The PDGFRα-laminin B1-keratin 19 cascade drives tumor progression at the invasive front of human hepatocellular carcinoma.

Human hepatocellular carcinomas (HCCs) expressing the biliary/hepatic progenitor cell marker keratin 19 (K19) have been linked with a poor prognosis and exhibit an increase in platelet-derived growth factor receptor α (PDGFRα) and laminin beta 1 (LAMB1) expression. PDGFRα has been reported to induce de novo synthesis of LAMB1 protein in a Sjogren syndrome antigen B (La/SSB)-dependent manner in a murine metastasis model. However, the role of this cascade in human HCC remains unclear. This study focused on the functional role of the PDGFRα-La/SSB-LAMB1 pathway and its molecular link to K19 expression in human HCC. In surgical HCC specimens from a cohort of 136 patients, PDGFRα expression correlated with K19 expression, microvascular invasion and metastatic spread. In addition, PDGFRα expression in pre-operative needle biopsy specimens predicted poor overall survival during a 5-year follow-up period. Consecutive histological staining demonstrated that the signaling components of the PDGFRα-La/SSB-LAMB1 pathway were strongly expressed at the invasive front. K19-positive HCC cells displayed high levels of α2ß1 integrin (ITG) receptor, both in vitro and in vivo. In vitro activation of PDGFRα signaling triggered the translocation of nuclear La/SSB into the cytoplasm, enhanced the protein synthesis of LAMB1 by activating its internal ribosome entry site, which in turn led to increased secretion of laminin-111. This effect was abrogated by the PDGFRα-specific inhibitor crenolanib. Importantly LAMB1 stimulated ITG-dependent focal adhesion kinase/Src proto-oncogene non-receptor tyrosine kinase signaling. It also promoted the ITG-specific downstream target Rho-associated coiled-coil containing protein kinase 2, induced K19 expression in an autocrine manner, invadopodia formation and cell invasion. Finally, we showed that the knockdown of LAMB1 or K19 in subcutaneous xenograft mouse models resulted in significant loss of cells invading the surrounding stromal tissue and reduced HepG2 colonization into lung and liver after tail vein injection. The PDGFRα-LAMB1 pathway supports tumor progression at the invasive front of human HCC through K19 expression.

ILEI requires oncogenic Ras for the epithelial to mesenchymal transition of hepatocytes and liver carcinoma progression.

In human hepatocellular carcinoma (HCC), epithelial to mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. We employed a model of EMT based on immortalized p19(ARF) null hepatocytes (MIM), which display tumor growth upon expression of oncogenic Ras and undergo EMT through the synergism of Ras and transforming growth factor (TGF)-beta. Here, we show that the interleukin-related protein interleukin-like EMT inducer (ILEI), a novel EMT-, tumor- and metastasis-inducing protein, cooperates with oncogenic Ras to cause TGF-beta-independent EMT. Ras-transformed MIM hepatocytes overexpressing ILEI showed cytoplasmic E-cadherin, loss of ZO-1 and induction of alpha-smooth muscle actin as well as platelet-derived growth factor (PDGF)/PDGF-R isoforms. As shown by dominant-negative PDGF-R expression in these cells, ILEI-induced PDGF signaling was required for enhanced cell migration, nuclear accumulation of beta-catenin, nuclear pY-Stat3 and accelerated growth of lung metastases. In MIM hepatocytes expressing the Ras mutant V12-C40, ILEI collaborated with PI3K signaling resulting in tumor formation without EMT. Clinically, human HCC samples showed granular or cytoplasmic localization of ILEI correlating with well and poorly differentiated tumors, respectively. In conclusion, these data indicate that ILEI requires cooperation with oncogenic Ras to govern hepatocellular EMT through mechanisms involving PDGF-R/beta-catenin and PDGF-R/Stat3 signaling.

Automated residue analysis of tetracyclines and their metabolites in whole egg, egg white, egg yolk and hen's plasma utilizing a modified ASTED system.

An automated analytical method is described allowing simultaneous determination of all tetracyclines and metabolites in whole egg, egg yolk, egg white and blood plasma of the hens. Sample pretreatment is restricted to homogenization and a dilution step. Clean-up is by on-line dialysis and on-line solid-phase extraction utilizing an extended ASTED system, followed by liquid chromatography with UV or fluorescence detection with post-column pH adjustment and confirmational analysis by LC-MS-MS. After feeding oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) to laying hens, not only residual parent compounds could be found in the eggs but also in vivo formed 4-epimers, isochlortetracycline (ICTC) and tentatively identified N-desmethyl metabolites of OTC, TC and ICTC.

Metabolites of oxytetracycline, tetracycline, and chlortetracycline and their distribution in egg white, egg yolk, and hen plasma.

4-epioxytetracycline and N-demethyloxytetracycline, as metabolites of oxytetracycline (OTC), 4-epitetracycline and N-demethyltetracycline, as metabolites of tetracycline (TC), and 4-epichlortetracycline, isochlortetracycline (ICTC), 4-epi-ICTC, and N-demethyl-ICTC, as metabolites of chlortetracycline (CTC), were detected in egg yolk and plasma obtained from feeding studies with either OTC, TC, or CTC. In egg white, only OTC, TC with its 4-epimer, and ICTC with its 4-epimer were detected in substantial concentrations. The ratios of epimerization and N-demethylation in the eggs did not change during the medication period. The samples were analyzed by an automated HPLC system (ASTED) with UV, fluorescence, or MS-MS detection.

Residues of veterinary drugs in eggs and their distribution between yolk and white.

Veterinary drugs and feed additives (especially some coccidiostats) can be absorbed by the digestive tract of laying hens and transferred to the egg. Physicochemical characteristics of these compounds determine their pharmacokinetic behavior and distribution to and within the egg. Traditionally the quite lipid soluble drugs and additives are expected to yield residues only in the fat-rich yolk. However, the quite lipid soluble drug doxycycline--as well as many other drugs--showed during long-term administration higher residues in white than in yolk. In a model study with 11 sulfonamides differing in pK(a) value and lipid solubility, their distribution in vivo between yolk and white was determined. Neither differences in pK(a) values nor those in lipid solubility could explain the distributions found. Binding to egg white macromolecules in vivo as an explanatory factor was tested with five sulfonamides, and no correlation between binding and the distribution of sulfonamides between white and yolk was found. Literature data on the distribution of drugs between egg white and yolk showed a reasonable consistency within drugs and a large variability among drugs (as could be expected). This larger database also did not provide a clue as to what factor determines the distribution of a drug between egg white and yolk when given to laying hens.

Analytical methods for the determination of aflatoxins in various food matrices at concentrations regarding the limits set in european regulations: development, characteristics, limits.

Methods for the determination of aflatoxins in paprika, peanut butter, pistachio paste, fig paste and baby food were developed. The methods employ an immunoaffinity cleanup step and reversed-phase liquid chromatography. All steps of the analysis were tested for their suitability for all matrices with focus on method robustness, simplicity, toxicology, environment, and user friendliness. Extraction procedures, chromatographic separation and post column derivatisation techniques were elaborated for this purpose. The methods were statistically validated in collaborative trials at currently established legal limits for aflatoxins and are in the process for adoption as official methods by CEN and AOAC.

Investigation of various extractants for the analysis of aflatoxin B1 in different food and feed matrices.

Various extractants were investigated concerning their suitability for aflatoxin B1 determinations in different matrices including spices, infant formula and animal feed employing an immunoaffinity clean-up procedure. It was shown that the use of aqueous acetonitrile extractants was limited due to the fact that dry sample material can absorb significant amounts of water from the extractant. This can result in recoveries that are too high and therefore in incorrect values for the aflatoxin concentration if aliquots are taken for further analysis. A correction of the results by recovery calculation using spiked blank material is unsuitable, since material from the same group of food (e.g. paprika powder) or feed can vary significantly in the recovery values. Therefore it is recommended that aqueous methanol extractants are used, since no significant interaction with matrix constituents was observed. In addition, aqueous acetone extractants are a useful alternative with some limitations.

Development of a gas chromatography-mass spectrometry method for the analysis of aminoglycoside antibiotics using experimental design for the optimisation of the derivatisation reactions.

A packed column GC-electron-capture detection method for the analysis of the aminoglycoside antibiotics kanamycin and gentamicin was adapted to capillary GC-MS. The analytes were derivatised using a two-step procedure involving trimethylsilylation of the hydroxyl groups with trimethylsilylimidazole and acylation of the amino groups with heptafluorobutyrylimidazole. Electron impact mass spectra of the resulting derivatives of kanamycin A and gentamicins C1, C1a and C2 are given and interpreted. The derivatisation procedure was optimised using experimental design. This chemometrical approach considers main effects as well as interactions of the influential parameters, thus conducting a more thorough investigation of the method than the common step-by-step approach. Optimisation using fractional factorial and Box Behnken Designs produced a derivatisation method featuring better yield than previously published methods while in many cases requiring less reagents and shorter reaction times.

Oxacillin residues in milk after drying off with Stapenor Retard TS.

Milk samples from 28 cows were analysed for residues of oxacillin after drying off with Stapenor Retard TS (oxacillin). Analysis was performed with an automated HPLC system consisting of an on-line solid-phase extraction and photochemical post-column derivatization with UV-detection at 300 nm. Although the time interval between treatment and parturition was less than the demanded 55 days, the maximum residue limit of 30 micrograms kg-1 was only exceeded in one case, in which the withdrawal time was 28 days.

Isotope dilution GC-MS of benzylpenicillin residues in bovine muscle.

The German provisional prevalidated official method for the determination of residues of benzylpenicillin in muscle (beef) which uses gas chromatography with nitrogen-specific detection (GC-NPD) was successfully adapted to gas chromatography-mass spectrometry (GC-MS) with selected-ion monitoring. The programmed temperature vaporisation injection technique used in the original method was substituted by on-column injection. An isotope dilution procedure for benzylpenicillin using 13C2-labelled benzylpenicillin as internal standard was developed to improve the reliability of the GC-MS results. The spectral overlap of this labelled standard and the analyte caused a non-linear relationship in calibration for which linear bracketing and single-point calibration were compared. Single-point calibration proved to be the superior method, showing less deviation from the 'true' value, while at the same time requiring fewer calibration measurements. A comparison of the results obtained by using (i) external standardisation only, (ii) a close analogue of the analyte (phenoxymethylpenicillin) and (iii) 13C2-labelled Pen G as internal standard showed, as was to be expected, that varying recoveries are best corrected by using the isotope-labelled standard.

Induction and characterization of multianalyte antibodies against penicillins in egg yolk.

Antibodies against penicillins were induced in eggs of laying hens after immunization with 6-aminopenicillanic acid (6-APA) coupled to key-hole limpet hemocyanin (KLH). Development of the antibody titer was monitored by an indirect enzyme-linked immunosorbent assay (ELISA), with 6-APA coupled to ovalbumin as antigen for coating microtiter plates. Different characteristics (time course, affinity) of the immune reaction were observed by testing eggs of individual hens. Titer values varied between 150 and 2000. Antibodies were isolated by polyethylene glycol precipitation and affinity chromatography, using a hapten sorbent with 6-APA as ligand. Glycine buffer, pH 3.0, was used to elute the immunoglobulins. Antibody specificity was determined in a competitive ELISA with 7 penicillins and the cephalosporin cephalexin as competitors. Cross reactivities for the penicillins were between 100 and 290% (6-APA = 100%). Cephalexin cross reacted only marginally (3%).

High-performance liquid chromatographic determination of penicillins by means of automated solid-phase extraction and photochemical degradation with electrochemical detection.

An automated precolumn exchange system for on-line solid-phase extraction (OPS-2) coupled to liquid chromatography with photochemical degradation and electrochemical detection was used for the determination of residual amounts of penicillin G, penicillin V, oxacillin, cloxacillin and dicloxacillin. A 5-10-fold increase in sensitivity was obtained when compared with direct UV detection of penicillins at 225 nm. The system is recommended for samples that have received an immunoaffinity clean-up. Analysis of bovine muscle tissue indicated, however, that this procedure could also be suitable for the determination of penicillin G at its maximum residue limit of 50 micrograms/kg even with conventional solvent partitioning for the first clean-up steps.

[HPLC determination of oxacillin, cloxacillin and dicloxacillin in bovine muscle with automated cleanup by on-line solid phase extraction]. / HPLC-Bestimmung von Oxacillin, Cloxacillin und Dicloxacillin in Muskelfleisch vom Rind mit automatischem Cleanup durch on-line Festphasenextraktion.

A high-performance liquid chromatographic (HPLC) method with automated cleanup by on-line solid phase extraction was developed for the determination of oxacillin, cloxacillin and dicloxacillin in bovine muscle tissue. The penicillins were extracted from the matrix with acetonitrile, transferred to phosphate buffer and automatically trapped on a RP-18 solid phase extraction column. After on-line elution onto the analytical HPLC-column, penicillins were quantified by UV (lambda = 225 nm) after isocratic separation. Mean recoveries at the MRL (0.3 mg/kg) were 92% for oxacillin, 92% for cloxacillin and 86% for dicloxacillin. The coefficients of variation for the three penicillins in the range of 0.15-0.6 mg/kg were 13.8%, 11.0%, and 7.3%, respectively. The applicability of this method for the determination of cloxacillin in milk was demonstrated.

[Fluorimetric determination of erythromycin residues in foods of animal origin after derivatization with FMOC and HPLC separation]. / Fluorimetrische Bestimmung von Erythromycin-Rückständen in Lebensmitteln tierischer Herkunft nach Derivatisierung mit FMOC und HPLC-Trennung.

A high-performance liquid chromatographic (HPLC) method for the determination of the macrolide antibiotic erythromycin in eggs, milk, swine muscle, kidney and liver was developed. The drug was extracted from the matrix with acetonitrile. The raw extract was purified by liquid-liquid partitioning and fractionation by reversed-phase HPLC for additional cleanup. Erythromycin was reacted in a pre-column procedure with 9-fluorenylmethylchloroformate (FMOC) to enable fluorimetric detection (excitation 255 nm, emission 315 nm) after isocratic separation on an analytical RP-18 HPLC column. Mean recoveries ranged from 99% at fortification levels of 0.03 mg/kg in egg to 38% at 0.06 mg/kg in liver. With the exception of liver all detection limits were below 0.01 mg/kg and precision for all other matrices and tested concentrations (0.015-0.09 mg/kg) better than 20% (coefficient of variation).

[Gas chromatographic method for the analysis of residues of seven penicillins in food of animal origin]. / Gaschromatographische Analysenmethode für Rückstände von sieben Penicillinen in Lebensmitteln tierischen Ursprungs.

A capillary gas chromatographic method is described for the determination of residues of benzylpenicillin, phenoxymethylpanicillin, methicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin in bovine muscle, liver, kidney, adipose tissue and milk. The samples are extracted with acetonitrile under slightly acidic conditions, the co-extracted water is separated with the addition of sodium chloride and dichloromethane and discarded. Clean-up is performed by liquid/liquid partitioning steps and anion exchange chromatography. The penicillin residues are methylated with diazomethane. After derivatization, only the extracts from liver and kidney needed further clean-up using cartridges with a polar diol sorbent. The gas chromatographic procedure is based on split/splitless injection, programmed temperature vaporization, separation on a methyl silicone fused silica column and nitrogen-specific thermionic detection. Internal standardization is used for quantification. The limits of detection for all penicillins are well below 3 microgram/kg in milk and all tissues. Recoveries of spiked samples at 3 and 10 micrograms/kg are in the range of 65-80% for milk and 50-70% for bovine tissues.

Capillary gas chromatographic method for determination of benzylpenicillin and other beta-lactam antibiotics in milk.

A capillary gas chromatographic method is described for determining residues of beta-lactam antibiotic residues in milk, with specificity for benzylpenicillin (penicillin G), phenoxymethylpenicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, and nafcillin. Residues are extracted from milk with acetonitrile. Samples are cleaned up by partitioning between aqueous and organic phases at different pH values. The penicillin residues are methylated with diazomethane to render them amenable to determination by gas chromatography on a methyl silicone fused silica column. Samples are introduced by split/splitless injection using a programmed temperature vaporization injector and are detected by nitrogen-selective thermionic detection. Internal standardization is used for quantitation. The limits of detection for all penicillins are well below 1 microgram/kg. Recoveries of spiked samples at 3 and 10 micrograms/kg are in the range of 42-85% (coefficients of variation 2-5%) and 41-92% (coefficients of variation 3-7%), respectively.

[Fluorometric determination of sulfanilamide and sulfaguanidine residues in animal tissues with high-performance-liquid chromatography]. / Fluorimetrische Bestimmung von Sulfanilamid- und Sulfaguanidinrückständen in tierischen Matrices durch Ionenaustausch-Hochleistungsflüssig-chromatographie.

A high-performance liquid chromatographic method is presented for the selective analysis of sulphanilamide (SA) and sulphaguanidine (SG) residues in animal tissues (muscle, liver), egg and milk at the 0.01-0.1 mg/kg level. A 25-g amount of the samples was extracted with 90 ml acetonitrile at pH 8.5 to which 2 ml saturated sodium carbonate had been added. After centrifugation, co-extracted water was separated by saturating the extract with sodium chloride and adding 50 ml dichloromethane. The residue of the dried and evaporated organic layer was partitioned between methanol, n-hexane and mobile phase (1 ml each). A 20-microliter volume of the aqueous layer was injected onto a Nucleosil 5 SA column [mobile phase 0.05 M sodium dihydrogenphosphate (adjusted to pH 2.0 with 85% orthophosphoric acid)-methanol, 85:15, v/v]. SA and SG were detected by their native fluorescence (excitation 275 nm, emission 340 nm) at retention times of 7 and 13 min, respectively, without interference of co-extractives. Parallel detection by ultraviolet absorption (275 nm) allowed on-line confirmation of the results. Recovery of spiked samples (0.1 mg/kg) was 80% (coefficient of variation, C.V. = 5%) for SA and 62% (C.V. = 6%) for SG. Concentrations of 0.27 mg/kg for SA and 0.05 mg/kg for SG were analyzed in muscle meat of a swine which received a single intramuscular injection of 2.1 g SA and 0.6 g SG and was slaughtered 24 h later.

Thin-layer chromatographic determination of erythromycin and other macrolide antibiotics in livestock products.

A method is described for determination of 4 macrolide antibiotics in livestock products. Erythromycin, tylosin, oleandomycin, and spiramycin were extracted from animal tissues, milk, and egg with acetonitrile at pH 8.5. Cleanup was done by adding sodium chloride and dichloromethane, evaporating the organic layer, and subsequent acid/base partitioning. After the antibiotics were separated by thin-layer chromatography (TLC), they were reacted with xanthydrol and could be detected as purple spots down to 0.02 mg/kg without interference by other commonly used therapeutic drugs (23 were tested). Anisaldehyde-sulfuric acid, cerium sulfate-molybdic acid, phosphomolybdic acid, and Dragendorff's reagent proved to be less sensitive as visualizing agents. For quantitation, TLC plates were scanned at 525 nm. Recoveries were between 71 and 96% for erythromycin and tylosin in liver, muscle, and egg at the 0.1-0.5 mg/kg level and 51% for erythromycin in milk at the 0.02 mg/kg level (coefficient of variation = 10-18%). Bioautography with Bacillus subtilis was used to confirm results, in addition to TLC analysis of derivatized antibiotics and liquid chromatography with electrochemical detection. Various derivatization procedures for erythromycin were investigated for improved ultra-violet or fluorescence detection in liquid chromatography.

[Gel chromatography (GPC) as a clean-up procedure in residue analysis of veterinary drugs]. / Untersuchungen zur Gelchromatographie (GPC) als Reinigungsverfahren in der Rückstandsanalytik von Tierarzneimitteln.

In this study we investigated the chromatographic behaviour of 40 veterinary drugs belonging to different classes (sulphonamides, nitrofurans, nitroimidazoles, antibiotics, anthelmintics, psychopharmacological substances, growth promoters) using the gel permeation media Sephadex LH-20, Bio Beads S-X 3 and Fractogel PGM 2,000. To allow the use of acetic acid in mobile phases all parts containing brass in the GPC equipment (Autoprep 1002 A) had to be exchanged for stainless steel components. The curves of elution with Sephadex LH-20 as stationary phase demonstrated good separation of the majority of drugs from co-extractives from liver, kidney, muscle, egg and milk by gravimetric measurement as well as by UV absorption. A clean-up of similar efficiency could however also be obtained with Sep-Pak silica cartridges, if final residue analysis was made by HPLC with UV detection.

[Chemical analysis of veterinary drugs in food. 1. General methods and gas chromatographic procedures]. / Chemische Analyse von Tierarzneimittelrückständen in Lebensmitteln. 1. Mitteilung: Allgemine Methodik und gaschromatographische Verfahren.

A review is presented that in the first section describes current techniques for preparation, extraction and clean up of food samples for the determination of veterinary drug residues by chemical methods. The second section gives an overview of gas chromatographic methods published up to 1983 for the analysis of meat, liver, kidney, egg and milk for residues of antibiotics and chemotherapeutic agents. Discussed are stability of the compounds, derivatization, detection and particular aspects of their metabolism. The structural formula is given for all drugs mentioned. The procedures for determination are summarized in a table, together with the most important analytical parameters.