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.
Citation: Shaeib F, Khan SN, Ali I, Najafi T, Maitra D, Abdulhamid I, et al. (2015) Melatonin Prevents Myeloperoxidase Heme Destruction and the Generation of Free Iron Mediated by Self-Generated Hypochlorous Acid. PLoS ONE 10(4): e0120737. https://doi.org/10.1371/journal.pone.0120737
Academic Editor: Pedro Tauler, University of the Balearic Islands, SPAIN
Received: November 19, 2014; Accepted: February 6, 2015; Published: April 2, 2015
Copyright: © 2015 Shaeib et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This work was supported by a grant to H.M.A-S. from National Institutes of Health RO1 HL066367, HL094230, and DK097153 and a grant from the Children’s Hospital of Michigan, Detroit, Michigan, USA. I.A. is a Senior Fulbright Research Scholar in Dr. Abu-Soud lab. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Melatonin (MLT) is naturally synthesized from the amino acid tryptophan in the pineal gland, but also produced by other non-endocrine organs (e.g., cerebellum, cerebral cortex, retina, skin, ovary, liver, pancreas, kidneys, and immune competent cells), and acts through two G-protein coupled receptors, MT1 and MT2 [1–4]. In humans, as in most vertebrates, MLT operates as a modulator of circadian rhythms, and displays an oscillatory pattern through its unique ability to function as a signal, which organisms use to synchronize their circadian systems [3, 5, 6]. Multiple studies have shown that when MLT is administered either exogenously in vivo or when added to cultured cells via regulation of cellular pathways [3, 7–11] MLT has the ability to scavenge a wide range of reactive oxygen species (ROS) through its distinct antioxidant and anti-inflammatory effects [3, 7–11].The effects and action mechanisms of MLT belong to or take part in many different cell types including inflammatory cells such as monocytes–macrophages, neutrophils, eosinophils, basophils, mast cells, and natural killer cells [10, 12]. Therefore, various doses of synthetic MLT supplements have been used to treat a variety of medical scenarios in which inflammation plays a role such as a weakened immune system due to stress, oxidative hemolysis of red blood cells, and cancer progression [13–15]. Recently, we have shown that MLT is a potent inhibitor of the inflammatory enzymes myeloperoxidase (MPO) and other related peroxidases (e.g. eosinophil peroxidase) [16–18].
Myeloperoxidase is a heme protein, present in the neutrophils, which utilizes chloride (Cl-) in the presence of H2O2 to generate HOCl [19, 20]. This process occurs through H2O2 reduction that leads to the formation of MPO Compound I (ferryl porphyrin π cation radical, Fe(IV) = O(+π•)), which oxidizes Cl- to HOCl . Myeloperoxidase compound I is also capable of oxidizing various organic and inorganic substrates by two successive 1e− transfers to generate compound II (MPO-Fe(IV) = O) and MPO-Fe(III), respectively. The rate limiting step in a typical peroxidase cycle is the reduction of compound II to MPO-Fe(III). Furthermore, physiological reductants such as superoxide, nitric oxide, MLT, and ascorbic acid are known to accelerate this process [17, 22–26]. Hypochlorous acid is a potent oxidant that is capable, under normal circumstances, of functioning as a powerful antimicrobial agent [19, 20]. However, under a number of pathological conditions such as inflammatory diseases, in which ROS production can become excessive, HOCl is capable of mediating tissue damage [19, 27]. Interestingly, many inflammatory disorders such as ovarian cancer and atherosclerosis, in which MPO/HOCl have been known to be elevated, are also associated with significant free iron accumulation [28–31]. Recently, we have highlighted the potential link between elevated HOCl and hemoprotein heme destruction, and subsequent generation of free iron [21, 32, 33]. Detailed mechanistic insight into how exogenously added or self-generated HOCl mediates the MPO heme moiety has recently been elucidated [32, 34]. Therefore, factors that influence rates of HOCl removal are of growing interest [20, 35–40]. Here, we examine the ability of MLT to prevent HOCl-mediated heme destruction and subsequent iron release. These findings may have therapeutic repercussions as they elucidate the mechanism behind the rationale for additional studies on MLT supplementation for patients with chronic inflammatory conditions in which MPO is elevated. Additionally, this work may open the door for the development of other treatment interventions in this patient population.
Materials and Methods
All the materials used were of the highest-grade purity and used without further purification. Sodium hypochlorite (NaOCl), H2O2, ammonium acetate (CH3COONH3), ferrozine, MLT, ascorbic acid, and dimethylformamide, were obtained from Sigma Aldrich (St. Louis, MO, USA).
MPO was initially purified from detergent extracts of human leukocytes by sequential lectin affinity and gel filtration chromatography [41–43]. Trace levels of contaminating eosinophil peroxidase (EPO) were then removed by passage over a sulfopropyl Sephadex column . Purity of isolated MPO was established by demonstrating a Reinheitszahl (RZ) value of greater than 0.85 (A430/A280), SDS−PAGE analysis with Coomassie Blue staining, and gel tetramethylbenzidine peroxidase staining to confirm no contaminating EPO activity. Enzyme concentration was determined spectrophotometrically utilizing extinction coefficients of 89,000 M-1 cm-1/heme of MPO .
H2O2-selective electrode measurements.
Hydrogen peroxide measurements were carried out using an H2O2-selective electrode (Apollo 4000 free radical analyzer; World Precision Instruments, Sarasota, FL, USA). Experiments were performed at 25°C by immersing the electrode in 3 ml of 0.2 M sodium phosphate buffer, pH 7.0. Experiments were carried out under two different conditions: sequential additions of 10 μM H2O2 to a continuously stirred buffer solution supplemented with 40 nM MPO and 100 mM Cl- in the absence and presence of 200 μM MLT during which the change of H2O2 concentration was continuously monitored.
The absorbance spectra were recorded using a Cary 100 Bio UV–visible photometer at 25°C, pH 7.0. Experiments were performed in 1 ml phosphate buffer solution supplemented with MPO (1.0–1.5 μM), 100 mM Cl-, and incremental additions of 180 μM of H2O2 (20 μM; 2 μl) in the absence and presence of increasing MLT concentrations (0–200 μM). After each H2O2 addition, the reaction mixture was left for 10 minutes for reaction completion and absorbance spectra were then recorded from 300 to 700 nm.
Free iron analysis.
Free iron release was measured colorimetrically using ferrozine, following a slight modification of a published method . To 100 μl of the sample (MPO–HOCl reaction mixture), 100 μl of ascorbic acid (100 mM) was added. After 5 min of incubation at room temperature, 50 μl of ammonium acetate (16%) and the same volume of ferrozine (16 mM) were added to the mixture and mixed well. Subsequently, the reaction mixture was incubated for 5 min at room temperature and the absorbance was measured at 562 nm. A standard curve prepared using ammonium Fe(III) sulfate was used for the calculation of free iron concentration. Final concentrations of the additives were as follows: ascorbic acid, 33.33 μM; ammonium acetate, 5.3%; and ferrozine, 5.3 μM.
High Performance Liquid Chromatography (HPLC).
HPLC analyses were performed using a Shimadzu HPLC system equipped with an SCL-10A controller, LC-10 AD binary solvent delivery pumps, SIL-10 AD autosampler, SPD-M10 A diode array detector, and an RF-10 A XL fluorescence detector. An Alltech 5 μm particle size column was used with a 4.6 x 150 mm reverse phase octadecylsilica (C18). To monitor the chromatogram, the RF fluorescence detector was set at 321 nm for excitation and 465 nm for emission and the SPD diode array detector was set at 400 nm. HPLC grade solvents were prepared as follows: solvent A, 0.1% TFA in water and solvent B, 0.1% TFA in 80% acetonitrile. Solvent gradients were set as follows: 0–10 min 55–65% B, 10–14 min 65–90% B, followed by reducing solvent B composition to 55% within 14–24 min. The column elution was carried out at a flow rate of 0.8 ml/min with a linear gradient of solvents. After treatment of MLT with MPO in presence of H2O2 for 24 hours, the reaction mixture was filtered through an Amicon Ultra-15 centrifugal filter unit with Ultracel-10 membrane (from Millipore) 3-kDa cut-off by centrifuging at 14,000 rcf rate for 30 min at 4°C; then 50 μl of filtered sample was injected for analysis. At the end of the run the system was equilibrated with 45% solvent A; each sample was analyzed in triplicate.
N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) synthesis.
The AFMK was synthesized following the method of Tan et al.  with slight modification. Briefly, 5 mg MLT were dissolved in 100 μL methanol and the reaction mixture was mixed with 500 μL H2O2 (30%). The formation of AFMK was followed by the increase in absorbance at 340 nm. AFMK was isolated and confirmed by HPLC analyses.
HOCl was prepared as previously described with some modifications . Briefly, a stock solution of HOCl was prepared by adding 1 ml of NaOCl solution to 40 ml of 154 mM NaCl and the pH was adjusted to around 3 by adding HCl. The concentration of active total chlorine species in solution, expressed as [HOCl]T (where [HOCl]T = [HOCl] + [Cl2] + [Cl3−] + [OCl−]) in 154 mM NaCl, was determined by converting all the active chlorine species to OCl− by adding a bolus of 40 μl of 5 M NaOH and measuring the concentration of OCl−. The concentration of OCl− was determined spectrophotometrically at 292 nm (ε = 362 M− 1 cm− 1). As HOCl is unstable, the stock solution was prepared on a daily basis, stored on ice, and used within 1 hour of preparation. For further experimentation, dilutions were made from the stock solution using 200 mM phosphate buffer, pH 7, to give working solutions of lower HOCl concentrations.
Melatonin prevents MPO inactivation by HOCl generated during MPO steady-state catalysis: The ability of MLT to prevent HOCl damage to MPO catalytic activity was determined by two methods. The first involved the use of an H2O2-selective electrode, which measured the first step in the MPO catalytic cycle in which H2O2 is consumed by MPO. The second method measured HOCl–mediated MPO heme destruction utilizing UV-Visible and free iron release using ferrozine assay.
The H2O2-selective electrode measurements revealed that addition of an aliquot of H2O2 (10 μM; 3.5 μL) to the continuously stirred reaction solution supplemented with 40 nM MPO and 100 mM Cl- demonstrated an instant consumption of H2O2, as previously reported [26, 35, 36]. Subsequent multiple additions of the same amount of H2O2 to the MPO/Cl- solution mixture caused MPO inhibition, as judged by the accumulation of H2O2 (amplitude of H2O2 signal) and a slower rate of its consumption (longer duration) (Fig. 1A). Under these circumstances, self-generated HOCl inhibited MPO through a mechanism that involves heme destruction, precluding the enzyme from functioning at maximum activity (80–90% inhibition)  (Fig. 1A). We used 40 nM MPO as the catalytic concentration. The pathophysiological effect of MPO was shown at concentrations of 5 nM . To examine whether MLT could prevent the feedback heme destruction mediated by HOCl, an identical experiment was repeated in the presence of a saturating amount of MLT. Addition of H2O2 (10 μM) to a continuously stirred buffer solution supplemented with 40 nM MPO, 100 mM Cl-, and 200 μM MLT caused a much slower rate of H2O2 consumption compared to the control solution (Fig. 1B), which indicated that MLT inhibited the MPO catalytic activity . The repeated addition of the same amounts of H2O2 to the MPO/Cl-/MLT reaction mixture showed that the degree of MPO inhibition remained the same for all five of the trials. Thus under these conditions, MLT protected the peroxidation activity of MPO, but inhibited the chlorinating activity of the enzyme by serving as 1 e- substrate for both MPO compounds I and II .
(A) A typical recording by an H2O2-selective electrode demonstrating the dramatic MPO feedback inhibition mediated by self-generated HOCl after addition of equal amounts of H2O2 (10 μM, 1–2 μl in 3 ml reaction mixture) five consecutive times (denoted by the arrows) to a continuously stirred phosphate buffer (200 mM, pH 7.4) containing 40 nM MPO and 100 mM Cl−, at 25°C. (B) Similar experiment was repeated in the presence of MLT (100 μM), showing a significant protection of peroxidation activity of MPO. Under these circumstances, MLT inhibits the chlorinating activity of MPO and no heme destructions have been observed . The data shown are representative of three independent experiments.
We next performed UV-visible photometry to correlate the degree of catalytic inhibition with HOCl-mediated heme destruction. As shown in the Fig. 2 inset; blue trace, MPO-Fe(III) as isolated displays a Soret absorbance peak centered at 430 nm, with three additional peaks at 573, 630, and 694 nm. Since the addition of a high molar ratio of H2O2 to MPO causes the conversion of MPO to Compound (III) (MPO-FeII-O2 complex) , the oxidation of the MPO heme moiety mediated by self-generated HOCl was monitored by sequential addition of H2O2 (20 μM; 3 μl) (180 μM H2O2 total) to the MPO-Fe(III)/Cl− mixture. With each incremental addition of H2O2, there was a proportional decrease in the MPO Soret peak, indicating that HOCl-mediated MPO feedback inhibition is associated with MPO heme destruction. After the last addition of H2O2 (180 μM total) solution to enzyme mixture, the spectrum recording showed a flattening in the Soret peak at 430 nm indicating MPO heme destruction (Fig. 2 inset; red trace). This flattening in the Soret peak region occurred solely in the presence of Cl−, signifying HOCl to be the major cause of MPO heme destruction. To confirm that MLT prevents HOCl-mediated MPO heme destruction, a fixed amount of MPO/Cl− mixture was preincubated with increasing concentrations of MLT prior to incremental additions of H2O2 to the reaction mixture. Fig. 2 shows the percentage recovery of MPO heme content, measured at 430 nm after the last addition of the incremental H2O2 to the enzyme solution, as a function of MLT concentration. In the presence of a saturating amount of MLT (>100 μM), spectral analysis indicated no losses in the heme content. Under these conditions, the MPO-H2O2 system utilized MLT as a 1e- substrate for the formation and subsequent decay of Compound II. The accumulation and stability of MPO Compound II (characterized by a Soret absorbance peak at 450 nm) during catalysis depended on the MLT concentration. In the presence of lower MLT levels, addition of limited amounts of H2O2 (10 μM) to the solution mixture caused immediate appearance of MPO Compound II, which then decayed to the ferric form in the next few seconds. In the presence of higher MLT concentrations (e.g. 100–400 μM), no significant change in absorbance was observed upon the addition of an H2O2 solution to the MPO mixture, indicating that the rate of MPO compound II decay exceeded the rate of formation, which was consistent with previous results . In the presence of 50 μM MLT, only 50% recovery was noted in the MPO Soret absorbance peak of the total enzyme. As shown in Fig. 2, the full protection of the MPO heme contents required the presence of a ratio 1:2 MLT: H2O2. Collectively, our results showed that heme destruction did not occur in the presence of MLT, where MPO began reducing H2O2 without generating HOCl, indicating that self-generated HOCl is the major cause of MPO inactivation.
Fixed amount of MPO (1 μM) was incubated with fixed amount of Cl- (100 mM) and increasing concentration of MLT (12–200 μM), and the reaction mixtures were incrementally received fixed amount of H2O2 (20 μM, total concentration of 180 μM). After reaction completion, the spectra of the reaction mixtures were scanned from 300–700 nm.
The percent recovery in MPO Soret peak (430 nm) plotted as a function of melatonin concentration. The full protection of the MPO heme contents required the presence of a ratio 1: 2 MLT: H2O2. The inset shows the absorbance spectra of MPO ferric form before (blue trace) and after the last incremental addition of H2O2 (red trace). The flattening in the MPO spectrum indicates MPO heme destruction. The data points are the average of three independent experiments.
To investigate how the flattening in the Soret absorbance peak at 430 nm in H2O2-treated samples is linked to MPO heme depletion and if MLT can prevent this finding, we studied the free iron release after H2O2 treatment in the absence and presence of saturating amounts of MLT. Compared to the free iron content of the untreated control, treatment with H2O2 led to a significant increase in free iron content (Fig. 3). Additionally, in the same figure, we noted around 25% free iron detection. This finding is likely secondary to the fact that not all iron was detached from the heme fragments, and therefore not able to be detected by the assay. The accumulation of free iron significantly decreased in the presence of saturating amounts of MLT, confirming the above spectrophotometric studies. Thus, MLT not only inhibits MPO catalytic activity, but also prevents heme destruction and subsequent free iron release mediated by self-generated HOCl.
MPO (1.2 μM) was incubated with 100 mM Cl− in the absence and presence of 400 μM MLT followed by the addition of aliquots of H2O2 (in increments of 20 μM) to the reaction mixture. The free iron released was measured using ferrozine assay as detailed under Materials and methods. No free iron was detected before the addition of H2O2. The data are the averages of three independent experiments with the error bars representing the standard error of measurement.
The protection of MPO heme destruction mediated by self-generated HOCl occurred at the expense of melatonin oxidation:
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) .
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).
However, incremental addition of H2O2 to the enzyme-MLT mixture resulted in MLT oxidation to a lower elution time indicating formation of MLT metabolites with lower hydrophobicity. The enzyme sample that was pre-incubated with 100 μM MLT and treated with 200 μM H2O2 (total) led to the production of two main MLT metabolites with elution times of 3.71 and 3.57 min with the first being the most abundant and attributed to the formation of hydroxyl melatonin metabolite (Fig. 3C). Although we provide no direct evidence for the hydroxyl melatonin formation, we do note corresponding 1e- heme reduction steps for MPO Compounds I and II in the presence of MLT. The MPO sample that was incubated with 100 μM MLT and treated with 400 μM H2O2 (total) led to the production of a MLT metabolite with elution time of 3.57 min, which was similar to elution time of AFMK alone (Fig. 4D). Consistent with these studies, Ximenes et al. similarly have observed two MLT metabolites (hydroxyl melatonin and AFMK) when MLT was incubated with MPO-H2O2 system or with stimulated neutrophils . Thus, MLT prevents MPO heme destruction either by directly scavenging HOCl and/or inhibiting MPO chlorinating activity.
In this work, we show that MLT largely prevents MPO catalytic inhibition, attributed to MPO heme destruction and the generation of free iron associated with HOCl synthesis through its function as a potent MPO inhibitor and/or a HOCl scavenger. Thus, MLT may contribute to the reduction of the inflammatory process not only by inhibiting MPO and consuming HOCl, but also by diminishing the release and accumulation of free iron.
Recently, we have characterized an irreversible inhibition that is related to MPO heme destruction and the generation of free iron, when appropriate concentrations of self-generated HOCl are reached in the enzyme milieu . These findings were recently confirmed by Paumann-Page et al . The accumulation of the released HOCl in the solution mixture permits the competition with H2O2 on the catalytic site of MPO, which is in this case is the heme prosthetic group . Hypochlorous acid interacts with both MPO-Fe(III) and Compound I and accelerates their conversion to Compound II [34, 54], or forms a relatively stable MPO-Fe(III)-OCl complex, which also converts to Compound II prior to heme destruction . Compound II is a long-lived intermediate, and thus would be notably susceptible to HOCl assault leading to heme destruction . In the absence of an MPO inhibitor, HOCl scavenger, or both, the degree of MPO heme destruction is significantly high in that only a small portion of the total enzyme (5–10%) is estimated to remain active after multiple cycles of HOCl synthesis . Our current results demonstrate that the degree of MPO heme pocket alterations (e.g., by changes in the hydrogen bonds) mediated by MLT is not only sufficient to affect the interaction of Compound I with Cl- preventing the generation of HOCl, but also prevents HOCl access to the heme moiety; thereby avoiding HOCl-dependent heme destruction. These results are consistent with our previous detailed kinetic studies, which showed the ability of MLT to inhibit MPO chlorinating activity despite the presence of high concentrations of Cl- . Melatonin competes with Cl- and switches the MPO catalytic activity from a 2e- oxidation to a 1e- oxidation pathway. Under these conditions, MPO did not generate HOCl but still consumed H2O2 at slower rates. H2O2-selective measurements showed that MLT presence inhibits MPO peroxidase activity. This observation appears relevant even in the presence of alternative substrates because peroxidases like MPO are not saturated under physiological conditions . These findings were also supported by theoretical modeling, which showed that indole compounds could be accommodated in the narrow regions of the active site pockets of MPO when the indole ring was situated parallel to the heme plane and close enough to the D pyrrole ring. Under the circumstances the side chain of the indole compound was directed toward the outside of the distal cavity .
It is clear from the MLT presence that the MPO chlorinating activity, but not H2O2, is implicated in MPO heme destruction and free iron release. This conclusion is consistent with previous studies by Paumann-Page et al. who showed that the MPO inactivation mediated by H2O2 is unlikely to take place in the presence of reducing substrates (100 mM Cl-) and under conditions in which the concentration of H2O2 does not accumulate . The amount of MLT used (100 μM) in the current work is sufficient to inhibit MPO. Studies on the effect of MLT on HOCl production by neutrophils and purified MPO have showed that the concentration of MLT that inhibited HOCl production by 50% (IC50) was estimated to be 18 μM and reduced to 4 μM when superoxide was removed by addition of superoxide dismutase . In contrast, the IC50 value, calculated from the initial rate of H2O2 consumption as a function of the MLT concentration was 3 μM . Our HPLC analysis showed that the protection of MPO heme destruction mediated by self-generated HOCl occurred at the expense of MLT oxidation, which depends on the concentration of H2O2 used. Ximenes et al. showed the elution of two MLT metabolites when MLT was exposed to neutrophils . In their system, the major and minor peaks were AFMK and a hydroxlyated melatonin metabolite, respectively. We similarly observed two peaks in our system; however, we believe the major peak was the hydroxylated intermediate when a lower concentration of H2O2 (1:2, MLT: H2O2) was used. In contrast, AFMK predominated when MPO was exposed to higher concentrations of H2O2 (1:4, MLT: H2O2). This alteration in the peroxidation turnover resulted in the reversal of the populations of the two MLT metabolites. Thus, prevention of MPO heme destruction depends on multiple factors including the bioavailability of HOCl, the presence of a capable 1e- substrate that can compete with Cl- switching the reaction from a 2e- to a 1e- oxidation pathway (e.g. ascorbic acid, superoxide, and nitric oxide), and the presence of HOCl scavengers.
Melatonin prevention of HOCl-mediated heme destruction is not limited to MPO, but also applies to other hemoprotein model compounds, such as hemoglobin, lactoperoxidase, catalase, as well as isolated human red blood cells [32–34, 52, 58, 59]. Earlier kinetic measurements have indicated that HOCl initially mediates the sequential formation of ferryl peroxidase-like intermediates, compounds I and II, followed by heme degradation [33, 34, 54, 60]. Hypochlorous acid can also mediate tetrapyrrole ring destruction independent of the iron molecule that resides in the porphyrin center . A general chemical mechanism that describes the tetrapyrrole ring destruction resulting from the direct attack of HOCl and generation of multiple heme degradation products is well documented [20, 21, 32, 33, 37]. Because of MLT's ability to inhibit MPO, destabilize the Compound II intermediate, and/or directly scavenge HOCl, MLT could be considered an ideal component for prevention against HOCl mediated oxidative damage.
Although experiments that utilized methionine or taurine as a scavenger of HOCl showed that they could prevent HOCl-mediated MPO heme destruction  similar to MLT, there are important differences in the fundamental aspects. Melatonin and its precursors, unlike other HOCl scavengers, display a high affinity towards transition metal binding (e.g. iron (III), copper and zinc), and subsequently reduce their cytoplasmic availability [61–63]. In addition, several in vivo studies have shown that administration of MLT directly or indirectly neutralizes a variety of ROS, resulting in the reduction of lipid peroxidation, protein oxidation, and DNA damage, thus helping the immune system [11, 62–65]. One other factor that distinguishes MLT from other HOCl scavengers (e.g. taurine, cystine, cysteine and uric acid) is that its oxidation products have no biologically harmful sequelae [18, 66]. Melatonin reacts with HOCl to produce 2-hydroxymelatonin  at a rate sufficient to protect catalase against inactivation by this molecule . Melatonin’s presence during MPO catalysis is associated with a significant diminution of free iron release, decrease in the intensity of the fluorescent heme degradation products, and reduction in different profiles of protein aggregation . In contrast, taurine reacts with HOCl to form a less active oxidant taurine chloramine. It is important, however, to note that while chloramines are less reactive than HOCl, they can still oxidize thiols, thioethers and heme proteins, and thus extend the reactivity of HOCl [66, 68, 69].
The association between enhanced MPO expression and increased levels of free iron is characteristic of many inflammatory disorders including cardiovascular diseases such atherosclerosis, pulmonary diseases such as cystic fibrosis, neurodegenerative diseases such as Alzheimer’s disease as well as arthritis, diabetes, and has been found to be a risk factor for various cancers [28, 29, 31, 70–75]. As free iron accumulates, it disturbs body processes by replacing certain vital minerals such as zinc, copper, and manganese in many enzymes, depleting vitamins such as vitamin E and D, and may lead to chronic infection and inflammation . Due to its properties as an excellent oxygen transporter, iron tends to stimulate the growth of tumor cells and bacteria [77–79]. Therefore, blocking the MPO chlorination machinery (MLT, tryptophan, and tryptophan analogs) [17, 35, 36] or scavenging HOCl (MLT, methionine, lycopine, taurine, and glutathione) might be a useful therapeutic approach in reducing free iron release in a wide variety of inflammatory conditions.
Conceived and designed the experiments: HMAS SP GS. Performed the experiments: FS SK IA TN DM. Analyzed the data: FS IA DM. Contributed reagents/materials/analysis tools: IA SP. Wrote the paper: FS SK HMAS.
- 1. Hardeland R. Melatonin: signaling mechanisms of a pleiotropic agent. Biofactors. 2009;35(2):183–92. Epub 2009/05/19. pmid:19449447.
- 2. Reiter RJ, Tan DX, Rosales-Corral S, Manchester LC. The universal nature, unequal distribution and antioxidant functions of melatonin and its derivatives. Mini Rev Med Chem. 2013;13(3):373–84. Epub 2012/11/30. pmid:23190034.
- 3. Reiter RJ, Tan DX, Fuentes-Broto L. Melatonin: a multitasking molecule. Prog Brain Res. 2010;181:127–51. Epub 2010/05/19. pmid:20478436.
- 4. Comai S, Gobbi G. Unveiling the role of melatonin MT2 receptors in sleep, anxiety and other neuropsychiatric diseases: a novel target in psychopharmacology. J Psychiatry Neurosci. 2014;39(1):6–21. Epub 2013/08/27. pmid:23971978; PubMed Central PMCID: PMC3868666.
- 5. Reiter RJ. Melatonin: the chemical expression of darkness. Mol Cell Endocrinol. 1991;79(1–3):C153–8. Epub 1991/08/01. pmid:1936532.
- 6. Reiter RJ. Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev. 1991;12(2):151–80. Epub 1991/05/01. pmid:1649044.
- 7. Dellegar SM, Murphy SA, Bourne AE, DiCesare JC, Purser GH. Identification of the factors affecting the rate of deactivation of hypochlorous acid by melatonin. Biochem Biophys Res Commun. 1999;257(2):431–9. Epub 1999/04/13. pmid:10198231.
- 8. Galano A, Tan DX, Reiter RJ. Melatonin as a natural ally against oxidative stress: a physicochemical examination. J Pineal Res. 2011;51(1):1–16. Epub 2011/07/15. pmid:21752095.
- 9. Galano A, Tan DX, Reiter RJ. On the free radical scavenging activities of melatonin's metabolites, AFMK and AMK. J Pineal Res. 2013;54(3):245–57. Epub 2012/09/25. pmid:22998574.
Mauriz JL, Collado PS, Veneroso C, Reiter RJ, Gonzalez-Gallego J. A review of the molecular aspects of melatonin's anti-inflammatory actions: recent insights and new perspectives. J Pineal Res. 2012. Epub 2012/06/26. https://doi.org/10.1111/j.1600-079X.2012.01014.x pmid:22725668.
- 11. Banerjee J, Maitra D, Diamond MP, Abu-Soud HM. Melatonin prevents hypochlorous acid-induced alterations in microtubule and chromosomal structure in metaphase-II mouse oocytes. J Pineal Res. 2012;53(2):122–8. Epub 2012/02/07. pmid:22304486.
- 12. Calvo JR, Gonzalez-Yanes C, Maldonado MD. The role of melatonin in the cells of the innate immunity: a review. J Pineal Res. 2013;55(2):103–20. Epub 2013/07/31. pmid:23889107.
- 13. Jung-Hynes B, Huang W, Reiter RJ, Ahmad N. Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells. J Pineal Res. 2010;49(1):60–8. Epub 2010/06/08. pmid:20524973; PubMed Central PMCID: PMC3158680.
- 14. Jung-Hynes B, Reiter RJ, Ahmad N. Sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer. J Pineal Res. 2010;48(1):9–19. Epub 2009/12/23. pmid:20025641; PubMed Central PMCID: PMC2948667.
- 15. Park SY, Jang WJ, Yi EY, Jang JY, Jung Y, Jeong JW, et al. Melatonin suppresses tumor angiogenesis by inhibiting HIF-1alpha stabilization under hypoxia. J Pineal Res. 2010;48(2):178–84. Epub 2010/05/08. pmid:20449875.
- 16. Tesoriere L, D'Arpa D, Conti S, Giaccone V, Pintaudi AM, Livrea MA. Melatonin protects human red blood cells from oxidative hemolysis: new insights into the radical-scavenging activity. J Pineal Res. 1999;27(2):95–105. Epub 1999/09/25. pmid:10496145.
- 17. Galijasevic S, Abdulhamid I, Abu-Soud HM. Melatonin is a potent inhibitor for myeloperoxidase. Biochemistry. 2008;47(8):2668–77. pmid:18237195.
- 18. Lu T, Galijasevic S, Abdulhamid I, Abu-Soud HM. Analysis of the mechanism by which melatonin inhibits human eosinophil peroxidase. Br J Pharmacol. 2008;154(6):1308–17. Epub 2008/06/03. pmid:18516076; PubMed Central PMCID: PMC2483384.
- 19. Davies MJ, Hawkins CL, Pattison DI, Rees MD. Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid Redox Signal. 2008;10(7):1199–234. Epub 2008/03/12. pmid:18331199.
- 20. Pennathur S, Maitra D, Byun J, Sliskovic I, Abdulhamid I, Saed GM, et al. Potent antioxidative activity of lycopene: A potential role in scavenging hypochlorous acid. Free Radic Biol Med. 2010;49(2):205–13. Epub 2010/04/15. pmid:20388538; PubMed Central PMCID: PMC3416054.
- 21. Maitra D, Byun J, Andreana PR, Abdulhamid I, Diamond MP, Saed GM, et al. Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release. Free Radic Biol Med. 2011;51(2):374–86. Epub 2011/05/10. pmid:21549834.
- 22. Abu-Soud HM, Hazen SL. Nitric oxide is a physiological substrate for mammalian peroxidases. J Biol Chem. 2000;275(48):37524–32. Epub 2000/11/25. pmid:11090610.
- 23. Abu-Soud HM, Hazen SL. Nitric oxide modulates the catalytic activity of myeloperoxidase. J Biol Chem. 2000;275(8):5425–30. Epub 2000/02/22. pmid:10681518.
- 24. Abu-Soud HM, Khassawneh MY, Sohn JT, Murray P, Haxhiu MA, Hazen SL. Peroxidases inhibit nitric oxide (NO) dependent bronchodilation: development of a model describing NO-peroxidase interactions. Biochemistry. 2001;40(39):11866–75. Epub 2001/09/26. pmid:11570887.
- 25. Maitra D, Abdulhamid I, Diamond MP, Saed GM, Abu-Soud HM. Melatonin attenuates hypochlorous acid-mediated heme destruction, free iron release, and protein aggregation in hemoglobin. J Pineal Res. 2012;53(2):198–205. Epub 2012/04/03. pmid:22462755.
- 26. Galijasevic S, Maitra D, Lu T, Sliskovic I, Abdulhamid I, Abu-Soud HM. Myeloperoxidase interaction with peroxynitrite: chloride deficiency and heme depletion. Free Radic Biol Med. 2009;47(4):431–9. Epub 2009/05/26. pmid:19464362; PubMed Central PMCID: PMC3416043.
- 27. Mohamed AO, Hashim MS, Nilsson UR, Venge P. Increased in vivo activation of neutrophils and complement in sickle cell disease. Am J Trop Med Hyg. 1993;49(6):799–803. Epub 1993/12/01. pmid:8279646.
- 28. Ong WY, Halliwell B. Iron, atherosclerosis, and neurodegeneration: a key role for cholesterol in promoting iron-dependent oxidative damage? Ann N Y Acad Sci. 2004;1012:51–64. Epub 2004/04/24. pmid:15105255.
- 29. Trinder D, Fox C, Vautier G, Olynyk JK. Molecular pathogenesis of iron overload. Gut. 2002;51(2):290–5. Epub 2002/07/16. pmid:12117898; PubMed Central PMCID: PMC1773304.
- 30. Defrere S, Lousse JC, Gonzalez-Ramos R, Colette S, Donnez J, Van Langendonckt A. Potential involvement of iron in the pathogenesis of peritoneal endometriosis. Mol Hum Reprod. 2008;14(7):377–85. Epub 2008/05/30. pmid:18508952.
- 31. Chau LY. Iron and atherosclerosis. Proc Natl Sci Counc Repub China B. 2000;24(4):151–5. Epub 2000/11/22. pmid:11087066.
- 32. Maitra D, Byun J, Andreana PR, Abdulhamid I, Saed GM, Diamond MP, et al. Mechanism of hypochlorous acid-mediated heme destruction and free iron release. Free Radic Biol Med. 2011;51(2):364–73. Epub 2011/04/07. pmid:21466849; PubMed Central PMCID: PMC3378337.
- 33. Souza CE, Maitra D, Saed GM, Diamond MP, Moura AA, Pennathur S, et al. Hypochlorous acid-induced heme degradation from lactoperoxidase as a novel mechanism of free iron release and tissue injury in inflammatory diseases. PloS one. 2011;6(11):e27641. pmid:22132121; PubMed Central PMCID: PMC3222650.
- 34. Maitra D, Shaeib F, Abdulhamid I, Abdulridha RM, Saed GM, Diamond MP, et al. Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway. Free Radic Biol Med. 2013;63C:90–8. Epub 2013/04/30. pmid:23624305.
- 35. Sliskovic I, Abdulhamid I, Sharma M, Abu-Soud HM. Analysis of the mechanism by which tryptophan analogs inhibit human myeloperoxidase. Free Radic Biol Med. 2009;47(7):1005–13. Epub 2009/07/15. pmid:19596067.
- 36. Galijasevic S, Abdulhamid I, Abu-Soud HM. Potential role of tryptophan and chloride in the inhibition of human myeloperoxidase. Free Radic Biol Med. 2008;44(8):1570–7. Epub 2008/02/19. pmid:18279680; PubMed Central PMCID: PMC2861567.
- 37. Abu-Soud HM, Maitra D, Byun J, Souza CE, Banerjee J, Saed GM, et al. The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride. Free Radic Biol Med. 2012;52(3):616–25. Epub 2011/12/06. pmid:22138102.
- 38. Carvalho LC, Estevao MS, Ferreira LM, Fernandes E, Marques MM. A new insight on the hypochlorous acid scavenging mechanism of tryptamine and tryptophan derivatives. Bioorg Med Chem Lett. 2010;20(22):6475–8. Epub 2010/10/12. pmid:20932760.
- 39. Yang YT, Whiteman M, Gieseg SP. Intracellular glutathione protects human monocyte-derived macrophages from hypochlorite damage. Life Sci. 2012;90(17–18):682–8. Epub 2012/04/05. pmid:22472425.
- 40. Schaffer SW, Azuma J, Mozaffari M. Role of antioxidant activity of taurine in diabetes. Can J Physiol Pharmacol. 2009;87(2):91–9. Epub 2009/02/24. pmid:19234572.
- 41. Proteasa G, Tahboub YR, Galijasevic S, Raushel FM, Abu-Soud HM. Kinetic evidence supports the existence of two halide binding sites that have a distinct impact on the heme iron microenvironment in myeloperoxidase. Biochemistry. 2007;46(2):398–405. Epub 2007/01/11. pmid:17209550.
- 42. Tahboub YR, Galijasevic S, Diamond MP, Abu-Soud HM. Thiocyanate modulates the catalytic activity of mammalian peroxidases. J Biol Chem. 2005;280(28):26129–36. Epub 2005/05/17. pmid:15894800.
- 43. Rakita RM, Michel BR, Rosen H. Differential inactivation of Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated antimicrobial system. Biochemistry. 1990;29(4):1075–80. Epub 1990/01/30. pmid:1692736.
- 44. Andrews PC, Krinsky NI. A kinetic analysis of the interaction of human myeloperoxidase with hydrogen peroxide, chloride ions, and protons. J Biol Chem. 1982;257(22):13240–5. Epub 1982/11/25. pmid:6292181.
- 45. Carter P. Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Anal Biochem. 1971;40(2):450–8. Epub 1971/04/01. pmid:5551554.
- 46. Rozov SV, Filatova EV, Orlov AA, Volkova AV, Zhloba AR, Blashko EL, et al. N1-acetyl-N2-formyl-5-methoxykynuramine is a product of melatonin oxidation in rats. J Pineal Res. 2003;35(4):245–50. Epub 2003/10/03. pmid:14521629.
- 47. Wang L, Bassiri M, Najafi R, Najafi K, Yang J, Khosrovi B, et al. Hypochlorous acid as a potential wound care agent: part I. Stabilized hypochlorous acid: a component of the inorganic armamentarium of innate immunity. J Burns Wounds. 2007;6:e5. Epub 2007/05/12. pmid:17492050; PubMed Central PMCID: PMC1853323.
- 48. Allen RC, Stephens JT Jr. Myeloperoxidase selectively binds and selectively kills microbes. Infect Immun. 2011;79(1):474–85. Epub 2010/10/27. pmid:20974824; PubMed Central PMCID: PMC3019908.
- 49. Maitra D, Abdulhamid I, Diamond MP, Saed GM, Abu-Soud HM. Melatonin attenuates hypochlorous acid-mediated heme destruction, free iron release, and protein aggregation in hemoglobin. Journal of pineal research. 2012;53(2):198–205. pmid:22462755.
- 50. Galijasevic S, Saed GM, Diamond MP, Abu-Soud HM. High dissociation rate constant of ferrous-dioxy complex linked to the catalase-like activity in lactoperoxidase. J Biol Chem. 2004;279(38):39465–70. Epub 2004/07/20. pmid:15258136.
- 51. Allegra M, Furtmuller PG, Regelsberger G, Turco-Liveri ML, Tesoriere L, Perretti M, et al. Mechanism of reaction of melatonin with human myeloperoxidase. Biochem Biophys Res Commun. 2001;282(2):380–6. Epub 2001/06/13. pmid:11401469.
- 52. Ximenes VF, Silva SO, Rodrigues MR, Catalani LH, Maghzal GJ, Kettle AJ, et al. Superoxide-dependent oxidation of melatonin by myeloperoxidase. J Biol Chem. 2005;280(46):38160–9. Epub 2005/09/09. pmid:16148002.
- 53. Paumann-Page M, Furtmuller PG, Hofbauer S, Paton LN, Obinger C, Kettle AJ. Inactivation of human myeloperoxidase by hydrogen peroxide. Arch Biochem Biophys. 2013. Epub 2013/09/17. pmid:24035742.
- 54. Furtmuller PG, Burner U, Jantschko W, Regelsberger G, Obinger C. The reactivity of myeloperoxidase compound I formed with hypochlorous acid. Redox Rep. 2000;5(4):173–8. Epub 2000/09/20. pmid:10994870.
- 55. van Dalen CJ, Whitehouse MW, Winterbourn CC, Kettle AJ. Thiocyanate and chloride as competing substrates for myeloperoxidase. Biochem J. 1997;327 (Pt 2):487–92. Epub 1997/11/14. pmid:9359420; PubMed Central PMCID: PMC1218820.
- 56. Hallingback HR, Gabdoulline RR, Wade RC. Comparison of the binding and reactivity of plant and mammalian peroxidases to indole derivatives by computational docking. Biochemistry. 2006;45(9):2940–50. Epub 2006/03/01. pmid:16503648.
- 57. Paumann-Page M, Furtmuller PG, Hofbauer S, Paton LN, Obinger C, Kettle AJ. Inactivation of human myeloperoxidase by hydrogen peroxide. Arch Biochem Biophys. 2013;539(1):51–62. Epub 2013/09/17. pmid:24035742; PubMed Central PMCID: PMC3808540.
- 58. Bonini MG, Siraki AG, Atanassov BS, Mason RP. Immunolocalization of hypochlorite-induced, catalase-bound free radical formation in mouse hepatocytes. Free Radic Biol Med. 2007;42(4):530–40. Epub 2007/02/06. pmid:17275685; PubMed Central PMCID: PMC1952183.
- 59. Aruoma OI, Halliwell B. Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase. Biochem J. 1987;248(3):973–6. Epub 1987/12/15. pmid:2829848; PubMed Central PMCID: PMC1148647.
- 60. Galijasevic S, Saed GM, Diamond MP, Abu-Soud HM. Myeloperoxidase up-regulates the catalytic activity of inducible nitric oxide synthase by preventing nitric oxide feedback inhibition. Proc Natl Acad Sci U S A. 2003;100(25):14766–71. Epub 2003/12/06. pmid:14657339; PubMed Central PMCID: PMC299800.
- 61. Tekbas OF, Ogur R, Korkmaz A, Kilic A, Reiter RJ. Melatonin as an antibiotic: new insights into the actions of this ubiquitous molecule. J Pineal Res. 2008;44(2):222–6. Epub 2008/02/22. pmid:18289175.
- 62. Gomez FJ, Raba J, Cerutti S, Silva MF. Monitoring melatonin and its isomer in Vitis vinifera cv. Malbec by UHPLC-MS/MS from grape to bottle. J Pineal Res. 2012;52(3):349–55. Epub 2012/01/10. pmid:22225625.
- 63. Gulcin I, Buyukokuroglu ME, Kufrevioglu OI. Metal chelating and hydrogen peroxide scavenging effects of melatonin. J Pineal Res. 2003;34(4):278–81. Epub 2003/03/29. pmid:12662350.
Reiter RJ, Korkmaz A, Ma S, Rosales-Corral S, Tan DX. Melatonin protection from chronic, low-level ionizing radiation. Mutat Res. 2011. Epub 2011/12/22. https://doi.org/10.1016/j.mrrev.2011.12.002 pmid:22185900.
- 65. Carrillo-Vico A, Guerrero JM, Lardone PJ, Reiter RJ. A review of the multiple actions of melatonin on the immune system. Endocrine. 2005;27(2):189–200. Epub 2005/10/12. pmid:16217132.
- 66. Ogino T, Than TA, Hosako M, Ozaki M, Omori M, Okada S. Taurine chloramine: a possible oxidant reservoir. Adv Exp Med Biol. 2009;643:451–61. Epub 2009/02/26. pmid:19239177.
- 67. Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI, Halliwell B. Evaluation of the antioxidant activity of melatonin in vitro. Free Radic Biol Med. 1996;21(3):307–15. Epub 1996/01/01. pmid:8855441.
- 68. Ximenes VF, Padovan CZ, Carvalho DA, Fernandes JR. Oxidation of melatonin by taurine chloramine. J Pineal Res. 2010;49(2):115–22. Epub 2010/06/16. pmid:20545824.
- 69. Grisham MB, Jefferson MM, Melton DF, Thomas EL. Chlorination of endogenous amines by isolated neutrophils. Ammonia-dependent bactericidal, cytotoxic, and cytolytic activities of the chloramines. J Biol Chem. 1984;259(16):10404–13. Epub 1984/08/25. pmid:6381484.
- 70. Ohshima H, Tatemichi M, Sawa T. Chemical basis of inflammation-induced carcinogenesis. Arch Biochem Biophys. 2003;417(1):3–11. Epub 2003/08/19. pmid:12921773.
- 71. Schiller J, Fuchs B, Arnhold J, Arnold K. Contribution of reactive oxygen species to cartilage degradation in rheumatic diseases: molecular pathways, diagnosis and potential therapeutic strategies. Curr Med Chem. 2003;10(20):2123–45. Epub 2003/07/23. pmid:12871089.
- 72. Van Der Vliet A, Nguyen MN, Shigenaga MK, Eiserich JP, Marelich GP, Cross CE. Myeloperoxidase and protein oxidation in cystic fibrosis. American journal of physiology Lung cellular and molecular physiology. 2000;279(3):L537–46. pmid:10956629.
- 73. Tzikas S, Schlak D, Sopova K, Gatsiou A, Stakos D, Stamatelopoulos K, et al. Increased myeloperoxidase plasma levels in patients with Alzheimer's disease. Journal of Alzheimer's disease: JAD. 2014;39(3):557–64. pmid:24217274.
- 74. Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arteriosclerosis, thrombosis, and vascular biology. 2005;25(6):1102–11. pmid:15790935.
- 75. Podrez EA, Abu-Soud HM, Hazen SL. Myeloperoxidase-generated oxidants and atherosclerosis. Free Radic Biol Med. 2000;28(12):1717–25. pmid:10946213.
- 76. Johnson S. The possible role of gradual accumulation of copper, cadmium, lead and iron and gradual depletion of zinc, magnesium, selenium, vitamins B2, B6, D, and E and essential fatty acids in multiple sclerosis. Med Hypotheses. 2000;55(3):239–41. Epub 2000/09/14. pmid:10985916.
- 77. Weinberg ED. Microbial pathogens with impaired ability to acquire host iron. Biometals. 2000;13(1):85–9. Epub 2000/06/01. pmid:10831229.
- 78. Weinberg ED. Modulation of intramacrophage iron metabolism during microbial cell invasion. Microbes Infect. 2000;2(1):85–9. Epub 2000/03/16. pmid:10717545.
- 79. Hantke K. Iron and metal regulation in bacteria. Curr Opin Microbiol. 2001;4(2):172–7. Epub 2001/04/03. pmid:11282473.