Chromatographic analysis of products of melatonin metabolism by rat liver microsomes. A - Boiled microsomes (0.5 mg protein/ml) incubated at 37°C for 60 min with 1 mM NADPH and 50 μM melatonin. B - Microsomes (0.5 mg protein/ml) incubated at 37°C for 60 min with 1 mM NADPH and 50 μM melatonin. See Methods section for detail description of incubation conditions and sample preparation for HPLC analysis. A Restec Allure C18 column (150×4.6 mm) was used for separation of melatonin metabolites. Elution was carried out isocratically with 15% acetonitrile and 0.1% acetic acid at flow rate of 0.75 ml/min and temperature of 40°C. The column eluent was monitored by absorbance at 265 nm. The insets show eluent fluorescence with an excitation wavelength of 285 nm and an emission wavelength of 365 nm. Peaks designated as 1, 2, 3, 4, 5, 6 and 7 correspond to Metabolite 1 (NAS; [M+H]⁺ at m/z=219; λ_max= 278 nm), Metabolite 2 (6-hydroxymelatonin; [M+H]⁺ at m/z=249; λ_max= 300 nm), Metabolite 3 (oxidation product of melatonin with m/z=231; λ_max=273 and 293 nm), Metabolite 4 (2-hydroxymelatonin; [M+H]⁺ at m/z=249, λ_max= 257 and 296 nm), Metabolite 5 (AFMK; [M+H]⁺ at m/z=265, λ_max= 234, 262 and 342 nm), Metabolite 6 (unknown product) and melatonin ([M+H]⁺ at m/z=233, λ_max=278 nm), respectively. See Methods section for detail description of LC-MS analysis.

Effect of melatonin concentration on rate of 6-hydromelatonin (A) and NAS (B) formation in mitochondria. The melatonin concentrations ranged from 0.1 to 1.5 mM. The concentration of mitochondrial protein in the reaction was 0.5 mg/ml. Reactions were conducted at 37°C for 60 min. A HPLC analysis with fluorescence detection (excitation: 285 nm, emission: 365nm) was used for quantification of the 6-hydroxymelatonin and NAS. Means ± SEM of three replicates are shown. Points are experimentally determined values, and the solid line is the computer-generated curve of best fit for a two-enzyme model. The insets show Eadie-Hofstee plots for O-demethylation and 6-hydroxylation of melatonin.

Melatonin metabolism mediated by cytochrome P450.

Effect of melatonin concentration on rate of 6-hydromelatonin (A) and NAS (B) formation in microsomes. The melatonin concentrations ranged from 0.1 to 1.5 mM. The concentration of microsomal protein in the reaction was 0.5 mg/ml. Reactions were conducted at 37°C for 60 min. A HPLC analysis with fluorescence detection (excitation: 285 nm, emission: 365nm) was used for quantification of the 6-hydroxymelatonin and NAS. Means ± SEM of three replicates are shown. Points are experimentally determined values, and the solid line is the computer-generated curve of best fit for a two-enzyme model. The insets show Eadie-Hofstee plots for O-demethylation and 6-hydroxylation of melatonin.

* P<0.05; ** P<0.01; *** P <0.001 (Significantly different from the corresponding values of control incubations).