Peak AAA fatty acid homolog contaminants present in the dietary supplement l-Tryptophan associated with the onset of eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA1-343) and linear (AAA2-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA1-343 and AAA2-343. We structurally characterized eight new AAA1-XXX/AAA2-XXX contaminants, where XXX represents the integer molecular ions of all the related homologs, differing by aliphatic chain length and isomer configuration. The contaminants were derived from the following fatty acids of the bacterial cell membrane, 5-methylheptanoic acid (anteiso-C8:0) for AAA1-315; n-octanoic acid (n-C8:0) for AAA2-315; 6-methyloctanoic acid (anteiso-C9:0) for AAA1-329; n-nonanoic acid (n-C9:0) for AAA2-329; 10-methyldodecanoic acid (anteiso-C13:0) for AAA1-385; n-tridecanoic acid (n-C13:0) for AAA2-385; 11-methyltridecanoic acid (anteiso-C14:0) for AAA1-399; and n-tetradecanoic acid (n-C14:0) for AAA2-399. The concentration levels for these contaminants were estimated to be 0.1-7.9 µg / 500 mg of an individual SD L-Trp tablet or capsule The structural similarity of these homologs to case-related contaminants of Spanish Toxic Oil Syndrome (TOS) is discussed.

Determination of ethyl glucuronide and fatty acid ethyl esters in hair samples.

Hair testing for alcohol biomarkers is an important tool for monitoring alcohol consumption. We propose two methods for assessing alcohol exposure through combined analysis of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) species (ethyl myristate, palmitate, stearate and oleate) in hair (30 mg). EtG was analysed by liquid chromatography-tandem mass spectrometry, while FAEEs were analysed by gas chromatography-tandem mass spectrometry using electron impact ionization. Both methods were validated according to internationally accepted guidelines. Linearity was proven between 3 and 500 pg/mg for EtG and 30-5000 pg/mg for FAEEs, and the limits of quantification were 3 pg/mg for EtG and 30 pg/mg for each of the four FAEEs. Precision and accuracy were considered adequate, processed EtG samples were found to be stable for up to 96 h left in the injector and processed FAEEs samples for up to 24 h. Matrix effects were not significant. Both methods were applied to the analysis of 15 authentic samples, using the cut-off values proposed by the Society of Hair Testing for interpretation. The results agreed well with the self-reported alcohol consumption in most cases, and demonstrated the suitability of the methods to be applied in routine analysis of alcohol biomarkers, allowing monitoring consumption using low sample amounts.

Global analysis of protein N-myristoylation and exploration of N-myristoyltransferase as a drug target in the neglected human pathogen Leishmania donovani.

N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen.

Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium for detection of alcohol abuse during pregnancy: Correlation study between both biomarkers.

This article presents results from 47 meconium samples, which were analyzed for fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) for detection of gestational alcohol consumption. A validated microwave assisted extraction (MAE) method in combination with GC-MS developed in the Institute of Forensic Science (Santiago de Compostela) was used for FAEE and the cumulative concentration of ethyl myristate, ethyl palmitate and ethyl stearate with a cut-off of 600ng/g was applied for interpretation. A simple method for identification and quantification of EtG has been evaluated by ultrasonication followed solid phase extraction (SPE). Successful validation parameters were obtained for both biochemical markers of alcohol intake. FAEE and EtG concentrations in meconium ranged between values lower than LOD and 32,892ng/g or 218ng/g respectively. We have analyzed FAEE and EtG in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. Certain agreement between the two biomarkers was found as they are both a very specific alcohol markers, making it a useful analysis for confirmation.

A SPME-GC/MS procedure for the determination of fatty acid ethyl esters in hair for confirmation of abstinence test results.

Fatty acid ethyl esters (FAEE), direct metabolites of ethanol, are suitable alcohol markers that can be detected in different tissues. The determination of FAEE in hair can help to evaluate social and excessive alcohol consumption. Due to the presence of FAEE in the hair of teetotalers, proving alcohol abstinence seems to be impossible. To verify these results, an solid phase micro extraction-gas chromatography/mass spectrometry procedure for the determination of the four FAEE: ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate in hair was validated with special focus on low concentration levels. Besides very high sensitivity (limits of detection between 0.005 and 0.009 ng/mg), good results for linearity, precision and accuracy, recovery and stability were achieved. In addition, 73 hair samples with measured ethyl glucuronide (EtG) concentrations between 4 and 10 pg/mg were analyzed for FAEE. By using the following cut-offs: EtG: 7 pg/mg, FAEE: 0.2 ng/mg a satisfying matching rate of 72.6% was found. This shows that FAEE can be determined to verify borderline EtG concentrations even in the context of abstinence tests. However, the diversified influencing factors on analyte concentrations in hair, which may explain the large deviations between EtG and FAEE results observed in some cases, have to be mentioned when interpret ambiguous results.

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids.

The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded ß,ß-carotenoid 3,3'-hydroxylase (CrtZ) and ß,ß-carotenoid 4,4'-ketolase (4,4'-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T0) (98.8%) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T1) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2% of total carotenoids), astaxanthin monoester (18.2%), and the free forms of astaxanthin (10.0%) and the other ketocarotenoids (17.5%), which indicated that artificial ketocarotenoids corresponded to 94.9% of total carotenoids (230 µg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8%) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.

Role of pagL and lpxO in Bordetella bronchiseptica lipid A biosynthesis.

PagL and LpxO are enzymes that modify lipid A. PagL is a 3-O deacylase that removes the primary acyl chain from the 3 position, and LpxO is an oxygenase that 2-hydroxylates specific acyl chains in the lipid A. pagL and lpxO homologues have been identified in the genome of Bordetella bronchiseptica, but in the current structure for B. bronchiseptica lipid A the 3 position is acylated and 2-OH acylation is not reported. We have investigated the role of B. bronchiseptica pagL and lpxO in lipid A biosynthesis. We report a different structure for wild-type (WT) B. bronchiseptica lipid A, including the presence of 2-OH-myristate, the presence of which is dependent on lpxO. We also demonstrate that the 3 position is not acylated in the major WT lipid A structures but that mutation of pagL results in the presence of 3-OH-decanoic acid at this position, suggesting that lipid A containing this acylation is synthesized but that PagL removes most of it from the mature lipid A. These data refine the structure of B. bronchiseptica lipid A and demonstrate that pagL and lpxO are involved in its biosynthesis.

Fatty acid ethyl esters in meconium are associated with poorer neurodevelopmental outcomes to two years of age.

OBJECTIVE: To determine the relationship between fatty acid ethyl esters (FAEE) in meconium and neurodevelopment in infants exposed to alcohol in utero at 6.5 months, 1 year, and 2 years of age. STUDY DESIGN: A secondary analysis of a prospective cohort of mothers at high risk and their infants recruited after admission to a labor and delivery unit. Mothers were screened for drug and alcohol use during pregnancy by clinical interview and urine screening. Meconium was analyzed for FAEE in 216 newborn infants. Outcome measures included the Bayley Scales of Infant Development Mental (MDI) and Psychomotor (PDI) Developmental Index scores in infants at 6.5 months, 1 year, and 2 years of age. RESULTS: After controlling for prenatal visits and maternal factors, increasing concentrations of FAEE were significantly associated with poorer mental and psychomotor development (beta +/- standard error) at all follow-up visits: ethyl myristate (MDI -2.46 +/- 1.24, P = .05; PDI -3.88 +/- 1.67, P = .02), ethyl oleate (MDI -1.94 +/- 0.65, P < .01; PDI -2.60 +/- 0.93, P < .01), ethyl linoleate (MDI -1.92 +/- 0.60, P < .01; PDI -2.28 +/- 0.84, P < .01), ethyl linolenate (MDI -1.99 +/- 0.74, P < .01; PDI -2.98 +/- 1.04, P < .01), and ethyl arachidonate (MDI -2.40 +/- 1.11, P = .03; PDI -3.32 +/- 1.51, P = .03). CONCLUSION: FAEE in meconium may be a marker for identifying newborns at risk for neurodevelopmental delay from alcohol exposure in utero.

Ethanol contamination leads to Fatty acid ethyl esters in hair samples.

The diagnosis of alcoholism is a topical subject of discussion; in fact, many studies have been published on the determination of biochemical markers useful to this target. Fatty acid ethyl esters (FAEE) are minor metabolites of ethanol, and their usefulness has been demonstrated by their detection in hair using a headspace solid-phase microextraction-gas chromatographic-mass spectrometric technique. Environmental contamination in the analysis of drugs of abuse is a well-known focus of discussion between scientists. In the same way, interference from the surroundings could be hypothesized in FAEE detection. To assess the influence of ethanol contamination, an in vitro experiment was performed, leaving hair in an atmosphere saturated with ethanol vapors for 15 days. The spontaneous production of FAEE was demonstrated by analyzing hair day by day. In fact, we observed a constant increase of ethyl myristate, palmitate, and stearate that reached very high concentrations at the end of the investigation. Although the experiment was managed in a stressed way and could not represent real life, its purpose was to focus the attention of researchers on the problem of hair contamination that can occur, for example, with ethanol-containing cosmetics. Therefore, care in interpretation must be taken into account, especially with such a volatile molecule.

Concentration of fatty acid ethyl esters in hair of alcoholics: comparison to other biological state markers and self reported-ethanol intake.

AIMS: In a variety of clinical and forensic situations long term use of alcohol must be monitored. In this project we explore the utility of fatty acid ethyl esters (FAEE) in this regard. Additionally, we propose a cut-off value of FAEE to distinguish teetotallers/moderate/social drinkers from alcoholics or individuals drinking at harmful levels. PATIENTS AND METHODS: FAEE levels from 18 alcohol-dependent patients in detoxification were contrasted with those of 10 social drinkers and 10 teetotallers. FAEE in hair were determined, using headspace solid phase microextraction and gas chromatography mass spectrometry. C(FAEE), as sum of the concentrations of four esters, was compared to a major FAEE, ethyl palmitate. PEth was measured in heparinized whole blood with a high pressure liquid chromatography (HPLC) method. Drinking validation criteria include self reports, phosphatidyl ethanol (PEth) in whole blood as well as the traditional markers of heavy drinking, gamma glutamyl transpeptidase (GGT), mean corpuscular volume (MCV) and carbohydrate deficient transferrin (CDT). RESULTS: Receiver-operating characteristic (ROC) curve analysis for C(FAEE), indicated a sensitivity of 100% and a specificity of 90% for a cut-off of 0.29 ng/mg. By using a cut-off of 0.4 ng/mg, C(FAEE) identified 94.4% correctly. C(FAEE) and ethyl palmitate were significantly associated (r = 0.945; P < 0.001) as were C(FAEE) and PEth (r = 0.527; P = 0.025). No significant correlation was found between C(FAEE) and total grams of ethanol consumed last month, blood-alcohol concentration at admission to the hospital, CDT, MCV, or GGT. Among the serum and blood markers, %CDT identified 47.1%, MCV 38.8% and GGT 72.2% of patients with chronic intake of higher amounts of ethanol correctly, whereas PEth achieved 100% accuracy. CONCLUSIONS: The data suggest that C(FAEE) is a potentially valuable marker of chronic intake of high quantities of ethanol. Furthermore, the results indicate that a reasonable and provisional FAEE cut-off to distinguish between social/moderate and heavy drinking/alcoholism in hair is 0.4 ng/mg.

Effect of hair care and hair cosmetics on the concentrations of fatty acid ethyl esters in hair as markers of chronically elevated alcohol consumption.

Fatty acid ethyl esters (FAEE) can be used as alcohol markers in hair. It was investigated in this study whether this diagnostic method is disturbed by hair care and hair cosmetics. Traces of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate were detected in all of 49 frequently applied hair care products by headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The highest concentration was 0.003% in a hair wax. From experiments with separated hair samples of alcoholics as well as from the evaluation of the FAEE concentrations and the data about hair care of 75 volunteers (alcoholics, social drinkers and teetotalers) follows that usual shampooing, permanent wave, dyeing, bleaching or shading are of minor importance as compared to the drinking amount and other individual features. However, false positive results were found after daily treatment with a hair lotion containing 62.5% ethanol, with a deodorant and with a hair spray. As an explanation, it is assumed that FAEE are formed in the sebum glands also after regular topical application of products with a higher ethanol content.

[Research on components of Cornus officinalis extracted by supercritical carbon dioxide].

OBJECTIVE: To study the chemical constituents of Cornus officinalis extracted by supercritical carbon dioxide fluid extraction (SFE). METHOD: The process was performed at 40 centigrade with pressures of 15 MPa for 2 hours and with CO2 fluid and gas at the flow rate of 22.0 kg x h(-1) and 18.0 kg x h(-1) respectively. The chemical constituents of the SFE extractions were determined by GC-MS. RESULT: The total amount of extractable substances or yields by SFE is 2.42% (mass). 31 Chemical constituents were identified and their relative contents were determined by normalization method of area. CONCLUSION: The major components identified in the extractions are 1,2-benzenedicarboxylic acid, butyl 2-methylpropyl ester, isopropyl myristate etc.

Fatty acid ethyl esters in hair as markers of alcohol consumption. Segmental hair analysis of alcoholics, social drinkers, and teetotalers.

BACKGROUND: Fatty acid ethyl esters (FAEEs) are products of nonoxidative ethanol metabolism. After incorporation in hair, they should be suitable long-term markers of alcohol abuse. METHODS: Hair samples from 19 alcoholics in a treatment program, 10 fatalities with verified excessive alcohol consumption, 13 moderate social drinkers who consumed up to 20 g ethanol/day, and 5 strict teetotalers were analyzed in 1-12 segments for four FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) by external degreasing with n-heptane, extraction with a dimethyl sulfoxide-n-heptane mixture, headspace solid-phase microextraction of the extracts, and gas chromatography-mass spectrometry with deuterated internal standards. The n-heptane washings were analyzed in the same way for FAEEs from the hair surface. RESULTS: The sum of the four ester concentrations in hair calculated for the proximal 0-6 cm segment was 2.5-13.5 ng/mg (mean, 6.8 ng/mg) for the fatalities, 0.92-11.6 ng/mg (mean, 4.0 ng/mg) for 17 of the alcoholics in treatment, 0.20-0.85 ng/mg (mean, 0.41 ng/mg) for the moderate social drinkers, and 0.06-0.37 ng/mg (mean, 0.16 ng/mg) for the teetotalers. In almost all cases the segmental concentrations increased from proximal to distal. There was no agreement between the self-reported drinking histories of the participants and the FAEE concentrations along the hair length. Ethyl oleate was the dominant ester in all samples. CONCLUSIONS: FAEEs are deposited in hair mainly from sebum. Despite large individual differences, FAEE hair concentrations can be used as markers for excessive alcohol consumption with relatively high accuracy.

A simplified and rapid high-performance liquid chromatographic assay for ketoprofen in isopropyl myristate.

A high-performance liquid chromatographic (HPLC) procedure for quantitating ketoprofen in isopropyl myristate (IPM), a compound widely used as a receptor medium in drug diffusion studies of topical aqueous-based formulations, is developed. Previously reported HPLC assays for ketoprofen in IPM have employed relatively complex and tedious methods for purifying the IPM prior to injection onto the HPLC column. The present assay method utilizes a direct injection of the IPM-based sample onto a new reversed-phase ODS column and employs ultraviolet detection at 265 nm. Propyl paraben is employed as the internal standard. The mobile phase consists of acetonitrile-methanol-water (36:54:10, v/v/v) at a flow rate of 1.2 mL/min. The calibration curves are linear (correlation coefficient r > or = 0.988) over concentration ranges of 0.625-10 micrograms/mL and 6.25-100 micrograms/mL. The within-day and between-day precision exhibit coefficients of variation of 1.3-3.3%, and the accuracy (reported as relative error of the mean) varies from -1.9% to 0.6%. The retention times for ketoprofen and propyl paraben are approximately 2.3 and 3.3 min, respectively. The total run time per sample is approximately 7 min. The minimum quantitatable concentration is approximately 0.625 microgram/mL. The assay is stability-indicating, rapid, reproducible, sensitive, and readily adaptable for assaying other non-steroidal anti-inflammatory drugs.

Possible fatty acyl pheromone precursors in Spodoptera littoralis. Search for 11- and 12-hydroxytetradecanoic acids in the pheromone gland.

Lipid extracts of Spodoptera littoralis pheromone glands submitted to acid methanolysis using: (i) sulfuric acid/methanol/benzene (0.1:4:2, by vol) at 90 degrees C for 1 h; (ii) 12 N HCI/methanol (1:2, vol/vol) at 90 degrees C for 1 h, or (iii) 14% BF3-MeOH at 90 degrees C for 1 h did not reveal the presence of either 11- or 12-hydroxytetradecanoic acid in the extracts, as concluded from the gas chromatography-mass spectrometry analyses. Under the above methanolysis conditions, a synthetic sample of methyl (14, 14, 14-2H3) 12-hydroxytetradecanoate remained unaltered. These results may indicate that formation of (E)-11-tetradecenoic acid from tetradecanoic acid does not occur in the pheromone gland by dehydration of an intermediate hydroxyacid. Acid methanolysis of a lipidic extract using BF3-MeOH led to the formation of a mixture of methoxy fatty acid methyl esters, identified by gas chromatography-mass spectrometry. These methoxy derivatives should arise from BF3-catalyzed addition of methanol to the double bond of the natural monounsaturated fatty acyl derivatives present in the gland. Thus, under the same conditions, a synthetic sample of methyl (Z)-11-tetradecenoate was partially transformed into methyl 11-methoxytetradecanoate and methyl 12-methoxytetradecanoate. This reaction might be a useful alternative procedure to obtain methoxy derivatives of olefins, which are very helpful for the structural characterization of the parent alkenes.

[Analysis of the volatile organic acids in 5 kinds of pericarpium Trichosanthis].

The volatile organic acids in the pericarps of Trichosanthes kirilowii, T. rosthornii, T. truncata, T. hupehensis and T. cucumeroides (Cucurbitaceae) were analyzed by methylation, GC and GC-MS-DS. The results showed that they were composed of fifteen long-chain fatty acids, such as palmitic, linolenic, linoleic, lauric, myristic acid, etc.

Evidence of myristylated disulfide-linked dimer of variant surface glycoprotein of Trypanosoma brucei-brucei.

1. Variant surface glycoprotein (VSGs) of Trypanosoma brucei-brucei may exist as a disulfide-linked dimer in both forms: myristylated (mfVSG) and non-myristylated (sVSG), as judge by fluorography and immunoblotting of SDS-PAGE under non-reducing conditions. 2. The dimeric VSG form is labeled with [3H]-myristic acid in our incorporation conditions. 3. AnTat 1.1 trypanosomes preincubated with tunicamycin and incubated with [3H]-myristic acid synthesized a labeled molecule that has an apparent molecular weight slightly smaller than the native form, and that also corresponds to a disulfide-linked dimer.

Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus.

The c-fyn proto-oncogene is a member of a family of closely related genes of which c-src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c-fyn gene product, the c-fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c-src and pp62c-yes, pp59c-fyn is able to form a stable complex with middle-T antigen, the transforming protein of polyomavirus. The transformation-defective middle-T mutant NG59, which is unable to associate stably with pp60c-src does not associate with pp59c-fyn. In contrast to pp60c-src, complex formation with middle-T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c-fyn. These findings lead us to suggest that middle-T mediated transformation may be a consequence of the deregulation of several members of the src-family of protein tyrosine kinases.