Amphetamines in hair by enzyme-linked immunosorbent assay

Human hair was collected from the occipital crown region of the head from several subjects; these hair samples were presumptively positive for amphetamines by a previously evaluated immunoassay. Hair was washed briefly with methanol to remove external contamination, then extracted with hot methanol for 2 h to recover the drugs. The extracts were evaporated to dryness, reconstituted in buffer, and analyzed using a new enzyme-linked immunosorbent assay (ELISA) technique adapted for the detection of amphetamines in hair. Gas chromatography-mass spectrometry was used as the reference technique. Cross-reactivity of several related compounds was evaluated by equating the inverse of the ligand concentration at 50% antibody binding to the affinity constant for each compound. The ratio of a compound's affinity constant to that for d-methamphetamine was used to derive percent crossreactivity. These experiments yielded values of 30.8% for d-amphetamine, 7.4% for I-methamphetamine, 4.3% for phentermine, 2.9% for I-amphetamine, and <1% for ephedrine, methylenedioxyamphetamine, and methylenedioxymethamphetamine. Cross-reactivity of unrelated compounds was found to be non-existent. The optimum cutoff concentration was determined by receiver operating characteristic curve analysis to be 300 pg/mg and the observed limit of detection was 60 pg/mg. Intra-assay precision at 300 pg/mg was 3.3% (coefficient of variation, CV), and the interassay CV was 10.5%. The sensitivity and specificity of the method were 83% and 92%, respectively.     

Interactive effects of subanesthetic ketamine and subhypnotic lorazepam in humans

Ketamine is an N-methyl-D-aspartate (NMDA) receptor antagonist with psychotogenic and dissociative effects in healthy humans. These cognitive and perceptual effects in humans are reportedly reduced by benzodiazepine premedication. This study assessed the interactive effects of a ketamine (i.v. bolus of 0.26 mg/kg followed by an infusion of 0.65 mg/kg per hour) and lorazepam 2 mg., PO, in humans. Twenty-three healthy subjects completed 4 test days involving the oral administration of lorazepam or matched placebo 2 h prior to the i.v. infusion of ketamine or placebo. Ketamine: 1) produced behaviors similar to the positive and negative symptoms of schizophrenia as assessed by the Brief Psychiatric Rating Scale (BPRS); 2) evoked perceptual alterations as measured by the Clinician-Administered Dissociative States Scale (CADSS); 3) impaired performance on the Wisconsin Card Sorting Test (WCST) and other tests sensitive to frontal cortical impairment; and 4) had amnestic effects. Lorazepam produced attention impairments, concrete proverb interpretations, and recall impairments. Lorazepam reduced ketamine-associated emotional distress and there was a non-significant trend for it to decrease perceptual alterations produced by ketamine. However, it failed to reduce many cognitive and behavioral effects of ketamine, including psychosis. Further, lorazepam exacerbated the sedative, attention-impairing, and amnestic effects of ketamine. There was no evidence of pharmacokinetic interaction between these medications. These data suggest that subhypnotic lorazepam and ketamine show a spectrum of interactive effects, ranging from antagonism to potentiation.     

Intravenous flumazenil following acute and repeated exposure to lorazepam in healthy volunteers: antagonism and precipitated withdrawal

The effects of i.v. administered flumazenil (3.0 mg) were studied in healthy male subjects who received pretreatment with p.o. placebo or lorazepam. The duration of placebo or lorazepam (3.0 mg single p.o. daily dose) pretreatment before a flumazenil or placebo injection was 1, 3, 7 or 14 days in four sequential groups of subjects. Initial administration of lorazepam produced a classic sedative profile of effects on various psychomotor/behavioral performance, observer-rated and subject-rated measures. Tolerance to repeated daily administration of lorazepam was suggested by a progressive diminution of performance disrupting effects. In subjects pretreated with placebo, flumazenil increased subject-ratings of dizziness over preinjection ratings. Flumazenil produced an immediate reversal of lorazepam effects in subjects who were not tolerant to lorazepam (1- and 3-day pretreatment groups). Flumazenil did not precipitate withdrawal symptoms in subjects who received a single administration of lorazepam. Precipitated withdrawal symptoms were evident after 3 and 7 days of lorazepam pretreatment, and there was a tendency toward precipitated withdrawal symptoms (that included one panic attack) after 14 days of lorazepam pretreatment. Precipitated withdrawal was characterized by an elevation in subject-rated symptoms including dizziness, tenseness, tachycardia, perceptual disturbance and sweating. Symptoms were maximal immediately after injection, usually mild in severity and usually resolved within 1 hr. There was no evidence of precipitated withdrawal on psychomotor/behavioral performance or observer ratings. The present study provides the strongest human experimental evidence to date that flumazenil can precipitate withdrawal symptoms after a history of repeated benzodiazepine exposure.     

Immunoassay of digoxin in hair

Digoxin analysis in blood is an essential tool for therapeutic drug monitoring in cardiology because compliance with the treatment is a critical issue for the patient. Unfortunately, in postmortem cases blood digoxin concentration is of poor quality because there is a possible drug redistribution in the corpse and because of digoxin-like factors present in some people's blood. On the other hand, no biological fluid can be obtained at the autopsy. The aim of the present study was to evaluate the ability of an immunological method to determine digoxin in hair, in order to confirm that hair analysis can provide information on digoxin use before death. We studied 35 elderly patients who had been taking digoxin (60-250 micrograms/day) for 1-5 years. Two decontamination procedures were tested: washing by dichloromethane or by water and methanol. Three extraction procedures were compared: crushing in a ball mill and chloroform/acetone: crushing and methanol; enzymatic digestion. Immunoassays were performed by a microparticulate enzyme immunoassay. Serum digoxin levels were also assayed when sampling hair. The best results were obtained after decontamination with water and methanol followed by enzymatic digestion. Hair digoxin concentrations range from 3.6 to 11.4 pg/mg. Those very low concentrations are probably due to low and narrow range serum digoxin levels (0.3-1.4 ng/ml). No correlation was found between hair and blood digoxin. A forensic case is presented with 5 pg/mg digoxin in hair.     

Quantification of dextropropoxyphene and its metabolite by HPLC in hair of overdose cases

Hair samples taken from 11 persons suspected to have died from an overdose of legal or illicit drugs were analysed. These hair samples were selected, because classical post-mortem toxicological investigations in blood revealed the presence of dextropropoxyphene (PPX) and its major metabolite norpropoxyphene (NPPX). For the hair analysis, hair strands were cut into segments of about 3 cm, washed with water and acetone, dried and pulverised. An aliquot of about 40 mg of those segments was incubated with acetate buffer pH 4.0 and beta-glucuronidase/arylsulfatase. After liquid-liquid extraction with hexane-3-methylbutanol (99:1), drugs were identified and measured by HPLC using piritramide as an internal standard. Preliminary assays showed that the limit of detection for PPX is below 1.0 ng/mg hair, the limit of detection for the metabolite NPPX being significantly higher (1.5 ng/mg). GC/MS, usually the method of choice for this kind of determination, was not chosen, because of the thermolability of PPX and its unspecific mass spectrum. From the hair of 11 persons, 24 segments were prepared and processed. Our results show that ten persons were found positive for PPX; moreover, when regarding the 24 segments, only three were found negative. PPX and NPPX concentrations were detected at maximal concentrations of 26.4 and 71.0 ng/mg hair respectively. Considering the ratio of PPX to NPPX for each person, we found more PPX than NPPX for three persons, whereas for seven persons we found more of the metabolite than its parent drug.     

Multiresidue determination of pesticides in apples and pears by gas chromatography-mass spectrometry

This paper describes a rapid, specific and sensitive multiresidue method for the routine analysis of several classes of pesticides used for the treatment of apples and pears, involving a rapid extraction procedure at pH 4.5 with a mixture of acetone-dichloromethane-hexane (50:20:30, v/v/v) and gas chromatography coupled to mass-selective detection, in order to achieve quantitative analysis down to their respective maximum residue limit. Extraction recoveries were between 55 and 98%. Limits of detection and limits of quantitation ranged respectively, from 0.01 to 0.05 mg/kg and from 0.02 to 0.1 mg/kg. Intra-assay relative standard deviation was less than 19% for all compounds. An excellent linearity was observed from these LOQs up to 500 mg/kg. Intermediate (inter-assay) precision and accuracy were satisfactory. The method has been applied to many fruit samples intended for commercialisation.     

Modeling and measurement of the spontaneous mutation rate in mammalian cells

In order to examine the respiratory effects of tonic-clonic seizures and their treatment with i.v. diazepam or lorazepam, we utilized a spontaneously breathing piglet seizure model. A tracheostomy, arterial catheter, and epidural electrodes were inserted and pigs were maintained under ketamine anesthesia. After baseline recordings, seizures were induced with a pentylenetetrazol (PTZ) bolus and a 20 min infusion (5-6 mg/kg/min). After 10 min of PTZ infusion, randomly assigned animals received diazepam (D; N = 7; 0.5 mg/kg), lorazepam (L; N = 7; 0.2 mg/kg), or 0.9% saline (C; N = 7; controls) by rapid peripheral vein injection. Minute ventilation (Ve), Pa(CO2), and the pressure change in response to airway occlusion at end-expiration (P0.1) were measured at standard intervals. All groups had comparable increases in respiratory drive during untreated seizures. Changes in Ve and P0.1 were reduced to at or below baseline values in groups D and L, but not C, from 2 to 45 min after treatment (P < 0.05). No significant changes were observed in Pa(CO2) after either intervention. Following anticonvulsants, the cumulative duration of seizures was significantly reduced in L and D groups, compared to C (P < 0.05). We conclude that increases in respiratory drive occur during tonic-clonic seizures induced with PTZ. Amelioration of seizure activity with lorazepam or diazepam results in a reduction in respiratory drive, but not respiratory failure, in this tracheostomized model.     

Determination of naringenin and its glucuronide conjugate in rat plasma and brain tissue by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method with ultraviolet detection was utilized for the investigation of the pharmacokinetics of naringenin and its glucuronide conjugate in rat plasma and brain tissue. Plasma and brain tissue were deproteinized by acetonitrile, then centrifuged for sample clean-up. The drugs were separated by a reversed-phase C18 column with a mobile phase consisting of acetonitrile-orthophosphoric acid solution (pH 2.5-2.8) (36:64, v/v). The detection limits of naringenin in rat plasma and brain tissue were 50 ng/ml and 0.4 microg/g, respectively. The glucuronide conjugate of naringenin was evaluated by the deconjugated enzyme beta-glucuronidase. The naringenin conjugation ratios in rat plasma and brain tissue were 0.86 and 0.22, respectively, 10 min after naringenin (20 mg/kg, i.v.) administration. The mean naringenin conjugation ratio in plasma was approximately four fold that in brain tissue.     

Determination of pharmaceuticals in plasma by capillary electrophoresis without sample pretreatment reproducibility, limit of quantitation and limit of detection

Pharmaceuticals in human plasma are determined on underivatized fused-silica capillaries by micellar electrokinetic capillary chromatography (MEKC) without sample pretreatment. Our best method to date uses as running buffer a sodium dodecyl sulfate (SDS) containing borate buffer (60 mM with 200 mM SDS) at pH 10. Between runs, proteins adsorbed to the capillary wall are removed by an acetonitrile and SDS-buffer rinsing regimen (50% v/v each). A day-to-day precision for relative peak areas of about 2% relative standard deviation (RSD; n > 40) has been reached. Different rinsing approaches are discussed (salts, enzyme-containing solutions, organic solvents, hydrofluoric acid). The separation system is tested in a concentration range between approximately 100 mg/L-10 mg/L. Correlations between the limit of quantitation, the limit of detection and the signal/noise are discussed. The applicability of the system is demonstrated for the pharmaceuticals acetaminophen, salicylic acid, sulfamethoxazole, tolbutamide, and trimethoprim.     

Nedaplatin and 5-FU combined with radiation in the treatment for esophageal cancer

A direct differentiation of the internal and external drug-deposition pattern into hair was made using two fluorescent dyes and fluorescence microscopy after systemic administration to mice or external exposure of untreated hair. Mice (23 days old, C57 and Balb/C) were administered either rhodamine or fluorescein intraperitoneally at varied doses on 3 consecutive days of 3 weeks, and hair was sampled 1 week later. Another group was given 10 mg/kg rhodamine or 100 mg/kg fluorescein and sampled at time points from 5 min to 168 hr. The time courses of external deposition of rhodamine and fluorescein into untreated hair were examined after hair was soaked in 0.1 mg/ml solutions at pH 3, pH 6, and pH 9 aqueous buffer or methanol. The hair was then extracted in pH 6 phosphate buffer or methanol for 24 hr. In vivo accumulation was distinguishable as fluorescent bands along the length of the hair for rhodamine and fluorescein. The pattern of in vivo deposition appears to arise from the rapid accumulation within the cortex and medulla, with little deposition evident in the cuticle. Neither phosphate buffer nor methanol washes affected the intensity of fluorescence in the hair. External loading of rhodamine into the hair resulted in staining of the junctions of cuticle scales. This pattern persisted even after 12 hr of solution exposure. Extraction with pH 6 phosphate buffer or methanol did not remove rhodamine. Fluoroscein followed a similar pattern, with maximum fluorescence when hair was loaded in pH 6 100mM phosphate buffer and nominal staining when loaded in pH 9 100 mM Tris buffer or methanol. Soaking the hair in pH 6 buffer, but not methanol, removed some fluorescein. These results demonstrate that compounds in the circulation can rapidly diffuse into the forming cortex and medulla, where rapid associations occur with elongating intermediate filaments specific to the medulla and cortex. These compounds can become significantly occluded within the mature matrix and are resistant to removal in aqueous or methanolic solutions.     

A field test comparison of hiking stick use on heartrate and rating of perceived exertion

A case is presented involving a young woman on several illicit drugs (heroin, cocaine and cannabis) as well as two medications and a solvent used for their anesthetic and narcotic properties: thiopental, ketamine and chloroform. This complex drug use was supported by hair analysis over a 10.5 cm segment of the hair taken at autopsy. The average measured concentrations in hair were: thiopental = 5.3 ng/mg, pentobarbital = 10.0 ng/mg, ketamine = 11.3 ng/mg norketamine = 1.0 ng/mg, diazepam = 1.2 ng/mg, nordiazepam = 0.1 ng/mg, 6-acetylmorphine = 4.4 ng/mg, morphine = 3.4 ng/mg, codeine = 1.2 ng/mg, cocaine = 5.5 ng/mg, benzoylecgonine = 1.5 ng/mg and methylecgonine ester = 1.0 ng/mg. While the ketamine/norketamine ratio is consistent with that already reported on drug detection in hair, the thiopental/pentobarbital ratio seems to be inverted.     

Detection of protein p53 in the stool of patients with colorectal cancer

BACKGROUND AND AIMS: Mutations of TP53, a tumor suppressor gene, are found in 60% to 70% of colorectal cancers. These mutations usually induce an overexpression caused by modification of the p53 protein conformation. The aim of this study was to investigate whether stool specimens of patients with colorectal cancer contain increased amounts of p53 protein. METHODS: p53 protein was measured using a sandwich enzyme immunoassay in the stool specimens of 52 patients: 25 with colorectal cancer, 4 with colorectal adenomas and 23 apparently free of gastrointestinal disease. Results were expressed as pg/mg of total protein. The presence of fecal occult-blood was searched using Hemoccult II and Hemolex (an immunochemical assay for human hemoglobin). RESULTS: Median concentrations of stool p53 protein were 16.6 pg/mg (range: 0-591 pg/mg) in patients with colorectal cancers, 39.1 pg/mg (range: 5-72 pg/mg) in patients with adenomas and 5.9 pg/mg (range: 0-65 pg/mg) in control subjects. Resection of colorectal cancers caused a marked decrease of stool p53 protein concentrations. When the cut-off value for stool p53 protein was set at 60 pg/mg of fecal protein (concentrations over the 95th percentile), the positivity of the assay was independent of tumor size and Astler-Coller stage, but weakly associated with rectal location of cancer. The sensitivity of stool p53 protein for colorectal cancer was 44%, and the specificity was 96%. In contrast, the sensitivity of Hemoccult II and Hemolex tests was 48% and 44%, whereas their specificity was 91% and 96%, respectively. CONCLUSION: The detection of p53 protein is achievable in stool, but this assay is not more efficient than fecal occult blood tests for detection of colorectal cancer.     

Automated and sensitive method for the determination of formoterol in human plasma by high-performance liquid chromatography and electrochemical detection

An automated high-performance liquid chromatography (HPLC) method for the determination of formoterol in human plasma with improved sensitivity has been developed and validated. Formoterol and CGP 47086, the internal standard, were extracted from plasma (1 ml) using a cation-exchange solid-phase extraction (SPE) cartridge. The compounds were eluted with pH 6 buffer solution-methanol (70:30, v/v) and the eluate was further diluted with water. An aliquot of the extract solution was injected and analyzed by HPLC. The extraction, dilution, injection and chromatographic analysis were combined and automated using the automate (ASPEC) system. The chromatographic separations were achieved on a 5 microm, Hypersil ODS analytical column (200 mm x 3 mm I.D.), using (pH 6 phosphate buffer, 0.035 M + 20 mg/l EDTA)-MeOH-CH3CN (70:25:5, v/v/v) as the mobile phase at a flow-rate of 0.4 ml/min. The analytes were detected with electrochemical detection at an operating potential of +0.63 V. Intra-day accuracy and precision were assessed from the relative recoveries of calibration/quality control plasma samples in the concentration range of 7.14 to 238 pmol/l of formoterol base. The accuracy over the entire concentration range varied from 81 to 105%, and the precision (C.V.) ranged from 3 to 14%. Inter-day accuracy and precision were assessed in the concentration range of 11.9 to 238 pmol/l of formoterol base in plasma. The accuracy over the entire concentration range varied from 98 to 109%, and precision ranged from 8 to 19%. At the limit of quantitation (LOQ) of 11.9 pmol/l for inter-day measurements, the recovery value was 109% and C.V. was 19%. As shown from intra-day accuracy and precision results, favorable conditions (a newly used column, a newly washed detector cell and moderate residual cell current level) allowed us to reach a LOQ of 7.14 pmol/l of formoterol base (3 pg/ml of formoterol fumarate dihydrate). Improvement of the limit of detection by a factor of about 10 was reached as compared to the previously described methods. The method has been applied for quantifying formoterol in plasma after 120 microg drug inhalation to volunteers. Formoterol was still measurable at 24 h post-dosing in most subjects and a slow elimination of formoterol from plasma beyond 6-8 h after inhalation was demonstrated for the first time thanks to the sensitivity of the method.     

Cytokine secretion by human fetal membranes, decidua and placenta at term

Cytokines such as monocyte chemotactic peptide-1 (MCP-1), interleukin-8 (IL-8), RANTES (Regulated on Activation and Normally T-cells Expressed and presumably Secreted) and interleukin-10 (IL-10) are thought to play pivotal roles in immune recognition, acceptance of the fetal allograft, maintenance of pregnancy and parturition. Their secretion and regulation within the third trimester uterus is, however, less well defined. We therefore investigated the release of these cytokines by third trimester amnion, chorion, placenta and decidua, and studied the influence of prostaglandin E2 (PGE2) infusion on their release in a dynamic placental cotyledon perfusion system. MCP-1 was released predominately by the chorion (78.2 +/- 7.3 pg/mg wet tissue weight; mean +/- SEM), decidua (112.4 +/- 5.2 pg/mg) and placenta (101.8 +/- 5.0 pg/mg) with low amounts from the amnion (1.3 +/- 0.4 pg/mg). High concentrations of IL-8 were released by the amnion (39.9 +/- 5.3 pg/mg), chorion (52.8 +/- 1.9 pg/mg), decidua (42.2 +/- 1.5 pg/mg) and placenta (45 +/- 1.3 pg/mg). Release of RANTES was not detectable from the amnion but was detected in moderate amounts from the chorion (6.0 +/- 1.2 pg/mg), decidua (15.2 +/- 1.4 pg/mg) and placenta (26.9 +/- 1.6 pg/mg). Low concentrations of IL-10 were secreted by the chorion (6.8 +/- 0.8 pg/mg), decidua (9.0 +/- 0.9 pg/mg) and placenta (3.3 +/- 0.3 pg/mg) with none detectable from the amnion. MCP-1, IL-8, RANTES and IL-10 were all released by perfused placental cotyledons. PGE2 stimulated release of MCP-1, IL-8 and IL-10 into the maternal and of MCP-1 and IL-8 into the fetal circulation of the placenta but had no effect on RANTES release. It is suggested that MCP-1 and IL-8 may be involved in the inflammatory process of parturition and IL-10 in the protection of the fetal allograft. In addition, PGE2 may have an important immunomodulatory role within the uterus at term.     

Fentanyl in hair. Chemical factors involved in accumulation and retention of fentanyl in hair after external exposure or in vivo deposition

The levels of fentanyl extractable from mouse hair after chronic systemic administration and the suitability of externally loaded hair samples for establishing control and comparison samples were determined. Additionally, the effects of chemical modification of specific polar functionalities within the hair protein matrix on the deposition and recovery of fentanyl in hair subjected to external loading were determined. BALB/c mice entering a second phase of synchronized hair growth were treated ip with fentanyl (0.02, 0.05, or 0.10 mg/kg) on Monday, Wednesday, and Friday for 3 weeks. At that time, fentanyl concentrations in hair, as determined by GC/MS, were 0.025-0.050 ng/mg of hair. Hair samples exposed to fentanyl in phosphate buffer (ionized drug) showed no significant accumulation of drug into the hair, as determined by loss of fentanyl from the loading solution or by extraction of the hair. Hair samples exposed to nonionized fentanyl in methanolic solution (10, 50, and 100 ng/ml) showed significant accumulation of drug in the hair and significant removal of drug from the incubation solution. Fentanyl removal from solution plateaued after 24 hr, suggesting equilibration between fentanyl in solution and fentanyl in the hair. A mass balance between drug lost from the incubation solution and drug recovered from hair samples suggests that 94% of accumulated fentanyl is tightly bound to the hair matrix or resides in water-inaccessible compartments within the hair. These results suggest that fentanyl accumulation after in vivo administration differs, in the nature of storage, from fentanyl accumulation from external solutions and that external spiking of hair may not provide suitable control samples. Chemical modification of hair protein functionalities (reaction with diazomethane to esterify carboxylic acid groups or with acetic anhydride and pyridine to acetylate amine and hydroxyl functionalities) led to reproducible protein structure modification, as demonstrated by Fourier transform-IR and by pH titration. Hair from BALB/c mice was used. The accumulation of fentanyl was examined in hair samples exposed to fentanyl in methanol or methylene chloride solutions (10 ng/ml, 24 hr). Fentanyl was recovered from hair by 24-hr extraction in phosphate buffer, pH 6. Esterification of hair resulted in significantly less uptake of nonionized fentanyl from a methanolic solution and significantly lower recovery of drug from hair, relative to untreated hair, suggesting that carboxylic acid functionalities are necessary for the incorporation of drug. Acetylation of hair resulted in increased removal of fentanyl from methylene chloride solutions and increased recovery of fentanyl. This is consistent with the creation or expansion of a less polar compartment. Fentanyl uptake from a methanolic solution was also greater in acetylated hair. These results demonstrate that solution-accessible ionizable functionalities of hair play a significant role in the accumulation and retention of nonionized fentanyl from organic solutions.     

Antagonism by benzodiazepines of the effects of serotonin-, but not norepinephrine-, uptake blockers in the learned helplessness paradigm in rats

Benzodiazepines are routinely administered in combination with antidepressant drugs. Such combination can induce pharmacodynamic or pharmacokinetic interactions resulting in either a potentiation or a reduction of the effects of one of the drugs. The present study was undertaken to determine the effects of benzodiazepines alone and in combination with two classes of antidepressant drugs: "specific" norepinephrine uptake blockers (desipramine, maprotiline) and specific serotonin uptake blockers (indalpine, fluvoxamine) in the learned helplessness paradigm in rats. The present results show that daily injection of diazepam (0.2-2 mg/kg) and lorazepam (0.06-0.25 mg/kg) did not reverse the helpless behavior. The reversal of helpless behavior induced by indalpine (1 mg/kg/day) or fluvoxamine (4 mg/kg/day) was antagonized dose-dependently by daily coadministration of benzodiazepines. In contrast, the effects of desipramine (24 mg/kg/day) or maprotiline (48 mg/kg/day) were not modified.     

A two-term MEDLINE search strategy for identifying randomized trials in obstetrics and gynecology

In this study, we examined whether endothelin (ET) plays a role in the short-term increase in mean arterial pressure (MAP) after nitric oxide synthase (NOS) inhibition with N(omega)-nitro-L-arginine methyl ester (L-NAME) in stroke-prone spontaneously hypertensive rats (SHRSPs). Experiments were performed by using Inactin-anesthetized male SHRSPs that were pretreated with chlorisondamine to block reflex autonomic cardiovascular effects. Injection of L-NAME (10 mg/kg, i.v.), but not D-NAME, produced rapid and marked increases (74 +/- 3 mm Hg) in MAP that were sustained for >1 h. In SHRSPs that were treated with the ET(A/B) receptor antagonist, L-754,142 (15 mg/kg + 15 mg/kg/h), L-NAME increased MAP by 45 +/- 4 mm Hg (p < 0.0001 compared with L-NAME alone). L-754,142 blocked pressor responses to big ET-1 by >90% but was without effect on pressor responses to norepinephrine. Plasma levels of ET-1 averaged 5 +/- 1 pg/ml in animals given vehicle and were slightly increased in animals given either L-NAME alone (7 +/- 2 pg/ml) or L-754,142 alone (7 +/- 2 pg/ml) but increased markedly when L-NAME and L-754,142 were given together (114 +/- 18 pg/ml). This may relate to an effect of L-754,142 to block ET-receptor-mediated clearance of ET-1. We conclude that ET plays a role in the short-term pressor response after NOS inhibition in SHRSPs.     

Quantitative determination of endogenous retinoids in mouse embryos by high-performance liquid chromatography with on-line solid-phase extraction, column switching and electrochemical detection

An isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid and all-trans-retinol in mouse embryos using on-line solid-phase extraction and column switching in combination with electrochemical detection has been developed. The method was validated using retinoids in albumin solutions and 13-cis-acitretin was used as internal standard. About 370 microliters of albumin solution was injected on a 10 x 2.1-mm I.D. pre-column packed with Bondapak C18, 37-53-micron particles. The proteins were washed to waste within 5 min using as mobile phase, a 1:3 dilution of mobile phase 2, which consisted of acetonitrile-methanol-2% ammonium acetate-glacial acetic acid (79:2:16:3, v/v). Components retained on the pre-column were back-flushed to and separated on the 250 x 4.6-mm I.D. Suplex pKb-100 analytical column using mobile phase 2. The retinoids were detected electrochemically at +750 mV using a coulometric electrochemical detector. The total analysis time was about 20 min. Recoveries were in the range of 86-103%. The mass limits of detection were about 10 pg and 25 pg for the retinoic acids and all-trans-retinol, respectively. The intra-assay precision, reported as relative standard deviation, was in general better than 4% (n = 6) for the four retinoids. Inter-assay precision was in the range 3-4% (n = 10). The method was applied for determination of endogenous retinoids in 9.5 day-old mouse embryos. A 340-microliter solution containing 100 microliters of embryo homogenate (1.64 embryos) was analyzed. The concentrations of all-trans-retinol and all-trans-retinoic acid were found to be 279 pg per embryo and 75.8 pg per embryo, respectively. The amount of 13-cis-retinoic acid and 9-cis-retinoic acid was below the detection limit.     

Genetically determined susceptibility to organophosphorus insecticides and nerve agents: developing a mouse model for the human PON1 polymorphism

Thin-layer chromatographic (TLC)-UV densitometric and gas-chromatographic-mass spectrometric detection (GC-MSD) methods were developed for simultaneous quantification of morphine and codeine in poppy capsules (Papaver somniferum). Morphine and codeine were isolated by extraction with chloroform: isopropanol (3:1, v/v) at pH = 8.5 and by solid-phase extraction on Snap-Cap cartridges at pH = 8.5. The TLC-UV densitometric quantification was performed by external standard method on silica gel plates using ethyl acetate: toluene: methanol: ammonia (68:17:10:5, v/v) as developing solvent and UV detection at 275 nm. For the GC-MSD analysis, the drugs were derivatized with acetic anhydride: pyridine (1:1, v/v) and separated on a 30 m HP5 capillary column. The quantification was performed using nalorphine as internal standard.     

Effect of valproate on the pharmacokinetics and pharmacodynamics of lorazepam

The pharmacokinetic-pharmacodynamic interaction between valproate and lorazepam was evaluated in this randomized, double-blind, placebo-controlled crossover study. Sixteen healthy male volunteers enrolled in the study to receive either divalproex sodium (500 mg every 12 hours) or matching placebo for 12 days in the first period, and then to receive the other regimen for an identical second 12-day period. In both periods, lorazepam (1 mg every 12 hours) was administered on days 6 through 9 and on the morning of day 10. Concomitant administration of divalproex sodium with lorazepam resulted in an 8%, 20%, and 31% increase in steady-state maximum plasma concentration, area under the concentration-time curve, and trough plasma concentrations of lorazepam, respectively. The apparent clearance of lorazepam through the formation of lorazepam glucuronide was reduced by 31% during coadministration of divalproex sodium. Pharmacokinetic properties of valproate did not change significantly in the ten available participants during coadministration of lorazepam. Sedation scales revealed no statistically significant differences in sedation between the two regimens. It is concluded that valproate increases plasma concentrations and reduces clearance of lorazepam, most likely by impairing hepatic glucuronidation, and that coadministration of lorazepam does not affect the steady-state pharmacokinetic properties of valproate.