Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C_16:0, C_16:1, C_18:0, C_18:1, C_18:2, C_18:3, C_20:0 and C_20:1. At high concentrations spore germination was inhibited by C_7:0, C_8:0, C_9:0, C_10:0, C_12:0, C_18:2 and C_18:3 and hyphal growth was merely retarded by C_5:0, C_6:0, C_6:2, C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_20:0 and C_20:1. The presence of C_15:0 at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C_16:0 and C_18-20 fatty acids. Low concentrations of C_5:0, C_6:0, C_6:2 and C_7:0 enhanced sporulation. Fatty acids C_5-12 as well as C_18:3, C_20:0 and C_20:1 decreased the ability of fungal colonies to infect G. mellonella while C_16:1 elevated it thus suggesting that C_16:1 may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C_6:0, C_6:2, C_7:0, C_9:0, C_12:0, C_16:1, C_18:2, C_18:3, C_20:0 and C_20:1; other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.     

Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum

Human meibum was targetly analyzed for the presence of intact wax esters (WEs) and related compounds by means of reverse-phase HPLC in combination with ion trap mass spectrometry. The major detected WEs were based on C_18:n (n = 1–4) unsaturated FAs ranking in the following order of abundance: C_18:1>C_18:2>C_18:3>C_18:4. The major fatty alcohols (FAls) found in WE were of saturated nature and varied from C_18:0 to C_28:0. The three most abundant species were C_18:1-FA esters of C_24:0, C_25:0, and C_26:0-FAl. Typically, a major compound based on C_18:1-FA and a saturated FAl was accompanied by a few related compounds based on a C_18:2, C_18:3, and C_18:4-FA. Contrary to previous reports, no epoxy-WEs or epoxy-FAs were detected in fresh and 1-year-old meibum samples. More than 20 (O-acyl)-ω-hydroxy-FAs (OAHFAs) were observed. The main detected OAHFAs were based on very long-chain ω-hydroxy-FA (C_30:1, C_32:1, and C_34:1) acylated through their ω-hydroxyls by a C_18:1-FA. Due to their amphiphilic anionogenic nature, OAHFAs may be responsible for stabilization of the tear film lipid layer by creating an interface between the vast pool of strictly nonpolar lipids of meibum (WEs, cholesteryl esters, etc.) and the aqueous subphase beneath it, a role previously attributed to phospholipids.     

Terpenoid and other extractives of western white pine bark

A detailed chemical analysis of the benzene extract of western white pine bark was conducted. The extract consisted of 13% phlobaphenes, 18% strong acids, 21% polar weak acids, 6.5% fatty acids, 9.5% resin acids, and 32% neutrals. The fatty acids consisted mainly of C_20:0, C_22:0, and C_24:0 acids. The resin acids were identified as: isopimaric, anticopalic, dehydroabietic, sandaracopimaric, abietic, 6,8,11,13-abietatetraen-18-oic and pimaric acids. The neutrals on saponification gave fatty acids, sterols, wax alcohols, nonsaponifiables, and other components. The esterified fatty acids consisted primarily of the C_16:0, C_18:0, C_20:0 and C_24:0 acids. The sterols included major amounts of sitosterol, campesterol, and stigmasterol, and traces of cholesterol. Over 70 individual compounds were isolated and identified from the nonsaponifiables. These included borneol, sesquiterpenes, diterpenes, steroidal ketones, as well as lanostane and serratane triterpenes. The characterization of12 new natural products or natural products isolated for the first time from Pinus species is reported.     

Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection

Positional and geometric isomers of mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms were separated on commercially available reversed-phase columns in gradient systems composed of acetonitrile and water, utilizing photodiode array detection. The biological samples were hydrolyzed with 2 M NaOH for 35–40 min at 85–90°C. After cooling, the hydrolysates were acidified with 4 M HCl and the free fatty acids were extracted with dichloromethane. The organic solvent was removed in a gentle stream of argon. The fatty acids were determined after pre-column derivatization with dibromacetophenone in the presence of triethylamine. The reaction components were mixed and reacted for 2 h at 50°C. Separations of derivatized fatty acids were performed on two C₁₈ columns (Nova Pak C₁₈, 4 μm, 250×4.6 mm, Waters) by binary or ternate gradient programs and UV detection at 254 and 235 nm. The geometric and positional isomers of some unsaturated fatty acids were substantially retained on the C₁₈ columns and were distinct from some saturated fatty acids, endogenous substances in biological samples or background interference. Only slight separation of critical pairs of cis-9 C_18:1/cis-11 C_18:1 and cis-6 C_18:1/trans-11 C_18:1 was obtained. A ternate gradient program can be used for complete fractionation of a mixture of conjugated linoleic acid isomers (CLA) from cis-9, cis-12 and trans-9, trans-12 isomers of C_18:2. The CLA isomers in the effluent were monitored at 235 nm. The CLA isomers were differentiated from saturated and unsaturated fatty acids using a photodiode array detector. The utility of the method was demonstrated by evaluating the fatty acid composition of duodenal digesta, rapeseed and maize oils.     

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C₁₈ reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C₁₈ reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.     

Sensitive fluorimetric determination of gentamicin sulfate in biological matrices using solid-phase extraction, pre-column derivatization with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of gentamicin in bacterial culture medium or plasma with increased sensitivity and improved separation of the C₁ component. Gentamicin was extracted from the biological matrix with high efficiency using carboxypropyl (CBA)-bonded silica. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C₁₈ reversed-phase chromatography allowed the fluorimetric detection of gentamicins C₁, C_1a and C₂. A fourth component, considered to be gentamicin C_2a, was partially resolved from the C₂ peak. Optimal conditions for the extraction and derivatization of gentamicin are described. The detection limit was below 50 μg/l, the assay was linear to 5 mg/1 and showed good reproducibility. It is concluded that pre-column derivatization with FMOC-Cl substantially improves the analysis of gentamicin compared with present methods based on reaction with o-phthaldialdehyde.     

Some variations in the composition of suberin from the cork layers of higher plants

The monomeric composition of the suberins from 16 species of higher plants was determined by chromatographic methods following depolymerization of the isolated extractive-free cork layers with sodium methoxide-methanol. 1-Alkanols (mainly C₁₈C₂₈), alkanoic (mainly C₁₆C₃₀), α,ω-alkanedioic (mainly C₁₆C24), ω-hydroxyalkanoic (mainly C₁₆C₂₁), dihydroxyhexadecanoic (mainly 10,16-dihydroxy- and 16-dihydroxyhexadecanoic), monohydroxyepoxyalkanoic (9,10-epoxy-18-hydroxyoctadecanoic), trihydroxyalkanoic (9,10, 18-trihydroxyoctadecanoic), epoxyalkanedioic (9,10-epoxyoctadecane-1,18-dioic) and dihydroxyalkanedioic (9,10-dihydroxyoctadecane-1 18-dioic) acids were detected in all species. The suberins differed from one another mainly in the relative proportions of these monomer classes and in the homologue content of their 1-alkanol, alkanoic, α,ω-alkanedioic and ω-hydroxyalkanoic acid fractions. C₁₈ epoxy and vic-diol monomers were major components (32–59%) of half of the suberins examined (Quercus robur, Q. ilex, Q. suber, Fagus sylvatica, Castanea sativa, Betula pendula, Acer griseum, Fraxinus excelsior) where as ω-hydroxyalkanoic and α,ω-alkanedioic acids predominated in those that contained smaller quantities of such polar C₁₈ monomers (Acer pseudoplatanus, Ribes nigrum, Euonymus alatus, Populus tremula, Solanum tuberosum, Sambucus nigra, Laburnum anagyroides, Cupressus leylandii). All species, however, contained substantial amounts (14–55 %) of ω-hydroxyalkanoic acids, the most common homologues being 18:1 (9) and 22: 0. The dominant α,ω-alkanedioic acid homologues were 16: 0 and 18: 1 (9) whereas 22: 0, 24: 0 and 26: 0, and 20: 0, 22: 0 and 24: 0 were usually the principal homologues in the 1-alkanol and alkanoic acid fractions, respectively. The most diagnostic feature of the suberins examined was the presence of monomers greater than C₁₈ in chain length; most of the C₁₆ and C₁₈ monomers identified in the suberins also occur in plant cutins emphasizing the close chemical similarity between the two anatomical groups of lipid biopolymer.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

Effect of cultural conditions on the lipid profile of Thiobacillus ferroxidas

The intensitive investigations on the lipid profile of Thiobacillus ferrooxidans at various culture ages suggest some correlations of the lipid constitutents with the membrane-bound iron oxidation system. Phosphatidic acid, phosphatidyl serine and phosphatidyl ethanolamine were the major polar components; hydrocarbon, triglyceride and diglyceride were the main neutral components. Major fatty acids were C_16:0, C_16:1, C_16:3, C_18:1, C_18:3, C_22:1 while C_20:1, C_20:2, C_12:0, C_14:2, C_18:0, C_18:2, C_20:0, C_22:0 were found in trace amounts which also depended upon the phase of the growth. One lipoamino acid was identified as ornithine lipid in the polar fraction. Each and every component varied to some extent at different growth phasesindicating relationship of these lipids to the iron oxidation system of the strain.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Rapid determination of lipids in healthy human lymphocytes

Lipids of human lymphocytes were determined from 20 ml of heparinized peripheral blood using thin-layer chromatography with a flame ionization detector, and gas chromatography.The weight per cent and μg per 10⁶ lymphocytes for cholesterol ester, triglyceride, free cholesterol and phospholipid were 11.1 and 5.2, 18.1 and 17.9, 15.1 and 8.5, and 55.7 and 44.2, respectively. Phospholipid was the major lipid component in human lymphocytes. Phospholipid was subfractionated into phosphatidylethanolamine, phosphatidylinositol plus phosphatidylserine, phosphatidylcholine and sphingomyelin in amounts of 25.2, 6.1, 46.9 and 22.9%, respectively.Total fatty acid composition was analyzed as: C_14:0, 13.3%; C_16:0, 20.9%; C_16:1, 6.5%; C_18:0, 19.6%; C_18:1, 18.8%; C_18:2, 7.1%; and C_20:4, 12.3%. Higher cholesterol ester and triglyceride and lower C_14:0 were characteristic of female lymphocytes when compared with male lymphocytes.The lipid composition quantitated by this method corresponded well with previously reported data.Thus, this method can be used clinically because of its simplicity and higher sensitivity.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

Application of Two‐Way Hierarchical Cluster Analysis for the Identification of Similarities between the Individual Lipid Fractions of Lucilia sericata

The composition of the cuticular and internal lipids of larvae and pupae of Lucilia sericata was studied using chromatographic techniques. The lipids from both stages of L. sericata had similar free fatty acid (FFA) profiles and also contained alcohols and cholesterol. The range of the number of C‐atoms detected for these classes of compounds was to some extent similar in larvae and pupae, but the relative amounts of each class differed between stages. Saturated as well as unsaturated FFAs with even and odd numbered C‐atom chains were present in both cuticular and internal lipids. The alcohol fractions of L. sericata were represented by free, straight‐chain primary alcohols containing an even number of C‐atoms. The lipid composition of male and female L. sericata adults and the hydrocarbon composition of all stages of L. sericata had previously been analyzed. To have a full overview of the lipid composition and to identify similarities or dissimilarities between the individual lipid fractions in this insect species, two‐way hierarchical cluster analysis (HCA) was performed using also the data from these previous publications. The content of FFA 18 : 1 (n‐9) was noticed to be very high in the cuticular fractions of larvae and pupae as well as in all internal fractions (male, female, larvae, and pupae) and low in the cuticular fractions of male and female imago. The contents of FFAs 16 : 0 and 16 : 1 (n‐9), cholesterol, and the n‐alkanes n‐C₃₁, n‐C₂₉, n‐C₂₇, n‐C₂₅, and n‐C₂₃ varied between particular fractions, whereas the amounts of other compounds were similar in all fractions.     

Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC

A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C₁₈ column using gradient elution in a water–methanol system. Detection was set at UV wavelength of 248 nm. A calibration curve ranging from 0.01 to 1.5 μg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01 μg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8 μg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at ?20 °C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T_1/2, 4.29 h; T_max, 2.70 h; C_max, 0.74 μg/mL; AUC_0–t and AUC_0–∞, 3.74 and 3.82 μg h/mL, respectively.     

Intra-injector methylation of free fatty acids from aerobically and anaerobically cultured Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus

Free fatty acids from the type strains of anaerobically and aerobically broth-cultured Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus cells were Soxhlet-extracted with hexane. The fatty acids were identified and quantified by gas chromatography and gas chromatography—mass spectrometry after intra-injector derivatization with trimethylanilinium hydroxide. This derivatization method, which we propose as suitable for routine use in clinical microbiology, is fast, accurate and sensitive, with low toxicity. Whereas the fatty acid content of A. actinomycetemcomitans was affected by the cultivation atmosphere, i.e. C_16:1, decreased under aerobic growth and C_16:0 increased, that of the closely related H. aphrophilus was more stable.     

Wangella harbinensis gen. nov., sp. nov., a new member of the family Micromonosporaceae

A novel endophytic actinomycete, designated strain NEAU-J3^T, was isolated from soybean root (Glycine max (L.) Merr) and characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain NEAU-J3^T fell within the family Micromonosporaceae. The strain was observed to form an extensively branched substrate mycelium, which carried non-motile oval spores with a smooth surface. The cell walls of strain NEAU-J3^T were determined to contain meso-diaminopimelic acid and galactose, ribose and glucose were detected as whole-cell sugars. The major menaquinones were determined to be MK-9(H₄) and MK-9(H₆). The phospholipids detected were phosphatidylcholine and phosphatidylethanolamine. The major cellular fatty acids were determined to be C_16:0, C_18:1 ω9c, C_18:0, C_17:0, C_17:1 ω7c, anteiso-C_17:0, C_16:1 ω7c and C_15:0. The DNA G + C content was 62.5 mol%. On the basis of the morphological and chemotaxonomic characteristics, phylogenetic analysis and characteristic patterns of 16S rRNA gene signature nucleotides, strain NEAU-J3^T is considered to represent a novel species of a new genus within the family Micromonosporaceae, for which the name Wangella harbinensis gen. nov., sp. nov. is proposed. The type strain of Wangella harbinensis is strain NEAU-J3^T (=CGMCC 4.7039^T = DSM 45747^T).     

Lipid Molecular Species of Lipomyces starkeyi

The molecular species of glycerides and phospholipids of the yeast Lipomyces starkeyi IFO 0678 harvested at 60 hr, corresponding to the late exponential phase, were analyzed by gas chromatography-mass spectrometry. The major triglyceride was C_16:0–C_18:1–C_18:1. The major molecular species of phospholipid were 1–C_16:0–2–C_18:1 and 1–C_18:1–2–C_18:2. Although phosphatidylcholine and phosphatidylethanol amine were composed of several kinds of molecular species, 1–C_16:1–2–C_18:1, 1–C_18:1–2–C_18:2 and l-C_16:0–2–C_18:1, phosphatidylserine was composed of almost exclusively 1–C_16:0-2-C_18:1. The lipid and the fatty acid compositions of the yeast harvested at the different growth phases were also investigated.     

Enantioselective determination of the hydroxylated metabolites of mexiletine in human plasma

Pre-column derivatization with o-phthaldialdehyde and N-acetyl-l-cysteine was used for liquid-chromatographic diastereomeric resolution of p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM), metabolites of mexiletine formed by aromatic and aliphatic hydroxylation, respectively. The resulting diastereomeric derivatives were resolved on a C₁₈ column and monitored by fluorescence detection. The diastereomeric elution order for both metabolites was determined on the basis of the circular dichroism spectra of each eluted fraction. Plasma samples (500 μl) showed recoveries greater than 75% for both the metabolites. Calibration curves in plasma samples were linear over the concentration ranges 10–500 and 20–1,000 ng/ml for each enantiomer of PHM and HMM, respectively. The limits of quantitation were found to be 10.0 and 5.0 ng/ml for both enantiomers of PHM and HMM. The within-day and between-day coefficients of variation were less than 10%. The assay was shown to be suitable for a pharmacokinetic study performed in a patient with ventricular arrhythmias following the short-term oral treatment of 200 mg t.i.d. of racemic mexiletine hydrochloride. Chirality 9:732–738, 1997. © 1997 Wiley-Liss, Inc.     

Identification of the alga known as <Emphasis Type="Italic">Nannochloropsis</Emphasis> Z-1 isolated from a prawn farm in Hainan,China as <Emphasis Type="Italic">Chlorella</Emphasis>

An alga known as “Nannochloropsis”, isolated from a prawn farm in Hainan, China, has been critically investigated and identified as Chlorella, a member of the Chlorophyceae based on fatty acid composition, ultrastructure, and 18S rDNA. Cells of this alga were spherical, measured by 1–6 μm in diameter and were enclosed in thin walls of approximately 0.04 μm thickness. They contained several small mitochondria, two to three thylakoids and had no vacuoles. There were many pyrenoids in the algal cells and their thylakoid lamellae were sparse and not translucent. Many lipid droplets were present in the cytoplasm. The total lipid content of this alga was 3% per gram dry weight and its major fatty acids were C_16:0, C_18:0, C_18:1, C_18:2, C_18:3 and C_20:0. Eicosapentaenoic acid (C_20:5, EPA) was not detected. The length of its 18S rDNA sequence was 1,712 bp. 18S rDNA sequence analyses indicated that this alga was a species of Chlorella.     

Leishmania species: fatty acid composition of promastigotes

The fatty acid composition of the total lipid fractions of five different Leishmania organisms grown on Eagle's medium was determined by gas chromatography. The major fatty acids identified in the total lipid fractions of L. donovani, L. tropica major, L. tropica minor, L. tropica (England strain), and L. enriettii were C_12:0, C_13:0, C_14:0, C_15:0, C_16:0, C_17:0, C_18:0, C_18:1, C_18:2, and C_18:3. The statistical differences among the fatty acid methyl esters of different Leishmania organisms are discussed.Gas chromatographic analysis of the fatty acid methyl esters of the total lipid fractions of the original Eagle's medium and the media after harvesting of various Leishmania species revealed the presence of C_18:3 fatty acid in the total lipid fraction of the medium of L. donovani and the complete absence of 18-carbon unsaturated fatty acids in the total lipid fraction of the medium of L. enriettii. The use of such differences in the differentiation of various Leishmania species is discussed.