Characterisation of novel defective thiopurine S-methyltransferase allelic variants.

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5'-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys(119)Thr, TPMT*9) and c.205C>G (p.Leu(69)Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln(179)His, TPMT*24) and c.634T>C (p.Cys(212)Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters (K(m) and V(max)) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu(69)Val and the p.Cys(212)Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys(119)Thr and the p.Gln(179)His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.

[Measurement of urinary free cotinine. Comparison with the level of expired air carbon monoxide]. / Dosage de la cotinine urinaire libre. Deuxième partie: comparaison avec le taux de monoxyde de carbone dans l'air expiré.

BACKGROUND AND AIM: Cotinine is a very reliable index for the estimation of active or passive smoking. Sampling from a single urine void is well accepted by smokers who are willing to stop. It is not possible to exclude modification of urine cotinine according to beverage intake. The aim of this study was to determine if urine cotinine concentration must necessarily be adjusted to creatinine or not, by making comparison with expired air carbon monoxide. MATERIAL AND METHODS: Carbon monoxide was measured in 53 smokers coming for the first time in a smoking cessation program. Urine cotinine was measured by HPLC-UV. The cut-off value for abstinence is 8ppm and 0.05 mg/L, repectively. Urine creatinine was determined using the Jaffe reaction. RESULTS: Mean CO level was 18.5 +/- 10.6 ppm and mean urine cotine was 1.45 +/- 0.86 mg/L. Eight smokers had CO 8 ppm. They should be considered as abstinent. However, only one of them had a cotinine under the detection limit. Urine creatinine varied in a large range (0.7 - 35 mmol/L). But, cotinine was only weakly correlated to creatinine (r = 0.279, p = 0.037). There was a highly significant correlation between cotinine and CO (0.649, p = 0.0001). The correlation of cotinine/creatinine versus CO was not significant (r = 0.249, p = 0.072). In order to take into account fluid intake, urine cotinine of each sample was adjusted as if creatinine was equal to the mean (8.3 mmol/L) of the group of subjects. The correlation observed with adjusted or non adjusted cotinine and CO (r = 0.640, p < 0.0001) was the same. CONCLUSION: Urine cotinine from a single void is an accurate index of tobacco smoking at the individual level. There is no need to adjust cotinine concentration, taking into account urine creatinine. Measurement of urine cotinine can be useful to manage smokers who deliberately wish to overcome tobacco dependence, offering the opportunity to provide an adequate level of nicotine substitutive therapy. It is also of peculiar importance to follow-up pregnant women and smokers for whom cessation is required after a clinical event. Finally, absence of cotinine in urine can be used to document abstinence from tobacco products.

[Urinary cotinine and nicotine metabolites measurement]. / Dosage urinaire de la cotinine et des métabolites de la nicotine.

UNLABELLED: According to the recent regulations (Circulaire DGS/DH du 3 avril 2000), tobacco dependence must be determined by the measurement of urine nicotine metabolites. Various assay methods are presently available. They were tested in order to evaluate their analytical performances and to determine how they can be used for the clinical management of smoking cessation. MATERIAL AND METHODS: Urine samples from a single void (n = 97) were obtained from active and abstinent smokers (with or without nicotine substitutive therapy). They were all analyzed by the various methods. Cotinine concentration was measured in six laboratories, using HPLC combined with UV detection according to a standardized procedure (Ann Biol Clin 2002 : 60 : 263-72). Immunoassay methods were also tested and the values obtained from urine samples were compared to urine cotinine measured by HPLC-UV. RESULTS: HPLC-UV: Urinary cotinine varied in a range from undetectable to 4 mg/L. An interlaboratory comparison was performed according to the Valtec procedure (calculation of equation of Deming, chart of differences). There was a good accordance between laboratories. Cotinine concentration was only slightly influenced by fluid intake, as shown by a poorly significant correlation between cotinine and creatinine (r = 0.23, p = 0.05). Homogeneous immunoassays: The two homogeneous immunoassays (Cotinine) from Thermo Electron and Cotinine Enzyme Immunoassay commercialized by Microgenics were highly correlated (r = 0.97). The correlation was not so strong with HPLC-UV (r = 0.86). Firstly, values were found higher with immunoassays because antibodies crossreact with 3-hydroxycotinine. Secondly, the ratio of immunoassays values to HPLC-UV values varied according to urine specimens. Finally, there was a highly significant correlation with urine creatinine (r = 0.40, p = 0.0001), thus indicating the influence of fluid intake. Heterogeneous immunoassay: The kit Metabolites of Nicotine commercialized by DPC France was tested on the analyzer Immulite, using a procedure specifically established for urine. Antibodies revealed a large spectrum of nicotine metabolites. Therefore, the values were much higher than those observed for the same urine samples with homogeneous immunoassays. CONCLUSION: HPLC-UV can be recommended for the measurement of urinary cotinine, as it was shown a good accordance between laboratories. The low detection limit is of interest for the diagnosis of Environmental Tobacco Smoking. Homogeneous immunoassays can be easily used for routine analysis as they can be performed directly on urine specimen. The results must be interpreted according to cut-off values specifically established according to homogeneous or heterogeneous immunoassays. Variability induced by fluid intake must be taken into account. The interest of the heterogeneous immunoassay needs to be confirmed for the diagnosis of Environmental Tobacco Smoking.

[Biomarkers of tobacco smoke exposure]. / Les marqueurs spécifiques du tabagisme.

Tobacco smoking is a major risk factor for cancer, cardiovascular diseases and respiratory illnesses. Smoking is increasing among children and adolescents with subsequent consequences on the health. Furthermore, maternal tobacco smoking during pregnancy adversely affects prenatal growth. Nicotine, the most important tobacco alkaloid, is responsible for maintaining tobacco addiction. According to a recent Circulaire de la direction générale de la santé, nicotine dependence should be determined through questionnaires and quantitative estimate of nicotine metabolites. Nicotine blood level fluctuates and urinary nicotine excretion is of short duration. Nicotine is intensively metabolized in the liver and oxidized into cotinine. Urinary measurement of cotinine appears to be highly related with the degree of intoxication and to allow the differentiation between non exposed and exposed non-smokers. In order to check the present application of nicotine metabolites measurement, a survey was conducted in 340 smoking cessation units. Forty percent physicians (n = 137) answered the survey. For 17% of them, the quantification of nicotine metabolites is included in their daily practise and for 79%, guidelines about cotinine measurement should be given in France. Sixty-seven biologists answered the survey. Recommendations for immunoassay and HPLC determination of cotinine should be given as reported by 66 and 44% of them respectively. Indeed, urinary cotinine measurement with high performance liquid chromatography is highly sensitive and specific. However, immunoassays are more convenient. These two approaches are presently under investigation in order to provide guidelines for optimal use in various clinical situations. Traditional measures for nicotine dependence are the number of cigarettes smoked per day, nicotine intake expressed as mg per day, Fagerstr m questionnaire, expired air carbon monoxide, thiocyanates and cotinine levels in biological fluids. Urinary cotinine measurement is the most useful for the follow-up of smoking cessation including adjustment of nicotine replacement therapy, especially after a clinical event or for the follow-up of smoking pregnant women. It allows the detection of passive smoke exposure in children who are hospitalized for recurrent respiratory illnesses.

[Fatal intoxication after accidental ingestion of viscous 2% lidocaine in a young child]. / Intoxication mortelle après ingestion accidentelle de xylocaine visqueuse à 2% chez une jeune enfant.

We report a case of fatal intoxication with 2% viscous lidocaine. A 18 month old infant was admitted after malaise and cardiorespiratory arrest at home. He was resuscitated, then seizures appeared before arrival at the hospital. Treatment was symptomatic, including cardiorespiratory resuscitation and administration of anticonvulsants. Identification of lidocaine and its metabolite monoethylglycinexylidide (MEGX) MEGX was performed after organic extraction by High Performance Liquid Chromatography (HPLC) with Diode Array Detection (DAD); the serum concentrations, determined by Fluorescence Polarisation Immuno Assay (FPIA), were: 1.1 micrograms/ml for lidocaine and 0.94 microgram/ml for MEGX (H + 7) and 0.30 microgram/ml for the lidocaine (Day + 1). Neurotoxic manifestations appear at lower concentrations than cardiotoxic symptoms which are correlated with plasma levels of lidocaine. The toxic symptoms begin with headache, hallucinations, seizure, coma, respiratory arrest and circulatory collapse. The toxic symptoms can persist even after the decrease of lidocaine concentration under therapeutic levels. There is no antidote and acute lidocaine toxicity is managed with supportive therapy (diazepam for seizures, intubation, chronotropic agents). Considering the gravity of these poisonings which remain rare, the 2% viscous lidocaine prescription is forbidden for children under 6 years old.

[Serial determination of crimidine by HPLC/SE/SM in a patient ingesting a "mouse trap"]. / Détermination sérique de la crimidine par CLHP/ES/SM chez un patient ayant ingere un "souricide foudroyant".

Crimidine (2 chloro, 4 methyl, 6 dimethyl amidopyrine) is a synthetic rodenticide which causes acute poisonings after oral ingestion in human. Major toxic effects are consciousness disorders, hypertonic coma and convulsions. Toxic level in human is about 5 mg/Kg. An intoxication case is reported. Five serums collected at different times were analyzed with HPLC/ES/MS. Crimidine was extracted with ethylacetate with recovery over 80%. Linearity was up to 800 micrograms/L. LOQ and LOD were 0.5 and 0.3 microgram/L respectively. The coefficients of variation were less than 10% for repeatability and reproductibility. Serum levels varied from 368 micrograms/L for H0 to 64 micrograms/L for H10 and elimination of crimidine was linear in time.

Determination serique de la crimidine par clhp/es/sm chez un patient ayant ingere un « souricide foudroyant ¼.

Crimidine (2 chloro, 4 methyl, 6 dimethyl amidopyrine) is a synthetic rodenticide which causes acute poisonings after oral ingestion in human. Major toxic effects are consciousness disorders, hypertonic coma and convulsions. Toxic level in human is about 5 mg/Kg. An intoxication case is reported. Five serums collected at different times were analyzed with HPLC/ ES/MS. Crimidine was extracted with ethylacetate with recovery over 80 %. Linearity was up to 800 µg/L. LOQ and LOD were 0.5 and 0.3 µg/L respectively. The coefficients of variation were less than 10 % for repeatability and reproductibility. Serum levels varied from 368 µg/L for H0 to 64 µg/L for H10 and elimination of crimidine was linear in time.

Intoxication mortelle après ingestion accidentelle de xylocaine visqueuse à 2% chez une jeune enfant.

We report a case of fatal intoxication with 2% viscous lidocaine. A 18 month old infant was admitted after malaise and cardiorespiratory arrest at home. He was resuscitated, then seizures appeared before arrival at the hospital. Treatment was symptomatic, including cardiorespiratory resuscitation and administration of anticonvulsants. Identification of lidocaine and its metabolite monoethylglycinexylidide (MEGX) MEGX was performed after organic extraction by High Performance Liquid Chromatography (HPLC) with Diode Array Detection (DAD); the serum concentrations, determined by Fluorescence Polarisation Immuno Assay (FPIA), were : 1,1 µg / ml for lidocaine and 0,94 µg / ml for MEGX (H+7) and 0,30 µg / ml for the lidocaine (Day+1). Neurotoxic manifestations appear at lower concentrations than cardiotoxic symptoms which are correlated with plasma levels of lidocaine. The toxic symptoms begin with headache, hallucinations, seizure, coma, respiratory arrest and circulatory collapse. The toxic symptoms can persist even after the decrease of lidocaine concentration under therapeutic levels. There is no antidote and acute lidocaine toxicity is managed with supportive therapy (diazepam for seizures, intubation, chronotropic agents). Considering the gravity of these poisonings which remain rare, the 2% viscous lidocaine prescription is forbidden for children under 6 years old.

High-performance liquid chromatographic method to screen and quantitate seven selective serotonin reuptake inhibitors in human serum.

A high-performance liquid chromatographic screening method (HPLC) is described for the determination of seven selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, milnacipran, paroxetine, sertraline, fluoxetine, citalopram, venlafaxine) and for three pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram and norfluoxetine). A tricyclic antidepressant, clomipramine, was used as an internal standard. The method consists of liquid extraction of serum after alcalinisation at pH 9.50, followed by chromatography on a Beckman C18 reversed-phase column. Compounds were detected at 200.4 nm. The standard curves were linear over a working range of 50-1,000 ng/ml for fluvoxamine, 15-1,000 ng/ml for fluoxetine, 25-500 ng/ml for norfluoxetine, 50-500 ng/ml for sertraline, 20-500 ng/ml for paroxetine, 25-550 ng/ml for citalopram, 25-750 ng/ml for desmethylcitalopram, 25-800 ng/ml for didesmethylcitalopram, 25-650 ng/ml for milnacipran, and 25-500 ng/ml for venlafaxine. The quantitation limits of the method were 15 ng/ml for fluoxetine, 20 ng/ml for paroxetine, 25 ng/ml for venlafaxine, norfluoxetine and citalopram, and its metabolites, 40 ng/ml for sertraline and 50 ng/ml for fluvoxamine. No interferences were noted with this sensitive and specific method which can be used for therapeutic drug monitoring.

Self-poisoning with Colchicum autumnale L. flowers.

UNLABELLED: A 44-year-old man ingested about 40 flowers of Colchicum autumnale L. The patient presented with nausea, vomiting, and abdominal pain 2 hours after ingestion and had diarrhea 14 hours after ingestion. Hematological values remained within normal range. Treatment was mainly supportive. The outcome was favorable. The intoxication was confirmed by high-performance liquid chromatography-mass spectrometry. Maximal colchicine levels were 4.34 ng/mL at 13 hours in plasma and 5.43 ng/mL at 16 hours in erythrocytes. CONCLUSION: We report one of the few symptomatic cases of Colchicum autumnale L. poisoning confirmed by toxicological analysis.

Human airway mucin glycosylation: a combinatory of carbohydrate determinants which vary in cystic fibrosis.

Human airway mucins represent a very broad family of polydisperse high molecular mass glycoproteins, which are part of the airway innate immunity. Apomucins, which correspond to their peptide part, are encoded by at least 6 different mucin genes (MUC1, MUC2, MUC4, MUC5B, MUC5AC and MUC7). The expression of some of these genes (at least MUC2 and MUC5AC) is induced by bacterial products, tobacco smoke and different cytokines. Human airway mucins are highly glycosylated (70-80% per weight). They contain from one single to several hundred carbohydrate chains. The carbohydrate chains that cover the apomucins are extremely diverse, adding to the complexity of these molecules. Structural information is available for more than 150 different O-glycan chains corresponding to the shortest chains (less than 12 sugars). The biosynthesis of these carbohydrate chains is a stepwise process involving many glycosyl- or sulfo-transferases. The only structural element shared by all mucin O-glycan chains is a GalNAc residue linked to a serine or threonine residue of the apomucin. There is growing evidence that the apomucin sequences influence the first glycosylation reactions. The elongation of the chains leads to various linear or branched extensions. Their non-reducing end, which corresponds to the termination of the chains, may bear different carbohydrate structures, such as histo-blood groups A or B determinants, H and sulfated H determinants, Lewis a, Lewis b, Lewis x or Lewis y epitopes, as well as sialyl- or sulfo- (sometimes sialyl- and sulfo-) Lewis a or Lewis x determinants. The synthesis of these different terminal determinants involves three different pathways with a whole set of glycosyl- and sulfo-transferases. Due to their wide structural diversity forming a combinatory of carbohydrate determinants as well as their location at the surface of the airways, mucins are involved in multiple interactions with microorganisms and are very important in the protection of the underlying airway mucosa. Airway mucins are oversulfated in cystic fibrosis and this feature has been considered as being linked to a primary defect of the disease. However, a similar pattern is observed in mucins from patients suffering from chronic bronchitis when they are severely infected. Airway mucins from severely infected patients suffering either from cystic fibrosis or from chronic bronchitis are also highly sialylated, and highly express sialylated and sulfated Lewis x determinants, a feature which may reflect severe mucosal inflammation or infection. These determinants are potential sites of attachment for Pseudomonas aeruginosa, the pathogen responsible for most of the morbidity and mortality in cystic fibrosis, and the expression of the sulfo- and glycosyl-transferases involved in their biosynthesis is increased by TNFalpha. In summary, airway inflammation may simultaneously induce the expression of mucin genes (MUC2 and MUC5AC) and the expression of several glycosyl- and sulfo-transferases, therefore modifying the combinatory glycosylation of these molecules.

[Study of passive smoking measured by urinary cotinine in maternal and child protective health centers in North-Pas-de-Calais]. / Etude du tabagisme passif par la mesure de la cotininurie en consultation de protection maternelle et infantile dans le Nord-Pas-de-Calais.

BACKGROUND: The aim of the study was to determine the circumstances of exposure to environmental tobacco smoke, to evaluate its importance by measurement of urinary cotinine, and to study the relationship with the children's medical history. POPULATION AND METHODS: It was a prospective investigation realized in 20 outpatient pediatric clinics. The parents answered a questionnaire to assess the child's exposure as well as the child's medical and surgical history. Cotinine was measured in urine samples collected during the visit. Concentrations > 6 ng/mL were considered to be positive. RESULTS: Two hundred and one children were included in the study (mean age 17 months, extremes: 1-72 months), 107 of whom were exposed to environmental tobacco smoke. Urinary cotinine was found to be positive in 27 cases (13%). There was a positive relation between passive tobacco exposure and positive urinary cotinine (P < 0.001). Eighty of 201 mothers and 135 of 185 fathers smoked. There was a relation between positive urinary cotinine and the mother's smoking, as well as with a history of upper respiratory tract infection (rhinitis, otitis media) or adenoidectomy. No relation was found between a history of bronchiolitis and passive smoking. CONCLUSIONS: Passive tobacco exposure is very frequently encountered in our region. Urinary cotinine, which can be easily measured, might constitute an efficient tool in order to convince the parents of the reality of passive smoking.

Tetrachloroethylene and trichloroethylene fatality: case report and simple headspace SPME-capillary gas chromatographic determination in tissues.

We describe a simple, precise, and sensitive assay of tetrachloroethylene and trichloroethylene in tissues, suitable both for emergency cases and forensic medicine. The method employs headspace solid phase microextraction-capillary gas chromatography and electron capture detection. The case is relative to a 45-year-old woman discovered unconscious in a laundry area. The concentrations of the solvents in tissues were determined and compared to other previously published fatalities.

[Hepatonephritis and massive ingestion of buprenorphine]. / Hépatonéphrite et ingestion massive de buprénorphine.

We present a case of a severe hepatitis associated with acute renal failure and anuria consequently to the ingestion of 112 mg of buprenorphine, 48 hours before. The normalisation of hepatic and renal functions is associated with discontinuation of buprenorphine administration and hemodialysis treatment. Buprenorphine seems to be directly responsible for this hepatonephritis as indicated by the high plasmatic levels of buprenorphine (224 ng/ml) and norbuprenorphine (30 ng/ml) never described until now. Buprenorphine toxicity could be due to the inappropriate ingestion mode (oral instead of sublingual) and could be increased by previous acetaminophen intake.

[Identification and quantitative determination of chlorinated++ solvents after solid phase microextraction. Applications in hospital toxicology]. / Identification et dosage de solvants chlorés après microextraction en phase solide (SPME). Applications en toxicologie hospitalière.

A simple method for the research and the identification of chlorinated solvents in biological fluids and tissues is described. The solvents are extracted using headspace solidphase microextraction and detected or measured by gas chromatography with electron capture detector. Three accidental poisonings are reported: one with carbon tetrachloride, one with trichlorethylene and the last one is a double intoxication with tetrachloroethylene and trichlorethylene.

The binding of surface proteins from Staphylococcus aureus to human bronchial mucins.

Colonization by Staphylococcus aureus is frequently observed in obstructive lung diseases, particularly in cystic fibrosis. It has been shown that the bacteria bind to mucins, the main constituent of bronchial secretions. The binding mechanism, however, remains unclear. We have investigated the interactions of two strains of S. aureus, one mucoid and one nonmucoid, with human bronchial mucins. Using a solution phase assay, the binding capacity of the two strains to radiolabelled bronchial mucins was assessed. The bacterial constituents were released by lysostaphin lysis and the surface components of the nonmucoid strain were extracted with the use of a detergent (3-([3-cholamidopropyl] dimethylammonio)-1-propane sulphonate (CHAPS)). All were analysed for mucin-binding using an overlay assay. The amount of mucins bound to the nonmucoid strain was threefold greater than that of the mucoid strain. In the lysostaphin extract from the mucoid strain, only a 57 kDa protein faintly bound 125I-labelled mucins, whereas three mucin-binding proteins (52, 57 and 71 kDa) were identified from the nonmucoid strain. Two surface proteins, one major at 60 kDa and one minor at 71 kDa, bound radiolabelled bronchial mucins and their binding was almost completely inhibited by ovine submaxillary mucin. These results indicate: 1) differences in the mucin-binding capacity from one strain of S. aureus to another; and 2) the presence of external and internal adhesins binding to human respiratory mucins in the nonmucoid strain.

Interactions between glycoconjugates from human respiratory airways and Pseudomonas aeruginosa.

Pseudomonas aeruginosa binds to different glycoconjugates in vitro. As six other bacteria, it binds to several glycolipids, mainly asialo GM1 and asialo GM2. Asialo GM1 has been reported to exist at the surface of cystic fibrosis cells. The binding of P. aeruginosa to asialo GM1 involves the pili, especially the C-terminal part of pilin that recognizes the GaINAc(beta 1,4) Gal sequence of asialo GM1.P. aeruginosa may also bind to sialylated membrane-bound glycoproteins. Human salivary and respiratory mucins are also recognized by P. aeruginosa. Mucins represent the main components of mucus. The peptide part (apomucin) of this broad family of secreted glycoproteins is encoded by several mucin genes. The apomucins are covered by a large number of carbohydrate chains that can be remarkably different and represent a mosaic of sites for attachment of microorganisms. The binding of P. aeruginosa to mucins involves outer membrane proteins and mucin carbohydrate chains that are structurally different from the carbohydrate recognized by pillin. Airway and salivary mucins secreted by patients suffering from cystic fibrosis (CF) show alterations in their carbohydrate moiety. The increased sulfation of airway mucins seems to correspond to a primary defect. Other abnormalities such as increased sialylation or fucosylation have also been detected. The binding of P. aeruginosa to airway or salivary mucins is increased in CF. However, the precise link between the carbohydrate alterations and the increased binding of P. aeruginosa to CF mucins remains to be elucidated.

Interaction of fimbriated and nonfimbriated strains of unencapsulated Haemophilus influenzae with human respiratory tract mucus in vitro.

Adherence to mucus may influence bacterial colonization of the respiratory tract. Clinical isolates of nontypable Haemophilus influenzae (NTHi) from the respiratory tract are often fimbriated. We wondered whether fimbriated strains have a different adherence from related nonfimbriated strains. A microtitre plate assay has been developed to study adherence of nontypable H. influenzae to mucus. Wells were coated by incubation either with sol phase of sterile mucoid secretions or with purified preparations of mucins. Two laboratory pairs of fimbriated (F+) and nonfimbriated (F-) nontypable H. influenzae, and six fresh clinical isolates of fimbriated nontypable H. influenzae each with nonfimbriated partners derived by serial passage on agar, were cultured to mid-log phase, washed, and then added to the wells. They were then incubated at 37 degrees C for 30 min before washing to remove unbound bacteria. Adherent bacteria were desorbed by agitation with 0.5% Tween 80 and a viable count performed. The two fimbriated laboratory strains (n = 12 and n = 17), and 5 of the 6 fimbriated clinical isolates were more adherent to sol phase than their respective nonfimbriated partners. Two nonfimbriated clinical isolates were more adherent to plastic than their fimbriated partners. A fimbriated laboratory strain was more adherent than its nonfimbriated partner both to a purified preparation of high molecular mass mucin and to the glycopeptide fraction of the same. We conclude that fimbriated strains of nontypable H. influenzae have increased adherence to sol phase of mucus and purified human respiratory tract mucin. The interactions of fimbriae with mucus are likely to be complex, and may involve both nonspecific and specific interactions.

Influence of iron depletion on growth kinetics, siderophore production, and protein expression of Staphylococcus aureus.

Growth rates, siderophore secretion, and bacterial proteins of two clinical isolates of Staphylococcus aureus were studied over 72 h of growth in iron-supplemented and iron-restricted chemically defined media. Under iron restriction the growth rates were decreased to different extents depending on the strain. Production of siderophore was detected in the mid-exponential and stationary phases of growth. The expression of iron-regulated proteins of 81, 23, and 17 kDa was time-dependent, associated with the same stage of growth, and might be involved in siderophore efficiency.

Determination of chloral hydrate and its metabolites (trichloroethanol and trichloracetic acid) in human plasma and urine using electron capture gas chromatography.

Capillary gas chromatography with electron capture detection is described for the quantification of chloral hydrate (CH) and is metabolites trichloroethanol (TCE) and trichloroacetic acid (TCA) in 0.1-1 mL of plasma samples. The method, with 2,2'-dichloroethanol (DCE) as internal standard, involved extraction of chloral hydrate and trichloroethanol with diethyl ether and methylation of trichloroacetic acid with 3-methyl-1-tolyltriazene (MTT), followed by diethyl ether extraction. The method has a detection limit of 5 ng/mL for CH and TCE and 10 ng/mL for TCA and also allows the determination of TCE-glucuronide in 0.1-1 mL of plasma samples. It exhibits good linearity and precision. The method was applied to samples of plasma from a neonate after a single dose of 40 mg/kg of chloral hydrate and from an adult after a single dose of 6.25 mg/kg.