Brain/blood ratios of methadone and ABCB1 polymorphisms in methadone-related deaths.

Methadone is an opioid that often leads to fatalities. Interpretation of toxicological findings can be challenging if no further information about the case history is available. The aims of this study were (1) to determine whether brain/blood ratios can assist in the interpretation of methadone findings in fatalities; (2) to examine whether polymorphisms in the gene encoding the P-glycoprotein (also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1)), which functions as a multispecific efflux pump in the blood-brain barrier, affect brain/blood ratios of methadone. Femoral venous blood and brain tissue (medulla oblongata and cerebellum) from 107 methadone-related deaths were analysed for methadone by gas chromatography-mass spectrometry. In addition, all the samples were genotyped for three common ABCB1 single nucleotide polymorphisms (SNPs rs1045642, rs1128503, and rs2032582) using ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS). In nearly all cases, methadone concentrations were higher in the brain than in the blood. Inter-individual brain/blood ratios varied (0.6-11.6); the mean ratio was 2.85 (standard deviation 1.83, median 2.35). Moreover, significant differences in mean brain/blood ratios were detected among the synonymous genotypes of rs1045642 in ABCB1 (p = 0.001). Cases with the T/T genotype had significantly higher brain/blood ratios than cases with the other genotypes (T/T vs. T/C difference (d) = 1.54, 95% CI [1.14, 2.05], p = 0.002; T/T vs. C/C d = 1.60, 95% CI [1.13, 2.29], p = 0.004). Our results suggest that the rs1045642 polymorphisms in ABCB1 may affect methadone concentrations in the brain and its site of action and may be an additional factor influencing methadone toxicity.

Crack path selection in orientationally ordered composites.

While cracks in isotropic homogeneous materials propagate straight, perpendicularly to the tensile axis, cracks in natural and synthetic composites deflect from a straight path, often increasing the toughness of the material. Here we combine experiments and simulations to identify materials properties that predict whether cracks propagate straight or kink on a macroscale larger than the composite microstructure. Those properties include the anisotropy of the fracture energy, which we vary several fold by increasing the volume fraction of orientationally ordered alumina (Al_{2}O_{3}) platelets inside a polymer matrix, and a microstructure-dependent process zone size that is found to modulate the additional stabilizing or destabilizing effect of the nonsingular stress acting parallel to the crack. Those properties predict the existence of an anisotropy threshold for crack kinking and explain the surprisingly strong dependence of this threshold on sample geometry and load distribution.

Bioprotection of microbial communities from toxic phenol mixtures by a genetically designed pseudomonad.

Pseudomonas sp. B13 SN45RE is a genetically engineered microorganism (GEM) that is able to simultaneously degrade mixtures of chloro- and methylaromatics ordinarily toxic for microbial communities via a designed novel ortho-cleavage pathway. The utility of the GEM was investigated in a laboratory scale sewage plant fed with mixtures of either 4-chlorophenol and 4-methyphenol or 3-chlorophenol and 4-methylphenol. In the model system the GEM significantly increased the rate and extent of degradation of the phenol mixtures. In the absence of the GEM, shock loads of the phenol mixtures (1 mM of each compound) reduced the numbers of culturable bacteria by three orders of magnitude, completely eliminated protozoa and metazoa, and caused a drastic decrease in oxygen consumption, whereas the presence of the GEM protected the indigenous microbial community and assured continued functioning of the sewage plant.

Tolerance to alprazolam after intravenous bolus and continuous infusion: psychomotor and EEG effects.

A study was undertaken to compare the time course of changes in psychomotor performance and spectral edge (SE) of the EEG and to relate these changes to alprazolam concentrations in a pharmacokinetic/pharmacodynamic tolerance model. Digit symbol substitution (DSS) tests were administered and EEG data were obtained for SE calculation in a four-way crossover study in four normal men. Each treatment consisted of a 2-minute bolus injection followed by an 8-hour infusion. Treatment A, placebo, consisted of a 50% propylene glycol injection followed by a saline infusion. Active drug treatments were: B, 0.5 mg alprazolam plus saline infusion; C, 2.0 mg alprazolam plus saline; and D, 1.0 mg plus 72 micrograms alprazolam/hr for a total dose of 1.576 mg in 8 hours. For both DSS and SE data, three distinct effect-concentration curves result from the three alprazolam treatments, with successive shifts to the right as dose increases. A tolerance rate constant (kt) of 0.15 hr-1 was calculated from the DSS vs. time data during the 8 hour alprazolam infusion. The Hill equation was altered by using kt in an exponential modification of EC50. The resulting tolerance model describes both DSS and SE vs. concentration data from the rapid-injection and continuous-infusion treatments.

Digital plethysmography for assessing erythrityl tetranitrate bioavailability.

The bioavailability or oral, sublingual, and chewable tablets or erythrityl tetranitrate (ETN) was evaluated in 15 normal men. In a randomized, complete crossover investigation with nitroglycerin and placebo controls, drug-induced changes in the diastolic amplitude response intensity were measured with a digital plethysmogram. Values for area under the response intensity curve (AUC), maximum response intensity (Imax), and time lapse from dosing to peak response (tmax) were obtained by computer processing and converted to intensity values and AUC segments for specific time intervals. Sublingual nitroglycerin induced a response (p less than 0.05) from placebo within the first hour. Although somewhat slower in reaching peak intensity, all forms of ETN induced significant responses over placebo (p less than 0.05) for 2 hr, with oral (swallowed) ETN different 6 hr. Our results indicate that all the ETN dosage forms were bioavailable, with the longest duration of effect by the oral form.

The pharmacokinetic-pharmacodynamic (Digit Symbol Substitution Test) relationship of flumazenil in a midazolam steady-state model in healthy volunteers.

To characterize the plasma concentration-effect relationship of flumazenil in the presence of a predefined midazolam level, a double-blind, placebo-controlled, randomized two-way crossover study was conducted in nine healthy male subjects. After reaching a criterion level of midazolam-induced depression of the Digit Symbol Substitution Test (DSST), volunteers received a dose of flumazenil (1.0 mg) or placebo over 1 minute, with the infusion of midazolam continued. Blood samples were collected, simultaneously with the DSST assessment, at predetermined intervals and were assayed for flumazenil and/or midazolam plasma concentrations. Pharmacokinetic-pharmacodynamic modeling techniques were used to estimate the equilibration rate constant (keo) between plasma concentration and effect for flumazenil; a sigmoidal maximum-effect model was used to relate the DSST score to the flumazenil plasma concentration. Flumazenil exhibited a rapid onset (the half-life of equilibration between drug concentration in the blood and drug effect was 3.3 minutes) and short duration of action (the flumazenil plasma concentration causing half-maximal effect was 7.4 ng/ml, which was reached about 1 hour after dosing). The results of this study also show the competitive nature of flumazenil as a midazolam antagonist.

Characterization of a gene cluster from Ralstonia eutropha JMP134 encoding metabolism of 4-methylmuconolactone.

A 2,585 bp chromosomal DNA segment of Ralstonia eutropha JMP134 (formerly: Alcaligenes eutrophus JMP134) which contains a gene cluster encoding part of the modified ortho-cleavage pathway encodes a putative transport protein for 4-methylmuconolactone, a novel 4-methylmuconolactone methylisomerase and methylmuconolactone isomerase. The putative 4-methylmuconolactone transporter, a protein with a calculated molecular mass of 45.8 kDa, exhibits sequence homology to other members of the major superfamily of transmembrane facilitators and shows the common structural motif of 12 transmembrane-spanning alpha-helical segments and the hallmark amino acid motif characteristic of the superfamily. Consistent with the novelty of the reaction catalyzed by 4-methylmuconolactone methylisomerase, no primary sequence homologies were found between this enzyme or its gene and other proteins or genes in the data banks, suggesting that this enzyme represents a new type of isomerase. The molecular mass of the native 4-methylmuconolactone methylisomerase was determined by gel filtration analysis to be 25 +/- 2 kDa. From the polynucleotide sequence of the gene, a molecular mass of 12.9 kDa was calculated and hence we predict a homodimeric quaternary structure. The high sensitivity of 4-methylmuconolactone methylisomerase to heavy metals and thiol-modifying reagents implicates the involvement of sulfhydryl groups in the catalytic reaction. The methylmuconolactone isomerase - calculated molecular mass 10.3 kDa - has a primary structure related to the classical muconolactone isomerases (EC 5.3.3.4) of Acinetobacter calcoaceticus, of two Pseudomonas putida strains and of Ralstonia eutropha JMP134, suggesting that these are all isoenzymes. Consistent with this proposal is the finding that the purified protein exhibits muconolactone-isomerizing activity.

Efficacy and tolerability of carbamazepine for agitation and aggression in dementia.

OBJECTIVE: The efficacy, safety, and tolerability of carbamazepine in the treatment of agitation and aggression associated with dementia were assessed. METHOD: In a 6-week, randomized, multisite, parallel-group study of 51 nursing home patients with agitation and dementia, individualized doses of carbamazepine were compared with placebo. Except for a physician monitor and a pharmacist, all participants were blind to treatment. The primary outcome measures were the Brief Psychiatric Rating Scale (BPRS) and Clinical Global Impression (CGI) global improvement rating. Secondary measures included measures of behavior, aggression, cognition, functional status, staff time, safety, and tolerability. Intent-to-treat analysis was performed. RESULTS: The modal carbamazepine dose at 6 weeks was 300 mg/day, and a mean serum level of 5.3 micrograms/ml was achieved. The study was terminated after a planned interim analysis showed that carbamazepine provided more benefit than did placebo. Over 6 weeks the mean total BPRS score decreased 7.7 points for the carbamazepine group and 0.9 for the placebo group, and the weekly scores showed a gradual divergence between the two groups. CGI ratings showed global improvement in 77% of the patients taking carbamazepine and 21% of those taking placebo. Secondary analyses confirmed that the positive changes were due to decreased agitation and aggression. The drug was generally well tolerated, and no change in cognition or functional status occurred. The perception of staff time needed to manage agitation showed a decrease for carbamazepine but not placebo. CONCLUSIONS: This controlled study showed significant short-term efficacy of carbamazepine for agitation with generally good safety and tolerability.

Nitric oxide-dependent and -independent hyperaemia due to calcitonin gene-related peptide in the rat stomach.

1. Calcitonin gene-related peptide (CGRP) potently enhances mucosal blood flow in the rat stomach. The aim of this study was to examine whether CGRP also dilates extramural arteries supplying the stomach and whether the vasodilator action of CGRP involves nitric oxide (NO). 2. Rat CGRP-alpha (0.03-1 nmol kg-1, i.v.) produced a dose-dependent increase in blood flow through the left gastric artery (LGA) as determined by an ultrasonic transit time technique in urethane-anaesthetized rats. Blockade of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME, 20 and 60 mumol kg-1, i.v.) significantly reduced basal blood flow (BF) in the LGA and attenuated the hyperaemic activity of CGRP by a factor of 2.8-4. D-NAME tended to enhance basal BF in the LGA but had no influence on the dilator activity of CGRP. The ability of vasoactive intestinal polypeptide to increase left gastric arterial blood flow remained unaltered by L-NAME. 3. L-NAME (20 and 60 mumol kg-1, i.v.) evoked a prompt and sustained rise of mean arterial blood pressure (MAP) and caused a slight decrease in the hypotensive activity of CGRP. In contrast, D-NAME induced a delayed and moderate increase in MAP and did not influence the hypotensive activity of CGRP. 4. Rat CGRP-alpha dilated the isolated perfused bed of the rat LGA precontracted with methoxamine and was 3 times more potent in this respect than rat CGRP-beta. The dilator action of rat CGRP-alpha in this preparation was not affected by L-NAME or D-NAME (40 microM). 5. L-NAME (60 micromol kg-1, i.v.) reduced gastric mucosal blood flow as assessed by laser Doppler flowmetry and diminished the hyperaemic activity of rat CGRP-alpha in the gastric mucosa by a factor of 4.5, whereas D-NAME was without effect.6. These data show that CGRP is a potent dilator of mucosal and extramural resistance vessels in the rat stomach. Its dilator action involves both NO-dependent and NO-independent mechanisms.

Withdrawal from controlled carbamazepine therapy followed by further carbamazepine treatment in patients with dementia.

BACKGROUND: The aim of this study was to assess the effects of withdrawal from placebo and carbamazepine administered for agitation associated with dementia and to assess safety, tolerability, and efficacy of subsequent ongoing carbamazepine therapy. METHOD: We previously reported the results of a 6-week, randomized, parallel-group study of placebo versus carbamazepine in 51 nursing home patients with dementia who were agitated; 47 subjects completed that study. This report first presents the results of withdrawal from that experimental treatment assessed by (blinded) observations 3 weeks later (N = 45 remaining). The primary outcome measure was the Brief Psychiatric Rating Scale. Secondary outcome measures addressed other aspects of behavior, cognition, function, safety, and tolerability. Patients were then treated with carbamazepine for an additional 6 weeks (N = 32 remaining) or 12 weeks (N = 25 remaining), with the same assessments performed. RESULTS: Patients who had previously shown behavioral improvement with carbamazepine therapy reverted to their baseline state after washout, whereas there was no change in the patients previously treated with placebo. There were no other significant effects of washout. During subsequent therapy with carbamazepine at a modal dose of 300 mg/day, there were 2 deaths and 4 other adverse events resulting in dropout. Neither of the deaths, and only 1 serious adverse experience, was judged to be related to carbamazepine. There were a variety of nonserious adverse experiences during the trial. Behavior ratings showed ongoing improvement in agitation and aggression, as well as in other aspects of psychopathology. CONCLUSION: The washout data provided independent confirmation of efficacy found in the prior placebo-controlled phase of this trial. Ongoing treatment was not associated with unexpected toxicity and was associated with improvement in measures of agitation and aggression that appeared to continue for up to 12 weeks. These findings confirm and extend results from earlier placebo-controlled studies.

Lack of carbamazepine toxicity in frail nursing home patients: a controlled study.

OBJECTIVE: To assess the effects of carbamazepine and placebo on measures of toxicity in frail nursing home patients. DESIGN: A nonrandomized, double-blind, placebo-controlled crossover study. SETTING: Two nursing homes in Rochester, New York. PARTICIPANTS: Twenty-five subjects (mean age 84.5 years) with dementia. INTERVENTION: Carbamazepine and placebo during two 5-week periods separated by a 2-week washout. The modal dose for each patient was 300 mg/day. MEASUREMENTS: Adverse experiences, comorbid events, and physical signs and symptoms were assessed continuously, and laboratory data were examined at the beginning and end of each treatment period. RESULTS: One subject died with a pneumonia (and elevated white blood cell count), and one subject developed probable carbamazepine-induced tics. There was a minimal drop in hematocrit and a slight elevation of liver enzymes, none of which was clinically significant. There was otherwise no excess of adversity or comorbid events on carbamazepine versus placebo, and there was no change in electrolytes or other laboratory measures. CONCLUSION: The findings indicate that carbamazepine in this dose range was well tolerated for the 5-week treatment period in this frail sample. These data supplement the literature regarding carbamazepine toxicity, which is devoid of controlled studies in older subjects.

Carbamazepine treatment of agitation in nursing home patients with dementia: a preliminary study.

OBJECTIVE: To determine the effects of carbamazepine versus placebo on ratings of behavior in agitated nursing home patients with dementia. DESIGN: Nonrandomized, placebo-controlled, crossover trial conducted in 25 patients in two nursing homes. INTERVENTION: Carbamazepine and placebo were administered during two 5-week periods separated by a 2-week washout. The carbamazepine dose was determined for each patient by a nonblinded physician who did not participate in ratings (modal dose 300 mg/day). MEASUREMENTS: The primary outcome measures were Brief Psychiatric Rating Scale scores and Clinical Global Impression of Change, rated by blind observers. Secondary measures of behavior, adversity, cognition, and functional status were also included. MAIN RESULTS: Median total Brief Psychiatric Rating Scale score decreased 7 points on carbamazepine versus 3 on placebo (P = 0.03). Sixteen subjects were rated as improved globally on carbamazepine versus four on placebo (P = 0.001). Secondary measures of behavior showed similar changes at significant or suggestive (P < 0.10) levels. One subject developed carbamazepine-induced tics, and one died with a pneumonia. There was minimal other adversity. CONCLUSION: This preliminary study suggests that carbamazepine in low doses can reduce agitated behaviors in some patients, with limited adversity resulting. Further research is required to confirm and extend this finding before it can be considered routine clinical practice.

Hysteroscopic tubal occlusion with silicone rubber.

A new method of sterilization for women is being investigated in several outpatient centers. The technique involves flowing liquid silicone rubber into the fallopian tubes. The silicone cures in place and forms a rubbery solid plug. The catalyzed liquid silicone is pumped through a special polysulfone guide assembly that fits through the operating channel of a standard hysteroscope. On the end of the guide is a hollow premolded silicone rubber tip that fits into the cornual ostium of the tube. When the silicone cures it also cross-links to this tip so that the tip becomes part of the plug. Results as of April 1, 1982, from 350 women in the Philadelphia center suggest that proper plug formation prevents pregnancy and that the method can be applied to about 85% of properly selected women.

Phylogeny and Abundance of Novel Denitrifying Bacteria Isolated from the Water Column of the Central Baltic Sea.

The Baltic Sea is an estuarine ecosystem where denitrification in the low oxic and anoxic parts of the deep water contributes significantly to the nitrogen budget. Seventy-six heterotrophic, denitrifying, strains have been isolated by four cultivation procedures from the water column of the Gotland Deep, the main anoxic basin of the Central Baltic. Phylogenetic positions of representative strains of 10 different genotypes, grouped beforehand by low molecular weight (LMW) RNA profiling, were estimated by 16S rRNA sequence analysis. The 10 genotypes consisted of two members of the alpha subclass of the Proteobacteria and eight members of the gamma subclass. The major fraction of the genotypes was considered to be novel species or even genera. The gamma-Proteobacteria were the most abundant of the denitrifying isolates (96% of the total isolates) with a predominance of Shewanella baltica (77%), whereas the alpha-Proteobacteria were represented by single isolates. The diversity spectrum of Baltic sea denitrifying isolates was rather distinct from that previously described for marine and freshwater environments. Denitrifying bacteria could be isolated from all depths of the water column with the highest diversity and abundance of genotypes detected in samples of the oxic-anoxic interface, the layer of high in situ denitrification. For success of isolation of phylogenetically divers denitrifiers, both sample origin and cultivation procedure were observed to have an impact.

Role of Microcolony Formation in the Protistan Grazing Defense of the Aquatic Bacterium Pseudomonas sp. MWH1.

A BSTRACTThe defense strategy of the aquatic bacterium Pseudomonas sp. MWH1 against flagellate grazing was investigated in chemostat and batch experiments. The influence of predation on the Pseudomonas population was studied in the absence and presence of a potential competitor ( Vibrio sp. CB5), as well as under starvation conditions and in a situation of unlimited growth. In the competition experiment the two bacterial strains were distinguished by immunofluorescence microscopy. When the Pseudomonas strain was cultured in the absence of the predator Ochromonas sp. DS, only mobile single cells were detectable. Grazing by this bacterivorous flagellate resulted in all experiments in the occurrence of a Pseudomonas subpopulation, which grew as floclike, suspended microcolonies. These microcolonies consisted of up to approximately 1,000 cells and were, because of their large size, protected against flagellate grazing. The microcolony subpopulation dominated the total Pseudomonas population in situations of high grazing pressure at a wide range of bacterial growth conditions. Thus, the formation of the microcolonies is interpreted as a successful grazing-defense strategy, which is effective under several growth conditions, allowing for the survival of the strain even when substrate depletion is combined with strong grazing pressure. Batch culture experiments demonstrated that the change in morphology of Pseudomonas sp. MWH1 is not controlled by growth rate, although no formation of microcolonies was observed after the addition of 0.2-&mgr;m-filtered flagellate cultures to Pseudomonas cultures, indicating that a chemical trigger released by the flagellate is not involved in the control of this defense mechanism.

Influence of isolation stress and inhibited protein biosynthesis on learning and memory in goldfish.

Goldfish, Carassius auratus auratus L. (Pisces, Cyprinidae), were trained by different kinds of training procedures under the influence of cycloheximide or puromycin, two inhibitors of the protein biosynthesis. After active avoidance training in a shuttle box an apparent amnesia was found only when the fish were exposed to a one day lasting isolation stress prior to training. If the animals were accustomed to isolation over a period of 20 days the inhibitors did not affect memory formation. After learning by positive reinforcement (food rewarded color discrimination) in groups under stress-free conditions, neither learning nor memory formation were impaired in spite of the presence of cycloheximide. It is suggested that the amnestic effect of the inhibitors is caused by isolation treatment. Lack of the additional stress, however, leads to memory formation.

Serum prolactin level increase in normal subjects following administration of perphenazine oral dosage forms: possible application to bioavailability testing.

Two pilot studies were performed to determine if oral phenothiazine products could generate a significant increase in serum levels of the hormone prolactin. The two studies employed three and four healthy normal male subjects, respectively. In the first study the subjects received a screening dose, a placebo, one 8-mg perphenazine tablet, and two 8-mg perphenazine tablets. In the second study, the subjects were dosed with two 10-mg amitriptyline tablets, one 10-mg amitriptyline tablet with one combination tablet containing 10 mg of amitriptyline and 4 mg of perphenazine, and two combination tablets, each containing 10 mg of amitriptyline and 4 mg of perphenazine. In both cases the drug treatments produced a significant rise in the serum prolactin levels versus a placebo or control. This increase was defined as a prolactin response. The possible utility of this response in bioavailability testing is discussed.

Predictive conversion of in vitro drug dissolution data into in vivo drug response versus time profiles exemplified for plasma levels of warfarin.

A mathematical approach to computing the in vivo drug response profiles (such as plasma level, pharmacological response, and urinary recovery versus time curves) corresponding to observed in vitro dissolution of drug dosage form versus time profiles is described. The method is exemplified for warfarin tablets,for which observed in vivo plasma level profiles are compared to corresponding predicted profiles computed from in vitro dissolution data. The potential of the method is demonstrated; approaches to its improvement, as well as its limitations, are discussed.

Artificial induction of lactation in cattle: Effect of modified treatments on milk yield, fertility, and hormones in blood plasma and milk.

Holsteins were divided into groups CON and IL, each with six dry cows and six heifers. Group CON calved in mid-summer when group IL was treated (kg body weight per day) with (a) progesterone (P; .25 mg) and estradiol-17beta (Ebeta), either .05 mg or .10 mg, for 7 days; (b) continued Ebeta at one-third the initial rate until udders were engorged; (c) then 12 injections (8-hr intervals) of TRH (each 200 mug) or saline; and (d) GnRH during lactation. Milk yield was not affected by Ebeta dose rate, TRH or GnRH. GnRH luteinized the persistent ovarian follicles in group IL, and pregnancy rates were 80% and 83% in groups IL and CON, respectively. Large differences (P < .01) between groups IL and CON were observed in plasma prolactin (IL-low), insulin (IL-high) and growth hormone (IL-low) wherein insulin was correlated (P < .01) negatively with milk yield between days 7 to 49 of lactation. Milk concentrations of P, Ebeta, estrone and estradiol-17alpha in group IL were no higher (P > .10) 14 days after the last injection of P or Ebeta than in group CON or in milk from the herd's bulk tank. The steroids were lowest in milk and plasma from ovariectomized cows. It was hypothesized that high insulin, as well as low prolactin and growth hormone, may contribute to inferior induced lactations.

Variables associated with peripartum traits in dairy cows. III. Effect of diets and disorders on certain blood traits.

BLood samples were collected from 89 Holstein cows on days 220 and 250 of gestation, within 24 hr prepartum and postpartum and on day 30 postpartum. Balanced diets which contained either chopped hay (29 cows), hay crop silage (HCS; 30 cows) or corn silage (CS; 30 cows) were fed from day 220 of gestation to day 30 postpartum. The purpose was to determine if variations in certain blood traits were indicative of peripartum and postpartum disorders. The blood traits evaluated were concentrations of plasma total protein, whole blood hemoglobin, packed cell volume, and white blood cells, and serum glutamic oxalacetic transaminase (SGOT), glucose, urea nitrogen, calcium, inorganic phosphorus, magnesium, potassium and sodium. No blood trait was useful to predict a disorder prior to its visual signs with one possible exception. Serum glucose and calcium were lower and SGOT and magnesium were higher peripartum which was prior to death of three cows from fat cow syndrome.