Circadian and other variation in epinephrine and norepinephrine among several human populations, including healthy blinded and sighted subjects and patients with leprosy.

Urinary levels of epinephrine and norepinephrine were studied in three populations: presumably healthy young men who were studied twice, 8 years apart, and in the same month to avoid seasonal influences; blinded but otherwise healthy males and females; and patients with leprosy. Determinations of epinephrine and norepinephrine were done on urine samples collected at 3-hr intervals for 72 hr from the first two groups and for 10 days from patients with leprosy. The same chemist, method, and protocol were used in all studies. In one study of the group of healthy young men, the effect of meal timing and fasting on the rhythms was investigated. From these studies several generalizations can be made. Highly statistically significant population circadian rhythms characterize both epinephrine and norepinephrine in all three groups. In general, the rhythm in norepinephrine was "noisier" than that in epinephrine; in the blind subjects, both variables were characterized by noisier rhythms than in the sighted. The overall levels of epinephrine were much higher in blind than in sighted subjects. The overall levels of norepinephrine were much higher in blind males than in blind females. In the studies done with the group of healthy young men, the overall levels of epinephrine and norepinephrine increased significantly over a 10-year period. The amplitude of the rhythm also increased significantly with time. The average age of the group was 27 years at the time of the first study and 40 years at the time of the second. The patients with leprosy also showed strong circadian variation in both epinephrine and norepinephrine, but, because many of the subjects were on medication, comparisons with the other groups are questionable. In general the data raise some questions about, but do not refute, the commonly held view that the amplitude and mesor of the circadian rhythm in epinephrine decrease with age. Additional work is needed to resolve this question completely.

Oxidation of 3,4-dihydroxyphenylalanine by connective tissue constituents. Identification of Mycobacterium leprae not related to phenolase activity.

The oxidation of 3,4-dihydroxyphenylalanine (DOPA) was studied by spectrophotometric methods at pH 6.8. In the presence of L- or D-DOPA, a color development occurred in the presence of the following substances as measured by increase in absorption both at 540 nm and 480 nm: hyaluronic acid, trypsinized human skin and umbilical cord extract, trypsin treated rat tissue from subcutaneous rat leproma, trypsin treated M. lepraemurium isolated from rat lepromata, and trypsinized M. leprae isolated from non-treated lepromatous leprosy cases. Normal human skin and connective tissue extract and nontrypsinized connective tissue of rat leprosy granuloma did not oxidize DOPA. While the trypsin-treated partially purified M. leprae suspension oxidized DOPA at both wave-lengths, the hyaluronidase-treated same suspension of M. leprae failed to oxidize these phenolic compounds. Mushroom tyrosinase oxidized D-DOPA, L-DOPA, epinephrine and norepinephrine at 480 nm. Hyaluronic acid also oxidized epinephrine and norepinephrine at both wave-lengths. Since it is known that M. leprae in the human host is closely associated with the presence of the acid mucopolysaccharides of the skin, and since acid mucopolysaccharides and skin constituents strongly oxidized DOPA, and since the hyaluronidase treated M. leprae failed to oxidize DOPA, it became evident that hyaluronic acid and not M. leprae is responsible for DOPA oxidation, and phenolase activity is not associated with the metabolism of M. leprae. Evidence is presented that DOPA is not a unique characteristic of the human leprosy bacillus. For instance, trypsin-treated murine leprosy bacilli from the rat strongly oxidized DOPA. The reaction of DOPA oxidation, therefore, must be rejected as a test for the identification of M. leprae. The obtained results confirmed the pertinent findings of Skinsnes and his co-workers.

Uptake of radioactive DOPA by Mycobacterium leprae in vitro.

Our previous studies demonstrated that Mycobacterium leprae contains a characteristic o-diphenoloxidase which converts a variety of phenolic compounds to quinones in vitro. This enzyme was not present in any other mycobacteria tested. The results reported here deal with the uptake and binding of radioactive DOPA by M. leprae. The leprosy bacilli incubated with tritium-labelled DOPA, readily took up the substrate. The binding of DOPA by the bacilli was markedly inhibited by diethyldithiocarbamate. The organisms also bound tritiated norepinephrine. Mycobacterium phlei which does not oxidize phenolic substrates failed to bind DOPA. Cultures of melanocytes which contain o-diphenoloxidase took up tritiated DOPA. Catecholamine metabolism is known to be important in myocardial cells. Cultures of turtle-heart cells did not oxidize DOPA to quinone; however, these cells bound the labelled substrate. A cell line of fibroblasts derived from armadillo skin neither oxidized nor took up DOPA. The results indicate that, like melanocytes and turtle-heart cells, M. leprae probably possesses specific receptor sites for the binding and subsequent metabolism of phenolic substrates.