Methodological considerations for the human platelet 5-HT2A receptor binding kinetic assay.

Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity K(D) during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein/ml (at 0.2 nM of ligand). Above 0.1 mg protein/ml, percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg/ml, well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.

Effects of diagnosis, race, and puberty on platelet serotonin levels in autism and mental retardation.

OBJECTIVE: To reevaluate platelet serotonin (5-HT) levels in autism, measuring and controlling for effects of race and puberty. The specificity of hyperserotonemia for autism versus cognitive impairment is also assessed. METHOD: Platelet 5-HT levels were measured in 77 individuals, aged 2 through 37 years, with autistic disorder; 65 normal controls; and 22 mentally retarded or otherwise cognitively impaired (MR/CI) prepubertal children. Effects of diagnosis, race, and pubertal status were evaluated by analysis of variance in separate pre- and postpubertal groups. 5-HT levels were expressed as ng/mL blood and ng/microL platelet volume. RESULTS: Among prepubertal children, significant effects of diagnosis (ng/mL; F2,109 = 5.9, p = .004) and race (F2,109 = 14.7, p < .0005) were found. Autistic youngsters had significantly higher 5-HT concentrations than controls, although the elevation (25%) was less than typically reported; MR/CI children had levels very similar to those of controls. White children had significantly lower 5-HT levels than black or Latino youngsters, regardless of diagnosis. Diagnosis and race effects were nonsignificant in the postpubertal group. Postpubertal subjects had lower 5-HT concentrations than prepubertal subjects (ng/mL; F1,114 = 28.5, p < .0005). CONCLUSIONS: The data underscore the importance of matching for race and pubertal status in neuropsychiatric research and suggest that the prevalence of hyperserotonemia in autistic individuals may have been overestimated because of a failure to control for both variables. Hyperserotonemia was not found in MR/CI youngsters without autistic features.

Serotonergic responsivity in eating disorders.

Evidence suggests that serotonin may play a role in the pathogenesis of eating disorders. In this ongoing study, serotonin-mediated physiological responses and whole-blood serotonin content are measured in young women with an eating disorder during the active phase of the illness and at the conclusion of inpatient treatment. The responsivity of central nervous system (CNS) serotonergic pathways is assessed by neuroendocrine challenge with a 60-mg oral dose of dl-fenfluramine, an indirect serotonin agonist, whereas the responsivity of the platelet serotonin2 (5-HT2) receptor complex is evaluated by measurement of the magnitude of serotonin-amplified platelet aggregation. Compared with normal controls, eating-disorder patients have exhibited a trend toward reduced prolactin responses to fenfluramine challenge at both the initial and followup assessments. Patients also have exhibited a substantially wider range of serotonin-amplified platelet aggregation responses than have controls; normal-weight bulimic patients have had significantly greater responses than both anorexic restrictors and normal subjects. These preliminary results suggest potential alterations in serotonin-mediated responses in eating-disorder patients that may vary with the diagnostic subgroup.