Enantiomeric separation of mirtazapine and its metabolite in rat plasma by reverse polar ionic liquid chromatography using fluorescence and polarimetric detectors connected in series

A simple and rapid reverse polar ionic LC method was developed and validated for simultaneous separation and determination of mirtazapine, an antidepressant drug, and its main metabolite N-desmethyl mirtazapine using fluorescence and polarimetric detectors connected in series. The chromatographic separation was achieved on Chirobiotic V column packed with vancomycin as a stationary phase in an isocratic mode of elution of methanol:glacial acetic acid:anhydrous triethyl amine (100:0.2:0.1, v/v/v) as a mobile phase. The compounds were detected by their excitation at 290nm and emission at 370nm using fluorescence detector while the optical rotation (+/-) of the enantiomers was identified by polarimetric detector. The analytes were extracted from rat plasma by precipitation of proteins and the average yield was 88-111% for mirtazapine and 85-123% for N-desmethyl mirtazapine. The method was linear over the concentration range of 20-5000ng/mL. The method was successfully applied on rat plasma spiked with the enantiomers of mirtazapine and N-desmethyl mirtazapine.     

Treatment of nocturnal asthma: the role of sustained-release theophylline and oral beta-2-mimetics

In two double-blind, multiple-dose cross-over studies the therapeutic effects of SR theophylline preparations given once each night (mean 11.2 mg/kg per day) versus twice daily in equal doses (mean 10.3 mg/kg per day) (study I) and SR-terbutaline in equal doses (mean 0.25 mg/kg per day) versus SR theophylline in unequally divided daily doses (mean 5.3 mg/kg morning dose, 10.6 mg/kg evening dose) study II) were compared in 19 patients with nocturnal asthma. At the end of each treatment period drug serum concentrations and PEFR were measured every 2 hr over a 24-hr period. With the twice-daily, equally divided regimen, serum theophylline concentrations were lower at night than during the day (mean 9.4 +/- 0.9 versus 11.3 +/- 1.0 mg/l). With the single evening administration, serum theophylline concentrations were considerably higher at night (Cmax 16.3 +/- 1.4 mg/l) and the circadian variation of PEFR was significantly reduced. PEFR was higher during night and early morning (283 +/- 14 versus 217 +/- 11 l/min, P less than 0.005). During daytime in study II, PEFR values were slightly higher with theophylline than terbutaline. There was no significant difference in peak flow between either treatment during the night and early morning. However, additional use of inhaled beta-2-mimetics because of asthmatic attacks occurred more often during terbutaline (79 times in 8/10 patients) than theophylline treatment (29 times in 5/10 patients). Symptom scores, number of attacks and side-effects clearly favor the theophylline regimen. We conclude that for patients with nocturnal asthma a once-nightly dose of SR theophylline can be sufficient for stabilization of the airways.     

Simultaneous determination of levomepromazine,midazolam and their major metabolites in human plasma by reversed-phase liquid chromatography

A sensitive and reliable high-performance liquid chromatographic (HPLC) assay is a prerequisite for pharmacokinetic analysis of continuous infusion of levomepromazine adjuvant to midazolam. We developed such a method to determine the levels of levomepromazine, midazolam and their major metabolites (levomepromazinesulfoxide, desmethyl-, didesmethyllevomepromazine, O-desmethyllevomepromazine and alpha-hydroxy-midazolam) simultaneously. Desmethylclomipramine was used as an internal standard (I.S.). The lower limit of quantification of this assay was set for levomepromazine 4.1 microg/l, levomepromazinesulfoxide 4.9 microg/l, O-desmethyllevomepromazine 18.4 microg/l, alpha-hydroxymidazolam 26.6 microg/l, midazolam 23.4 microg/l, didesmethyllevomepromazine 15.8 microg/l, and desmethyllevomepromazine 6.6 microg/l. The between- and within day assay variations were commonly below 5%. The recovery in human plasma for the different analytes varied between 85 and 11%. The accuracy of this assay varied between 95 and 105% for the different concentrations. The linearity of this assay was set between 25 and 800 microg/l (r(2)>0.999 of the regression line). The first results of pharmacokinetic analysis of midazolam indicated that half-life varied between 1.1 and 1.9 h. Pharmacokinetic analysis using a one-compartment model of levomepromazine revealed that the apparent volume of distribution was 4.1+/-2.4 l per kg lean body mass and the metabolic clearance was 309+/-225 l per hour per 70 kg. This assay proved to be robust and reproducible. It can reliably be used for further study of the pharmacokinetics of continuous infusion of levomepromazine.     

Determination of total mycophenolic acid and its glucuronide metabolite using liquid chromatography with ultraviolet detection and unbound mycophenolic acid using tandem mass spectrometry

Two simple, sensitive and reproducible methods for determination of total mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) as well as unbound MPA (fMPA) was developed by the use of HPLC-UV and LC-MS/MS methods, respectively. For the total MPA/MPAG method, the analytes were extracted using Isolute C(2) solid-phase extraction (SPE) cartridges and analyzed at 254 nm over a Zorbax Rx C(8) column (150 mm x 4.6 mm, 5 microm). The mobile phase was a gradient mixture of methanol and water (containing 0.1% (v/v) phosphoric acid). The total run time was 18 min and the extraction recovery was 77% for MPA and 84% for MPAG. The method was precise and accurate with a lower limit of quantification (LLOQ) of 0.5 mg/l for MPA and 5.0 mg/l for MPAG. For the fMPA method, plasma was subjected to ultrafiltration followed by SPE using C(18) cartridges. Analytical column was the same as the HPLC-UV method and the mobile phase was a gradient composition of methanol:0.05% formic acid with a flow rate of 0.6 ml/min for the first 3 min and 0.7 ml for the last 4 min. The chromatographic method separated MPA from its metabolites MPAG and Acyl-MPAG. Mass transitions in negative ionization mode for MPA and the internal standard, indomethacin were m/z: 319-->190.9 and m/z: 356-->312.2, respectively. The assay was linear in the concentration range of 1-1000 microg/l for fMPA with a LLOQ of 1 microg/l and an accuracy of >95%. The two methods reported have an adequate degree of robustness and dynamic concentration range for the measurement of MPA, MPAG and fMPA for therapeutic drug monitoring purposes or pharmacokinetics investigations.     

Direct and simultaneous analysis of loxoprofen and its diastereometric alcohol metabolites in human serum by on-line column switching liquid chromatography and its application to a pharmacokinetic study

A simple, rapid, and accurate column-switching liquid chromatography method was developed and validated for direct and simultaneous analysis of loxoprofen and its metabolites (trans- and cis-alcohol metabolites) in human serum. After direct serum injection into the system, deproteinization and trace enrichment occurred on a Shim-pack MAYI-ODS pretreatment column (10 mm x 4.6 mm i.d.) by an eluent consisting of 20 mM phosphate buffer (pH 6.9)/acetonitrile (95/5, v/v) and 0.1% formic acid. The drug trapped by the pretreatment column was introduced to the Shim-pack VP-ODS analytical column (150 mm x 4.6 mm i.d.) using acetonitrile/water (45/55, v/v) containing 0.1% formic acid when the 6-port valve status was switched. Ketoprofen was used as the internal standard. The analysis was monitored on a UV detector at 225 nm. The chromatograms showed good resolution, sensitivity, and no interference by human serum. Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 15 and over 95%, respectively, in the concentration range of 0.1-20 microg/ml. With UV detection, the limit of quantitation was 0.1 microg/ml, and good linearity (r = 0.999) was observed for all the compounds with 50 microl serum samples. The mean absolute recoveries of loxoprofen, trans- and cis-alcohol for human serum were 89.6 +/- 3.9, 93.5 +/- 3.2, and 93.7 +/- 4.3%, respectively. Stability studies showed that loxoprofen and its metabolites in human serum were stable during storage and the assay procedure. This analytical method showed excellent sensitivity with small sample volume (50 microl), good precision, accuracy, and speed (total analytical time 18 min), without any loss in chromatographic efficiency. This method was successfully applied to the pharmacokinetic study of loxoprofen in human volunteers following a single oral administration of loxoprofen sodium (60 mg, anhydrate) tablet.     

The role of sub-optimal preovulatory oestradiol secretion in the aetiology of premature luteolysis during the short oestrous cycle in the cow

Premature regression of the corpus luteum, following the first post partum ovulation, is often preceded by sub-optimal preovulatory oestradiol secretion and accompanied by elevated levels of oxytocin receptors early in the luteal phase. We have investigated the role of preovulatory oestradiol in the control of subsequent oxytocin receptor concentration and activity by treating ovariectomised cows, over a simulated 48 h follicular phase, with high (600 microg per day) medium (300 microg per day) or low (150 microg per day) levels of oestradiol. These doses of oestradiol generated mean+/-S.E.M. plasma oestradiol concentrations of 12.1+/-1.0, 4.9+/-0.5 and 2.9+/-0.4 pg ml(-1), respectively. In Study 1 (n=4 per group), we found that by day 4 following oestrus there was a significant (P< 0.05) effect of the level of oestradiol on the inhibition of oxytocin binding activity measured in endometrial biopsy samples. This had fallen to mean+/-S.E.M. concentrations of 25+/-2 fmol per mg protein in the high group, 47+/-8 fmol per mg protein in the medium group and 65+/-12 fmol per mg protein in the low group. In Study 2, cows (n=3 per group) were treated with the same three levels of oestradiol followed by treatment with increasing levels of progesterone from days 3 to 6 following oestrus, generating mean+/-S.E.M. plasma concentrations of 2.17+/-0.18 ng ml(-1) by day 6. On day 6, there was a significant (P< 0.01) effect of the level of oestradiol on PGF(2alpha) release in response to oxytocin challenge. High, medium and low oestradiol groups exhibiting mean+/-S.E.M., increase plasma PGF(2alpha) metabolite concentrations of 10.0+/-2.2, 21.3+/-4.3 and 41.3+/-1.2 pg ml(-1), respectively, during the hour after oxytocin administration. From these results, we postulate that at the first post partum ovulation a low level of preovulatory oestradiol can result in the early generation of a luteolytic mechanism during the subsequent luteal phase due to impaired inhibition of oxytocin receptors allowing increased PGF(2alpha) release.     

Liquid chromatographic method for the determination of lidocaine and monoethylglycine xylidide in human serum containing various concentrations of bilirubin for the assessment of liver function

A high-performance liquid chromatographic method is described for determination of lidocaine (2-(dietyloamino)-N-(2,6-dimetylofenylo) acetamid) and its metabolite, monoethylglycine xylidide (MEGX), in human serum containing various concentration of bilirubin. Lidocaine and its metabolite were extracted from human serum using dichloromethane. After separation of the layers and freezing at -32 degrees C, the organic layer was decanted and evaporated under a stream of nitrogen. The sample was dissolved in the mobile phase (12% acetonitrile in 15mM potassium dihydrogen orthophosphate, pH 3.0), and after separation on a Supelcosil LC-8-DB column, the analytes were measured by ultraviolet detection at 205nm. Trimethoprim (TMP) was used as the internal standard. The recovery of the examined analytes ranged from 95.7 to 97.9% for lidocaine and from 98.0 to 99.9% for MEGX. The lower limit of quantification (LLOQ) was established at 200microg/l for lidocaine and at 10microg/l for MEGX. The choice of suitable conditions for chromatographic separation of lidocaine and its metabolite MEGX allowed the elimination of the influence of endogenous bilirubin on the result of analysis.     

Steady state pharmacokinetics, metabolism and pharmacodynamics of theophylline in children after unequal twice-daily dosing of a new sustained-release formulation

This was an open-label study in 19 children aged 9-13 years, weighing 27-44 kg, with bronchial asthma. Twenty-four-hour steady-state concentrations of theophylline and its metabolites 1,3-dimethyl uric acid, 3-methyl xanthine and 1-methyl uric acid were assessed after daily dosing of 600 mg (ca 18 mg/kg/day) of the sustained-release theophylline micro-pellet sprinkle system BY158K, for 4 days. The dosing regimen used was an unequal twice-daily dose of 200 mg in the morning after breakfast and 400 mg in the evening after dinner. Twenty-four-hour peak expiratory flow (PEF) profiles were compared before treatment and at steady-state, along with lung function parameters after bronchial provocation. Mean values +/- SD (n = 16) of the steady-state characteristics were Cmin 6.8 +/- 2.1 mg/l, Cmax 14.5 +/- 4.8 mg/l and Cav 10.5 +/- 2.9 mg/l, the plateau time was 11.7 +/- 4.8 hr and peak-trough fluctuation and swing were 72 +/- 21 and 118 +/- 52%, respectively. There was an excellent reproducibility of theophylline pre-dose levels at corresponding time points of the 24-hr sampling period [r = 0.864 (p less than 0.001)]. Mean values +/- SD of the 24 hr average serum metabolite levels were 0.9 +/- 0.2 mg/1 for 1,3-dimethyl uric acid, 0.6 +/- 0.1 mg/1 for 3-methyl xanthine and 0.4 +/- 0.1 mg/1 for l-methyl uric acid. Lung function (n = 17) following bronchial provocation, improved in 10 children after theophylline treatment of 4 days, remained stable in 2 patients and deteriorated in 5 patients. Serum theophylline profiles and PEF profiles ran largely in parallel over the 24-hr period. Six children exhibited typical theophylline induced side-effects, headache (n = 3), nausea (n = 4), dizziness (n = 1), vomiting (n = 4), sleep disturbances (n = 1), pallor (n = 1) and tremor (n = 1), necessitating in 3 children one dose omission/reduction (n = 2) or subsequent dose reduction (n = 1). It has been shown that a twice daily dosing regimen with unequal doses of anhydrous theophylline (BY158K) is well suited to this population of fast metabolisers. The patients were well protected throughout the day, including the critical early morning hours.     

In vitro metabolism of BYZX in human liver microsomes and the structural elucidation of metabolite by liquid chromatography-mass spectrometry method

In vitro phase I metabolism of BYZX, a novel central-acting cholinesterase inhibitor for the treatment of the symptoms of Alzheimer's disease, was studied in human liver microsomes (HLM) and the metabolite formation pathways were investigated by chemical inhibition experiments and correlation analysis. The residual concentration of substrate and the metabolite formed in incubate were determined by HPLC method. The calibration curves of BYZX were linear over the concentration range from 5.07 microM to 200.74 microM. The relative standard deviations of within day and between day were less than 5% (n=5). The limit of detection (LOD) was 0.18 microg/mL (S/N=3) and the limit of quantification (LOQ) was 0.55 microg/mL (R.S.D.=5.2%, n=5). The determination recoveries of BYZX were in the range of 98.2-104.8%. The apparent K(m) of BYZX in HLM was 53.25+/-17.2 microM, the V(max) was 0.94+/-0.77 microM/min/mg protein, and the intrinsic clearance value (Cl(int)) was 0.018+/-0.02 mL/min/mg protein. Ketoconazole and cyclosporin A were the most potent inhibitors on BYZX metabolism in HLM with IC(50) being 0.89 microM and 18.17 microM, respectively. And the inhibition constant (K(i)) of ketoconazole was 0.42 microM. The metabolite of BYZX was N-des-ethyl-BYZX elucidated by LC-MS-MS. The results demonstrated that the developed HPLC method was reliability, simple technique, and was applicable to be used for the researches of in vitro metabolism of BYZX. CYP3A4 was the major isozyme responsible for BYZX metabolism; N-dealkylation was the major metabolic pathway of BYZX. The predominant metabolite of BYZX was N-des-ethyl-BYZX detected in vitro phase I metabolism in HLM.     

A sensitive liquid chromatographic method for the spectrophotometric determination of urinary trans,trans-muconic acid

Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg OasisMAX cartridge was used to clean-up 50 microl of urine sample and gradient elution was performed on a Zorbax SB-C(18) column (30 degrees C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean+/-S.D.microg/g creatinine) were 90+/-34 for smokers (n=26), 49+/-39 for non-smokers (n=21) and 23+/-18 for non-smoking hospital staff (n=14). A correlation coefficient, r=0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 microg/l.     

Determination of paeoniflorin in rat hippocampus by high-performance liquid chromatography after intravenous administration of Paeoniae Radix extract

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of paeoniflorin in rat hippocampus was developed in this study. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Zorbax SB-C(18) column, a mobile phase of methanol-water (32:68, v/v), and detection by ultraviolet (UV) absorption at 233 nm. The lower limits of quantitation (LLQ) were 1 microg/ml for paeoniflorin. The calibration curve for paeoniflorin was linear (r = 0.9999) over the concentration range of 1-50 microg/ml. The coefficients of variation of intra- and inter-day assays were 7.00, 0.58, 1.46% and 5.48, 1.79, 1.70% at concentrations of 1, 10, 50 microg/ml, respectively. The recoveries of paeoniflorin from rat hippocampus were 98.28 +/- 2.14, 98.96 +/- 1.48, and 95.34 +/- 0.92 at concentrations of 1, 10 and 50 microg/ml, respectively. Stability studies showed that paeoniflorin was stable at temperatures of 2-8 degrees C in methanol for at least 20 days. The method was applied to determine the time course of paeoniflorin in rat hippocampus, following the administration of a 60 mg/kg i.v. dose of paeoniflorin in Paeoniae Radix extract to a male Wistar rat.     

A LC-MS/MS method to quantify the novel cholesterol lowering drug ezetimibe in human serum, urine and feces in healthy subjects genotyped for SLCO1B1

Ezetimibe (Ezetrol) is a novel cholesterol lowering drug which disposition is not fully understood in man. We developed a selective and high-sensitive assay to measure serum concentration-time profiles, renal and fecal elimination of ezetimibe in pharmacokinetic studies. Ezetimibe glucuronide, the major metabolite of ezetimibe was determined by enzymatic degradation to the parent compound. Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-hydroxychalcone as internal standard and liquid chromatography coupled via an APCI interface with tandem mass spectrometry (LC-MS/MS) for detection. The chromatography (column XTerra) MS, C(18), 2.1 mm x 100 mm, particle size 3.5 microm) was done isocratically with acetonitrile/water (60/40, v/v; flow rate 200 microl/min). The MS/MS analysis was performed in the negative ion mode (m/z transition: ezetimibe 408-271, internal standard 223-117). The validation ranges for ezetimibe and total ezetimibe were as follows: serum 0.0001-0.015 microg/ml and 0.001-0.2 microg/ml; urine and fecal homogenate 0.025-10 microg/ml and 0.1-20 mg/ml, respectively. The assay was successfully applied to measure ezetimibe disposition in two subjects genotyped for the hepatic uptake transporter SLCO1B1.     

Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 microM (0.13-39.44 microg/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 microM (0.26-60 microg/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus C(18) column, 5 microm, 150 x 4.6 mm, and a Waters C(18), 4 microm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6+/-0.5, 18.1+/-1.1 and 29.0+/-0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.     

Intranasal midazolam in piglets: pharmacodynamics (0.2 vs 0.4 mg/kg) and pharmacokinetics (0.4 mg/kg) with bioavailability determination

Intranasal midazolam was studied in two series of piglets: series 1, n = 20 (18 +/- 3 kg), a randomized double blind pharmacodynamic study to compare doses of 0.2 mg/kg and 0.4 mg/kg; series 2, n = 9 (42 +/- 8 kg), a pharmacokinetic study with a 0.4 mg/kg dose administered either intravenously (i.v.) or intranasally (i.n.) in a cross-over protocol with a one-week wash-out period between each. In series 1, midazolam caused significant anxiolysis and sedation within 3 to 4 min, without a significant difference between 0.2 and 0.4 mg/kg doses for any of the studied parameters. In series 2, after intranasal midazolam administration of 0.4 mg/kg, plasma concentrations attained a maximum (Cmax) of 0.13 +/- 0.04 mg/l at 5 min (median Tmax) and remained higher than 0.04 mg/l until 60 min. The bioavailability factor (F) in this study was F = 0.64 +/- 0.17 by the intranasal route. The terminal half-life (T1/2 lambda z) = 145 +/- 138 min was comparable with the i.v. administration half-life (158 +/- 127 min). In conclusion, optimal intranasal midazolam dose in piglets was 0.2 mg/kg, which procures rapid and reliable sedation, adapted to laboratory piglets.     

Quantification of free mycophenolic acid and its glucuronide metabolite in human plasma by liquid-chromatography using mass spectrometric and ultraviolet absorbance detection

The immunosuppressant drug mycophenolic acid (MPA) and its major metabolite, mycophenolic acid glucuronide (MPAG), are highly bound to albumin. An HPLC-tandem-MS (HPLC/MS/MS) and an HPLC-UV assay were developed to measure free (unbound) concentrations of MPA and MPAG, respectively. Ultrafiltrate was prepared from plasma (500 microl) by ultrafiltration at 3000 x g for 20 min (20 degrees C). Both MPA and MPAG were isolated from ultrafiltrate (100 microl) by acidification and C18 solid-phase extraction. Free MPA was measured by electrospray tandem mass spectrometry using selected reactant monitoring (MPA: m/z 338.2--> 206.9) in positive ionisation mode. Chromatography was performed on a PFPP column (50 mm x 2 mm, 5 microm). Total analysis time was 7 min. The assay was linear over the range 1-200 microg/l with a limit of quantification of 1 microg/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 microg/l) were 94-99% and < 7%, respectively. Free MPAG was chromatographed on a C18 Nova-Pak column (150 mm x 3.9 mm, 5 microm) using a binary gradient over 20 min. The eluent was monitored at 254 nm. The assay was linear over the range 1-50 mg/l with the limit of quantification at 2.5 mg/l. The inter-day accuracy and imprecision of quality controls (5, 20, 45 mg/l) was 101-107% and < 8% (n = 4), respectively. For both methods no interfering substances were found in ultrafiltrate from patients not receiving MPA. The methods described have a suitable dynamic linear range to facilitate the investigation of free MPA and MPAG pharmacokinetics in transplant patients. Further, this is the first reported HPLC-UV method to determine free MPAG concentrations.     

Effects of gender on exercise-induced growth hormone release.

We examined gender differences in growth hormone (GH) secretion during rest and exercise. Eighteen subjects (9 women and 9 men) were tested on two occasions each [resting condition (R) and exercise condition (Ex)]. Blood was sampled at 10-min intervals from 0600 to 1200 and was assayed for GH by chemiluminescence. At R, women had a 3.69-fold greater mean calculated mass of GH secreted per burst compared with men (5.4 +/- 1.0 vs. 1.7 +/- 0.4 microg/l, respectively) and higher basal (interpulse) GH secretion rates, which resulted in greater GH production rates and serum GH area under the curve (AUC; 1,107 +/- 194 vs. 595 +/- 146 microg x l(-1) x min, women vs. men; P = 0.04). Compared with R, Ex resulted in greater mean mass of GH secreted per burst, greater mean GH secretory burst amplitude, and greater GH AUC (1,196 +/- 211 vs. 506 +/- 90 microg x l(-1) x min, Ex vs. R, respectively; P < 0.001). During Ex, women attained maximal serum GH concentrations significantly earlier than men (24 vs. 32 min after initiation of Ex, respectively; P = 0.004). Despite this temporal disparity, both genders had similar maximal serum GH concentrations. The change in AUC (adjusted for unequal baselines) was similar for men and women (593 +/- 201 vs. 811 +/- 268 microg x l(-1) x min), but there were significant gender-by-condition interactive effects on GH secretory burst mass, pulsatile GH production rate, and maximal serum GH concentration. We conclude that, although women exhibit greater absolute GH secretion rates than men both at rest and during exercise, exercise evokes a similar incremental GH response in men and women. Thus the magnitude of the incremental secretory GH response is not gender dependent.     

Water extract of the root of Lindera strychnifolia slows down the progression of diabetic nephropathy in db/db mice

We examined a possible preventive effect of Linderae radix (LR), the root of Lindera strychnifolia, on the progression of diabetic nephropathy. Water extract of Linderae radix (LR extract) was orally administered to the C57BL/KsJ-db/db (db/db) mice, a model of genetic diabetes, at a dose of 730 mg/kg/day for 12 week. The LR extract treatment did not affect glucose metabolism and systolic pressure. However, it resulted in a better renal function as evaluated by creatinine clearance (Ccr) and serum creatinine than the control; Ccr and serum creatinine were progressively worsened in controls (0.13+/-0.01 (l/day) and 0.69+/-0.04 (mg/dl), respectively) whereas unchanged in the treated group (0.24+/-0.03 (l/day), p<0.05 and 0.53+/-0.04 (mg/dl), p<0.05, respectively). Kidneys of the LR extract-treated group showed glomeruli with greater area and cell population, smaller glomerular sclerotic index, and less fibrosis in glomeruli, where apoptotic rate of glomerular cells were decreased compared with the control kidneys. Furthermore, renal TGF-beta(1) expression was decreased in the LR extract-treated group. These findings suggest that the LR therapy can be a novel therapeutic approach against diabetic nephropathy.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

Suppression of ethanol and lipopolysaccharide-induced liver injury by extracts of Hydrangeae Dulcis Folium in rats

In female SD rats that were injected with 4 g/kg BW ethanol p.o. followed by a 5 mg/kg BW lipopolysaccharide (LPS) i.v. injection, serum glutamic pyruvic transaminases (GPT) activity increased to about eight times that of normal rats. In this model, rats that had been fed a diet containing 1% Hydrangeae Dulcis Folium (HDF) extracts for fifteen days showed significantly lower serum GPT activity (380.0+/-58.2 IU/l) than the control group (3527.0+/-774.1 IU/l). HDF's efficacy was far superior to milk thistle in this model (2950.0+/-915.9 IU/l). When mouse macrophages were treated with HDF extracts at 50 microg/ml, TNF-alpha production induced by LPS was suppressed to about 10% of the control. Rat serum TNF-alpha levels induced by LPS was decreased to 58.7% of the control by administering 1000 mg/kg BW HDF extract p.o. These results indicate that HDF prevents alcohol-induced liver injury through the inhibition of TNF-alpha production.     

CCK inhibits the orexigenic effect of peripheral ghrelin

CCK and ghrelin exert antagonistic effects on ingestive behavior. The aim of the present study was to investigate the interaction between ghrelin and CCK administered peripherally on food intake and neuronal activity in specific hypothalamic and brain stem nuclei, as assessed by c-Fos-like immunoreactivity (c-FLI) in nonfasted rats. Ghrelin (13 microg/kg body wt) injected intraperitoneally significantly increased the cumulative food intake when measured at 30 min and 1 h after injection, compared with the vehicle group (2.9 +/- 1.0 g/kg body wt vs. 1.2 +/- 0.5 g/kg body wt, P < 0.028). Sulfated CCK octapeptide (CCK-8S) (2 or 25 microg/kg body wt) injected simultaneously blocked the orexigenic effect of ghrelin (0.22 +/- 0.13 g/kg body wt, P < 0.001 and 0.33 +/- 0.23 g/kg body wt, P < 0.0008), while injected alone, both doses of CCK-8S exerted a nonsignificant trend to reduce food intake. Ghrelin (13 microg/kg body wt ip) markedly increased the number of c-FLI-positive neurons per section in the arcuate nucleus (ARC) compared with vehicle (median: 31.35 vs. 9.86, P < 0.0001). CCK-8S (2 or 25 microg/kg body wt ip) had no effect on neuronal activity in the ARC, as assessed by c-FLI (median: 5.33 and 11.21 cells per section), but blocked the ghrelin-induced increase of c-fos expression in this area when both peptides were administered simultaneously (median: 13.33 and 12.86 cells per section, respectively). Ghrelin at this dose had no effect on CCK-induced stimulation of c-fos expression in the paraventricular nucleus of the hypothalamus and the nucleus of the solitary tract. These results suggest that CCK abolishes ghrelin-induced food intake through dampening increased ARC neuronal activity.