Acute Liver Failure after Ingestion of Fried Rice Balls: A Case Series of Bacillus cereus Food Poisonings.

Bacillus cereus foodborne intoxications and toxicoinfections are on a rise. Usually, symptoms are self-limiting but occasionally hospitalization is necessary. Severe intoxications with the emetic Bacillus cereus toxin cereulide, which is notably resistant heat and acid during cooking, can cause acute liver failure and encephalopathy. We here present a case series of food poisonings in five immunocompetent adults after ingestion of fried rice balls, which were massively contaminated with Bacillus cereus. The patients developed a broad clinical spectrum, ranging from emesis and diarrhoea to life-threatening acute liver failure and acute tubular necrosis of the kidney in the index patient. In the left-over rice ball, we detected 8 × 106Bacillus cereus colony-forming units/g foodstuff, and cereulide in a concentration of 37 µg/g foodstuff, which is one of the highest cereulide toxin contaminations reported so far from foodborne outbreaks. This report emphasizes the potential biological hazard of contaminated rice meals that are not freshly prepared. It exemplifies the necessity of a multidisciplinary approach in cases of Bacillus cereus associated food poisonings to rapidly establish the diagnosis, to closely monitor critically ill patients, and to provide supportive measures for acute liver failure and-whenever necessary-urgent liver transplantation.

Dietary exposure to furan of the Austrian population.

In the period from 2007 to 2017 furan levels of foods were analysed by the Austrian Agency for Health and Food Safety. Based on these analytical data and the Austrian consumption data the dietary exposure of children and adults to furan was estimated by using a deterministic approach. For the adult population the mean and 95th percentile dietary exposures to furan were estimated at 0.31 µg/kg bodyweight per day and at 0.72 µg/kg bodyweight per day, respectively. The mean dietary exposure of children was estimated at 0.18 µg/kg bodyweight per day and is thus only about half as high as for Austrian adults. At the 95th percentile the dietary exposure of children was estimated at 0.49 µg/kg bodyweight per day. The main contributor to the total dietary exposure for adults is coffee followed by convenience products and for children the main contributors are grain products as well as convenience products, bread and snacks. Based on the BMDL10 of 0.064 mg/kg bodyweight per day for the development of cholangiofibrosis, the MOE-calculation revealed that the current levels of dietary exposure to furan are of concern for Austrian adult high consumers. The MOE-calculation, based on the BMDL10 of 1.31 mg/kg bodyweight per day for the development of hepatocellular adenomas, indicated a health concern for Austrian children and adults.

LC-MS/MS analysis of neonicotinoid insecticides in honey: methodology and residue findings in Austrian honeys.

An analytical method for the simultaneous determination of residues of eight neonicotinoid insecticides and two metabolites in honey using LC-MS/MS was developed and validated. Two approaches of sample preparation were investigated, with the final method involving acetonitrile extraction and subsequent cleanup by dispersive solid-phase extraction (QuEChERS type). Validation was based on quintuplicate analysis at three fortification levels and showed satisfactory recoveries (60-114%) and high precision (RSDs between 2.7 and 12.8%). Low limits of detection and quantification could be achieved for all analytes ranging from 0.6 to 5 µg/kg and from 2 to 10 µg/kg, respectively. Investigations of Austrian honey samples revealed the presence of acetamiprid, thiacloprid, and thiamethoxam residues in honey; however, no sample exceeded the maximum residue limits. On average, flower honey samples contained neonicotinoid residues in higher quantities compared to forest honey samples.

The use of tumour necrosis factor alpha-blockers in daily routine. An Austrian consensus project.

To define relevant disease parameters and their respective limits indicating the initiation of TNF-alpha-blockers in individual patients. Subsequently, to analyze retrospectively patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) or ankylosing spondylitis (AS), who started TNF-alpha inhibition in 2006. Points to consider, regarded relevant for individual treatment decisions as well as their assessment methods, were ascertained by experts' consensus applying the Delphi technique. Subsequently, these parameters' thresholds with respect to the initiation of a TNF-alpha-blocker were identified. Thereafter, the rheumatologists representing 12 centres all over Austria agreed to retrospectively analyze their patients started on a TNF-alpha-blocker in 2006. Experts' opinion regarding disease parameters relevant to initiate TNF-alpha-blockers in RA patients only slightly differed from those applied in clinical trials, but the parameters' threshold values were considerably lower. For PsA patients, some differences and for AS patients, considerable differences between experts' opinion and clinical studies appeared, which held also true for decisive parameters' means and thresholds. Six hundred and fifty patients, started on TNF-blockers in 2006, could be analyzed retrospectively, 408 RA patients (53.3 years mean, 340 females), 93 PsA patients (48.9 years mean, 59 males) and 149 AS patients AS (42.2 years mean, 108 males), representing approximately 25% of all Austrian patients initiated on a TNF-blocker in this respective year. Far more individualized, patient-oriented treatment approaches, at least in part, are applied in daily routine compared with those derived from clinical trials or recommendations from investigative rheumatologists.

Absolute quantitation of beta-lactoglobulin by protein liquid chromatography-mass spectrometry and its application to different milk products.

The absolute quantitation of proteins in biological matrixes is of great interest in many fields and can be accomplished by different methodologies. Here, a method for the absolute quantitation of the whey protein beta-lactoglobulin using protein liquid chromatography coupled to mass spectrometry is reported. The developed approach was characterized in detail and applied to the determination of beta-lactoglobulin contents in various milk products. A special focus was placed on the recovery rates of the isolation procedure and on robust quantitation by LC-MS. For these purposes protein internal standards were employed. The observed recovery rates of beta-lactoglobulin from various samples ranged from 100% for whole milk to just over 50% for a strongly processed yogurt-based baby food product. The influence of processing was investigated in greater detail, showing that an increasing intensity of the applied heat treatment resulted in an increasing loss of beta-lactoglobulin. LC-MS quantitation at the protein level proved to be highly suitable, avoiding a potentially problematic digestion step. The use of an appropriate internal standard to compensate for sample losses during sample workup was shown to be essential for obtaining accurate results.

Investigation of the lactosylation of whey proteins by liquid chromatography-mass spectrometry.

Heat treatment of milk induces a reaction between the milk proteins and lactose, resulting in lactosylated protein species. The lactosylation of the two major whey proteins alpha-lactalbumin and beta-lactoglobulin was investigated by reversed phase liquid chromatography-mass spectrometry (LC-MS). Three sample series, consisting of aqueous model solutions of each whey protein separately and in mixture and whole milk, were heated for different time periods, and the progression of the lactosylation reaction was monitored. The observed degrees of lactosylation and the reaction kinetics showed that the lactosylation of beta-lactoglobulin was not influenced by the presence of other components, whereas the lactosylation of alpha-lactalbumin was enhanced in whole milk compared to the aqueous model systems. An in-depth evaluation of the LC-MS data yielded information regarding changes of physicochemical properties of the whey proteins upon lactosylation. Whereas retention time shifts indicated changes in hydrophobicity for both alpha-lactalbumin and beta-lactoglobulin, changes in the charge state distribution denoting conformational alterations were observed only for beta-lactoglobulin. The analysis of different liquid and solid milk products showed that the lactosylation patterns of the whey proteins can be used as indicators for the extent of heat treatment.

Peptide enantiomer separations: influence of sequential isomerism and the introduction of achiral glycine moieties on chiral recognition.

The influence of sequential isomerism and the introduction of achiral, conformationally flexible glycine moieties into a peptide chain on the chiral recognition mechanism of a cinchona alkaloid based chiral selector has been evaluated. For this purpose, enantiomers of N-terminally protected alanine-glycine di- and tripeptides were separated by liquid chromatography-mass spectrometry on a corresponding chiral stationary phase (CSP). To obtain complementary information, the reversed phase retention behaviour of the various peptides was also evaluated and subsequently used to further elucidate the chromatographic characteristics of the CSP. For peptides that contained glycines in the N-terminal region chiral recognition was compromised, while glycines located at the C-terminus had no or little negative effect.

Stereoselective peptide analysis.

The stereochemistry of a peptide determines its spatial features and can profoundly influence its chemical properties and biological activity. Thus, the analysis of the stereochemical properties of a peptide is an important aspect of its characterisation. For such investigations a "selector" that engages in stereoselective interactions with the peptide analytes is often used. A substantiated knowledge of the underlying molecular recognition mechanism will therefore be helpful in understanding existing and developing new stereoselective analysis systems. After a short introduction concerning the fundamentals of peptide stereoisomers and their biological implications, the stereoselective peptide analysis methods described in the literature are comprehensively reviewed. The characteristics and applications of the employed methods based on various techniques including chromatography (pressure- and electrokinetically driven), capillary electrophoresis, nuclear magnetic resonance spectroscopy and mass spectrometry are discussed. The various selectors that have been utilised to discriminate peptide enantiomers and/or diastereomers are described concurrently. The review concludes with an overview of combinations and comparisons of techniques that have been applied to the analysis of peptide stereoisomers and constitute a trend for further developments.

Enantiomer discrimination of peptides by tandem mass spectrometry: influence of the peptide sequence on chiral recognition.

The enantiomer discrimination of peptides by electrospray ionization tandem mass spectrometry is described. A cinchona alkaloid derivative, tert-butylcarbamoylquinine, is used as chiral selector. The chiral selector forms diastereomeric complexes with the peptide enantiomers in the liquid phase (methanolic solution), which are then transferred to the gas phase, where their dissociation behaviour is studied in an ion-trap mass spectrometer. Different degrees of dissociation of the diastereomeric complexes allow for the discrimination of the peptide enantiomers. The influence of the peptide sequence on enantiomer discrimination is discussed and molecular recognition information is derived by comparing the results obtained for related peptides. For dipeptides, small amino acid residues at the N-terminus and bulky side chains at the C-terminus were found to enhance chiral recognition, while for tripeptides the effects were rather irregular.

Enantiomer discrimination by mass spectrometry: noncovalent interactions of an N-derivatized dipeptide with various cinchona alkaloid derivatives and comparison with enantioselective liquid-phase separations.

The enantiomer discrimination properties of cinchona alkaloid derived chiral selectors (CSs) towards a dipeptide analyte are examined by electrospray ionization mass spectrometry. The complexes formed between the CSs and the analyte enantiomers owing to various noncovalent interactions are analyzed and the magnitudes of enantiomer discrimination are determined from the complexes' mass spectrometric intensities. The influence of different structural features of the CSs on enantioselectivity is discussed. The enantiomer discrimination results obtained by mass spectrometry are compared with those from related liquid chromatography enantiomer separations. A certain coherence between the chromatographic and mass spectrometric enantioselectivities could be established and the enantiomer discrimination patterns, i.e., the relative binding strengths, were identical for the two techniques. Thus, the use of mass spectrometry as a screening tool in the development of new CSs for chromatographic applications seems feasible.

Liquid chromatographic-mass spectrometric separation of oligoalanine peptide stereoisomers: influence of absolute configuration on enantioselectivity and two-dimensional separation of diastereomers and enantiomers.

This contribution describes the chromatographic separation of peptide stereoisomers. Thereby, one focus is laid on the influence of the absolute configurations of peptide enantiomer pairs on their enantioselective separation. Three different N-terminal protecting groups and three different chiral stationary phases (CSPs) based on cinchona alkaloid derivatives were employed and oligoalanine di-, tri- and tetra-peptides were used as model set. The absolute configurations of the individual enantiomeric pairs were found to profoundly influence both the elution order and the enantioselectivity. The stereoselective molecular recognition mechanism was observed to be dependent on the combination of configuration and the chosen protecting group and CSP. As the CSPs on their own exhibited insufficient diastereoselectivity, a two-dimensional liquid chromatography-mass spectrometry (LC-MS) system was developed for the separation of both diastereomers and enantiomers of peptides in the second part of this study. Diastereomers were separated by reversed phase (RP) and the resulting enantiomeric pair fractions were transferred to a CSP for enantioseparation. All eight stereoisomers of a tripeptide (Ala-Ala-Ala) and 9 out of 10 stereoisomers of a tetrapeptide (Ala-Ala-Ala-Ala) could be successfully resolved.

Chiral recognition of peptide enantiomers by cinchona alkaloid derived chiral selectors: mechanistic investigations by liquid chromatography, NMR spectroscopy, and molecular modeling.

The chiral recognition mechanism of a cinchona alkaloid based chiral selector for N-protected peptide enantiomers was investigated. A chiral stationary phase derived from this selector was employed for liquid chromatographic enantiomer separations. It showed exceptionally high enantiomer discrimination for the (all-R)- and (all-S)-enantiomers of dialanine (alpha = 20), while a pronounced loss of chiral recognition occurred upon the insertion of an additional alanine residue into the peptide backbone. This reduction of enantioselectivity was investigated in great detail by NMR spectroscopy of complexes of the chiral selector and the analyte enantiomers accompanied by molecular modeling studies. Investigation of intramolecular NOEs provided the conformational states of the free and complexed forms of the selector. The analysis of complexation-induced shifts yielded information on intermolecular interactions and allowed us to propose binding models, which were further supported by the observation of intermolecular NOEs, indicating the relative arrangements of selector and analytes. Stochastic molecular dynamics simulations were able to reproduce the chromatographic retention orders and energy differences, as well as the intermolecular NOEs. The computational data were used to evaluate the intermolecular forces responsible for analyte binding. In addition, the relative contributions of the fragments of the chiral selector to the enantioselective binding event were assessed. A spatial arrangement of the chiral selector and the analyte allowing the primary ionic interaction as well as hydrogen bonding and pi-pi-stacking to take place simultaneously was found to be essential to obtain very high enantioselectivities.

Micro-HPLC and standard-size HPLC for the separation of peptide stereoisomers employing an ion-exchange principle.

Standard-size (4 mm ID) and micro-HPLC columns (0.5 mm ID) packed with a quinine-based ion-exchange type chiral stationary phase are comparatively evaluated for the separation of peptide enantiomers with up to six amino acid residues. The results show that downscaling the separation system in order to gain the advantages of miniaturized HPLC is possible without sacrificing separation power. Further, five different N-terminal protections (3,5-dinitrobenzoyl, 2,4-dinitrophenyl, 3,5-dinitrobenzyloxycarbonyl, carbazole-9-carbonyl, and 9-fluorenylmethoxycarbonyl) of the analytes are investigated regarding their effect on enantioselectivity. A comparison between a hydro-organic and a polar-organic mobile phase is also reported. The enantiomers of the peptides containing one to four amino acid residues were baseline resolved, while for the penta- and hexamers only partial separations were possible. In addition, all four stereoisomers of alanylalanine could be baseline separated.

Direct high-performance liquid chromatographic separation of peptide enantiomers: study on chiral recognition by systematic evaluation of the influence of structural features of the chiral selectors on enantioselectivity.

All-R/all-S enantiomers of oligoalanines (Ala(n), n = 1-10) with N-terminal protection group have been separated by HPLC on chiral stationary phases based on various cinchona alkaloid selectors. Structure-enantioselectivity relationships derived by extensive selector structure optimization provided insights into binding mechanisms and chiral recognition. Their interpretation was supported by X-ray crystal structures of amino acid and dipeptide, respectively, in complex with chiral selector. Optimized selectors have bulky elements representing steric barriers and deep binding pockets that afforded very high enantioselectivities; e.g., for the all-R and all-S enantiomers of N-(3,5-dinitrobenzoyl)alanylalanine, an alpha-value of 20.0 (corresponding to deltadeltaG of -7.43 kJ/mol) was obtained with a chiral stationary phase based on 6'-(neopentoxy)-9-O-tert-butylcarbamoylcinchonidine. Further, a chiral stationary phase based on 1,4-bis(9-O-quinidinyl)phthalazine was able to distinguish between the all-R and all-S enantiomers of hepta- to decaalanine peptides with enantioselectivity values between 1.8 and 1.9, corresponding to deltadeltaG of -1.46 and -1.59 kJ/mol, respectively.

Electrolyte and additive effects on enantiomer separation of peptides by nonaqueous ion-pair capillary electrophoresis using tert.-butylcarbamoylquinine as chiral counterion.

Nonaqueous ion-pair capillary electrophoresis separations of N-protected (all-R)/(all-S) alanine peptide enantiomers with up to six amino acid residues using tert.-butylcarbamoylquinine as selector and employing the partial filling technique are presented. The effects of various conditional parameters on separation were studied, namely chemical nature of the capillary wall, solvent composition of the background electrolyte (BGE), acid-base-ratio (equivalent to apparent pH), ionic strength and selector concentration. The influence of the solvent composition (methanol-ethanol ratios) on resolution turned out to be rather complex. The separation of the peptide enantiomers was strongly altered by small changes in pH and ionic strength. An increase of the selector concentration was found to offer an easy way for enhancing enantioselectivity, although some drawbacks, e.g., elongation of run times, have to be considered. A method was developed that allowed the separation of N-3,5-dinitrobenzoyl oligoalanine enantiomers containing 1-6 amino acid residues in one run. Like in a recent high-performance liquid chromatography (HPLC) study, separation selectivity thereby decreased from 1.541 (Ala), 1.340 (Ala(2)), 1.054 (Ala(3)), 1.029 (Ala(4)), 1.024 (Ala(5)) to 1.020 (Ala(6)). In addition, all four stereoisomers of N-2,4-dinitrophenyl- and N-3,5-dinitrobenzyloxycarbonyl-protected alanylalanine could be baseline-resolved.