A METHOD FOR MEASURING THE NARCOTIC ACTION OF CHEMICALS USING THE GRAIN WEEVIL, CALANDRA GRANARIA

A method of measuring narcotic potency is described. Grain weevils ( Calandra granaria ) are exposed to the narcotic vapour in 2l. round-bottomed flasks illuminated from the bottom, and narcotic potency measured as number of grain weevils so affected as to be unable to climb the glass walls. Some common anaesthetics have been examined as an illustration of this method. Results are fitted by the probit plane
Y= A + b ₁ log α+ b ₂ log t ,
where Y = a function of the number of insects affected (probit); α= the thermodynamic concentration of the narcotic vapour; t = time of exposure of insects to the narcotic vapour. The values of the coefficients b ₁ and b₂ are discussed in relation to the biological action of the narcotic vapours.     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell I. Separation and partial characterization of altered lipids

Alterations induced by carbon tetrachloride poisoning in fatty acids of liver microsomal lipids were studied. Thin layer chromatography of fatty acid methyl esters prepared from liver microsomal lipids, revealed, in the CCl₄-treated rats, the presence of a component (the “D” spot) with an R_f value lower than that of the methyl esters. The lipids recovered from this component showed a marked diene conjugation absorption when examined spectrophotometrically over the UV range, while the lipids recovered from the spot of the methyl esters showed no absorption of conjugated dienes.Studies carried out with labelled carbon tetrachloride indicated that compounds present in the “D” spot contained 28% of ¹⁴C applied to the chromatoplate. The spot of the methyl esters (the “M” spot) contained 42% of ¹⁴C applied to the chromatoplate. However, specific activity of the “D” spot was about 1000 times greater than specific activity of the “M” spot.The lipids recovered from either the “D” spot or the spot of the methyl esters were analyzed separately by gas-liquid chromatography (GLC) with an electron capture detector (ECD). It was found that the lipids recovered from the “D” spot showed no response, while those recovered from the spot of the methyl esters exhibited the response of the ECD, which was similar to that observed with the unfractionated fatty acid methyl esters. The lack of the response of the ECD for compounds in the “D” spot appears to be due to the fact that they cannot be eluted from the column.On the basis of the analytical results, it can be postulated that the “D” spot contains compounds formed by a chain termination addition reaction of free radicals derived from CCl₄ (probably trichloromethyl free radicals) to fatty acid free radicals containing conjugated dienes. On the other hand, the spot of the methyl esters appears to contain also, together with unmodified fatty acids, the fatty acids in which a simple addition of CCl₄ free radicals to double bonds has occurred.     

Extraction of mycobacterial mycolic acids and other long-chain compounds by an alkaline methanolysis procedure

Treatment of whole organisms with methanolic tetramethylammonium hydroxide and toluene, followed by addition of iodomethane in dimethylformamide, released long-chain compounds and fatty acids, as their methyl esters, from representative strains of Mycobacterium. Two-dimensional thin-layer chromatography was used to analyze methanolysates for the presence of the methyl esters of mycolic acids which are characteristic high molecular weight 3-hydroxy-2-alkyl fatty acids.     

Agonistic interactions, cuticular and hemolymphatic lipid variations during the foraging period in spider females Brachypelma albopilosa (Theraphosidae)

Agonistic behaviour and lipid state were examined in tarantula Brachypelma albopilosa females during the foraging period. Modulation of the agonistic behaviour of females was not connected to their body size. Results show that the agonistic pattern of females differed significantly from the predation pattern at the behavioural and lipid levels. Aggressive-foraging females had low predation behaviour. Quantitative lipid changes were observed in relation to agonistic behaviour and predation. The total lipid index was studied by colorimetric methods, and lipid compounds were analyzed by gas chromatography-mass spectrometry in cuticle and hemolymph of females. The lipid components were free fatty acids, methyl esters, cholesterol, and long-chain aliphatic hydrocarbons. Methyl esters were much more abundant in cuticular lipids; unsaturated free fatty acids (linoleic and oleic acids) and methyl esters (methyl linoleate and methyl stearate) predominated in the hemolymph. Spider aggression was positively correlated with lipid concentration (cholesterol, fatty acids, methyl esters and hydrocarbons) in the hemolymph and the levels of cuticular fatty acids. Lipid levels are hypothesized to have evolved as a regulatory factor of predation and agonistic behaviours in tarantula females.     

Lipids of Rhizopus arrhizus Fischer

The lipids of Rhizopus arrhizus Fischer mycelia and sporangiospores were extracted, isolated, and separated by thin-layer, liquid, and gas chromatography. Structural confirmations of the compounds were made by a gas chromatographmass spectrometer combination. The n-heptane fraction contained squalene (1%) as a major hydrocarbon constituent. Other major lipid classes detected were free fatty acids, naturally occurring methyl esters of fatty acids, triglycerides, sterols, and polar lipids. The polar lipids (44.4%) were found in the highest concentrations, and the triglycerides (22.1%), sterols (16.7%), and free fatty acids (11.7%) were present in lesser concentrations. This is the first report of naturally occuring methyl esters of long-chain fatty acids being present in fungal mycelium. There appears to be a preference for incorporation of unsaturated acids into the complex lipids, with the exception of the triglycerides. The major saturated fatty acids in the mycelium were palmitic (C(16)) and arachidic (C(20)), whereas the major unsaturated acids were oleic (C(18:1)) and linoleic (C(18:2)), respectively.     

Changes in Esters of Fatty Acids of Rhizopus arrhizus During Germination and Growth

Natural esters of fatty acids of Rhizopus arrhizus Fischer increased during the first 48 hr of growth. During the 48- to 72-hr period, there was a shift in concentration from the methyl esters to the ethyl esters of fatty acids.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Effect of Conidiobolus coronatus on the Cuticular and Internal Lipid Composition of Tettigonia viridissima Males

Conidiobolus coronatus is an entomopathogenic fungus which has a potential as a biological control agent of insects. The cuticular and internal lipid composition of infected and noninfected Tettigonia viridissima males were analyzed by GC/MS. A total of 49 compounds were identified in the infected and noninfected males, including fatty acids, fatty acid methyl esters (FAMEs), n‐alkanes, alcohols, sterols, and other organic compounds. The most abundant components of the cuticular and internal lipids of the insects were fatty acids. After exposure to C. coronatus, the cuticular lipids of the T. viridissima males contained 17 free fatty acids from C(8) to C(22), while the cuticular lipids of the noninfected insects contained only 15 fatty acids from C(12) to C(24). The cuticular and internal lipids of both the infected and the noninfected males also contained five FAMEs from C(15) to C(19), seven n‐alkanes from C(25) to C(34), five alcohols from C(16) to C(25), five sterols, and the following six other organic compounds: azelaic acid, phenylacetic acid, glutaric acid, benzoic acid, sebacic acid, and glycerol. The compounds which were present only in the cuticular lipids of the infected males could be due to fungal infection.     

Preparation and evaluation of the in vitro erythroid differentiation induction properties of some esters of methyl 3,4-O-isopropylidene-beta-D-galactopyranoside and 2,3-O-isopropylidene-D-mannofuranose.

Two series of glycide esters of short fatty acids, designed for avoiding intramolecular transesterification, were prepared and tested for in vitro erythroid differentiation induction activities using the K562 cell line as experimental system. The 6-O-isobutiryl and pivaloyl derivatives of methyl 3,4-O-isopropylidene-beta-D-galactopyranosides as well the same 1-O-esters of 2,3-O-isopropylidene-alpha- and beta-D-mannofuranose exhibit biological activities much higher that the corresponding acids and could be proposed as possible agents to modulate production of embryo-fetal hemoglobins by human erythroid cells.     

Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacyglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.     

Properties and metabolism of 2alkylalkanoates. 3. absorption of methyl and ethyl 2-methylpalmitate

The recovery from rat and rabbit tissues of fed methyl-(14)C and ethyl-2-(14)C 2-methylpalmitate with unaltered specific activity has demonstrated the existence of mechanisms for the absorption and deposition of both methyl and ethyl esters of fatty acids, at least for 2-methylpalmitate. In thoracic duct-cannulated rats, approximately 9% of the fed compounds was recovered from the lymph during the first 24 hr, the rate of recovery reaching a maximum between 6 and 8 hr. In the rabbit, the fed, unaltered esters in plasma were transported principally by means of the low density lipoproteins. Only trace amounts of the unaltered esters were subsequently detected in the blood and tissue lipids after feeding, however, even during the period of maximal absorption; moreover, in contrast to at least one report by others, further analyses for methyl or ethyl esters of other fatty acids has shown that such esters of short-chain alcohols constitute no more than a trace amount (0.004-1.03%) of the lipids extracted from a wide variety of mammalian tissues. The possibility remains that even these trace amounts of esters arose as artifacts of autolysis, extraction, or assay.     

Evidence in the formation of conjugated linoleic acids from thermally induced 9t12t linoleic acid: a study by gas chromatography and infrared spectroscopy

Thermally induced isomerisation leading to the formation of conjugated linoleic acids (CLAs) has been observed for the first time during the thermal treatment of 9t12t fatty acid triacylglycerol, and methyl ester. Fifteen microlitre portions of the triacylglycerol sample containing 9t12t fatty acid (trilinoelaidin) were placed in micro glass ampoules and sealed under nitrogen, then subjected to thermal treatment at 250 °C. The glass ampoules were removed at regular time intervals, cut open, and the contents were analysed by infrared spectroscopy using a single reflectance attenuated total internal reflectance crystal accessory. The samples were then subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were analysed by gas chromatography. The same procedure was repeated with methyl ester samples containing 9t12t fatty acid (methyl linoelaidate). Each sample was subjected to infrared measurements and gas chromatographic analysis after appropriate dilution in heptane.The results show that the thermally induced isomerisation of 9t12t fatty acids from both triacylglycerol molecules and methyl esters give identical CLA profiles as those found for the thermally induced isomerisation of 9c12c fatty acids. The infrared spectrometry provides additional evidence confirming the formation of CLA acids during thermal treatment. A mechanism for the formation of the CLAs from 9t12t fatty acid molecules is also formulated for the first time. This mechanism complements the pathways of formation of CLAs from 9c12c fatty acids during thermal treatment.     

Quantitative analysis of fatty acid precursors in marine samples: direct conversion of wax ester alcohols and dimethylacetals to FAMEs

To apply fatty acid analyses to the study of foraging ecology and diet determination, all compounds that may be deposited as fatty acids in a predator must be quantified in the prey. These compounds include the usual fatty acids in acyl lipids, but also the alcohols of wax esters and the vinyl ethers of plasmalogens. In routine fatty acid analysis, samples are extracted and transesterified (methylated), resulting in the formation of fatty acid methyl esters (FAMEs); however, fatty alcohols and dimethylacetals (DMAs) are also generated if wax esters or plasmalogens are present. Here, we present a new method using a modified Jones' reagent to oxidize these alcohols and DMAs to free fatty acids (FFAs). These FFAs are then easily methylated and quantitatively recombined with FAMEs from the same sample. This generates a fatty acid signature of prey that is equivalent to that which the predator has available for deposition upon digestion of that prey. This method is validated with alcohol and DMA standards. Its application to typical marine samples is also presented, demonstrating the change in effective fatty acid signature after inclusion of fatty acids derived from wax esters and plasmalogens.     

Hydrocarbons,phenols and sterols of the testa and pigment strand in the grain of Hordeum distichon

Material from the testa of decorticated barley grains contained hydrocarbons, esters, triglycerides, free sterols, 5-n-alkylresorcinols, and traces of free alcohols, carbonyl compounds, and various polar, acidic materials. The hydrocarbon fraction was mainly a series of n-alkanes, extending at least from C₁₁ to C₃₆, in which the C₂₉ and C₃₁ components were prominent. Two minor series of alkanes were also present. Sometimes a trace of an unsaturated hydrocarbon was detected. The ester fraction contained sterols and alkanols esterified by fatty acids, which differed in relative amounts from the fatty acids found in the triglycerides. The triglycerides were thought to have leached from within the grain. At least five free sterols were present, including sitosterol and campesterol. The 5-n-alkylresorcinols were at least twelve members of a homologous series, of which four, C₂₅, C₂₇, C₂₉, and C₃₁, made 98% of the total. Members of the series with even numbers of carbon atoms were also present. It is suggested that they are partly responsible for excluding microorganisms from the interior of the grain. The testa membrane, with the associated pigment strand, contained an estolide of fatty acids and various hydroxyacids, a polysaccharide component, and uncharacterized material.     

Identification of ecdysone 22-long-chain fatty acyl esters in newly laid eggs of the cattle tick Boophilus microplus.

The five major apolar ecdysone esters present in newly laid eggs of the cattle tick Boophilus microplus have been purified by h.p.l.c. The quantities of the apolar esters present in the eggs were increased by administration of ecdysone to the mature females. G.c.-m.s. analysis, as their methyl esters, of the fatty acids released from the apolar ecdysone derivatives by alkali, coupled with positive-ion fast-atom-bombardment m.s. of the intact ecdysone esters, showed that the compounds consisted of a series of fatty acyl esters of ecdysone. The position of esterification of the ecdysone was established by p.m.r. spectroscopy. The combined data show that the novel apolar derivatives of ecdysone consist of the 22-palmitate, -palmitoleate, -stearate, -oleate, and -linoleate esters respectively. Confirmation was obtained by comparison with synthetic ecdysone 22-palmitate. The significance of the ecdysone fatty acyl esters as a possible source of free hormone during embryogenesis is discussed.     

Observation of the terminal methyl group in fatty acids of the linolenic series by a new 1H NMR pulse sequence providing spectral editing and solvent suppression. Application to excised frog muscle and rat brain

A new 1H NMR pulse sequence is described that combines water suppression with the selective observation of signals from coupled spin systems. The pulse sequence is easy to set up and compensates for pulse width inhomogeneity in the biological sample. Suppression of the water signal is achieved by pulses that return the water spins to their equilibrium position; spectral editing is based on the J modulation present in spin-echo spectra and its inhibition by coherent decoupling at one of the resonances of the spin system of interest. The pulse sequence, which was designed for 1H NMR spectroscopy of tissue, was tested at 470 MHz on excised frog muscle and rat brain. The lactate methyl resonance of caffeine-treated frog sartorius muscle was observed selectively by irradiation at the position of its alcoholic proton. The terminal methyl signal of linolenic acid, along with other fatty acids of the linolenic series (first double bond in the omega-3 position), was observed selectively by irradiation at the position of its omega-1 methylene group. 1H NMR spectra of rat brain were edited to reveal the terminal methyl of either linolenic series or all other fatty acids. The results suggest that the terminal methyl groups of fatty acids of the linolenic series (mostly docosahexaenoic acid, 22:6) have higher mobility than those of all other fatty acids.     

Dimeric epoxy fatty acid methyl esters: formation,chromatography and mass spectrometry

The formation of dimers is reported from the thermal treatment of a series of epoxy fatty acid methyl esters. These compounds were isolated from the reaction mixture by steric exclusion chromatography and were subsequently characterised by their high resolution electron impact and ammonia chemical ionisation mass spectra. The spectra were consistent in each case with the presence of a mixture of four possible positional isomers each containing an ether bridge linking a pair of fatty acid methyl esters across the carbon chains, with a keto group on a carbon adjacent to the bridge on one of the esters.     

Skin surface lipids of the mink

Skin surface lipids from mink (Mustela vison) were collected in acetone and analyzed by thin-layer chromatography and gas chromatography. The principal components were wax monoesters (92%), cholesteryl esters (5%), free fatty acids (1%), fatty alcohols (1%) and cholesterol (1%). The fatty acids and alcohols contained in these lipids were composed principally of homologous series of straight chained omega 7-unsaturated structures (C16-C24), accompanied by lesser proportions of homologous series of saturated (C14-C22) and omega 9-unsaturated (C18-C22) structures.     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.     

Elimination of n-butylated hydroxytoluene methylation during fatty acid analysis by gas chromatography

An improved method for fatty acids analysis with optimum recovery of highly polyunsaturated fatty acids methyl esters in biological systems is presented. The method is based on transesterification of phospholipid and triacylglycerols to fatty acid methyl esters using a commercially available reagent, Methyl-Prep II. Without proper precautions, as much as 50% of n-butylated hydroxytoluene (BHT) added to prevent oxidation of polyunsaturated fatty acids, could be methylated during the transesterification step. Methylated BHT elutes close to 14:0 (myristic acid) and no longer functions as an antioxidant, but the modified conditions virtually eliminate the methylation of BHT. Sample extraction and methylation was completed in 30 min at room temperature. A chelator (diethylenetriamine-pentaacetic acid; DTPA) is also added to prevent peroxidation of metal catalyzed free radical chain reactions. The standard deviations of the major fatty acids from multiple human plasma samples prepared on different days were less than 5%. The recovery of arachidonic acid, 20:4, from plasma was improved using the new method, and the recovery for docosahexaenoic acid, 22:6, spiked to human plasma was found to be 99%.