A specific practical radioimmunoassay suited to determinations of melatonin in both tissues and body fluids is described. The rabbit antibody employed was raised to an antigen formed by condensation between N-acetylserotonin and the Mannich adduct of bovine serum albumin and formaldehyde. Substitution was shown by protonmagnetic resonance spectroscopy to occur exclusively at die 4 position of the indole nucleus. The antibody reacted with a variety of N-acetylated indoles, and absolute specificity was dependent upon the extraction procedure and column (Lipidex 5000) chromatography.In addition to the usual reliability criteria, the validity of the assay was checked by gas chromatography-mass spectrometry using [ 2 H 3 ]melatonin as an internal standard, the preparation of which is described. The occurrence of melatonin in the plasma of man, sheep, rat and chicken was confirmed, and its presence in the plasma of the pig (22-76 pg/ml), donkey (24-128 pg/ml), cow (20-320 pg/ml), camel (29-221 pg/ml) and a scincid lizard (20-500 pg/ml) established. A nocturnal rise in plasma melatonin content occurred in all species.Melatonin was found in the plasma of ewes 2-12 weeks after pinealectomy, but the nocturnal rise was abolished. The results establish a nyctohemeral variation in plasma melatonin in a wide variety of species, and indicate that sources of melatonin other than the pineal may assume precedence following pinalectomy. (Endocrinology 101: 119, 1977)

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We investigated an enzymatic colorimetric procedure for quantification of plasma 1,5-anhydro-D-glucitol (AG) with respect to its reproducibility and application for evaluation of diabetes mellitus. Assay specificity is critically dependent on the two-layer ion-exchange column chromatography procedure to remove glucose from the sample. For female nondiabetic subjects (n = 110, ages 22-36 years) the median plasma AG concentration was 143 mumol/L (range 70-209 mumol/L); there was no relation to body mass index (BMI), fasting plasma glucose, or insulin, but an inverse association was noted for age. For a group of older subjects (n = 69, 70-85 years), no association between AG concentrations and sex, age, BMI, or various medical conditions was found. In diabetic subjects (n = 170) a significant inverse nonlinear relation existed between plasma AG and glycohemoglobin (GHb) such that at GHb > 8.5%, AG concentrations were typically < 50 mumol/L. The findings confirm that plasma AG, in the presence of normal renal function, is a reliable marker for hyperglycemia.