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Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

Shaeib F, Khan SN, Ali I, Najafi T, Maitra D, Abdulhamid I, Saed GM, Pennathur S, Abu-Soud HM - PLoS ONE (2015)

Bottom Line: Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron.Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl.Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Obstetrics and Gynecology, the C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

No MeSH data available.


Related in: MedlinePlus

HPLC analysis shows MLT oxidation thereby preventing MPO heme destruction and generation of free iron.A) HPLC trace for MLT (elution time 3.98 min) dissolved in DMF (elution time 3.31 min) and phosphate buffer (elution time 2.48 min). B) Addition of MPO and Cl- causes no significant change in MLT peak intensity and/or retention time. C) Addition of H2O2 (sequential addition of 20 μM, total 200 μM) results in a significant shift in MLT retention time elution time (3.71 min) as well as the appearance of a small peak around 3.57 min. D) Increasing levels of H2O2 (400 μM) resulted in the domination of the MLT metabolite eluted at 3.57 min showing the retention and absorbance properties of AFMK (elution time 3.57 min) as shown in panel (E).
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pone.0120737.g004: HPLC analysis shows MLT oxidation thereby preventing MPO heme destruction and generation of free iron.A) HPLC trace for MLT (elution time 3.98 min) dissolved in DMF (elution time 3.31 min) and phosphate buffer (elution time 2.48 min). B) Addition of MPO and Cl- causes no significant change in MLT peak intensity and/or retention time. C) Addition of H2O2 (sequential addition of 20 μM, total 200 μM) results in a significant shift in MLT retention time elution time (3.71 min) as well as the appearance of a small peak around 3.57 min. D) Increasing levels of H2O2 (400 μM) resulted in the domination of the MLT metabolite eluted at 3.57 min showing the retention and absorbance properties of AFMK (elution time 3.57 min) as shown in panel (E).

Mentions: Finally, HPLC analysis (anion exchange) was utilized to investigate in depth the mechanism by which MLT presence prevents MPO heme destruction mediated by self-generated HOCl. Using this method, we observed an accumulation of two major MLT metabolites when concentrations of MLT used were sufficient to produce dramatic effects on the rates of Compound II formation, duration, and decay. The population of these metabolites is varied and dependent on the H2O2 concentration used (Fig. 4). HPLC analyses were conducted under five different conditions: MLT alone; MPO (40 nM) pre-incubated with MLT (100 μM) alone; the solution mixture of MPO pre-incubated with MLT (100 μM) which received sequential additions of 20 μM H2O2 (to total either 200 or 400 μM H2O2); and finally AFMK alone. After reaction completion, the reaction mixtures were filtered to eliminate MPO and the supernatants were then injected into the HPLC system. Under our experimental conditions phosphate buffer and DMF were eluted at 2.48 and 3.31 min, respectively (Fig. 4). MLT alone was eluted at 3.95 min (Fig. 4A) while AFMK alone was eluted at 3.57 min (Fig. 4E), and both were identified by their characteristic spectra observed from the photodiode array detector at 222 and 236 nm, respectively. Pre-incubation of MLT (100 μM) with a catalytic amount of MPO (40 nM) and Cl- (100 mM), in the absence of H2O2, fails to generate any detectable MLT metabolite (Fig. 4B). HPLC analysis also indicated that the relatively short incubation times of H2O2 at the different concentrations employed (200–400 μM) in the experiment for 2h has no effect on the MLT (100 μM) moiety (data not shown). However, long incubation of high concentration of H2O2 (30%) with MLT in the presence of methanol generates AFMK (Fig. 4E) [46].


Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

Shaeib F, Khan SN, Ali I, Najafi T, Maitra D, Abdulhamid I, Saed GM, Pennathur S, Abu-Soud HM - PLoS ONE (2015)

HPLC analysis shows MLT oxidation thereby preventing MPO heme destruction and generation of free iron.A) HPLC trace for MLT (elution time 3.98 min) dissolved in DMF (elution time 3.31 min) and phosphate buffer (elution time 2.48 min). B) Addition of MPO and Cl- causes no significant change in MLT peak intensity and/or retention time. C) Addition of H2O2 (sequential addition of 20 μM, total 200 μM) results in a significant shift in MLT retention time elution time (3.71 min) as well as the appearance of a small peak around 3.57 min. D) Increasing levels of H2O2 (400 μM) resulted in the domination of the MLT metabolite eluted at 3.57 min showing the retention and absorbance properties of AFMK (elution time 3.57 min) as shown in panel (E).
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getmorefigures.php?uid=PMC4383586&req=5

pone.0120737.g004: HPLC analysis shows MLT oxidation thereby preventing MPO heme destruction and generation of free iron.A) HPLC trace for MLT (elution time 3.98 min) dissolved in DMF (elution time 3.31 min) and phosphate buffer (elution time 2.48 min). B) Addition of MPO and Cl- causes no significant change in MLT peak intensity and/or retention time. C) Addition of H2O2 (sequential addition of 20 μM, total 200 μM) results in a significant shift in MLT retention time elution time (3.71 min) as well as the appearance of a small peak around 3.57 min. D) Increasing levels of H2O2 (400 μM) resulted in the domination of the MLT metabolite eluted at 3.57 min showing the retention and absorbance properties of AFMK (elution time 3.57 min) as shown in panel (E).
Mentions: Finally, HPLC analysis (anion exchange) was utilized to investigate in depth the mechanism by which MLT presence prevents MPO heme destruction mediated by self-generated HOCl. Using this method, we observed an accumulation of two major MLT metabolites when concentrations of MLT used were sufficient to produce dramatic effects on the rates of Compound II formation, duration, and decay. The population of these metabolites is varied and dependent on the H2O2 concentration used (Fig. 4). HPLC analyses were conducted under five different conditions: MLT alone; MPO (40 nM) pre-incubated with MLT (100 μM) alone; the solution mixture of MPO pre-incubated with MLT (100 μM) which received sequential additions of 20 μM H2O2 (to total either 200 or 400 μM H2O2); and finally AFMK alone. After reaction completion, the reaction mixtures were filtered to eliminate MPO and the supernatants were then injected into the HPLC system. Under our experimental conditions phosphate buffer and DMF were eluted at 2.48 and 3.31 min, respectively (Fig. 4). MLT alone was eluted at 3.95 min (Fig. 4A) while AFMK alone was eluted at 3.57 min (Fig. 4E), and both were identified by their characteristic spectra observed from the photodiode array detector at 222 and 236 nm, respectively. Pre-incubation of MLT (100 μM) with a catalytic amount of MPO (40 nM) and Cl- (100 mM), in the absence of H2O2, fails to generate any detectable MLT metabolite (Fig. 4B). HPLC analysis also indicated that the relatively short incubation times of H2O2 at the different concentrations employed (200–400 μM) in the experiment for 2h has no effect on the MLT (100 μM) moiety (data not shown). However, long incubation of high concentration of H2O2 (30%) with MLT in the presence of methanol generates AFMK (Fig. 4E) [46].

Bottom Line: Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron.Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl.Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Obstetrics and Gynecology, the C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

No MeSH data available.


Related in: MedlinePlus