Human plasma contains factors beside MEL, which interfere in the direct RIA and which show a high individual variation. As illustrated in Figure 1, the assay of 12 individual plasma samples (freed from MEL by C18-extraction) showed a variation in tracer binding of 37.3% associated with a general low binding. The presence of 3% PEG in the incubation mixture decreased the individual variation to only 6.9% and increased the tracer binding about two-fold. The smallest detectable MEL concentration in the RIA (difference from zero-standard by two standard deviations) was 0.3pg/tube corresponding to 1.5pg/ml plasma. A typical standard curve is shown in Figure 2. Intra-assay variations for 15, 55 and 150 pg/ml were 10.2,7.1 and 9.4%, respectively. Inter-assay variations for these concentrations were 15.2, 8.8 and 11.8%, respectively. The antiserum shows a very high specificity for MEL as indicated in Table 1. In the RIA, MEL added to individual plasma samples in different concentrations could be quantitatively recovered. Day time levels (late afternoon) were in general below 5 pg/ml. The measurement of the circadian patterns of plasma MEL concentrations by the direct RIA and the RIA with prior C18-extraction of plasma samples showed very similar results for both assay variants (correlation: r = 1.0). The validation of the direct RIA by gas chromatographic-mass spectrometry (GCMS) showed a nice correlation of r = 0.95 (Figure 3). The use of the less polar 125I-2-iodo-MEL (2) as tracer in the direct RIA often resulted in an overestimation of individual plasma MEL concentrations particularly of the low day time levels (data not shown).

Individual Plasma Samples

Figure 1. Effect of polyethylene glycol (PEG) on the measurement of melatonin-free individual plasma samples. Individual samples were freed of melatonin by stripping with octadecylsilica (C18) and assayed in the direct RIA with and without 3% PEG 6000. Mean of all samples (each in duplicate): 4350 ± 1620 (SD) for control; 8932 ± 618 (SD) for PEG.

Sleeping Sanctuary

Sleeping Sanctuary

Salvation For The Sleep Deprived The Ultimate Guide To Sleeping, Napping, Resting And  Restoring Your Energy. Of the many things that we do just instinctively and do not give much  of a thought to, sleep is probably the most prominent one. Most of us sleep only because we have to. We sleep because we cannot stay awake all 24 hours in the day.

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