Phospholipase D1 activity is crucial for cytosolic phospholipase A2 -dependent prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells.

In bone, prostaglandin E2 (PGE2) is highly osteogenic and formed by osteoblasts, a key modulatory event in the regulation of bone cell activity. MC3T3-E1 cells are widely used as an in vitro model of osteoblast function. It is still not clear which pathways contribute to the release of AA in these cells. In this study we have focussed on the contribution of phospholipase D (PLD) enzymes to osteoblastic PGE2 formation after stimulation with endothelin-1 (ET-1). Using specific inhibitors of PLD1 and PLD2 we could show that PGE2 formation was strictly dependent on PLD1 but not PLD2 activity and cytosolic phospholipase A2 (cPLA2) was activated by triggering through PLD1. We have identified diacyl glycerol (DAG) as a possible effector molecule which may serve as a triggering signal for PKC activation and subsequent cPLA2 phosphorylation.

Identification and Characterization of the Stability of Hydrophobic Cyclolinopeptides From Flaxseed Oil.

Flaxseed (linseed) is a cultivar of the spring flowering annual plant flax (Linum usitatissimum) from the Linaceae family. Derivatives of this plant are widely used as food and as health products. In recent years, cyclic peptides isolated from flaxseed and flaxseed oil, better known as cyclolinopeptides (CLPs), have attracted the attention of the scientific community due to their roles in the inhibition of osteoclast differentiation or their antimalarial, immunosuppressive, and antitumor activities, as well as their prospects in nanotechnology and in the biomedical sector. This study describes the detection, identification, and measurement of CLPs in samples obtained from nine different flaxseed oil manufacturers. For the first time, Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer was used for CLP identification together with RP-HPLC. The routine analyses were performed using RP chromatography, measuring the absorption spectra and fluorescence detection for identifying tryptophan-containing peptides using the native fluorescence of tryptophan. In addition, existing protocols used for CLP extraction were optimized and improved in a fast and cost-efficient way. For the first time, 12 CLPs were separated using methanol/water as the eluent with RP-HPLC. Finally, the stability and degradation of individual CLPs in the respective flaxseed oil were examined over a period of 60 days at different temperatures. The higher temperature was chosen since this might reflect the cooking practices, as flaxseed oil is not used for high-temperature cooking. Using HPLC-MS, 15 CLPs were identified in total in the different flaxseed oils. The characterization of the peptides via HPLC-MS highlighted two types of CLP profiles with a substantial variation in the concentration and composition of CLPs per manufacturer, probably related to the plant cultivar. Among the observed CLPs, CLP-O, CLP-N, and CLP-B were the least stable, while CLP-C and CLP-A were the most stable peptides. However, it is important to highlight the gradual degradation of most of the examined CLPs over time, even at room temperature.

A Novel Class of Defensive Compounds in Harvestmen: Hydroxy-γ-Lactones from the Phalangiid Egaenus convexus.

When threatened, the harvestman Egaenus convexus (Opiliones: Phalangiidae) ejects a secretion against offenders. The secretion originates from large prosomal scent glands and is mainly composed of two isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1), a ß-hydroxy-γ-lactone. The compounds were characterized by GC-MS of their microreaction derivatives, HRMS, and NMR. After the synthesis of all four possible stereoisomers of 1, followed by their separation by chiral-phase GC, the absolute configurations of the lactones in the Egaenus secretion was found to be (4S,5R)-1 (90%) and (4S,5S)-1 (10%). Hydroxy-γ-lactones represent a new class of exocrine defense compounds in harvestmen.

Methyl-ketones in the scent glands of Opiliones: a chemical trait of cyphophthalmi retrieved in the dyspnoan Nemastoma triste.

The homologous and phylogenetically old scent glands of harvestmen-also called defensive or repugnatorial glands-represent an ideal system for a model reconstruction of the evolutionary history of exocrine secretion chemistry ("phylogenetic chemosystematics"). While the secretions of Laniatores (mainly phenols, benzoquinones), Cyphophthalmi (naphthoquinones, chloro-naphthoquinones, methyl-ketones) and some Eupnoi (naphthoquinones, ethyl-ketones) are fairly well studied, one open question refers to the still largely enigmatic scent gland chemistry of representatives of the suborder Dyspnoi and the relation of dyspnoan chemistry to the remaining suborders. We here report on the secretion of a nemastomatid Dyspnoi, Nemastoma triste, which is composed of straight-chain methyl-ketones (heptan-2-one, nonan-2-one, 6-tridecen-2-one, 8-tridecen-2-one), methyl-branched methyl-ketones (5-methyl-heptan-2-one, 6-methyl-nonan-2-one), naphthoquinones (1,4-naphthoquinone, 6-methyl-1,4-naphthoquinone) and chloro-naphthoquinones (4-chloro-1,2-naphthoquinone, 4-chloro-6-methyl-1,2-naphthoquinone). Chemically, the secretions of N. triste are remarkably reminiscent of those found in Cyphophthalmi. While naphthoquinones are widely distributed across the scent gland secretions of harvestmen (all suborders except Laniatores), methyl-ketones and chloro-naphthoquinones arise as linking elements between cyphophthalmid and dyspnoan scent gland chemistry.

Ionomycin induces prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells via mechanisms independent of its ionophoric nature.

Ionomycin and A23187 are divalent cation ionophores with a marked preference for calcium. Studies using these ionophores have almost exclusively interpreted their results in the light of calcium elevation. It was the aim of this study to investigate the effects of ionomycin in osteoblatic MC3T3-E1 cells that are not attributable to its ionophoric properties. Thus, we have found that in contrast to A23187, ionomycin shows similar effects on prostaglandin E2 formation as bradykinin and endothelin-1, being potentiated by extracellular nickel and inhibited by cholera toxin and pertussis toxin. Our data strongly suggest that inomycin, at least in part, exerts its effects via specific binding to a G-protein coupled receptor, thereby evoking downstream cellular events like arachidonate release with subsequent prostaglandin formation.

"Quinone Millipedes" Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions across the Julidae.

The defensive chemistry of juliformian millipedes is characterized mainly by benzoquinones ("quinone millipedes"), whereas the secretions of the putative close outgroup Callipodida are considered to be exclusively phenolic. We conducted a chemical screening of julid secretions for phenolic content. Most species from tribes Cylindroiulini (15 species examined), Brachyiulini (5 species examined), Leptoiulini (15 species examined), Uncigerini (2 species examined), Pachyiulini (3 species examined), and Ommatoiulini (2 species examined) had non-phenolic, in most cases exclusively benzoquinonic secretions. In contrast, tribes Cylindroiulini, Brachyiulini, and Leptoiulini also contained representatives with predominantly phenol-based exudates. In detail, p-cresol was a major compound in the secretions of the cylindroiulines Styrioiulus pelidnus and S. styricus (p-cresol content 93 %) and an undetermined Cylindroiulus species (p-cresol content 51 %), in the brachyiulines Brachyiulus lusitanus (p-cresol content 21 %) and Megaphyllum fagorum (p-cresol content 92 %), as well as in an undescribed Typhloiulus species (p-cresol content 32 %, Leptoiulini). In all species, p-cresol was accompanied by small amounts of phenol. The secretion of M. fagorum was exclusively phenolic, whereas phenols were accompanied by benzoquinones in all other species. This is the first incidence of clearly phenol-dominated secretions in the Julidae. We hypothesize a shared biosynthetic route to phenols and benzoquinones, with benzoquinones being produced from phenolic precursors. The patchy taxonomic distribution of phenols documented herein supports multiple independent regression events in a common pathway of benzoquinone synthesis rather than multiple independent incidences of phenol biosynthesis.

Calcium-independent phospholipases A2 in murine osteoblastic cells and their inhibition by bromoenol lactone: impact on arachidonate dynamics and prostaglandin synthesis.

CONTEXT: Bromoenol lactone (BEL) is an inhibitor of group VI phospholipases (iPLA2s), but has been shown to have severe side effects. OBJECTIVE: iPLA2 characterization in osteoblasts and effect of BEL on prostaglandin (PG) E2 formation. METHODS: iPLA2 expression: RT-PCR, Western Blotting. PGE2 formation: GC-MS after stimulation, treatment with inhibitors or gene silencing. Arachidonate (AA) reacylation into phospholipids, inhibitor reaction products, PGHS-1 modification proteomic analysis: HR-LC-MS/MS. AA accumulation: (14)C-AA. RESULTS: iPLA2ß and iPLA2γ were expressed and functionally active. BEL inhibition up to 20 µM caused AA accumulation and enhanced PGE2 formation, followed by a decrease at higher concentrations. BEL reacted with intracellular cysteine and GSH leading to GSH depletion and oxidative stress. DISCUSSION: Initial PGE2 enhancement after BEL inhibition is due to iPLA2-independent accumulation of AA. GSH depletion caused by high BEL concentrations is responsible for the decrease in PGE2 production. CONCLUSION: BEL must be used with caution in a cellular environment due to conditions of extreme oxidative stress.

Potentiation of endothelin-1-induced prostaglandin E2 formation by Ni(2+) and Sr(2+) in murine osteoblastic MC3T3-E1 cells.

Cation recognition mechanisms beyond calcium-sensing receptors are still largely unexplored and consequently there is surprisingly little information on linking of this primary event to key metabolic features of different cell systems, such as arachidonic acid metabolism. However, information on the modulatory role of extracellular cations in cellular function is scarce. In this study we have demonstrated, that Ni(2+) and Sr(2+) potentiate endothelin-1 induced prostaglandin E2 formation in the osteoblastic cell line, MC3T3-E1, even in the absence of extracellular calcium. The effect is strictly dependent of receptor-mediated signal transduction processes evoked by endothelin-1 and arachidonate release involves cytosolic phospholipase A2 activity. The ligation sites, at least for Ni(2+) are extracellular. The data suggest a novel activation mechanism for arachidonate release and subsequent prostaglandin formation that does not require calcium.

Chemical basis of unwettability in Liacaridae (Acari, Oribatida): specific variations of a cuticular acid/ester-based system.

Oribatid mites of the family Liacaridae comprise a large number of species with smooth and shiny body surfaces that display extraordinary anti-wetting properties. The principle of liacarid unwettability is not related to micro-structured surfaces as present in many Oribatida ("Lotus effect") but the formation of raincoat-like lipid layers covering the epicuticle. We here conducted a comparative study on the chemistry of cuticular lipid layers in a selection of Liacaridae, including representatives of all major Central European genera, Liacarus, Dorycranosus, Adoristes, and Xenillus. Cuticular lipids of unwettable individuals were removed from mite bodies by hexane extraction, and were analyzed by GC-MS. Basically, two chemically distinguishable systems were found. Type I: cuticular lipids of Liacarus subterraneus, L. coracinus, L. nitens, Dorycranosus curtipilis, and Xenillus tegeocranus contained different carboxylic acids (C8-, C10-, C10:1-, C10:2-acids) and their corresponding di-glycerides in species-specific combinations. Type II: Adoristes ovatus exhibited a system of cuticular lipids composed of esters of pentanoic- and heptanoic acids with C14-, C15-, C16- and C17-alcohols. Interestingly, the chemistry of surface lipids did not reflect the morphology of the cuticle in the species investigated. Smooth and shiny cuticles, though exhibiting a specific pattern of round or slit-like pores, were found in representatives of Liacarus, Dorycranosus (all of which exhibiting cuticular chemistry of type I) and Adoristes (exhibiting cuticular chemistry of type II). Xenillus, possessing a rough, cerotegumental cement layer-covered surface, showed type I-chemistry. The acid-esters systems herein investigated are considered characteristic for the cuticular chemistry of Liacaridae or a lineage of these, and provide first insights into the comparative chemistry of the inner (=lipid) layer of the oribatid cerotegument.

Molecular characterisation of group IVA (cytosolic) phospholipase A2 in murine osteoblastic MC3T3-E1 cells.

Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell activity, is preceded by release of the precursor arachidonic acid from phospholipid stores. The main routes of arachidonate liberation may involve phospholipase enzymes such as group IVA phospholipase A2 which is believed to be the main effector in many cell system due to its preference for arachidonate-containing lipids. MC3T3-E1 cells are non-transformed osteoblasts and are widely used as an in vitro model of osteoblast function. In these cells there is still no clarity about the main release pathway of arachidonic acid. Besides cytosolic phospholipase A2, phospholipase C and D pathways may play a key role in arachidonate release. Despite the crucial role of osteoblastic prostgalandin synthesis information on the occurrence of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. We have characterised group IVA phospholipase A2 at the mRNA in these cells as a constitutively expressed enzyme which is cytosolic and translocates to the membrane upon endothelin-1 stimulation. Using immunopurification combined with Western blotting and high-resolution mass spectrometry, the enzyme was also identified at the protein level. Using specific gene silencing we were able to show that osteoblastic cytosolic phospholipase A2 is crucially involved in ET-1-induced prostaglandin formation.

On the enigmatic scent glands of dyspnoan harvestmen (Arachnida, Opiliones): first evidence for the production of volatile secretions.

While considerable knowledge on the chemistry of the scent gland secretions from the opilionid suborders Laniatores and Cyphophthalmi has been compiled, it is the Palpatores (Eupnoi and Dyspnoi) where chemical data are scarce. In particular, the Dyspnoi have remained nearly unstudied, mainly due to their reported general reluctance to release secretions as well as to the phenomenon of production of insoluble-and inaccessible-solid secretion. We here show that at least certain nemastomatid Dyspnoi, namely all three species of genus Carinostoma, indeed produce a volatile secretion, comprising octan-3-one, 6-methyl-5-hepten-2-one and acetophenone in species-specific combinations. In all Carinostoma spp., these volatiles are embedded in a semi-volatile, naphthoquinone matrix (mainly 1,4-naphthoquinone and 6-methyl-1,4-naphthoquinone). In detail, acetophenone and traces of naphthoquinones characterize the secretions of Carinostoma carinatum. A mixture of octan-3-one, 6-methyl-5-hepten-2-one and large amounts of naphthoquinones were found in C. elegans, and 6-methyl-5-hepten-2-one together with small amounts of naphthoquinones in the secretions of C. ornatum. So far, exclusively naphthoquinones had been reported from a single dyspnoan hitherto studied, Paranemastoma quadripunctatum.

Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro.

2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood-brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting.

LPA-induced suppression of periostin in human osteosarcoma cells is mediated by the LPA(1)/Egr-1 axis.

Lysophosphatidic acid (LPA), a naturally occurring bioactive phospholipid, mediates a multitude of (patho)physiological events including activation of mitogen-activated protein kinases (MAPKs). As LPA may induce cellular reponses in human osteosarcoma, the present study aimed at investigating expression of various LPA receptors, LPA-mediated activation of MAPK via G-protein coupling, and expression of early response genes in a cellular model for human osteosarcoma. We show that MG-63 cells express three members of the endothelial differentiation gene (Edg) family of G-protein coupled receptor transcripts (LPA(1-3)) but only two (LPA(4/5)) out of three members of the non-Edg family LPA receptor transcripts. Stimulation of MG-63 cells with LPA or synthetic LPA receptor agonists resulted in p42/44 MAPK phosphorylation via LPA(1)-LPA(3) receptors. Using pharmacological inhibitors, we show that LPA-mediated phosphorylation of p42/44 MAPK by LPA receptor engagement is transmitted by G(αi)-dependent pathways through the Src family of tyrosine kinases. As a consequence, a rapid and transient upregulation of the zinc finger transcription factor early growth response-1 (Egr-1) was observed. Egr-1 expression was strictly mediated via G(αi)/Src/p42/44 MAPK pathway; no involvement of the G(αq/11)/PLC/PKC or the PLD/PI3 kinase/Akt pathways was found. LPA-induced expression of functional Egr-1 in MG-63 cells could be confirmed by electrophoretic mobility shift assay. LPA-induced Egr-1 upregulation was accompanied by a time-dependent decrease of periostin (previously called osteoblast-specific factor 2), a cell adhesion protein for pre-osteoblasts. Silencing of LPA(1) and/or Egr-1 in MG-63 cells reversed LPA-mediated suppression of periostin. We here demonstrate a crosslink between Egr-1 and periostin in cancer cells, in particular in human osteosarcoma.

Discrimination of Oribotritia species by oil gland chemistry (Acari, Oribatida).

The chemical composition of secretions from opisthonotal (oil) glands in four species of the oribatid mite genus Oribotritia (Mixonomata, Euphthiracaroidea, Oribotritiidae) was compared by means of gas chromatography--mass spectrometry. The secretions of all, O. banksi (from North America) and three Austrian oribotritiids (O. berlesei, O. hermanni, O. storkani), are shown to be based on certain unusual compounds, the iridoid monoterpenes chrysomelidial and epi-chrysomelidial and the diterpene ß-springene. These components probably represent general chemical characteristics of oribotriid oil glands. Their relative abundance in the secretions along with further components (mainly saturated and unsaturated C(13)-, C(15)-, C(17)-hydrocarbons, and the tentatively identified octadecadienal) led to well-distinguishable, species-specific oil gland secretions profiles. In addition a reduced set of "Astigmata compounds" (sensu Sakata and Norton in Int J Acarol 27:281-291, 2001)--namely the two monoterpenes neral and geranial--could be detected in extracts of O. banksi nevertheless indicating the classification of euphthiracaroids within the (monophyletic) group of "Astigmata compounds-bearing"-Oribatida. These compounds are considered to be apomorphically reduced in all Austrian species. Our findings emphasize the potential of chemosystematics using oil gland secretion profiles in the discrimination of morphologically very similar, syntopically living or even cryptic oribatid species.

A versatile electrophoric derivatisation reagent for negative ion chemical ionisation mass spectrometry: o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride.

The synthesis of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride, is described. The reagent was tested against selected primary and secondary amino compounds, as well as phenolic and aliphatic hydroxyl compounds as analytical targets. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. Since the reagent bears a pentafluorobenzyl ester group, resulting negative ion chemical ionisation mass spectra were expectedly dominated by dissociative resonance electron capture typically observed with these compounds, additionally showing neutral loss of carbon dioxide and ammonia (in the case of primary amines). The reagent is suitable for detecting the target compounds with high sensitivity, as exemplified for the analysis of amphetamine and methylphenidate from human plasma where chromatographic background is drastically reduced by a shift in detected m/z and retention time and lower limits of quantification at 7.8 pg/mL (amphetamine) and 4.5 pg/mL (methylphenidate) can be obtained. The choice of two or three target quantification masses allows selective detection and adjustment of lowest background interference. No carryover effect was observed for the derivatives of amphetamine and methylphenidate.

15-Deoxy-Δ12,14-prostaglandin J2 induces Cox-2 expression in human osteosarcoma cells through MAPK and EGFR activation involving reactive oxygen species.

Prostaglandins (PGs), important modulators in bone biology, may also contribute to tumor formation and progression in human osteosarcoma. 15-Deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), a metabolite of PGD(2) and PPARγ-ligand, exerts a panel of biological activities via receptor-dependent and -independent mechanisms. As inducible cyclooxygenase-2 (Cox-2) is a candidate inflammatory marker in human osteosarcoma and a rate-limiting enzyme in PG biosynthesis, this study aimed at investigating intracellular redox status and signaling cascades leading to Cox-2 induction in human MG-63 osteosarcoma cells. 15d-PGJ(2) induced the accumulation of reactive oxygen species (ROS) that in turn may lead to upregulation of Cox-2 via two different routes in a PPARγ-independent manner. First, phosphorylation of p38 MAPK directly enhances Cox-2 expression by promoting mRNA stability. Second, 15d-PGJ(2) induces activation of epidermal growth factor receptors and downstream activation of Cox-2 via phosphorylation of p42/44 MAPK. Glutathione precursor molecules reversed enhanced ROS levels and Cox-2 expression. Functional activity of Cox-2 expression was tested by measurement of PGE(2) and PGF(2α). The synthetic compound 9,10-dihydro-15d-PGJ(2) lacking the α,ß-unsaturated carbonyl group in the cyclopentenone ring did not exhibit the cellular responses observed with 15d-PGJ(2). We conclude that the electrophilic carbon atom of 15d-PGJ(2) is responsible for alterations in intracellular redox status and Cox-2 expression.

Mouse brain plasmalogens are targets for hypochlorous acid-mediated modification in vitro and in vivo.

Plasmalogens, 1-O-alk-1'-enyl-2-acyl-sn-glycerophospholipids, are significant constituents of cellular membranes and are essential for normal brain development. Plasmalogens, which contain a vinyl ether bond at the sn-1 position, are preferential targets for hypochlorous acid (HOCl), generated by myeloperoxidase (MPO) from H(2)O(2) and chloride ions. Because MPO is implicated in neurodegeneration, this study pursued two aims: (i) to investigate the reactivity of mouse brain plasmalogens toward HOCl in vitro and (ii) to obtain in vivo evidence for MPO-mediated brain plasmalogen modification. Liquid chromatography coupled to hybrid linear ion trap-Fourier transform-ion cyclotron resonance mass spectrometry revealed plasmalogen modification in mouse brain lipid extracts at lower HOCl concentrations as observed for diacylphospholipids, resulting in the generation of 2-chloro fatty aldehydes and lysophospholipids. Lysophosphatidylethanolamine accumulation was transient, whereas lysophosphatidylcholine species containing saturated acyl residues remained stable. In vivo, a single, systemic endotoxin injection resulted in upregulation of cerebral MPO mRNA levels to a range comparable to that observed for tumor necrosis factor-α and cyclooxygenase-2. This inflammatory response was accompanied by a significant decrease in several brain plasmalogen species and concomitant in vivo generation of 2-chlorohexadecanal. The present findings demonstrate that activation of the MPO-H(2)O(2)-chloride system under neuroinflammatory conditions results in oxidative attack of the total cerebral plasmalogen pool. As this lipid class is indispensable for normal neuronal function, HOCl-mediated plasmalogen modification is likely to compromise normal synaptic transmission.

Wearing a raincoat: exocrine secretions contain anti-wetting agents in the oribatid mite, Liacarus subterraneus (Acari: Oribatida).

Liacarus subterraneus is a large, soil-dwelling oribatid mite species that possesses a conspicuously shiny, clean and not wettable cuticular surface. The exocrine cuticular chemistry of this species was investigated by means of gas chromatography-mass spectrometry. Besides a fraction of hydrocarbons and a terpene, hexane extracts of whole mite bodies exhibited free carboxylic acids and their glycerides as main components. The compounds were arranged in three distinct extract profiles. Based on data from individual extracts, (1) the majority (more than 3/4) of specimens showed large amounts of 1,2-dioctanoyl-glycerol (and three other related esters) but no (or only traces of) free carboxylic acids. (2) In about 1/8 of extracts, free acids (mainly octanoic (caprylic) acid) and glycerides were detected. This second type of profile highly varied with respect to the relative abundance of acids and esters. (3) The third profile (in about 7% of specimens) exclusively exhibited free acids and no (or only traces of) glycerides. In addition, a few extracts exhibited no components at all. The extract compounds most likely originate from the lipid layer of the cerotegument of L. subterraneus. The cuticle of individuals that possessed extractable cerotegumental compounds (profile I, II, III) exhibited strong water repellent properties, while the cuticle of individuals that possessed no components in their extract did not. After hexane extraction, water repellent properties got lost. The distinct extract profiles detected most likely portray the stepwise generation of an anti-wetting, exocrine surface lipid layer of glycerides: If this layer is lost, fatty acids may be discharged again (profile III) and may subsequently esterify (profile II) to larger and more stable esters (diacyl-glycerols), eventually building up the "raincoat" (mainly profile I) of L. subterraneus.

Chrysomelidial in the opisthonotal glands of the oribatid mite, Oribotritia berlesei.

Gas chromatographic-mass spectrometric analyses of whole body extracts of Oribotritia berlesei, a large-sized soil-dwelling oribatid mite, revealed a consistent chemical pattern of ten components, probably originating from the well-developed opisthonotal glands. The three major components of the extract were the iridoid monoterpene, (3S,8S)-chrysomelidial (about 45% of the extract), the unsaturated hydrocarbon 6,9-heptadecadiene, and the diterpene beta-springene (the latter two, each about 20-25% of the extract). The remaining minor components (together about 10% of the extract) included a series of hydrocarbons (tridecene, tridecane, pentadecene, pentadecane, 8-heptadecene, and heptadecane) and the tentatively identified 9,17-octadecadienal. In contrast, analysis of juveniles showed only two compounds, namely a 2:1 mixture of (3S,8S)-chrysomelidial and its epimer, epi-chrysomelidial (3S,8R-chrysomelidial). Unexpectedly, neither adult nor juvenile secretions contained the so-called astigmatid compounds, which are considered characteristic of secretions of oribatids above moderately derived Mixonomata. The chrysomelidials, as well as beta-springene and octadecadienal, are newly identified compounds in the opisthonotal glands of oribatid mites and have chemotaxonomic potential for this group. This is the first instance of finding chrysomelidials outside the Coleoptera.

Quantitative determination of fluvastatin in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry using [18O2]-fluvastatin as an internal standard.

A sensitive and specific method for the quantitative determination of fluvastatin in human plasma is presented. The drug was isolated from plasma by extractive alkylation with pentafluorobenzyl bromide and further derivatized to the bis-trimethylsilyl derivative. [18O2]-Fluvastatin was prepared from unlabelled fluvastatin and used as an internal standard. Gas chromatography/mass spectrometry under negative ion chemical ionization conditions was used for quantitative measurement of the drug, using m/z 554.26 and 558.26 for target and internal standard, respectively. Calibration graphs were linear within a range of 2 and 512 ng mL(-1) plasma. Intra-day precision was 0.94% (2 ng mL(-1)), 2.53% (8.2 ng mL(-1)), 2.16% (81.9 ng mL(-1)) and 3.26% (409.6 ng mL(-1)); inter-day variability was found to be 1.64% (2 ng mL(-1)), 0.97% (8.2 ng mL(-1)), 1.97% (81.9 ng mL(-1)) and 2.01% (409.6 ng mL(-1)). Intra-day accuracy showed deviations of 0.6% (2 ng mL(-1)), 0.37% (8.2 ng mL(-1)), -1.52% (81.9 ng mL(-1)) and -1.67% (409.6 ng mL(-1)); inter-day accuracy was of -1.64% (2 ng mL(-1)), -1.13% (8.2 ng mL(-1)), -2.28% (81.9 ng mL(-1)) and -0.46% (409.6 ng mL(-1)). The stable isotope labelled standard was found to be stable under the analytical conditions.