Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes

Summary  The induction of cytochrome P450 (CYP450) and Phase II conjugating enzymes by prototypical hepatic enzyme inducers was studied in adult male rat hepatocytes. Hepatocytes were suspended and cultured in diluted Matrigel? in a basal serum-free Dulbecco’s modified Eagle medium and exposed to the prototypical liver enzyme inducers, 3-methylcholanthrene, phenobarbital, hydrocortisone, and clofibrate for 48 h. Total RNA and microsomes were isolated and prepared, respectively, at 72 h. The expression of CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A1, CYP3A2, CYP4A1, fatty acyl-CoA oxidase, uridine diphosphate-glucuronosyltransferase, glutathione-S-transferase, and sulfotransferase was determined at the mRNA level with reverse transcriptase polymerase chain reaction (RT-PCR). The expression of CYP1A1, CYP2B1, CYP2C11, CYP2E1, and CYP4A1 was also measured at the apoprotein level by Western immunoblotting. Using these culture and expression analysis techniques, we have found that the expression of these metabolic enzymes can be maintained in culture for up to 7 d at the mRNA and apoprotein levels. In addition, hepatocytes were found to respond to chemical enzyme inducers with marked increases in enzyme expression at either the mRNA or protein level and in a concentration-related fashion. Cells were responsive to enzyme induction as early as 24 h after initial plating. The results obtained from this investigation indicate that the presence of diluted Matrigel? (at a concentration of 0.35 mg/ml), the use of low concentrations of insulin (1 μM), hydrocortisone (0.1 μM), and serum-free culture medium can maintain the differentiated phenotype and responsiveness of cultured hepatocytes to chemical-induced metabolic enzyme expression. Under the conditions used in this study, enzyme induction in adult male rat hepatocytes shows close agreement with enzyme induction observed in the livers of rats exposed to these or similar prototypical enzyme inducers. Rat hepatocytes cultured in the presence of diluted Matrigel? coupled with enzyme mRNA expression analysis with RT-PCR are proven to be a valuable and important in vitro toxicological approach to assess the chemical-induced changes in expression of liver CYP450 and Phase II conjugating enzymes.

Analysis of conjugated linoleic acid isomers and content in french cheeses

Abstract  Conjugated linoleic acid (CLA) occurs in food as a result of microbial enzymatic reactions, free radical-type oxidation, and heat treatment. CLA is found in animal products, such as meat and dairy products, especially in cheeses. The CLA composition of 12 different French cheeses was determined by a combination of different analytical methods: reversed-phase high-performance liquid chromatography (RP-HPLC), gas chromatography-mass spectrometry (GC-MS), GC-Fourier transform infrared (GC-FTIR), and silver nitrate thin-layer chromatography (AgNO3-TLC). New isomers (Δ8,10- and Δ11,13-octadecadienoic acids with all possible cis and trans configurations) that co-eluted with previously identified isomers (Δ9c,11t-; Δ9t,11c-; Δ10c,12t-; Δ10t,12c-; Δ11c,13c-; Δ9c,11c-; Δ10c,12c-; Δ9t,11t-; Δ10t12t-octadecadienoic acids) were detected. Δ9c,11t-Octadecadienoic acid was the major CLA isomer in these cheeses. All isomers were present in each product, whatever the production process. However, CLA content in the cheeses varied from 5.3 to 15.80 mg/g of cheese fat, which depended primarily on the origin of the milk (season, geography) and somewhat on the production process.

Analysis of cesium extracting solvent using GCMS and HPLC

Abstract  A high-level waste (HLW) remediation process scheduled to begin in 2007 at the Savannah River Site is the Modular Caustic Side Solvent Extraction (CSSX) Unit (MCU). The MCU will use a hydrocarbon solvent (diluent) containing a cesium extractant, a calix[4]arene compound, to extract radioactive cesium from caustic HLW. The resulting decontaminated HLW waste or raffinate will be processed into grout at the Saltstone Production Facility (SPF). The cesium containing CSSX stream will undergo washing with dilute nitric acid followed by stripping of the cesium nitrate into a very dilute nitric acid or the strip effluent stream and the CSSX solvent will be recycled. The Defense Waste Processing Facility (DWPF) will receive the strip effluent stream and immobilize the cesium into borosilicate glass. Excess CSSX solvent carryover from the MCU creates a potential flammability problem during DWPF processing. Bench-scale DWPF process testing was performed with simulated waste to determine the fate of the CSSX solvent components. A simple high performance liquid chromatography (HPLC) method was developed to identify the modifier (which is used to increase Cs extraction and extractant solubility) and extractant within the DWPF process. The diluent and triocytlamine (which is used to suppress impurity effect and ion-pair disassociation) were determined using gas chromatography mass spectroscopy (GCMS). To close the organic balance, two types of sample preparation methods were needed. One involved extracting aqueous samples with methylene chloride or hexane, and the second was capturing the off gas of the DWPF process using carbon tubes and rinsing the tubes with carbon disulfide for analysis. This paper addresses the development of the analytical methods and the bench-scale simulated waste study results.

Analysis of Cerebrospinal Fluid from Clinically Healthy Iranian Fat-tailed Sheep

Analysis of Acylcarnitine Ester for Neonatal Screening of Inborn Errors of Metabolism Using Tandem Mass-spectrometry

Summary.  Tandem mass-spectrometry has been introduced worldwide into neonatal screening programs for the quantitative analysis of acylcarnitine species and amino acids for the diagnosis of organic acidurias, defects of fatty acid oxidation, and amino acidopathies, respectively. Since April 2002 more than 200000 newborn infants have been screened in Austria using tandem mass-spectrometry. In this cohort 37 infants with amino acidopathies and 38 infants with fatty acid oxidation defects or organic acidurias have been diagnosed. The overall incidence of these disorders is 0.036%. The analysis of acylcarnitine species using tandem mass-spectrometry has enabled the diagnosis of infants with inborn errors of metabolism prior to clinical presentation and to initiate therapy early enough to prevent long-term sequelae and to reduce mortality in these disorders.

Analysis of Rodgersia aesculifolia Batal. Rhizomes by Microwave-Assisted Solvent Extraction and GC–MS

Abstract   Rodgersia aesculifolia Batal. rhizome is an effective antibacterial and antivirus Chinese medicinal plant. In this work focused microwave-assisted solvent extraction (FMASE) coupled with gas chromatography–mass spectrometry has been used for analysis of Rodgersia aesculifolia Batal. rhizomes. The main compound classes in the extract were fatty acids (67.22%), aliphatic hydrocarbons (9.02%), and steroids (7.02%). Oleic acid, linoleic acid, and palmitic acid were the most abundant compounds. Compared with Soxhlet extraction, the much shorter extraction time and the similar components in the extract make FMASE an excellent alternative for extraction of the components of plant rhizomes.

Analyse des anaeroben Benzolabbaus: Vergleich von In-situ-Mikrokosmen, Elektronenakzeptorbilanzen und Isotopenfraktionierungsprozessen

Kurzfassung  Geochemische und isotopenchemische Methoden wurden zur Charakterisierung des anaeroben Benzolabbaus am Standort eines ehemaligen Hydrierwerkes (Zeitz, Deutschland) genutzt. Im Abstrom des Schadenszentrums wurde hierfür die vertikale Struktur der Benzolfahne in verschiedenen Tiefen untersucht. Durch Einsatz von [13C6]Benzol in In-situ-Mikrokosmen konnte anhand der Transformation des markierten Kohlenstoffs in die Biomasse eindeutig das Abbaupotenzial nachgewiesen werden. Das Fetts?urespektrum sowie deren Isotopensignaturen deuten auf eine Besiedlung durch komplexe mikrobielle Gemeinschaften hin, die in unterschiedlicher Weise am Benzolabbau beteiligt sind. Die Sulfatsenke in der Schadstofffahne deutet auf überwiegend sulfatreduzierende Abbaubedingungen mit einem Abbaupotenzial von etwa 1,7 mmol/l Benzol hin. Anhand der Isotopensignaturen und Konzentrationen des DIC wurde in der Schadstofffahne ein Abbau von 2,2 mmol/l Benzol abgesch?tzt. Eine Berechnung des Benzolabbaus anhand der Isotopenfraktionierungsmethode ergibt einen Abbau von etwa 3,0 mmol/l. Die quantitativen geochemischen und isotopenchemischen Absch?tzungen liegen in der gleichen Gr??enordnung und zeigen einen signifikanten Benzolabbau im Aquifer.