Lipids of Chlamydomonas reinhardtii. Incorporation of [14C]Acetate, [14C]Palmitate and [14C]Oleate into Different Lipids and Evidence for Lipid-Linked Desaturation of Fatty Acids

Chlamydomonas reinhardtii, parent strain (ssf), was pulse-labelled with [14C]acetate, [14C]-palmitate or [14C]oleate. Lipids were separated by TLC and HPLC. Radioactivity was measured in each class of lipids and in its fatty acids and molecular species. After 1 hour of incubation with acetate, the label was incorporated mainly into phosphatidylglycerol (PG), diacylglyceryl(N,N,N-trimethyl)homoserine (DGTS), digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG). Saturated, monoene and diene fatty acids were strongly labelled. Within 10 hours of incubation in the absence of labelled precursor, the label shifted from monoenes and dienes to trienes and tetraenes. The transfer of radioactivity from mono- to polyunsaturated MGDG and DGDG molecular species suggests a lipid-linked desaturation of the C-l position (and, in MGDG, also of the C-2 position) of these prokaryotic lipids. In the eukaryotic DGTS, all the species present were labelled simultaneously. On incubation with [14C]-palmitate or [14C]oleate, most of the label appeared in DGTS. Palmitate was immediately incorporated into the polyene species of DGTS, while oleate first appeared in the monoene species and then shifted to the polyene species. From these results it is concluded that, in DGTS, the acyl groups in the C-l position (mostly 16:0) were rapidly exchanged, while those in the C-2 position (mostly C18) became desaturated to give 18:3(5,9,12) and 18:4(5,9,12,15) acid

Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These cannabinoids include ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), ∆9-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-∆9-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and ∆9-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal™ device. The advantages of performing a liquid-liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5min on a Kinetex™ column packed with 2.6-μm core-shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000™ triple quadrupole LC-MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50pg/ml and from 500 to 80pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method's LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collectio

Lipids of Chlamydomonas reinhardtii. Analysis of Molecular Species and Intracellular Site(s) of Biosynthesis

Membrane lipids of Chlamydomonas reinhardtii were separated into the major components, MGDG, DGDG, SQDG, DGTS, PG, PE, PI, and the molecular species of each lipid were isolated and analyzed. The fatty acid composition was determined for the total lipid, the particular lipid classes and the molecular species. The positional distribution of fatty acids between the C-1 and C-2 position of the glycerol moiety was also determined. MGDG, DGDG, SQDG, PG and probably PI were found to be of plastidic (prokaryotic) origin, while DGTS and PE were found to be of cytoplasmic (eukaryotic) origin. Prokaryotic lipids mainly contained 18:3(9,12,15), 18:2,16:4 and 16:3, while DGTS and PE were rich in 18:3(5,9,12), 18:4(5,9,12,15), 18:2 and 18:1(11) fatty acids. From the fact that each lipid class was characterized by an individual pattern of molecular species, we conclude that during their biosynthesis, all the lipids act individually as substrates for the lipid-linked desaturation of fatty acids. Moreover, our results suggest that in Chlamydomonas, 18:3(5,9,12) and 18:4(5,9,12,15) are formed in the cytoplasm using DGTS and PE as substrate

Mesure des modules viscoélastiques d'un polypropylène lors d'un refroidissement et d'un changement de phase

International audienceLa rhéologie d'un polypropylène de grade injection a été mesurée sur un rhéomètre plan - plan lors d'un refroidissement depuis l'état fondu jusqu'au début de la solidification. Ce type de donnée est important en particulier lorsque l'on s'intéresse aux contraintes résiduelles prenant naissance à la solidification. Un outillage plan - plan rainuré a été monté sur un rhéomètre asservi en contrainte. Le rainurage limite le phénomène de glissement qui interviendrait sur un matériau solidifié. On a ainsi pu mesurer les températures de transition lors de la cristallisation. Un point de gel a pu être mis en évidence au début de la cristallisation pour des vitesses de refroidissement de 2°C par minute. Le dispositif a également pu être évalué dans le sens de la fusion du matériau solidifié. Des expériences complémentaires ont été conduites en calorimétrie pour valider les températures de transitions obtenues.Des vitesses de refroidissement plus élevées ont permis de mesurer l'évolution des modules en fonction des conditions de refroidissement = The rheology of an injection grade polypropylene was measured in a plate plate rheometer during cooling from the melt to the beginning of solidification. These results are particularly important when we are interested by the construction of residual stresses during solidification. A grooved tool was mounted on a tresscontrolled rheometer. The grooves limit the slippage which happened on a solid. We measured the transition temperature during crystallisation. A gel point is observed for a cooling rate of 2°C by minute. The fixture is also evaluated for melting. Differential Scanning Calorimetry was also used for validation of the transition temperatures. Higher cooling rates permitted the measurement of modulus versus cooling conditions

A Fatal Overdose of Cocaine Associated with Coingestion of Marijuana, Buprenorphine, and Fluoxetine. Body Fluid and Tissue Distribution of Cocaine and Its Metabolites Determined by Hydrophilic Interaction Chromatography-Mass Spectrometry (HILIC-MS)

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 μL pure acetonitrile and 5 μL was directly injected onto a Waters Atlantis™ HILIC 150- × 2.1-mm, 3-μm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below ± 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 μg/L) were also discovered. A relatively high concentration of Δ9-THC was measured both in peripheral blood (8.2 μg/L) and brain cortex (13.5 μg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoid

A systematic review of passive exposure to cannabis

Passive exposure to cannabis smoke may induce effects on behavior and psychomotor skills, and have legal consequences, including the risk of being falsely considered as a cannabis user. This can become a concern, especially in occupational contexts or when driving vehicles. In order to enable a differentiation between a passive and an active exposure to cannabis and to limit the likeliness to be detected positive following passive exposure, this review identified specific biomarkers of passive exposure in urine, blood, oral fluid, hair, and sebum. Out of 958 papers identified on passive exposure to cannabis, 21 were selected. Although positive tests had been observed in all matrices following extremely high passive exposure, some distinctive features were observed in each matrix compared to cannabis active use. More specifically, in everyday life conditions, 11-nor-delta-9-THC-carboxylic acid (THC-COOH) urinary level should be detected below the positivity threshold used to confirm active smoking of cannabis, especially after normalization to creatinine level. Measuring delta-9-tetrahydrocannabinol (THC) and THC-COOH in blood is an appropriate alternative for appraising passive exposure as low and very low concentrations of THC and THC-COOH, respectively, should be measured. In hair, oral fluid (OF) and sweat/sebum emulsion, no THCCOOH should be detected. Its presence in hair argues for regular cannabis consumption and in OF or sweat for recent consumption. The experts should recommend to persons who have to demonstrate abstinence from cannabis to avoid heavily smoky and unventilated environments

Codeine accumulation and elimination in larvae, pupae, and imago of the blowfly Lucilia sericata and effects on its development

The aim of this study was to evaluate the reliability of insect larvae as samples for toxicological investigations. For this purpose, larvae of Lucilia sericata were reared on samples of minced pig liver treated with different concentrations of codeine: therapeutic, toxic, and potentially lethal doses. Codeine was detected in all tested larvae, confirming the reliability of these specimens for qualitative toxicology analysis. Furthermore, concentrations measured in larvae were correlated with levels in liver tissue. These observations bring new elements regarding the potential use of opiates concentrations in larvae for estimation of drug levels in human tissues. Morphine and norcodeine, two codeine metabolites, have been also detected at different concentrations depending on the concentration of codeine in pig liver and depending on the substance itself. The effects of codeine on the development of L. sericata were also investigated. Results showed that a 29-h interval bias on the evaluation of the larval stage duration calculated from the larvae weight has to be considered if codeine was present in the larvae substrate. Similarly, a 21-h interval bias on the total duration of development, from egg to imago, has to be considered if codeine was present in the larvae substrat