Determination of the Benzodiazepine Plasma Concentrations in Suicidal Patients Using a Radioreceptor Assay

Abstract

Impairments in memory and psychomotor function appear to be induced by benzodiazepines not only after long-term use, but also after administration of a single dose. Because it is known on which neurotransmitter system the benzodiazepines exert their action, the use of a quantitative radioreceptor assay (RRA) can be a useful tool in studying the interrelationship between the neurochemical and memory processes. The RRA measures the sum of the main compound(s) and all active metabolites present, where it relates the biological activity to the pharmacodynamic effect instead of relating it to the plasma levels of the individual compounds. To correlate the loss of memory with the benzodiazepine concentration, plasma concentrations were determined in suicidal patients. From suicidal patients (n = 84), the benzodiazepines in plasma were measured with a direct radioreceptor assay using tritiated flunitrazepam as the labelled ligand. The receptor material was a lyophilized preparation from calf cortex. Furthermore, the samples were subjected to high-performance liquid chromatographic (HPLC) analysis, and the HPLC data were converted to diazepam equivalents using cross-reactivities of the individual compounds. Patients who had ethanol residues in their plasma were excluded from this correlation experiment. The data (n = 40) obtained with the two analytical techniques were compared and correlated to assess the validity of the radioreceptor assay in establishing the relationship between the loss of memory and the total amount of benzodiazepines present. The cumulative amount of diazepam determined with the RRA and the sum of compounds determined with the HPLC method, after correction using the cross-reactivities, were plotted and correlated using regression analysis. Regression analysis showed an x variable of 0.75 and a correlation coefficient of 0.67. The intercept was not significantly different from zero (P = 0.49, t-test), whereas the slope was significantly different from zero (P < 0.01). Benzodiazepines can be directly determined in plasma using this radioreceptor assay. The data obtained from HPLC analysis were easily converted to diazepam equivalents using the cross-reactivities. A discrepancy between the data obtained from the two analytical techniques, however, indicates that certain metabolites are present, which were not quantitated in the HPLC analysis, but were measured in the radioreceptor assay. Therefore, the radioreceptor assay proved to be a valuable tool for the assessment of clinical effects, such as the demonstration of the loss of memory in suicidal patients after a benzodiazepine overdose.