The impact of rifaximin on the hospital burden and infections in patients with hepatic encephalopathy: a retrospective observational study.

Background and study aims: Advanced liver disease frequently culminates in hepatic encephalopathy (HE), which can be classified as covert or overt HE, with subtle or clinically obvious changes respectively. 30-40% of patients with cirrhosis develop overt HE, which negatively affects the patients' quality of life. Next to lactulose, rifaximin-α has been prescribed as a second line therapy to treat and reduce the risk of recurrence of overt HE. In this study, we aimed to evaluate the effect of rifaximin-α therapy, both on the number of occurring infections and on the evolution in hospital admissions of patients with overt HE. Patients and methods: A total of 66 cirrhotic patients, treated for at least 6 months with rifaximin-α at AZ Maria Middelares, between October 1st 2014 and January 1st 2020, were included in the study analysis. Medical records of all patients were evaluated over a period of 6 months prior and after initiation of rifaximin-α therapy. Results: Data analysis revealed that the included cirrhotic patients were severely ill, with a mean model for end-stage liver disease (MELD) score of 21, and a median Child Pugh score of 11. Among these patients, rifaximin-α treatment significantly downgraded the total number of infections, with a main effect on respiratory infections. Furthermore, rifaximin-α therapy led to a significant decrease in HE-related, as well as in other liver-related hospital admissions. Conclusions: This study confirms the potential value of rifaximin-aα in reducing the number of developing infections and hospital admissions in a severely ill cirrhotic patient population.

Scapular positioning in overhead athletes with and without shoulder pain: a case-control study.

Abnormalities of scapular positioning are considered important risk factors for developing shoulder disorders. This study analyses the scapular positioning pattern in a group of overhead athletes with and without shoulder pain. In a multi-center blinded case-control study, 36 shoulder pain athletes (19 men, 17 women), were compared with 36 unimpaired athletes free of shoulder pain, matched for gender, age, hand dominance and body mass index. The blinded assessor performed visual observation, the measurement of the distance between the acromion and the table, inclinometry and the kinetic medial rotation test for dynamic scapular control in random order. Athletes with shoulder pain demonstrate scapular asymmetry in the sagittal plane, observed visually as anterior tilting on the painful side. Athletes with shoulder pain show a lack of scapular motor control on their painful side in contrast to their pain-free side. No scapular positioning or motor control differences were found in athletes with or without shoulder pain.

Metabolism pathway of vinorelbine (Navelbine) in human: characterisation of the metabolites by HPLC-MS/MS.

The biotransformation of vinorelbine (VRL), an anti-neoplastic vinca-alkaloid derivate already marketed for nonsmall cell lung cancer and advanced breast cancer as an i.v. form and currently registered in several countries as an oral form, was investigated in human. Biological specimen from several human sources constituted the material for the metabolic identification in human. An isocratic liquid chromatographic system composed of 40 mM ammonium acetate (pH 3) and acetonitrile was used for separation of the potential metabolites of VRL. Tandem mass spectrometry with positive electrospray ionisation was used to enable the structural identification of the metabolites. A total of 17 metabolites (12 directly obtained from VRL and 5 involving sequential step pathways) were characterised with proposed structures for most of the metabolites. All metabolites went through phase I reactions by the way of deacetylation, dealkylation, oxidation and hydroxylation. No conjugates were observed. Despite the high number of metabolites quantified, VRL was the major compound observed whatever the matrix. Most of the metabolites rapidly disappeared from blood, except 4-O-deacetyl vinorelbine which was slowly cleared. Most of the enzymatic pathways involved in the metabolites strongly suggested the major role of cytochrome P450 in the biotransformation of vinorelbine.

Development of a sensitive liquid chromatography method coupled with a tandem mass spectrometric detection for the clinical analysis of vinflunine and 4-O-deacetyl vinflunine in blood, urine and faeces.

A sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and fully validated for the simultaneous quantification of vinflunine (VFL) and its pharmacologically active metabolite, 4-O-deacetyl vinflunine (DVFL). The two compounds, as well as vinblastine (used as internal standard), were deproteinised from blood and faeces, analysed on a cyano type column and detected on a Micromass Quattro II system in the positive ion mode after ionisation using an electrospray ion source. In blood, linearity was assessed up to 200 ng/ml for vinflunine and 100 ng/ml for 4-O-deacetyl vinflunine. The lower limit of quantification was validated at 250 pg/ml for both compounds. In other biological media, the linearity was assessed within the same range; the limit of quantification was adjusted according to the expected concentration levels of each compound. This method was first developed in order to identify the structures and to elucidate the metabolic pathway of vinflunine. Thanks to its high sensitivity and specificity, the method has enabled the quantification of vinflunine and 4-O-deacetyl vinflunine in blood at trace levels, and has contributed to the knowledge of vinflunine metabolism by monitoring up to 10 metabolites.

Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for on-line sample clean-up.

A new automated method for the quantitative analysis of cyproterone acetate (CPA) in human plasma has been developed using on-line solid phase extraction (SPE) prior to the LC-MS/MS determination. The method was based on the use of a pre-column packed with internal-surface reversed-phase material (LiChrospher RP-4 ADS, 25 mm x 2 mm) for sample clean-up coupled to LC separation on an octadecyl silica stationary phase by means of a column switching system. A 30 microl plasma sample volume was injected directly onto the pre-column using a mixture of water, acetonitrile and formic acid (90:10:0.1 (v/v/v)) adjusted to pH 4.0 with diluted ammonia as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase consisting of water, methanol and formic acid (10:90:0.1 (v/v/v)). The dispensing flow rates of the washing liquid and the LC mobile phase were 300 microl min(-1). Medroxyprogesterone acetate (MPA) was used as internal standard. The MS ionization of the analytes was achieved using electrospray (ESI) in the positive ion mode. The pseudomolecular ionic species of CPA and MPA (417.4 and 387.5) were selected to generate daughter ions at 357.4 and 327.5, respectively. Finally, the developed method was validated according to a new approach using accuracy profiles as a decision tool. Very good results with respect to accuracy, detectability, repeatability, intermediate precision and selectivity were obtained. The LOQ of cyproterone acetate was 300 pg ml(-1).

A structured strategy for assessing chemical risks, suitable for small and medium-sized enterprises.

BACKGROUND: A previous study carried out in 20 Belgian companies, especially small and medium-sized enterprises (SMEs), showed that prevention advisors did not use any structured approaches to assess chemical risk. They used their personal judgement and the information contained in manufacturers' Safety Data Sheets to assess the risk. OBJECTIVE AND METHOD: The purpose of the Regetox network is to provide companies with a global approach for assessing chemical health risks. The structured approach proposed consists of two successive steps of increasing complexity. For the first step, we chose a method developed by the INRS (France), the 'ranking of potential risk', which allows the safety officer or staff member to identify hazards and to set priorities among all the supplied products used in the workplace. For the second step, we applied the COSHH method and EASE model established by the UK Health & Safety Executive for assessing 'chemical risk by reference to occupational exposure limits'. The INRS and COSHH Essentials methods were chosen because they define hazards using R-phrases of the European classification system and assess the exposure by using simple information that is easy to collect in the workplace and toxicological databases. A feasibility study conducted in two enterprises shows the usefulness of this approach. In addition to the intrinsic limitations of the methods, the approach showed some limitations related to the inaccuracy of the manufactured safety data sheets and to the collection of the basic information needed for ranking potential risks. CONCLUSION: The use of the Regetox approach needs training of prevention advisors and a strategy involving employers, staff members and workers in collecting basic information and managing chemical risks. Under these conditions, Regetox seems to be a useful tool for chemical risk assessment in SMEs.

Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography-electrospray ionization tandem mass spectrometry.

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation and programmed cell death. Qualitative and quantitative analysis of these second messengers requires sensitive and specific analytical methods to detect endogenous levels of individual ceramide species and to differentiate between them. Nine synthetic ceramides were separated by liquid chromatography coupled to tandem mass spectrometry on a C18 bonded silica column. The lipids were eluted in gradient elution mode using a mixture of water, acetonitrile and 2-propanol as mobile phase. They were detected by reaction monitoring performed on positive ion electrospray generated ions. Collision-induced fragmentations conducted on ceramides produced a well characteristic product ion at m/z 264, making multiple reaction monitoring (MRM) well suited for various ceramides quantitative measurements. After optimization of the extraction step, the proposed methodology was able to identify and quantify different ceramide species issued from human cancer cells. The method could be validated for C16, C18 and C20 ceramides, quantified at the nanogram level. The validation exhibits good results with respect to linearity, accuracy and precision.

New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urine and faeces.

A new sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and validated for the simultaneous quantitation of vinorelbine, its main metabolite, 4-O-deacetylvinorelbine and two other minor metabolites, 20'-hydroxyvinorelbine and vinorelbine 6'-oxide. All these compounds, including vinblastine (used as internal standard) were deproteinised from blood, plasma and faeces (only diluted in urine), analysed on a cyano column and detected on a Micromass Quattro II system in the positive ion mode after ionisation, using an electrospray ion source. Under tandem mass spectrometry conditions, the specific product ions led one to accurately quantify vinorelbine and its metabolites in all biological fluids. In whole blood, linearity was assessed up to 200 ng/ml for vinorelbine and up to 50 ng/ml for the metabolites. The limit of quantitation was validated at 250 pg/ml for both vinorelbine and 4-O-deacetylvinorelbine. In the other biological media, the linearity was assessed within a same range and the limit of quantitation was adjusted according to the expected concentrations of each compound. This method was initially developed in order to identify the metabolite structures and to elucidate the metabolic pathway of vinorelbine. Thanks to its high sensitivity, this method has enabled the quantitation of vinorelbine and all its metabolites in whole blood over 168 h (i.e., 4-5 elimination half lives) whilst the previous liquid chromatographic methods allowed their measurement for a maximum of 48-72 h. Therefore, using this method has improved the reliability of the pharmacokinetic data analysis of vinorelbine.

Nitazoxanide: pharmacokinetics and metabolism in man.

OBJECTIVES: Nitazoxanide (N), a new broad-spectrum parasiticidal agent, is rapidly deacetylated to tizoxanide (T). The objective of the study was to determine if metabolites other than T are present in the plasma and excreted after single dose oral administration of radiocarbon-labelled N in healthy subjects. METHODS: Six healthy volunteers received a single 500 mg oral dose of N labelled with 2.92 MBq radiocarbon. The radioactivity in blood, plasma, urine, feces and expired air was monitored at scheduled intervals for up to 10 days. Selected samples were assayed by HPLC for T and submitted to metabolite identification by mass spectrometry. In vitro experiments were also conducted (incubation with animal and human microsomes, deacetylation kinetics). Plasma and bile samples obtained in a patient treated with N for sporozoal infection were also assayed for T. RESULTS: Elimination of radiocarbon occurred both in the urine (31.5% of the dose on average) and in the feces (66.2% on average). T and T-glucuronide contributed 15% of total urine radioactivity. N was found to deacetylate extremely rapidly to T in plasma (half-life of about 6 minutes at 37 degrees C) as well as in presence of liver microsomes. T was the only species obtained by incubation with human microsomes while rat microsomes yielded hydroxylated T in addition. The main species identified in human plasma, urine and bile was T-glucuronide, the identification of which was confirmed by comparison with an authentic sample. No species other than T was detected in feces, indicating intensive intestinal deconjugation, while radioactivity and absorbance detectors showed largely unresolved clusters.

[Quality specifications and allowable standards for validation of methods used in clinical biochemistry]. / Analyses de biologie médicale: spécifications et normes d'acceptabilité à l'usage de la validation de techniques.

The purpose of this work is to provide, for a large number of analysis in the field of clinical biochemistry, appropriate criteria for the evaluation of the performance of in vitro diagnostic methods. Based on a first set of data established in 1986 and the experience cumulated by organisers in charge of internal and external quality assessment surveys, an expert group has proposed acceptable limits for a large list of analysis (n = 116). Data are reported and presented in tables divided into 7 chapters including: general biochemistry, enzymes, proteins, tumour markers, hormones, drugs (and toxic), and urinary analysis. For each analysis are given: analytical and reference ranges, three concentration levels for control specimens to be used during evaluations and the range of values within which they can be chosen, reproducibility and repeatability limits expressed as CV%. Maximal tolerable systematic error and inaccuracy are given for control and biological specimens and compared to those obtained using a reference or validated method. These data are essential for evaluations using the protocol designed by the SFBC and can serve as quality criteria for the choice and validation of in vitro diagnostic systems.

GC-MS determination of anabolic steroids after multi-immunoaffinity purification.

For many years, EC regulations have prohibited the use of anabolic agents in food-producing animals. Multiple screening methods have been published, but some lack specificity and some are difficult to apply when screening for unknowns in surveillance programmes. This paper presents a new and powerful technique, combining multiresidue immunoaffinity chromatography and GC-MS, for the simultaneous identification and semi-quantification of various anabolic steroids in urine and faeces samples of bovine origin. It should reduce the cost, time and effort of screening by limiting the number of tedious clean-up steps and analyses required. A preliminary extraction step is applied to the individual biological specimens: solid-phase extraction followed by enzymatic digestion in the case of urine samples and a single liquid extraction step for faeces. This step is followed by a first clean-up step involving both a solid-phase column and a rapid RP-HPLC separation. The individual biological fractions (urine or faeces) are further purified on a multiresidue immunoaffinity chromatographic gel (MIAC-steroids-CER) so as to decrease interferences due mainly to background signals. A final trimethylsilyl derivatization is followed by the analysis of the biological samples by a sensitive and specific GC-MS procedure.

Quantitative determination of several synthetic corticosteroids by gas chromatography-mass spectrometry after purification by immunoaffinity chromatography.

A study was conducted to test a multiresidue analytical procedure for detecting and quantifying several corticosteroids on which the European Union imposes maximum residue limits (MRLs). Primary extracts from different matrices (liver, milk, urine, faeces) were first purified on C18 cartridges. A new immunoaffinity clean-up step was included. The immunoaffinity gel was used to purify several corticosteroids simultaneously with enrichment of the corresponding fractions. The extracts were treated with an aqueous solution of pyridinium chlorochromate to fully oxidise all corticosteroids and to facilitate their extraction with dichloromethane. After evaporation, the final extract was reconstituted with toluene before injection into the GC-MS apparatus. The analysis was performed in the CI-negative ionisation mode using ammonia as the reactant gas. The estimated detection and quantification limits were, respectively, 0.25 and 0.5 ppb or lower. Overall, the method is reproducible to within 20%. Recovery is between 50 and 80% according to the corticosteroid.

Laboratory medicine in France. A jeopardized situation.

The expenses for health care in France have risen considerably during the present decade, ranking third after USA and Canada in the Western world. In spite of the very low cost of laboratory medicine (2.4% of the total expenditure in 1995), clinical laboratories have undergone a severe squeeze, due to two limiting factors; a decrease in the ordering of laboratory tests from private physicians and a reduction in the total expenses for laboratory services from the Social Security. Consequently, there has been unemployment of technical and secretarial staff and severe restriction in investment for buying new equipment. However, hospital laboratories will manage to assume their challenge in developing robotics, automation, molecular pathology techniques and expert systems. Private laboratories, in spite of their efforts to follow the technological advances in automation, will survive thanks to consolidation of regional networks that operate in a cooperative rather than competitive mode. Therefore, the challenge will be not in the adaptation of clinical laboratories, but in the limitation of overspending at the national level and in modification of the behaviour of irresponsible citizens accustomed to spending freely on health care services.

Automated technical validation--a real time expert system for decision support.

Dealing daily with various machines and various control specimens provides a lot of data that cannot be processed manually. In order to help decision-making we wrote specific software coping with the traditional QC, with patient data (mean of normals, delta check) and with criteria related to the analytical equipment (flags and alarms). Four machines (3 Ektachem 700 and 1 Hitachi 911) analysing 25 common chemical tests are controlled. Every day, three different control specimens and one more once a week (regional survey) are run on the various pieces of equipment. The data are collected on a 486 microcomputer connected to the central computer. For every parameter the standard deviation is compared with the published acceptable limits and the Westgard's rules are computed. The mean of normals is continuously monitored. The final decision induces either an alarm sound and the print-out of the cause of rejection or, if no alarms happen, the daily print-out of recorded data, with or without the Levey Jennings graphs.

An atypical aortic atherosclerotic lesion in cynomolgus monkeys during hypercholesterolemia: a protection by smooth muscle cells against advanced lesions?

This study focuses on the fortuitous discovery of an atypical atherosclerotic lesion in four of 49 male adult cynomolgus monkeys (macacus fascicularis) which were maintained for a long time at a high level of hypercholesterolemia, and in seven of 19 female cynomolgus monkeys examined from the second to the 24th week of hypercholesterolemic diet: this lesion was in formation or already mature during this period of diet. This atypical lesion was formed by a collagen and elastic network surrounding synthetic smooth muscle cells without fibrofatty or fibrous plaques. Lipids were occasionally seen in the inner intima. The lesion appeared early (from the third week of diet). Once established, its morphology did not change. It became more extensive, but was not complicated by lipid overload in spite of prolonged, permanent hypercholesterolemia. This response to hypercholesterolemia is interesting because the activity of the smooth muscle cells differs from that observed in the classic lesion: they intervene earlier, their replication is very marked and rapid, their elastin secretion is greater and remains constant over time, and their phagocytic properties are reduced. This experimental study examines the installation and the maintenance of this lesion and raises the problem of its origin.

Reduction and glucuronidation of naftazone by human and rat liver microsomes.

Reduction and glucuronidation of the vasoprotectant drug, naftazone, by human and rat liver microsomes and by recombinant UDP-glucuronosyltransferases (UGT) stably expressed in V79 cells were studied. The oxo group was first reduced in the presence of NADPH or NADH, and was subsequently readily glucuronidated on the phenolic moiety leading to a 1 beta-O-glucuronide, as revealed from MS and by proton and 13C-NMR. Glucuronide extracted from the urine of rats treated with the drug presented the same structure. In all enzyme systems tested, NADH was the most efficient electron donor, when compared with NADPH. The reaction was strongly inhibited by quercitrin, a specific inhibitor of carbonyl reductase. Attempts to isolate the reduced intermediate were unsuccessful because of its marked instability. In humans, a large interindividual variation for the formation of glucuronide was observed with microsomes of seven different liver samples (3.98 +/- 3.22 nmol/min.mg). In rat, glucuronidation of reduced naftazone was strongly induced (12-fold) by 3-methylcholanthrene and, to a lesser extent (2.6-fold) by phenobarbital, but was not affected by clofibrate. In addition, liver microsomes from Gunn rats, which present a genetic defect in bilirubin and phenol UGTs could not form glucuronide of reduced naftazone. The drug, after addition of NADH, was a substrate of the human liver recombinant UGT1*6 that presents a strict specificity toward planar phenolic substances, but not that of UGT2B4 and UGT2B1 expressed in V79 fibroblasts. The reducing step by the endogenous V79 cellular reductase was rate-limiting. In conclusion, the powerful inducing effect exerted by 3-methylcholanthrene, the lack of glucuronidation in the Gunn rat and the ability of UGT1*6 encoded by the UGT1 gene to glucuronidate reduced naftazone suggest that, in humans and in the rat, the compound is metabolized by a UGT isoform (UGT1*6 and the rat orthologous form) belonging to family 1, with a restricted specificity toward the drug.

A data management program for the Electra 800 automatic analyser.

The Electra 800 automatic coagulation analyser rapidly performs most chronometric coagulation tests with high precision. To facilitate data handling, software, adaptable to any PC running under MS-DOS, was written to manage the analyser. Data are automatically collected via the RS232 interface or can be manually input. The software can handle 64 different analyses, all entirely 'user defined'. An 'electronic worksheet' presents the results in pages of ten patients. This enables the operator to assess the data and to perform verifications or complementary tests if necessary. All results outside a predetermined range can be flagged and results can be deleted, modified or added. A patient's previous files can be recalled as the data are archived at the end of the day. A 120 Mb disk can store approximately 130,000 patient files. A daily archive function can print the day's work in alphabetical order. A communication protocol allows connection to a mainframe computer. This program and the user's manual are available on request, free of charge, from the authors.

Computer program for connection and data management of an automated coagulation system: the KC 10.

In Europe, the KC 10, manufactured by Amelung Germany, is one of the instruments most commonly found in coagulation laboratories. For facilitating the work of technical validation, we wrote a software adapted to any IBM or compatible PC running under MS-DOS, to manage the analyser performance. Data are automatically collected via the BCD interface from the analyser or keyed in for the other techniques. The software deals with 64 different analyses entirely 'user defined'. An 'electronic worksheet' presents the results, by page of ten patients. This enables the laboratory technician to assess the coherence of the various data and to perform verifications or complementary tests if necessary. As an option, a blinking asterisk can signal all results outside predetermined range. By moving the cursor through the table, a test result can be deleted, modified or added. A function displays the patient's previous files in a window because the data are recorded in long-term archives at the end of the day. This long-term recording allows a search of previous files to decide additional tests if the patient is unknown. A daily archive function classifies and prints the whole day's work in alphabetical order. A protocol of communication allows connection to a mainframe Bayer-Technicon computer. This program and the user's manual are free, available on request from address above.

Identification and characterization of 19-nortestosterone in urine of meat-producing animals.

To monitor the illegal use of 19-nortestosterone as an anabolizing agent in meat-production, the Belgian Institute of Veterinary Expertise applies a strategy of urine control by radioimmunoassay, positive samples being confirmed by thin-layer chromatography. We have evaluated this control strategy, using gas chromatography-mass spectrometry to confirm the presence of 19-nortestosterone, or its metabolite oestrane-diol, in positive samples from radioimmunoassay. Our results show that the effective way of proceeding remains reliable in cattle, for mature and immature males as well as non-pregnant females, and in pigs, for pregnant and non-pregnant sows. The possible presence of endogenous 19-nortestosterone in cattle, in pregnant cows urine, and in pigs, in boars and in cryptorchid pigs, impedes the control of the use of 19-nortestosterone on these samples. False-positive (not confirmed by gas chromatography-mass spectrometry) results were produced by radioimmunoassay in the urine of castrated pigs and sheep.