Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.

Dietary exposure of the Belgian population to emulsifiers E481 (sodium stearoyl-2-lactylate) and E482 (calcium stearoyl-2-lactylate).

A dietary exposure assessment of food emulsifiers E481 (sodium stearoyl-2-lactylate) and E482 (calcium stearoyl-2-lactylate) in the Belgian population was performed. Nationally representative food consumption data from the Belgian National Food Consumption Surveys 2004 (BNFCS2004) and 2014 (BNFCS2014) were used for calculations. A conservative approach (combining individual food consumption data with the maximum permitted level (MPL) of foods (tier 2), was compared with more refined estimates (combining individual food consumption data with actual concentrations measured in food products available on the Belgian market (tier 3)). Estimated daily intakes were compared to the acceptable daily intake (ADI) of the stearoyl-2-lactylates. The results of tier 2 demonstrated that 92% of the children (3-9 years), 53% of the adolescents (10-17 years), 15% of the adults (18-64 years) and 26% of the elderly (64-98 years) had a potential intake higher than the ADI. When replacing the MPL with maximum analysed concentration levels in foods, daily intake estimates decreased dramatically. The estimated daily intake of the food emulsifiers was below the ADI for all age groups, except for a small percentage of children (1.9%) for which the intake exceeded the ADI. The main contributors to the exposure of E481 and E482 were bread, rolls and fine bakery wares.

Treating murine Kala-azar with a Mayan plant induces immunochemical changes.

Pentalinon andrieuxii Muell Arg is a Mexican-Central American plant anciently used by local people to treat cutaneous leishmaniasis. We evaluated a hexane extract of the root we called PAE for its chemical content and for its immunochemical and in vitro activity against Leishmania donovani and healing of experimental Kala-azar. Chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) identified hexadecanoic acid, hexadecanoic acid ethyl ester, 9, 12-octadecadienoic acid ethyl ester, octadecanoic acid ethyl ester, 9-octadecenoic acid ethyl ester and diethyl phthalate as the main compounds present in PAE. We also demonstrated PAE kills promastigotes and amastigotes in vitro and significantly reduces parasite loads in liver and spleen of infected Balb/c mice. PAE induces expression of NFkB/AP-1 transcription factors and production of IL-2 and IFN-γ by spleen cells of PAE treated but not in the untreated control mice. Furthermore, there were not IL-6, IL-10 nor TNF production in macrophages treated in vitro with PAE. We developed an affordable extract of P. andrieuxii effective to treat experimental Kala-azar in Balb/c mice.

Co-metabolism of substrates by Bacillus thuringiensis regulates polyhydroxyalkanoate co-polymer composition.

Polyhydroxyalkanoate (PHA) production by Bacillus thuringiensis EGU45 was studied by co-metabolism of crude glycerol (CG) (1%, v/v), glucose (0.05-0.5%, w/v) and propionic acid (0.05-0.5%, v/v) under batch (shake flask) culture conditions. Glycerol+PA combination resulted in 15-100mg/L PHA co-polymers with a HV content of 33-81mol%. The addition of NH4Cl (0.5%, w/v) to CG+PA enhanced PHA production by 1.55-fold, with a HV content of 58-70mol%. The time period of incubation of PA to the feed: CG+glucose was optimized to be 3h after initiation of fermentation. The PHA contents were found to be stable at 1900-2050mg/L up scaling from 0.4 to 2.0L feed material. Biochemical characterization through GC-MS of PHA co-polymer revealed the presence of 3-hydroxydecanoate (3-HDD), 3-hydroxyoctadecanoate (3HOD), 3-hydroxyhexadecanoate (3HHD).

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.

Contrasting the crospovidones functionality as excipients for direct compression

Specific values of technological properties of excipients allow the establishment of numerical parameters to define and compare their functionality. This study investigates the functionality of Polyplasdones XL and XL10. Parameters studied included tablet disintegration profiles, compactibility profiles and powder flow. The results allowed the establishment of quantitative surrogate functionalities of technological performance, such as absolute number, and as a value relative to the known microcrystalline cellulose type 102. Moreover, the establishment of an explicit functionality to improve the technological performance of two diluents and a model drug was investigated, as was setting up of these functionalities, as quantitative values, to determine the input variables of each material and its probable functionality in a drug product. Disintegration times of pure Polyplasdone XL and its admixtures were around half that of Polyplasdone XL10. The improvement in tablet compactibility was 25-50% greater for Polyplasdone XL10 than Polyplasdone XL. Crospovidones proportions of up to 10% have little effect on the flow properties of other powders, although pure Polyplasdone XL10 and its admixtures display compressibility indexes about 20% greater than Polyplasdone XL. The observed results are in line with a smaller particle size of Polyplasdone XL10 compared to Polyplasdone XL.

Os valores específicos de propriedades tecnológicas de excipientes permitem o estabelecimento de parâmetros numéricos para definir e comparar a sua funcionalidade. Este estudo investiga a funcionalidade dos excipientes. Os parâmetros estudados foram perfis de desintegração dos comprimidos, perfis de compactação e fluxo de pó. Os resultados permitiram expressar o desempenho tecnológico através de valores absolutos e valores relativos à conhecida celulose microcristalina tipo 102. Do mesmo modo, permitiram estabelecer uma funcionalidade explícita para melhorar o desempenho tecnológico de dois diluentes e um fármaco modelo. A criação destas funcionalidades, como valores quantitativos, permite conhecer as variáveis de entrada de cada material e sua provável funcionalidade em um medicamento. Os tempos de desintegração do Poliplasdone XL e das suas misturas são cerca da metade do observado para as misturas com o Poliplasdone XL10. Melhoria da compressão de comprimidos que contêm Polyplasdone XL10 é 25-50% maior do que o Polyplasdone XL. Crospovidonas em proporções de até 10% têm pouco efeito sobre as propriedades de fluxo dos outros pós embora o Poliplasdone XL10 e suas misturas exibam índices de compressibilidade cerca de 20% maior do que o Poliplasdone XL. Os resultados observados estão em sintonia com o menor tamanho de partícula do Poliplasdone XL10, em comparação com o Poliplasdone XL.

Development of a quantitative GC-FID method for the determination of stearoyl-lactylates (E481/482) in foods.

Sodium and calcium salts of stearoyl-lactylates (SLs) are food emulsifiers especially used in bread and bakery products to improve texture. They should be used at the lowest level at which the desired technological effect is achieved in a specific food category and at amounts not exceeding the maximums set by European Commission Regulation No. 1129/2011. In order to be able to evaluate whether these emulsifiers are used correctly but also to evaluate whether the commercial additive formulations comply with legislation, a quantitative GC-FID method was developed. An internal standard (nonadecanoyl-1-lactylate) was synthesized in-house and pure ester standards were isolated from commercial additive formulations. The method showed a limit of detection of 0.04 and a limit of quantification of 0.12 mg esters ml⁻¹. The commercial additive formulations analysed proved to be complex mixtures of free lactic and fatty acids together with only 50-60% esters. Besides SLs important amounts of palmitoyl-lactylates were present. Different food matrices (with low- and high-fat contents) were spiked with commercial SL formulations and recoveries ranged between 85% and 109%. Determination of SLs in commercial foods (such as bakery and bread) indicated that pre-treatment with amylase was essential to determine accurately the SL content due to the interaction of SL with the amylose.

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.

Free radical mediated formation of 3-monochloropropanediol (3-MCPD) fatty acid diesters.

The present study was conducted to test the hypothesis that a free radical was formed and mediated the formation of 3-monochloropropanediol (3-MCPD) fatty acid diesters, a group of food contaminants, from diacylglycerols at high temperature under a low-moisture condition for the first time. The presence of free radicals in a vegetable oil kept at 120 °C for 20 min was demonstrated using an electron spin resonance (ESR) spectroscopy examination with 5,5-dimethylpyrroline-N-oxide (DMPO) as the spin trap agent. ESR investigation also showed an association between thermal treatment degree and the concentration of free radicals. A Fourier transform infrared spectroscopy (FT-IR) analysis of sn-1,2-stearoylglycerol (DSG) at 25 and 120 °C suggested the possible involvement of an ester carbonyl group in forming 3-MCPD diesters. On the basis of these results, a novel free radical mediated chemical mechanism was proposed for 3-MCPD diester formation. Furthermore, a quadrupole-time of flight (Q-TOF) MS/MS investigation was performed and detected the DMPO adducts with the cyclic acyloxonium free radical (CAFR) and its product MS ions, proving the presence of CAFR. Furthermore, the free radical mechanism was validated by the formation of 3-MCPD diesters through reacting DSG with a number of organic and inorganic chlorine sources including chlorine gas at 120 and 240 °C. The findings of this study might lead to the improvement of oil and food processing conditions to reduce the level of 3-MCPD diesters in foods and enhance food safety.

Aerobic biodegradation of 8:2 fluorotelomer stearate monoester and 8:2 fluorotelomer citrate triester in forest soil.

Aerobic biodegradation of 8:2 fluorotelomer stearate (FTS) and 8:2 fluorotelomer citrate triester (TBC) was evaluated in a forest soil in closed bottle microcosms. Loss of parent, production of 8:2 fluorotelomer alcohol (8:2 FTOH), which is released along with stearic acid (SA) by microbial ester linkage, and subsequent metabolites from FTOH degradation were monitored for up to 7months. Soil microcosms were extracted with ethyl acetate followed by two heated 90/10 v/v acetonitrile/200mM NaOH extractions. Cleavage of the ester linkage in the 8:2 FTS occurred (t1/2∼28d), producing 8:2 FTOH and various levels of subsequent metabolites. Quantifying the generation of SA from ester cleavage in FTS was complicated by the natural production and degradation of SA in soil, which was probed in an additional FTS and SA study with the same soil that had been stored at 4°C for 12months. In the latter study, FTS degraded faster (t1/2∼5d) such that SA production well above soil background levels was clearly observed along with rapid subsequent SA degradation. Cold storage was hypothesized to enrich fungal enzymes, which are known to be effective at hydrolytic cleavage. 8:2 TBC biotransformation was slow, but evident with the production of PFOA well above levels expected from known FTOH residuals. Slower degradation of TBC compared to FTS is likely due to steric hindrances arising from the close proximity of three 8:2 FT chains on the citrate backbone limiting the enzyme access.

Mass spectrometry analysis of influenza virus reassortant clones does not reveal an influence of other viral proteins on S-acylation of hemagglutinin.

Hemagglutinin (HA) of influenza virus is S-acylated with stearate at a transmembrane cysteine and with palmitate at two cytoplasmic cysteines. The amount of stearate varies from 35 (in avian strains) to 12% (in human strains), although the acylation region exhibits only minor or even no amino acid differences between HAs. To address whether matrix proteins and neuraminidase affect stearoylation of HA, we used mass spectrometry to analyze laboratory reassortants containing avian virus HA and the internal proteins from a human virus. Only minor fluctuations in the amount of stearate were observed, implying that other viral proteins do not affect acylation of HA.

[LC-MS analysis of commercial sodium stearoyl lactylate components].

The components of commercial sodium stearoyl lactylate (SSL), purchased in Japan, were determined and identified using thin layer chromatography (TLC) and liquid chromatography with mass spectroscopy (LC-MS). Stearoyl lactate (SL) and stearoyl-2-lactylate (SLL) were purified using TLC and silica gel chromatography to obtain standards. The results show that SSL consisted of lactic acid (8.4%), stearic acid (15%), SL (57%), and SLL (13%). The total amounts of free lactic acid, lactic acid derived from SL and lactic acid derived from SLL were determined using LC-MS. The mean value was approximately equal to that determined using the JECFA method. This is the first study to determine and identify the components of SSL purchased in Japan, using TLC and LC-MS.

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.

An infrared spectroscopic method for quantitative analysis of fatty alcohols and fatty acid esters in machinery oils.

A new infrared spectroscopic method suitable for determining total fatty alcohol and fatty acid ester concentrations in industrial oils has been developed. Oil samples were diluted with toluene (1:3 w/w), the toxicity and volatility of which are relatively low compared with more commonly used IR solvents, like carbon tetrachloride or carbon disulfide. Mixture standards were prepared from dodecanol, tetradecanol, octadecanol, methyl stearate and methyl palmitate. Some analytical and statistical tests were performed on the developed method. The recoveries and the repeatability of the method proved to be sufficient for the quantitative determination of fatty alcohol and fatty acid ester additives in industrial oils. Reproducibility testing in another laboratory also produced satisfactory results. The developed method also proved to be relatively quick and simple. This method was developed to satisfy industry's need to determine the concentrations of these oil additives, and it has already been applied successfully in machinery oil analysis.

Langmuir monolayer approaches to protein recognition through molecular imprinting.

Surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM) have been employed to investigate ferritin adsorption to binary surfactant monolayers of cationic dioctadecyldimethylammonium bromide (DOMA) and non-ionic methyl stearate (SME). Surfactant molar ratios, miscibility, and lateral mobility were controlled to define the number, size, and distribution of "binding sites" for ferritin, which under the low ionic strength conditions investigated, adsorbed to the monolayers predominantly through electrostatic interactions. Successive adsorption/desorption cycles revealed that fluid monolayers, capable of laterally restructuring during the initial protein adsorption event, bound up to 60% more ferritin (dependent on SME:DOMA ratios) as compared to monolayers that were immobilized on a hydrophobic support during this first adsorption step. The enhanced binding of ferritin to fluid monolayers was accentuated in films having non-ionic SME as the principal component. These findings support the premise that the surfactants reorganize to form favorable interactions with an adsorbing protein, leading to protein specific charge patterns, or templates, in the films. Template assessment, however, was complicated by the presence of an irreversibly bound protein fraction, which AFM revealed to be locally ordered protein clusters.

L-ascorbic acid microencapsulated with polyacylglycerol monostearate for milk fortification.

Efficiency was examined of microencapsulating L-ascorbic acid by polyglycerol monostearate (PGMS), and changes in the chemical and sensorial aspects of L-ascorbic acid and/or iron-fortified milk during storage were evaluated. The selected core materials were ferric ammonium sulfate and L-ascorbic acid. The highest efficiency (94.2%) of microencapsulation was found with the ratio of 5:1 as the coating to core material. The release of ascorbic acid from the microcapsules increased sharply from 1.6 to 6.7% up to 5 d of storage. The TBA value was the lowest in the milk sample with added encapsulated iron and unencapsulated L-ascorbic acid up to 5 d of storage in comparison with the other treated samples. A sensory analysis showed that most aspects were not significantly different between the control and fortified samples encapsulated with ascorbic acid after 5 d of storage. The results indicate that L-ascorbic acid microencapsulated with PGMS can be applied to fortify milk and acceptable milk products can be prepared with microencapsulated L-ascorbic acid and iron.

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.