Development and validation of a high throughput direct radioimmunoassay for the quantitative determination of serum and plasma melatonin (N-acetyl-5-methoxytryptamine) in mice

Abstract

A simple, sensitive and specific high throughput radioimmunoassay (RIA) for the quantitative determination of total melatonin (N-acetyl-5-methoxytryptamine) was developed. This method allows the analysis of melatonin in different biological fluids with small sample volumes (e.g. mice and rats), and wide working range. With the preparation of matrix-specific calibrators called "Equalizing Reagent" the influence of results due to different composition between standards and sample matrix was reduced. This reagent is produced by use of the respective biological liquid. Endogenous melatonin is removed by adsorption to activated charcoal. The melatonin-free biological liquid is then used to equalize the assay matrix of standards and untreated samples. Finally, all samples including the standards are digested by use of a protease to reduce non-specific binding, for example to albumin or albumin-like molecules. High-affinity specific antibodies were produced by immunization of rabbits with 5-methoxytryptamine-bovine serum albumin. The review of cross reactions to ten structurally similar compounds showed that the antibody has a high specificity for melatonin. This direct RIA uses a [(125)I]-melatonin tracer for the determination of melatonin. 5-methoxytryptamine was synthesized by direct iodination with [(125)I]-Bolton-Hunter-Reagent. The required acceptance criteria for validation parameters were fulfilled. The flexible standards cover a working range from 12 to 4000 pg/mL with a sample volume of 50 microL (e.g. working range from 3 to 1000 pg/mL with a sample volume of 200 microL). The limit of detection in mouse serum and mouse plasma was 9 pg/mL and 7 pg/mL, respectively. The recovery of melatonin in mouse serum was 108% and in mouse plasma 99%. The variation coefficients of the assay, within and between runs, ranged between 7 and 13% in mouse serum and between 5 and 8% in mouse plasma. Based on the determination of a 24-h profile of melatonin in mouse samples a characteristic diurnal rhythm of melatonin was observed. The wide working range makes it possible to analyse low and high melatonin concentrations. The validated direct RIA was compared with established sample preparation methods such as liquid-liquid- and solid-phase-extraction followed by RIA. The correlation for methanol extraction is y=1.1x-0.9, R(2)=0.98, P<0.001 and C(18)-extraction y=0.8x-0.03, R(2)=0.99, P<0.001. The distinct advantage of the direct assay of melatonin is that complicated extraction steps can be avoided. The realized advantages of the direct RIA when compared to a commercially available melatonin RIA are its low sample volume and ease of implementation. Exemplary, plasma melatonin was determined at C3H, C57BL, wild-type and melatonin receptor (MT1-/- and MT2-/-) knockout mice kept under L:D=12:12 cycles. The results have indicated that at all mouse strains have plasma melatonin content, with higher levels during the night and lower levels during the day. Because of different melatonin concentrations the direct RIA convince by its low detection limit and wide working range. The determination of serum and plasma melatonin in mice by ELISA or HPLC-technique previously failed due to the required use of a high sample volume and the low sensitivity. In summary, it can be concluded that the developed and validated direct RIA meets the requirements for the determination of melatonin and is especially suitable for the analysis of melatonin in different mouse strains.