Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland

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Abstract
This study details the procedures involved in measuring a number of the components of indole metabolism within the pineal gland. Tryptophan, 5‐hydroxytryptophan, serotonin, N‐acetylserotonin, melatonin, 5‐hydroxyindoleacetic acid, N‐acetyltransferase (NAT) activity, and hydroxyindole‐O‐methyltransferase activity were measured by a combination of three techniques, which included microassays, radioimmunoassay, and high‐performance liquid chromatography. Determination of the melatonin synthetic pathway components within the same gland reduces the number of animals needed for studies and allows correlations between these constituents to be calculated. Very high degrees of correlation (r=0.91−0.99) are seen between those compounds which exhibit significant rhythms (serotonin, NAT activity, N‐acetylserotonin, and melatonin) when group means are compared. When correlations are calculated on a per‐animal basis throughout the experiment, moderate to high degrees of correlation (r=0.57−0.79) are found among those components that exhibit rhythms. However, when correlations are determined on a per‐animal basis at each time point, no significant correlations are found. One hypothesis accounting for these differences may be that changes occur in indole metabolism within the same pineal gland over a period of a few minutes.