Kinetic profiles of diacetoxyscirpenol and two of its metabolites in blood serum of pigs

Orally administered diacetoxyscirpenol (2 mg/kg of body weight) was rapidly absorbed into the blood serum of pigs; within 1 h, the highest amounts of diacetoxyscirpenol (9.6 to 21.9 ng/ml) were detected. Two metabolites of diacetoxyscirpenol were identified by gas chromatography-mass spectroscopy as monoacetoxyscirpenol and scirpenetriol. The three trichothecenes were present in the blood serum of pigs for only 24 h, indicating a rapid metabolism of these compounds.     

Dynamic Patterns of serum metabolites in fulminant hepatic failure pigs

Fulminant hepatic failure (FHF) is still an intractable disease associated with serious metabolic disorder. Investigating the dynamic changes of serum metabolites during the development of FHF would facilitate revealing the pathogenesis and also promote its treatment. Therefore, this study characterized the dynamic metabonome of serum from FHF Pigs using ultra performance liquid chromatography?Cmass spectrometry. Based on multiple statistical analysis of the resulting dataset, three types of up-regulated and one type of down-regulated patterns were delineated. Each pattern demonstrated distinct trends at different stages during the whole process of FHF, implying the differential clinical significance of them. Specifically, aromatic amino acids (Pattern 1) and lysophosphatidylcholines (LPCs) (Pattern 4) might be good markers for evaluating the severity of FHF, while some conjugated bile acids, long chain acylcarnitines (Pattern 2) and Glycocholic acid (Pattern 3) could indicate liver injury in the early stage. Inspired from the PCA plot that the pathogenetic condition of FHF aggravated with sampling time, a linear discriminant analysis (LDA) model based on phenylalanine and LPC 18:1 were further constructed for evaluating the severity of FHF. The leave-one-out cross-validation accuracy of 91.67% for the training set and the prediction accuracy of 92.31% for the external validation set confirmed its excellent performance. In conclusion, findings obtained from the present study, including four types of Dynamic Patterns of serum metabolites during FHF development and an LDA model for evaluating the severity of FHF, will be of great help to the research and management of FHF in the future.     

Jejunal morphology and blood metabolites in tail biting,victim and control pigs

Tail biting has several identified feeding-related risk factors. Tail biters are often said to be lighter and thinner than other pigs in the pen, possibly because of nutrition-related problems such as reduced feed intake or inability to use nutrients efficiently. This can lead to an increase in foraging behavior and tail biting. In this study, a total of 55 pigs of different ages were selected according to their tail-biting behavior (bouts/hour) and pen-feeding system to form eight experimental groups: tail-biting pigs (TB), victim pigs (V) and control pigs from a tail-biting pen (Ctb) and control pen (Cno) having either free access to feed with limited feeding space or meal feeding from a long trough. After euthanasia, a segment of jejunal cell wall was cut from 50 cm (S50) and 100 cm (S100) posterior to the bile duct. Villus height, crypt depth and villus : crypt ratio (V : C) were measured morphometrically. Blood serum concentration of minerals and plasma concentration of amino acids (AA) was determined. Villus height was greater in Cno than Ctb pigs in the proximal and mid-jejunum (P < 0.05), indicative of better ability to absorb nutrients, and increased with age in the proximal jejunum (P < 0.001). Serum mineral concentration of inorganic phosphate (P_i) and calcium (Ca) was lower in Ctb compared with Cno pigs, and that of P_i in V compared with all the other pigs. Many non-essential AA were lower in pigs from tail-biting pens, and particularly in victim pigs. Free access feeding with shared feeding space was associated with lower levels of essential AA in blood than meal feeding with simultaneous feeding space. Our data suggest that being a pig in a tail-biting pen is associated with decreased jejunal villus height and blood AA levels, possibly because of depressed absorption capacity, feeding behavior or environmental stress associated with tail biting. Victim pigs had lower concentrations of AA and P_i in plasma, possibly as a consequence of being bitten.     

Simultaneous quantitation of chlormethiazole and two of its metabolites in blood and plasma by gas—liquid chromatography

A simple and rapid gas chromatographic method for the quantitation of chlormethiazole and two of its pharmacologically active metabolites, 5-acetyl-4-methylthiazole and 5-(1-hydroxyethyl)-4-methylthiazole, in plasma and blood is described. The total analysis time is less than 20 min for a single sample. The method requires 50–500 μl of plasma or 1 ml of blood. The compounds are detected with a nitrogen—phosphorus detector. An internal standard technique is used for the quantitation. Calibration data are linear over the range 32–2376 ng of chlormethiazole and a similar range of the metabolites in plasma. The method may be used for pharmacokinetic studies.     

Production and characterization of antibody against diacetoxyscirpenol.

Antibodies against diacetoxyscirpenol (DAS) were obtained from rabbits after immunizing them with hemisuccinate or hemiglutarate derivatives of DAS conjugated to bovine serum albumin (BSA). DAS-hemiglutarate-BSA was found to be a much better immunogen than DAS-hemisuccinate-BSA. Competitive radioimmunoassay revealed that the antisera obtained from rabbits after immunization with DAS-hemiglutarate-BSA showed high specificity toward DAS. The concentrations causing 50% displacement of radioactive DAS by unlabeled DAS, 4-monoacetoxyscirpenol (MAS), and 15-MAS were found to be 1.5, 130, and 300 ng per assay, respectively. Thus, the cross-reactivities for 4-MAS and 15-MAS are ca. 87 and 300 times weaker than that of DAS. Practically no cross-reaction (less than 5% displacement) was observed when deoxynivalenol, T-2 toxin, deoxyverrucarol, and scirpentriol were tested at a concentration of 2,000 ng/ml.     

Field-adapted assays for chloroquine and its metabolites in urine and blood

Many types of field malaria studies require the on-site detection of chloroquine (CQ) in body fluids to determine the history of CQ ingestion, the patient's compliance with chemoprophylaxis or the treatment regime used, as well as to identify patterns of CQ use in the community. The three general approaches to field-adapted CQ assay are colorimetry, thin-layer chromatography and ELISA. In each case, CQ may be estimated by the visual comparison of samples and standards, or more precisely quantified by using the appropriate photometric device. The available methods are discussed and compared and, as Fred Churchill describes, not only represent a wide variety of alternatives for CQ detection and quantification for the malariologist engaged in clinical research but also serve as prototype models for the development of similar methods for other antiparasitic drugs.     

A comparative proteomic analysis of blood serum for developmental stages in pigs

This study aimed to differentiate genes at developmental stages of pigs from 0 to 150 days of age, to build up a protein database and to find candidate genetic markers for growth traits. The analysis of two‐dimensional electrophoresis and matrix‐assisted laser‐desorption/ionization mass spectrometry separated 252 protein segments. After successfully blasting the peptide sequences, the analysis confirmed 37 differentially expressed proteins that increased from birth to 150 days of age (type A), whereas the type B proteins presented the inverse pattern. The type C proteins included proteins that were expressed continuously throughout the developmental periods. A total of 319 primer sets for 33 genes were designed to find genetic variants using pooled DNA samples of Yorkshire pigs. Amplification products for all primer sets produced approximately 20 000 clones that were sequenced, and 48 candidate SNP sites were finalized for genotyping. A total of 475 animals were used for high throughput genotyping analysis. Among these, phenotype data of all 475 animals were collected for average daily gain, backfat thickness and days to 90 kg, whereas feed conversion data were collected for 300 animals and body measurement traits (starting weight, ending weight, body length, wither height and chest depth) were collected for 209 animals. Association analysis found significant statistical differences between the animals having genotypes of 13 SNPs (g.78935883C>T, g.147629986C>T, g.98266037T>C, g.214707340G>A, g.88350299C>T, g.17180956C>T, g.17181024C>T, g.2350283A>G, g.138361311C>T, g.44996379C>T, g.44996247A>C, g.107715245C>T, g.4149631C>T) for the various measured traits. The identified genetic polymorphisms, of which one was novel (g.214707340G>A), may serve as candidate molecular markers to change population means for the targeted growth traits.     

Mass fragmentography. Identification of chlorpromazine and its metabolites in human blood by a new method

Chlorpromazine and some of its metabolites have been identified in human blood with the combination of gas chromatography and mass spectrometry. A new method, mass fragmentography has been elaborated. It is based upon a continuous recording of up to three mass numbers characteristic of a single substance or a group of compounds. With this technique both a high sensitivity and a high selectivity which can be changed according to wish are achieved. Compounds are identified by their retention times and the fact that all mass numbers are represented in characteristic relative intensities. Refocusing on other characteristic fragments and/or the molecular ion confirms the identity. Through repeated refocusing “a partial mass spectrum” of a compound can be established even when the amounts present are too small for the scanning of a complete spectrum.With this method chlorpromazine, and its desmethylated and didesmethylated metabolites have been identified in plasma. The latter two metabolites were obtained also after treatment of the plasma with β-glucuronidase, as was 2-chlorophenothiazinylpropionic acid, which was found in quantities that allowed the scanning of a complete mass spectrum. In red blood cells the two desmethylated metabolites could be identified with mass fragmentography only after treatment with β-glucuronidase. The use of the method is discussed, particularly with regard to blood levels of drugs as related to therapeutic effects and side effects.     

Development and validation of a rapid HPLC method for the simultaneous determination of triethylenetetramine and its two main metabolites in human serum

A validated method for the determination of triethylenetetramine, a selective copper-chelator currently undergoing clinical trials for the treatment of diabetic heart failure, and its two major metabolites, N(1)-acetyltriethylenetetramine and N(1),N(10)-diacetyltriethylenetetramine in human serum using HPLC is reported. The method used 9-flouorenylmethylchloroformate chloride to label all three analytes. The fluorescence labeled analytes were then separated chromatographically using a reversed phase C18 column under a gradient elution program and detected spectrofluorometrically at 317 nm with excitation at 263 nm. Application of the method is demonstrated by pharmacokinetic measurement in one healthy volunteer taking the drug orally.     

Cancer-specific MALDI-TOF profiles of blood serum and plasma: Biological meaning and perspectives

MALDI-TOF mass-spectrometry has become a popular tool of cancer research during the last decade. High throughput and relative simplicity of this technology have made it attractive for biomarker discovery and validation across various platforms in blood serum/plasma. Many technical approaches have been developed for plasma/serum profiling including protein-chip based SELDI-TOF mass-spectrometry, purification of serum on magnetic beads, analysis of carrier-associated fraction and mass-spectrometric immunoassays. Extensive data about the identity of differential features detected on mass-spectra up to now makes it possible to draw conclusions about potency and perspectives of MALDI-TOF mass-spectrometry in this field. A great majority of identified differentially expressed proteins are either house-keeping or inflammatory proteins as well as their modifications or fragments. Discriminating ability of mass-spectra is likely to be based on differential modification and fragmentation patterns of abundant serum proteins reflecting activity of enzymes including proteases and their inhibitors.     

Kinetic pulse-labeling study of Fusarium culmorum. Biosynthetic intermediates and dead-end metabolites

A kinetic pulse-labeling method was utilized in Fusarium culmorum to detect plausible biosynthetic intermediates and differentiate them from dead-end metabolites. The ultimate test to demonstrate a precursor relies on feeding experiments. We now report the detection of four new metabolites, one of them (compound 1) behaves as a dead-end metabolite, whereas compounds 2, 3, and 4 seem to be putative intermediates: they metabolize with time just when 3-acetyldeoxynivalenol (3-ADN) and/or sambucinol (SOL) start to be produced. Feeding experiments confirmed these results: compound 1 is not converted to 3-ADN or SOL, and compounds 2-4 are precursors to 3-ADN. In addition 3 is a precursor to SOL.     

Determination of theophylline in serum and saliva in the presence of caffeine and its metabolites

Because of marked variability in its metabolic clearance and its narrow therapeutic range (10–20 μg/ml) investigation of each patient's clearance of theophylline is desirable. The author reports here a rapid reversed-phase high-performance liquid chromatographic (HPLC) method to determine, within 3 min, the theophylline in serum and saliva in the 0.1–50 μg/ml range. A fast HPLC column, 10 × 4.6 mm, packed with 3-μm spherical ODS packing is used with acetonitrile—methanol—buffer pH 4.7 (4:7:89) to achieve separation of theophylline from paraxanthine and matrix components. Since theophylline is a major pediatric bronchodilator, the feasibility of assay in saliva was investigated as an alternative route for determining the clearance in stressed asthmatic children. Using this method it was found that the ratio of theophylline in simultaneous serum and saliva samples is very consistent over time in the same person (± 3.99%), but inter-individually this consistency is reduced ten-fold. Simultaneous serum and saliva samples need be taken only once to obtain the ratio and the kinetics followed further with salivary samples only.