A metabolomic perspective of melatonin metabolism in the mouse

Abstract

Metabolism of melatonin (MEL) in mouse was evaluated through a metabolomic analysis of urine samples from control and MEL-treated mice. Besides identifying seven known MEL metabolites (6-hydroxymelatonin glucuronide, 6-hydroxymelatonin sulfate, N-acetylserotonin glucuronide, N-acetylserotonin sulfate, 6-hydroxymelatonin, 2-oxomelatonin, 3-hydroxymelatonin), principal components analysis of urinary metabolomes also uncovered seven new MEL metabolites, including MEL glucuronide, cyclic MEL, cyclic N-acetylserotonin glucuronide, cyclic 6-hydroxymelatonin; 5-hydroxyindole-3-acetaldehyde, di-hydroxymelatonin and its glucuronide conjugate. However, N(1)-acetyl-N(2)-formyl-5-methoxy-kynuramine and N(1)-acetyl-5-methoxy-kynuramine, known as MEL antioxidant products, were not detected in mouse urine. Metabolite profiling of MEL further indicated that 6-hydroxymelatonin glucuronide was the most abundant MEL metabolite in mouse urine, which comprised 75, 65, and 88% of the total MEL metabolites in CBA, C57/BL6, and 129Sv mice, respectively. Chemical identity of 6-hydroxymelatonin glucuronide was confirmed by deconjugation reactions using beta-glucuronidase and sulfatase. Compared with wild-type and CYP1A2-humanized mice, Cyp1a2-null mice yielded much less 6-hydroxymelatonin glucuronide (approximately 10%) but more N-acetylserotonin glucuronide (approximately 195%) and MEL glucuronide (approximately 220%) in urine. In summary, MEL metabolism in mouse was recharacterized by using a metabolomic approach, and the MEL metabolic map was extended to include seven known and seven novel pathways. This study also confirmed that 6-hydroxymelatonin glucuronide was the major MEL metabolite in the mouse, and suggested that there was no interspecies difference between humans and mice with regard to CYP1A2-mediated metabolism of MEL, but a significant difference in phase II conjugation, yielding 6-hydroxymelatonin glucuronide in the mouse and 6-hydroxymelatonin sulfate in humans.

Figure 1

Metabolomic analysis of control and MEL-treated mouse urines. WT mice (n = 4) were treated with 4 mg/kg MEL (ip), and 24-h urines were collected for analysis. A, Separation of control and MEL-treated mouse urine samples in a PCA scores plot. The t(1) and t(2) values represent the scores of each sample in principal component 1 and 2, respectively. B, Loadings S-plot generated by OPLS analysis. The y-axis is a measure of the relative abundance of the ions, and the x-axis is a measure of the correlation of each ion to the model. This loading plot represents the relationship between variables (ions) and observation groups (control and MEL treated) with regard to the first and second components present in A. MEL metabolites are labeled.

Figure 2

LC-MS/MS structural elucidation of MEL metabolites in mouse urine. Urine samples from WT mice were collected for 24 h after ip administration of 4 mg/kg MEL. MS/MS fragmentation was conducted with collision energy ramping from 10 to 40 eV. Major daughter ions from fragmentation were interpreted in the inlaid structural diagrams. A, MS/MS fragmentation of 6-HMEL glucuronide. B, MS/MS fragmentation of MEL glucuronide. C, MS/MS fragmentation of NAS glucuronide. D, MS/MS fragmentation of 2-OMEL. E, MS/MS fragmentation of cNAS glucuronide. F, MS/MS fragmentation of cMEL. G, MS/MS fragmentation of di-HMEL (m/z 265⁺), retention time at 2.66 min. H, MS/MS fragmentation of AFMK (m/z 265⁺), retention time at 3.79.

Figure 3

Representative chromatograms of major MEL metabolites and their relative quantification. WT mice (C57/BL6 strain) were treated with 4 mg/kg MEL (ip). Urine samples were collected 24 h after MEL treatment, and analyzed by UPLC-MS. Screening and identification of major metabolites were performed by using MetaboLynx software based on accurate mass measurement. The identities of metabolites were presented in Table 1 and Fig. 7. A, Chromatograms of major MEL metabolites in positive ion mode. B, Chromatograms of major MEL metabolites in negative ion mode. C, Relative quantification of MEL metabolites in positive ion mode. D, Relative quantification of MEL metabolites in negative ion mode. The abundance of each metabolite was represented as a relative peak area (area percent ± sd, n = 4) by calculating its percentage in the total peak area of MEL and its urinary metabolites.

Figure 4

β-Glucuronidase (G0251) and sulfatase (S9626 and S9754) activity analysis. A, Deconjugation of phenolphthalein β-d-glucuronide by β-glucuronidase. The deconjugation system included 1 mm phenolphthalein β-d-glucuronide, 40 U/ml β-glucuronidase, with or without 1 mm SAL, 200 mm sodium acetate buffer, and the total volume was 1 ml. The incubation proceeded at 37 C for 30 min with shaking and terminated by adding 1 ml 1 m NaOH and tested by spectrophotometry at 540 nm. B, Deconjugation of p-nitrocatechol sulfate by sulfatase. The deconjugation system included 1 mm p-nitrocatechol sulfate, 40 U/ml sulfatase, with or without 1 mm SAL, 200 mm sodium acetate buffer, and the total volume was 1 ml. The incubation proceeded at 37 C for 30 min with shaking and terminated by adding 1 ml 1 m NaOH and tested by spectrophotometry at 515 nm. C, Deconjugation of SaMT by sulfatase. The deconjugation system included standard SaMT, 40 U/ml sulfatase or β-glucuronidase, with or without 1 mm SAL, 200 mm sodium acetate buffer, and the total volume was 1 ml. The incubation proceeded at 37 C for 6 h with shaking and terminated by adding 2 ml ethyl acetate/tert-butyl methyl ether. The deconjugation mixture was extracted and 6-HMEL monitored by LC-MS/MS.

Figure 5

Deconjugation of urinary MEL metabolites. The deconjugation system included 50 μl MEL-treated mouse urine, 40 U/ml sulfatase or β-glucuronidase, with or without 1 mm SAL, 200 mm sodium acetate buffer, and the total volume was 1 ml. The deconjugation mixtures were incubated for 6 h at 37 C. The incubation was terminated by the addition of 2 ml ethyl acetate/tert-butyl methyl ether (1:1). The deconjugation mixture was extracted, and 6-HMEL, NAS, and MEL were monitored by LC-MS/MS. A, Relative level of 6-HMEL in deconjugation system with S9626. B, Relative level of NAS in deconjugation system with S9626. C, Relative level of MEL in deconjugation system with S9626. D, Relative level of 6-HMEL in deconjugation system with G0251. E, Relative level of NAS in deconjugation system with G0251. F, Relative level of MEL in deconjugation system with G0251.

Figure 6

Major urinary MEL metabolites in WT, Cyp1a2-null, and hCYP1A2 mice. MEL (4 mg/kg) was administered ip, and mice were immediately housed in separate metabolic chambers. Urines were collected over a 24-h period after MEL administration and analyzed by UPLC-MS. A, Relative level of 6-HMEL glucuronide in mouse urine. B, Relative level of NAS glucuronide in mouse urine. C, Relative level of MEL glucuronide in mouse urine. Each metabolite in the WT group was regarded as 100%. *, P < 0.05, compared with WT and hCYP1A2 mice (n = 4).

Figure 7

MEL metabolic pathways in mouse. CYP1A2 transforms MEL (I) to 6-HMEL (II), which can be further metabolized dominantly to glucuronide metabolite (VII) and minor sulfate metabolite (VIII). Besides C6 hydroxylation, MEL undergoes O-demethylation to NAS (III), which is further conjugated to its glucuronide (IX) and sulfate (X). MEL can also be converted to MEL glucuronide (IV), 2-OMEL (V), 3-HMEL (XI), di-HMEL (VI), cNAS glucuronide (XIII), cMEL (XIV), c6-HMEL (XII), and 5-HIAL (XV). AFMK (XVI) and AMK (XVII) pathways are insignificant.