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Title: Radioreceptor assay and radioimmunoassay of selected benzodiazepines in urine samples from racing greyhounds
Author: Burnett, Josephine Elizabeth Caroline
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1989
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Radioreceptor assay and radioimmunoassay of selected benzodiazepines in urine samples from racing greyhounds. Radioreceptor assay is a quick and relatively simple analytical method which can discriminate between classes of drugs with a high degree of specificity. As it is receptors which interact and "detect" the drugs, the particular class of drugs detected is dependant on the radiolabelled ligand which is displaced from the receptor. This eliminates the need to obtain a pure preparation of one particular type of receptor which contrasts with radioimmunoassay. With this method the antibodies have to be raised against a specific drug and then purified in order that crossreactivity with drugs from other pharmacological classes does not occur. Also radioreceptor assay will only detect the pharmacologically active compounds in the biological sample. This is advantageous as only they will be acting in the body to cause an effect, so only they need to be measured. Prior to the analysis of greyhound urine samples, various aspects of the receptor assay were determined to ensure optimum results were obtained under an established set of conditions. Rat brain tissue was centrifuged in order to isolate the synaptosomal fraction containing the benzodiazepine receptors. The protein content of this fraction was estimated together with determination of the actual number of receptors and their affinity for benzodiazepines. As the brain tissue was collected in batches and stored until required, the length of time it could be kept at -20°C was determined together with the length of time prepared synaptosomal fractions could be stored under similar conditions. The limits for the incubation period which enabled enough time for an equilibrium to be established between the benzodiazepine/radiolabelled benzodiazepine and the receptor were determined. These also allowed for the separation of large numbers of samples before disruption occurs naturally. Separation of bound and free radioligand was achieved by filtration. The filters were washed with sufficient assay buffer to minimise the non-specific binding whilst maintaining an almost instantaneous separation time. Radioreceptor assay of a variety of benzodiazepines, and their pharmacologically active metabolites, was carried out to assess the affinity of each one for the benzodiazepine receptor and therefore enabling their potency to be calculated. The very specific nature of the benzodiazepine-radioreceptor assay for benzodiazepines was tested by assaying a wide variety of non-benzodiazepines. Relatively high concentrations of these other drugs were used, compared to the diazepam standards, to increase the possibility of cross reaction with the benzodiazepine receptor. Once the experimental laboratory conditions had b ee n established, a pilot study was carried out with the greyhounds to uncover any practic al problems relating to oral dosing and sample collection. This study also allowed the laboratory procedures to be tested with actual greyhound urine samples containing diazepam. The major studies were carried out with triazolam and flunitrazepam, both short acting benzodiazepines. They both have half-lives of only 2 to 3 hours in dogs and consequently can only be det ec te d in samples taken within a few hours of dosing. Also the concentration of drug present in each sample is less as triazolam and flunitrazepam are more potent than diazepam so lower doses are required to obtain a similar effect. To increase the sample content of the amount of benzodiazepines and their metabolites whi c h can be detected by radioreceptor assay, the urine underwent mild hydrolysis with J3-glucuronidase. This rele as es the metabolites from the glucuronide conjugate. By increasing the amount of pharmacologically active metabolites in the sample this serves to increase the sensitivity of the assay. In order to increase the sensitivity of the receptor assay even more, the samples were extracted with a variety of organic solvents. Howe ve r the in cr ease in assay time due to the inclusion of this extra step was not balanced by the increase in detection efficiency. Therefore the laboratory protocol to assay the samples included centrifugation of samples, to deposit any particulate matter, rather than a full extraction procedure. In order to confirm the presence of benzodiazepines in the urine samples, they were also analysed by radioimmunoassay, both before and after hydrolysis. The statistical comparison of the receptor a ssay and the radioimmunoassay results demonstrated the relative non-specificity of the benzodiazepine antibody used in RIA compared to the relative high specificity of the benzodiazepine receptor. The differing affinities of triazolam and flunitrazepam metabolites for the receptor had been previously determined and as the proportion in which they are excreted is known from the literature, it was possible to re-calculate the receptor assay results. Using only the increase in benzodiazepines detected after hydrolysis, accurate pharmacokinetic data could be obtained. As only a single oral dose was given with only serial urine samples collected, such data is limited to the calculation of the elimination rate constants and the half-lives of triazolam and flunitrazepam in greyhounds. Re-assay of triazolam urine samples after they had been stored at -20°C for 12 months gave results which were statistically compared to previous results from the assay of samples within two days of collection. This was to determine the stability of the samples under such conditions and has implications in cases where samples cannot be analysed on receipt and have to be stored for some time.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available