Vina R. Spiehler

Naloxone enhancement of memory.

Naloxone enhanced retention when systemically administered to male F344 rats after training in a one-trial inhibitory avoidance task. Further, the memory-enhancing ability of naloxone appears to be opiate receptor dependent, because it was antagonized by morphine. Naloxone also improved retention of rats in an active avoidance task, indicating that the effect of naloxone is not task specific. The influence of naloxone on retention was time dependent in both tasks. The results showed that the drug must be present for a considerable period beginning soon after the onset of memory consolidation in order to be effective. For inhibitory avoidance, it was necessary to administer naloxone immediately after training, and because of its short duration of action, again 30 min later. In the active avoidance task, naloxone was effective only when given both immediately before and, as in the inhibitory avoidance task, within 30 min after the eight acquisition trails. These results provide strong evidence that naloxone influences memory and suggest that endogenous opioid systems are involved in memory storage processes.

Brain Concentrations of Cocaine and Benzoylecgonine in Fatal Cases

Since cocaine in blood rapidly hydrolyzes to benzoylecgonine, cocaine concentrations determined in postmortem blood may not reflect the presence or concentration of cocaine in the body at the time of death. The interpretative value of the determination of cocaine and benzoylecgonine in brain tissue was investigated. Cocaine and benzoylecgonine were quantitated by coextraction and formation of the propyl derivative of benzoylecgonine followed by selected ion monitoring gas chromatography/mass spectrometry (GC/MS) using electron ion impact ionization. Cocaine and benzoylecgonine were found to be evenly distributed throughout the brain. Cocaine and benzoylecgonine concentrations were stable in frozen brain tissue (-4 degrees C) on reanalysis after 1 to 3 months of storage, and in refrigerated tissue (10 degrees C) after 30 days of storage. Blood, brain, and liver concentrations of cocaine and benzoylecgonine in 37 cocaine overdose cases and 46 cases in which cocaine was incidental to the cause of death were reviewed. The ratios of cocaine/benzoylecgonine in the toxic cases (brain mean 14.7 and blood mean 0.64) were clearly different from those found in the incidental cases (brain mean 0.87 and blood mean 0.27). The brain/blood ratios of cocaine and benzoylecgonine concentrations generally were characteristic of the time elapsed since cocaine dosing. In cocaine overdose cases, the mean ratio was 9.6 for cocaine and 0.36 for benzoylecgonine. These are within the range found in animal studies for brain/blood ratios of cocaine and benzoylecgonine 0.5 to 2 h after cocaine administration. In incidental cases, the brain/blood ratios were mean 2.5 for cocaine and 1.4 for benzoylecgonine.(ABSTRACT TRUNCATED AT 250 WORDS)

3H-dihydromorphine binding in brain regions of young and aged rats

Abstract Opiate receptor-ligand binding was investigated in brains of young and aged female F344 rats. A significant reduction in the density of binding sites for 3H-dihydromorphine was observed in the thalamus and midbrain of aged rats. Evidence of two binding sites was observed in the anterior cortex of young rats, whereas aged rats exhibited only a single site. The occurrence of pituitary tumors in the old female rats did not affect 3H-dihydromorphine binding. The highest density of dihydromorphine binding sites was found in the diencephalon, and the lowest density in the hippocampus. Receptor densities in the anterior cortex, striatum, amygdala, frontal poles and midbrain were intermediate, and few, if any, high affinity binding sites for dihydromorphine were found in the pons-medulla or posterior cortex.

Unconjugated Morphine in Blood by Radioimmunoassay and Gas Chromatography/Mass Spectrometry

Morphine, the active metabolite of heroin, is rapidly inactivated by glucuronidation at the 3 carbon. Unconjugated (pharmacologically active) morphine was measured in postmortem blood by radioimmunoassay using an antibody-coated tube kit. The kit shows less than 0.2% cross-reactivity with codeine and morphine-glucuronide. Unconjugated morphine concentrations were confirmed by gas chromatography/mass spectrometry (GC/MS) using deuterated morphine as the internal standard. The blood was precipitated with 10% trichloroacetic acid (TCA) and concentrated hydrochloric acid (HCl), centrifuged, and decanted. The supernatant was then either diluted (unhydrolyzed) or heated to 100 degrees C, 30 min (hydrolyzed), followed by a wash with 4:1 chloroform:isopropranol. The upper aqueous layer was then saturated with sodium bicarbonate (NaHCO3) and extracted with 4:1 chloroform:isopropranol. The organic layer was evaporated, derivatized with trifluoroacetic anhydride (TFA), and analyzed by selected ion monitoring (SIM) GC/MS. Comparison of the results for unconjugated morphine by radioimmunoassay and unhydrolyzed morphine by GC/MS gave a correlation coefficient of r = 0.98, n = 100. Unconjugated morphine ranged from 0 to 100% of total morphine with a mean of 42%, n = 200, for heroin or morphine involved deaths. Review of 56 putative rapid deaths gave a mean of 68% unconjugated morphine with a range of 26 to 100%. The ratio of unconjugated to total morphine was found to be stable in postmortem blood after more than a year of storage at room temperature, within the precision of the method.

Memory, opiate receptors, and aging ☆

Abstract In a series of studies, we investigated differences in retention of avoidance responses in young and aged rats. The aged rats showed poorer retention performance of an inhibitory avoidance response, but better performance of an active avoidance response. In a swim escape task, when training trials were given close together in time, the aged rats showed impaired acquisition as compared to younger animals. We then investigated the effects of naloxone on learning and retention performance in young and old rats. Our findings indicated that in some cases, the direction of the opiate antagonist effect depends upon the age of the animal. With low footshock, naloxone either impairs or has no effect on the retention performance of old rats, but with high footshock, naloxone enhances their retention performance indicating that age-related changes in opioid systems may underly some behavioral differences between young and aged animals Therefore, we examined, in vitro , opiate receptor binding in various brain regions of young and old rats. Analysis of dihydromorphine binding indicated that opiate receptor concentrations in aged rats are reduced in some brain regions and that these changes are different in aged male and female rats. Studies of in vivo bindings of opiate receptors have yielded relationships between receptor changes and differences in response to footshock in the flinch-jump test. These findings suggest that age-related alterations in brain opioid systems may underlie some of the changes in learning and memory processes that occur with age.

Effect of naloxone on antinociceptive activity of phenoxybenzamine

Abstract Phenoxybenzamine was found to have antinociceptive activity in the mouse writhing syndrome test. The percent protection against writhing was dose related from 2.0 to 164.6 μM/kg phenoxybenzamine (ED-50, 37.1 ± 2.0 μM/kg). The dose-response curve for phenoxybenzamine was shifted to the left when morphine was administered. Administration of morphine, 1.12 μM/kg, reduced the ED-50 for phenoxybenzamine to 4.5 ± 1.3 μM/kg. Administration of naloxone shifted the dose response curve for phenoxybenzamine to the right. Naloxone 0.03 μM/kg increased the ED-50 for phenoxybenzamine to 102.5 ± 22.7 μM/kg. Chronic treatment with phenoxybenzamine did not change the antinociceptive response to phenoxybenzamine. The α-adrenergic blockers, phenotolamine and tolazoline showed weak antinociceptive activity which was not blocked by naloxone.

Comparison of GC-MS and EIA Results for the Analysis of Methadone in Oral Fluid

The purpose of these studies was to evaluate the performance characteristics of the Cozart Microplate Enzyme Immunoassay (EIA) for the determination of methadone in oral fluid from patients in a drug misuse treatment program. Oral fluid specimens were collected using the Cozart RapiScan Collection system from 198 donors who were receiving treatment for their addiction and were monitored for drug misuse. Oral fluid specimens were also collected from forty volunteer donors who were not drug users. The specimens were analyzed in the laboratory by EIA and then analysed for methadone and its main metabolite EDDP by gas chromatography-mass spectrometry (GC-MS). A total of 103 samples were confirmed positive for methadone. The Cozart Microplate EIA for d-Methadone in oral fluid using a cutoff of 30 ng/mL in diluted oral fluid had a sensitivity of 91.3% +/- 2.8% and a specificity of 100% +/- 1.0% vs. GC-MS.

Digoxin concentrations in fatal cases.

Analytical methods for the determination of digoxin in pharmaceutical and autopsy specimens using colorimetry [1], gas liquid chromatography [2ߝ4], fluorometry [1,5], and polarography [6] have been reported. These classic methods, however, suffered from lack of sensitivity and specificity in the therapeutic (1.0 to 1.4 μg/litre) [7] and borderline toxic (2.0 μg/litre or greater) ranges, making detection of this drug possible only when large amounts remained unadsorbed in the stomach or excreted via the kidneys. The introduction of radioimmunoassay in 1969 by Smith et al [7] and Smith and Haber [8] made possible the detection of digoxin in postmortem biological fluids and tissue samples. Radioimmunoassay for the determination of serum digoxin is now the most extensively used radioisotope test in many nuclear laboratories and may be the most commonly requested drug assay by hospital physicians.

Digoxin-Like Immunoreactive Substance in Postmortem Blood of Infants and Children

A digoxin-like immunoreactive substance (DLIS) has been reported in the serum of infants not receiving digoxin. This study was undertaken to determine if DLIS is present in the postmortem blood and tissues of infants or children and whether the endogenous substance could interfere with forensic toxicological analysis in suspected overdose. Ninety blood specimens taken from the heart at autopsy of children or infants were screened for DLIS using commercial radioimmunoassay kits. The average age at death in these cases was 8.6 months, the median age was 2 months. DLIS equivalent to 0.25 to 2.0 ng/mL digoxin was found in one third of the cases. The incidence of positive findings was 5/6 stillborns, 10/45 Sudden Infant Death Syndrome (SIDS), 10/15 deaths as a result of infection, 4/7 homicides, 1/8 deaths caused by congenital defects, and 0/9 accidental deaths. The body distribution of DLIS was investigated and highest levels were found in the liver. Findings of DLIS in blood were correlated with renal failure, (elevated vitreous urea nitrogen), electrolyte imbalance, and liver trauma. Apparent concentrations were in the equivalent therapeutic range of digoxin and would not be confused with accidental or intentional overdose with digoxin.

Setting Cutoff Concentrations for Immunoassay Screening of Postmortem Blood

The objective of this study was to establish the optimum immunoassay cutoff concentrations for screening postmortem blood from coroners cases for drugs of abuse with a coated tube radioimmunoassay (RIA) to ensure that the results with the coated tube RIA would be equal to or better than those with the previously used double antibody RIA. Immunoassay results (positive or negative) blood were compared to confirmed results on those cases by GC/MS alone or in combination with GLC using either a NPD or FID detector. Four to seven potential cutoff concentrations were evaluated for the drug classes opiates, amphetamines, cocaine and metabolites, and barbiturates. Specimens were 350 postmortem blood specimens and liver homogenates. The cutoffs chosen for the coated tube RIA using this approach were 5 ng/mL morphine, 25 ng/mL methamphetamine, 500 ng/mL benzoylecgonine, and 500 ng/mL secobarbital. These cutoffs corresponded to a sensitivity and specificity of 94% and 96% for opiates, 93% and 86% for amphetamines, 91% and 96% for cocaine and metabolites and 91% and 87% for barbiturates. The double antibody RIAs were run on the same specimens with cutoffs of 20 ng/mL morphine, 50 ng/mL methamphetamine, 50 ng/mL benzoylecgonine and 1000 ng/mL phenobarbital. The sensitivity and specificitys for the double antibody immunoassay were: > 99% and 96% for opiates, 83% and 89% for amphetamines, 98% and 97% for cocaine, 79% and 95% for barbiturates.