratios and fragment ion ratios from comparable dissociation pathways from MS(2) through MS(4) experiments. The results suggest a remodeling pathway in which the amide linked fatty acid of the reducing glucosamine favors a 3-hydroxyhexadecanoic acid substituent for growth conditions at 25 degrees C versus a 3-hydroxyoctadecanoic acid substituent for growth conditions at 37 degrees C.     

Liquid chromatography-mass spectrometry of hydroxy and non-hydroxy fatty acids as amide derivatives.

A useful method for analyzing fatty acids by liquid chromatography-mass spectrometry with an atmospheric-pressure chemical-ionization interface system has been developed. The sensitivity of six kinds of palmitamide derivatives monitored by a single ion of [M+H]+ was, in decreasing order: N-n-propylamide greater than anilide greater than N,N-diethylamide, amide greater than N,N-diphenylamide greater than N-1-naphthylamide. Individual fatty acids were identified from a mixture of amide derivatives of authentic fatty acids from C16:0 to C30:0 on a mass chromatogram. This method was used to detect both hydroxy and non-hydroxy fatty acids. Many kinds of fatty acid, including hydroxy fatty acids of the rat brain, were detected in a single run.     

Graphene and graphene oxide: two ideal choices for the enrichment and ionization of long-chain fatty acids free from matrix-assisted laser desorption/ionization matrix interference

In this work, graphene (G) and graphene oxide (GO) were utilized to enrich and ionize long-chain fatty acids. All together five long-chain fatty acids were selected as models here, n-dodecanoic acid (C12), n-tetradecanoic acid (C14), n-hexadecanoic acid (C16), n-octadecanoic acid (C18), and n-eicosanoic acid (C20). Due to the large surface area and strong interaction force of G or GO, all the five long-chain fatty models were effectively enriched by G or GO. On the other hand, the excellent electronic, thermal, and mechanical properties enable G and GO to be prefect energy receptacles for laser radiation, which make the ionization steps more effective. Eventually, the promoted G and GO methodology can sensitively detect the five long-chain fatty acid models from real biological samples even at low concentrations. Meanwhile, by adopting our promoted methodology, the detection of long-chain fatty acids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was demonstrated to be simple, sensitive, fast, cost effective and high throughput, which is meaningful as to practical usage.     

Characterization of complex,heterogeneous lipid A samples using HPLC‐MS/MS technique III. Positive‐ion mode tandem mass spectrometry to reveal phosphorylation and acylation patterns of lipid A

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low‐energy collision‐induced dissociation conditions of an electrospray quadrupole time‐of‐flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed‐phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive‐ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter‐ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4′ phosphate group) were simultaneously present in the sample. Continuous series of high‐abundance B‐type and low‐abundance Y‐type inter‐ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative‐ion mode, they were very similar in the positive‐ion mode. The complementary use of positive‐ion and negative‐ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1‐monophosphorylated lipid A species.     

Elucidation of the molecular structure of lipid A isolated from both a rough mutant and a wild strain of Aeromonas salmonicida lipopolysaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry

The chemical structure of lipid A, isolated by mild acid hydrolysis from a rough mutant and a wild strain of Aeromonas salmonicida lipopolysaccharide, was investigated using electrospray ionization quadrupole time-of-flight (QqToF) hybrid tandem mass spectrometry and showed a great degree of microheterogeneity. The chemical structure of the main constituent of this heterogeneous mixture was identified as a beta-D-(1 --> 6) linked D-glucosamine disaccharide substituted by two phosphate groups, one being bound to the non-reducing end at position O-4' and the other to the position O-1 of the reducing end of the D-glucosamine disaccharide. The location of the fatty acids linked to the disaccharide backbone was established by identifying diagnostic ions in the conventional QqToF-MS scan. Low-energy collision tandem mass spectrometry analysis of the selected precursor diagnostic ions confirmed, unambiguously, their proposed molecular structures. We have established that myristyloxylauric (C14:0(3-O(12:0))) acid residues were both N-2' and O-3' linked to the non-reducing end of the D-GlcN residue, and that two 3-hydroxymyristic (C14:0(3-OH)) acid chains acylated the remaining positions of the reducing end. The MS and MS/MS data obtained allowed us to determine the complex molecular structure of lipid A. The QqToF-MS/MS instrument has shown excellent superiority over a conventional quadrupole-hexapole-quadrupole tandem instrument which failed to fragment the selected precursor ion.     

气相色谱-质谱联用辅助鉴定细菌内毒素标准品菌种来源的纯度

为分析内毒素标准品菌种来源的纯度,建立了采用气相色谱-质谱联用法、以N,O-双三甲基硅三氟乙酰胺作为硅烷化试剂对待测物进行衍生、对内毒素标准品所含的3-羟基脂肪酸种类进行检测的方法。色谱柱为DB-5 (60 m×0.25 mm i.d.);载气为氦气,恒压,柱前压206 kPa;进样器温度250 ℃;柱箱初始温度为90 ℃,保持5 min,以5 ℃/min的升温速率升至280 ℃,保持5 min。进样量为1 μL,不分流进样。质谱离子源为电子轰击离子源（EI）,离子源温度为250 ℃,接口温度为280 ℃。通过对内毒素标准品和大肠杆菌、绿脓杆菌、去离子水中3-羟基脂肪酸种类的比较,探讨了通过3-羟基脂肪酸种类来辅助判断内毒素标准品菌种来源的问题。结果显示,来源为大肠杆菌的9000 EU/支内毒素国家标准品中只含有3-羟基十四烷酸;20 EU/支的内毒素工作标准品中除含有3-羟基十四烷酸外,还含有3-羟基十二烷酸,说明其含有非大肠杆菌的细菌。     

Analysis of deprotonated acids with silicon nanoparticle-assisted laser desorption/ ionization mass spectrometry

Chemically modified silicon nanoparticles were applied for the laser desorption/negative ionization of small acids. A series of substituted sulfonic acids and fatty acids was studied. Compared to desorption ionization on porous silicon (DIOS) and other matrix-less laser desorption/ionization techniques, silicon nanoparticle-assisted laser desorption/ionization (SPALDI) mass spectrometry allows for the analysis of acids in the negative ion mode without the observation of multimers or cation adducts. Using SPALDI, detection limits of many acids reached levels down to 50 pmol/μl. SPALDI of fatty acids with unmodified silicon nanoparticles was compared to SPALDI using the fluoroalkyl silylated silicon powder, with the unmodified particles showing better sensitivity for fatty acids, but with more low-mass background due to impurities and surfactants in the untreated silicon powder. The fatty acids exhibited a size-dependent response in both SPALDI and unmodified SPALDI, showing a signal intensity increase with the chain length of the fatty acids (C12-C18), leveling off at chain lengths of C18-C22. The size effect may be due to the crystallization of long chain fatty acids on the silicon. This hypothesis was further explored and supported by SPALDI of several, similar sized, unsaturated fatty acids with various crystallinities. Fatty acids in milk lipids and tick nymph samples were directly detected and their concentration ratios were determined by SPALDI mass spectrometry without complicated and time-consuming purification and esterification required in the traditional analysis of fatty acids by gas chromatography (GC). These results suggest that SPALDI mass spectrometry has the potential application in fast screening for small acids in crude samples with minimal sample preparation.     

Determination of the acyl moieties of the antibiotic complex A40926 and their relation with the membrane lipids of the producer strain.

Structures of the fatty acid residues characterizing the various components of A40926 were determined by gas chromatography/mass spectrometry on the methyl esters obtained by methanolysis of the complex. The results confirm the residues previously assigned to Factor A (n-undecanoic acid) and B (10-methyl-undecanoic acid) and establish the residues of Factor A1 (9-methyl-decanoic acid), B1 (n-dodecanoic acid), RS1 (8-methyl-nonanoic acid), RS2 (n-decanoic acid), and RS3 (n-tridecanoic acid). As the Actinomadura species contain in their mycelia large quantities of C15-C17 fatty acid residues as membrane phospholipids, these mycelia were saponified and the fatty acids obtained were analyzed as above. There is a close correlation between the fatty acid content of A40926 complex and that of the longer homologues in the producer mycelia.