Norepinephrine augmented in vitro growth of uropathogenic E. coli in Type 2 diabetes mellitus and its suppression by silodosin (alpha blocker).

Norepinephrine is secreted under conditions of stress in humans. The ability of bacteria to sense mammalian hormone may have a role in propagation of infection. The present study investigated the effect of norepinephrine on in vitro growth of uropathogenic E. coli (UPEC) and the effect of silodosin on norepinephrine-induced changes. The spot urine samples were collected from 56 individuals (14 diabetic patients with UTI, 14 diabetic without UTI, 14 non-diabetic UTI and 14 healthy volunteer controls) for the measurement of urinary norepinephrine concentrations. The concentration of norepinephrine, as found in urine of human subjects, was reproduced in artificial urine medium to study the growth of UPEC. The norepinephrine concentration showing maximum growth response was selected to study the effect of silodosin on the growth inhibition of UPEC. Result showed significantly elevated urinary norepinephrine in diabetic patients with and without UTI and also in nondiabetic UTI groups. The norepinephrine concentration equivalent to that in diabetic UTI patients enhanced the growth of UPEC. Furthermore, silodosin (0.32 µM) inhibited the growth of the UPEC.

[The study of naphthyzin present in material evidence and biological fluids].

The optimal conditions for isolation of naphazoline from naphthyzin preparations and biological fluids with chloroform at pH 9.18 are described. The compound of interest was identified with the use of color and precipitation reactions, IR and UV spectroscopy, thin-layer and gas chromatography, and chemical methods including high performance liquid chromatography, chromatodensitometry, and UV spectroscopy. The results obtained by the three methods are comparable.

Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine.

Amitraz (N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-N-methyl-methanimidamide) is an alpha-2 adrenergic agonist used in veterinary medicine primarily as a scabicide- or acaricide-type insecticide. As an alpha-2 adrenergic agonist, it also has sedative/tranquilizing properties and is, therefore, listed as an Association of Racing Commissioners International Class 3 Foreign Substance, indicating its potential to influence the outcome of horse races. We identified the principal equine metabolite of amitraz as N-2,4-dimethylphenyl-N'-methylformamidine by electrospray ionization(+)-mass spectrometry and developed a gas chromatographic-mass spectrometric (GC-MS) method for its detection, quantitation, and confirmation in performance horse regulation. The GC-MS method involves derivatization with t-butyldimethylsilyl groups; selected ion monitoring (SIM) of m/z 205 (quantifier ion), 278, 261, and 219 (qualifier ions); and elaboration of a calibration curve based on ion area ratios involving simultaneous SIM acquisition of an internal standard m/z 208 quantifier ion based on an in-house synthesized d(6) deuterated metabolite. The limit of detection of the method is approximately 5 ng/mL in urine and is sufficiently sensitive to detect the peak urinary metabolite at 1 h post dose, following administration of amitraz at a 75-mg/horse intravenous dose.

Determination of xylazine and its metabolites by GC-MS in equine urine for doping analysis.

Xylazine and its main metabolites were detected in equine urine after a single-dose intravenous administration of 0.98 and 1.01 mg/kg body weight xylazine, respectively, in two horses, in order to be used for equine doping control routine analysis. The urine levels of the parent drug and its metabolites were determined using gas chromatography-mass spectrometry (GC-MS). Xylazine is metabolised rapidly, down to a concentration level of about 1.0 microg/ml after 1-3h administration. Seven metabolites were identified in urine. 4-Hydroxy-xylazine, the major metabolite, could be traced for 25 h and it is regarded as the long-term metabolite of xylazine in horse. 2,6-Dimethylaniline was, for the first time, reported as metabolite in equine.

Extraction of clenbuterol from calf urine using a molecularly imprinted polymer followed by quantitation by high-performance liquid chromatography with UV detection.

A method for the extraction of clenbuterol from calf urine samples using a molecularly imprinted polymer (MIP) has been developed. The aim was that the final extracts from the MIP should allow quantitation of clenbuterol down to 0.5 ng/mL urine using HPLC with UV detection. The MIP was produced using brombuterol as a template and the selectivity of the MIP, for clenbuterol, was tested against a non-imprinted polymer (produced without template) and was found to be high. After loading of 5 mL diluted centrifuged urine, selective binding was established in acetonitrile-acetic acid (98:2). For further elution of interferences, 0.5 M ammonium acetate buffer pH 5 and 70% acetonitrile in water was used. Clenbuterol was eluted using 1% trifluoroacetic acid in methanol, which was evaporated and reconstituted in buffer. Results from the HPLC analyses showed that the extraction of clenbuterol using MIP is linear in the range 0.5-100 ng/mL with good precision (4.3% for 0.6 ng/mL and 2.1% for 6.0 ng/mL) and accuracy (96.7% for 0.6 ng/mL and 96.7% for 6.0 ng/mL). The recoveries were 75%. The results show that the method offers a selectivity and sensitivity that make the quantitation of 0.5 ng clenbuterol/mL urine by HPLC-UV possible and a competitive alternative to state-of-the-art routine analytical methods.

Severe intoxication with the veterinary tranquilizer xylazine in humans.

Xylazine (Rompun, Proxylaz) is a veterinary tranquilizing agent. A case of self-injection of 1.5 g xylazine by a 27-year-old farmer is reported. He subsequently became comatose, hypotensive, bradycardic, and mildly glycemic. An intensive supportive therapy including intubation and ventilation was required. The patient made a full recovery over the next 30 h. The largest concentrations measured were 4.6 mg/L in plasma, 446 mg/L in gastric fluid, and 194 mg/L in urine. The calculated plasma half-life was 4.9 h. Kinetic data correlated with clinical symptoms. Qualitative and quantitative analyses of xylazine were done by thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. These methods allow the detection of small amounts substance in stomach, plasma, and urine. Liquid-liquid extraction was used for the isolation of drug. The sensitvity is high, and with these methods, a rapid analysis is possible. Xylazine intoxications in humans are rare. We describe the management of acute poisoning and present a review of xylazine toxicity in humans.

Pharmacokinetics of NS-49, a phenethylamine class alpha 1A-adrenoceptor agonist. 1st communication: absorption and excretion in rats after a single administration of 14C-NS-49.

The absorption and excretion of NS-49 ((R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride, CAS 137431-04-0), a phenethylamine class alpha 1A-adrenoceptor agonist, were studied in rats after a single administration of 14C-NS-49. In addition, the protein binding of this drug was investigated in vivo and in vitro. After oral administration of 14C-NS-49 (1 mg/kg) to male rats, the radioactivity concentrations in the blood and plasma reached maximums within 1 h, then decreased biexponentially with respective elimination half-lives of 25.4 and 11.9 h. Most of the plasma radioactivity was due to unchanged NS-49, indicating of the poor metabolism of this drug in rats. The results of the in situ absorption study using the intestinal loop method showed that 14C-NS-49 was well absorbed from the small intestine. Systemic availability was high (86%), as determined by a comparison of the areas under the plasma concentration-time curves of unchanged NS-49 for oral and intravenous administrations. Food affected the absorption of NS-49. There were no significant sex-related differences in the plasma concentration profiles after the intravenous administration of 14C-NS-49 (p > 0.05). NS-49 was primarily eliminated by renal excretion, 76% and 62% of the dose being excreted unchanged in the urine after intravenous and oral administrations, respectively. The absorption rate, determined on the basis of the urinary excretion of radioactivity, was 83%, being almost the same as the systemic availability. First-pass metabolism of NS-49, therefore, is considered to be very limited in rats. The excretion of radioactivity in the bile within 48 h after the oral administration of 14C-NS-49 (1 mg/kg) was 5.9% of the dose, and the excretion of radioactivity in the exhaled air after the intravenous administration (0.2 mg/kg) was negligible. The percentage of 14C-NS-49 bound to serum proteins in vitro was less than 15% in all the animal species tested. The percentage of radioactivity bound to rat serum proteins after the oral administration of 14C-NS-49 (1 mg/kg) was 16-21%.

[The neurobiology of depression]. / Neurobiologie de la dépression.

Neurobiology dominates efforts to understand depression. This psychiatric illness is thought to result from dysfunctions in monoaminergic systems affecting norepinephrine, serotonin and dopamine. Abnormalities are linked to functional deficit of monoamines at several effector sites. Findings include reduced cerebrospinal fluid and urinary concentrations of metabolites, decreased plasma concentrations of precursors, modifications of receptor density and clinical effectiveness of drugs which increase neurotransmission in depressed patients. The original hypothesis of affective disorder envisaged a single transmitter model, but neuroscientific developments highlight the complexity of the central nervous system. Considerable evidence supports the hypothesis of combined alterations of monoaminergic functions and other systems like neuropeptides and neuroendocrine functions.

Isotachophoretic determination of bisoprolol, clonidine, disopyramide and tolazoline in human fluids.

Separation and determination of bisoprolol, clonidine, disopyramide and tolazoline in control serum and in human urine was investigated by capillary isotachophoresis. The drugs were separated by using the cationic electrolyte system. viz., sodium acetate buffer (pH 4.64) (c1 = 10 mM)-beta-alanine. The compounds were almost totally isolated from serum by solid-phase extraction using a Sep-Pak C18 cartridge. The recovery of compounds varied from 87 to 99%. The linear calibration range was studied to apply the method to real human fluids. The limit of determination of the drugs was 40.0 micrograms/ml serum. The limit of determination by direct sampling for bisoprolol is 3 micrograms/ml urine.

Do pseudoephedrine or phenylpropanolamine improve maximum oxygen uptake and time to exhaustion?

OBJECTIVE: To study the effects of over-the-counter dosages of the pure alpha 1-agonists pseudoephedrine (PSE) and phenylpropanolamine (PPA) on selected parameters of exercise performance, and to establish a range of corresponding drug levels in the urine of the athletes who use these drugs. DESIGN: Placebo-controlled, randomized, double-blinded, multiple-dose trial. SETTING: The National Institute of Fitness and Sport, the Department of Family Medicine, Indiana University, and the Sports Medicine Lab, Department of Pathology, Indiana University, Indianapolis, Indiana. PARTICIPANTS: A convenience sample of 20 male cyclists, aged 18-35, from the local cycling community. Inclusion criteria required cycling at least 50 miles a week, no chronic medical problems, and not taking any medications. Subjects were recruited by local ads and word of mouth. INTERVENTION: Patients were randomized to one of two groups of 10 subjects. Each subject in both groups performed three separate bicycle ergometer tests after ingestion of varying dosages of alpha 1-agonists. One group performed tests after receiving placebo, 0.33 mg/kg PPA, and 0.66 mg/kg PPA, whereas the other group received placebo, 1 mg/kg PSE, and 2 mg/kg PSE. A minimum 1-week washout period was required between tests. Urine for drug testing was collected 1 h before, immediately afterward, and the next morning after testing. Drug testing was performed by gas GC/MCD at a facility approved by the International Olympic Committee. MAIN OUTCOME MEASURES: Maximum oxygen uptake (VO2max), time to exhaustion, urine drug levels of PSE and PPA, peak blood pressures (BPs), peak pulse, and Borg scale (rating of perceived exertion or RPE). MAIN RESULTS: In the PPA group, the 0.33-mg/kg dose resulted in insignificant changes in peak systolic BP (+5.4 mm Hg, p = 0.260), peak diastolic BP (-1.6 mm Hg, p = 0.622), peak pulse (-2.2 beats/min, p = 0.12), peak Borg (RPE = -0.10 (p = 0.823), time to exhaustion (-16.9 s, p = 0.287), and VO2max (+0.50 ml/kg/min, p = 0.71). No significant change was noted in any study variable at the 0.66-mg/kg PPA dose, and some effects were dissimilar to the lower PPA dose effects. Peak systolic BP increased 2.8 mm Hg (p = 0.617), diastolic BP decreased 1.6 mm Hg (p = 0.634), peak pulse increased 1.4 beats/min (p = 0.504), peak Borg RPE decreased 0.80 (p = 0.210), time to exhaustion decreased 2.6 s (p = 0.861), and VO2max decreased 2.92 ml/kg/min (p = 0.14). In the 1-mg/kg PSE group, there was a significant increase in peak systolic BP (+10.6 mm Hg, p = 0.029). No significant changes occurred in peak diastolic BP (+2.4 mm Hg, p = 0.333), peak pulse (+2.2 beats/min, p = 0.306), peak RPE (+0.2, p = 0.62), time to exhaustion (+21.4 s, p = 0.289), and VO2max (+2.29 ml/kg/min, p = 0.31). In the 2-mg/kg PSE dose trial, there were insignificant changes in peak systolic BP of +2.4 mm Hg (p = 0.559), +3.8 mm Hg in peak diastolic BP (p = 0.106), +1.6 beats/min in peak pulse (p = 0.586), -0.1 in peak Borg RPE scales (p = 0.76), -10.4 s in time to exhaustion (p = 0.41), and +1.79 ml/kg/min in VO2max (p = 0.43). Urine drug levels in those subjects receiving 1 mg/kg PSE ranged from 7-55 micrograms/ml before performance and 30-128 micrograms/ ml after performance to 7-35 micrograms/ml the next morning. Levels in those receiving 2 mg/kg ranged from 5-160 micrograms/ml before performance and 44-200 micrograms/ml after performance to 8-44 micrograms/ ml the next day. In the PPA 0.33-mg/kg dose trials, the levels ranged 1-36 micrograms/ml before performance and 9-50 micrograms/ml after performance to < 1-14 micrograms/ml the next morning. In the PPA 0.66-mg/kg dose trials, the levels were 4-52 micrograms/ml before performance, 8-80 micrograms/ml after performance, and 6-74 micrograms/ml the next day. CONCLUSIONS: We found no significant differences between trials in maximum oxygen uptake (VO2max), peak or progression of Borg Scale (RPE), maximum systolic and diastolic BPs, peak pulse, or t

Paracetamol (acetominophen) sulphoconjugation in man: no correlation with tyramine sulphoconjugation.

There is considerable evidence that subjects vulnerable to endogenous depression excrete less tyramine sulphate after an oral dose of free tyramine than controls (the tyramine test). In this study, 26 psychiatric inpatients, exhibiting a wide range of responses to the test, and 10 normal controls were challenged with oral doses of paracetamol and tyramine on two separate occasions. Urinary output of paracetamol sulphate and paracetamol glucuronide in all subjects was monitored but there were no significant correlations with tyramine sulphate output. Thus, the output of these metabolites appears to be under complex control, and paracetamol cannot be substituted for tyramine in the "tyramine test". The basic deficit responsible for low values in the tyramine test is unlikely to stem from sulphate depletion or a generalised disturbance of the sulphation system, and remains obscure.

Quantitative analysis of S3341 in human plasma and urine by combined gas chromatography-negative ion chemical ionization mass spectrometry: 15 month inter-day precision and accuracy validation.

A new quantitative assay for the determination of S3341, an alpha-2 agonist antihypertensive drug, has been developed using combined gas chromatography-negative ion chemical ionization mass spectrometry. The [M]-. ions from TFA derivatives of S3341 (m/z 276) and the internal standard (2H4)S3341 (m/z 280) are monitored simultaneously by selected ion monitoring. For S3341 concentrations ranging from the limit of detection (0.2 ng ml-1 using 1 ml of plasma) to 5 ng ml-1, the average assay precision (CV) is approximately 7% while the average assay accuracy (percentage of error) is 4%. Validation of the day-to-day precision and accuracy was realized after analysing control plasma samples (n = 295) concurrently with the biological samples collected during the pharmacokinetic studies conducted over 15 months. The average day-to-day precision (CV) and accuracy (percentage of error) are 10% and 6% respectively, thus indicating that this assay procedure routinely provides reliable analytical data.

Gas chromatographic negative ion mass spectrometric assay of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug.

The quantification in plasma and urine of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug is described using a sensitive gas chromatographic negative ion mass spectrometric method with ammonia as moderating gas. After a two-step extraction, derivatization is carried out with 3,5-bis(trifluoromethyl)benzoyl chloride and the abundance of the molecular ion (m/z 420) obtained is compared with that of the tetradeuterated standard (m/z 424). The low background due to the high mass and negative ion detection provides a detection limit of about 1 pg per injection. Oral administration of 1 or 2 mg S-3341 to patients gives a maximum concentration of 3.3 +/- 0.7 ng ml-1 and 7.6 +/- 2.0 ng ml-1 at 1.8 +/- 0.6 h and 1.4 +/- 0.7 h and an average elimination half-life of 6.7 h.