Ibrahim Abdulhamid

Melatonin Is a Potent Inhibitor for Myeloperoxidase

Myeloperoxidase (MPO) catalyzes the formation of potent oxidants that have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Melatonin plays an important part in the regulation of various body functions including circadian sleep rhythms, blood pressure, oncogenesis, retinal function, seasonal reproduction, and immunity. Here, we demonstrate that melatonin serves as a potent inhibitor of MPO under physiological-like conditions. In the presence of chloride (Cl-), melatonin inactivated MPO at two points in the classic peroxidase cycle through binding to MPO to form an inactive complex, melatonin-MPO-Cl, and accelerating MPO compound II formation, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO compound II without any sign of compound I accumulation. This behavior indicates that melatonin binding modulates the formation of MPO intermediates and their decay rates. The Cl- presence enhanced the affinity of MPO toward melatonin, which switches the enzyme activity from peroxidation to catalase-like activity. In the absence of Cl-, melatonin served as a 1e- substrate for MPO compound I, but at higher concentration it limited the reaction by its dissociation from the corresponding complex. Importantly, melatonin-dependent inhibition of MPO occurred with a wide range of concentrations that span various physiological and supplemental ranges. Thus, the interplay between MPO and melatonin may have a broader implication in the function of several biological systems. This dual regulation by melatonin is unique and represents a new means through which melatonin can control MPO and its downstream inflammatory pathways.

Potent Antioxidative Activity of Lycopene: A Potential Role in Scavenging Hypochlorous Acid

Lycopene, a carotenoid found in tomatoes, is a proven antioxidant that may lower the risk of certain disorders including heart disease and cancer. Hypochlorous acid (HOCl) is an oxidant linked to tissue oxidation in cardiovascular disease and other inflammatory disorders through its ability to modify proteins, deoxyribonucleic acid, ribonucleic acid, and lipids. Here we show that lycopene can function as a potent scavenger of HOCl at a wide range of concentrations that span various pathophysiological and supplemental ranges. The oxidation of lycopene by HOCl was accompanied by a marked change in color, from red to colorless, of the lycopene solution, suggesting lycopene degradation. HPLC and LC-MS analysis showed that the exposure of lycopene to increasing concentrations of HOCl gave a range of metabolites resulting from oxidative cleavage of one or more C=C. The degree of degradation of lycopene (as assessed by the number and chain lengths of the various oxidative metabolites of lycopene) depends mainly on the ratio of HOCl to lycopene, suggesting that multiple molecules of HOCl are consumed per molecule of lycopene. Collectively, this work demonstrates a direct link between lycopene and HOCl scavenging and may assist in elucidating the mechanism of the protective function exerted by lycopene.

Reaction of hemoglobin with HOCl: Mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl(-)) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl(-) was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl(-)) and hydrogen peroxide (H₂O₂). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV-visible spectra and H₂O₂-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H₂O₂ (10 µM), precluding the enzyme from functioning at maximum activity (80-90% inhibition). MPO heme destruction occurred only in the presence of Cl(-). Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO-Compound I-Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.

Mechanism of hypochlorous acid mediated heme destruction and free iron release

Here, we show that hypochlorous acid (HOCl), a potent neutrophil-generated oxidant, can mediate destruction of free heme (Ht) and the heme precursor, protoporphyrin IX (PPIX). Ht displays a broad Soret absorbance peak centered at 365 and 394 nm, indicative of the presence of monomer and μ-oxo-dimer. Oxidation of Ht by HOCl was accompanied by a marked decrease in the Soret absorption peak and release of free iron. Kinetic measurements showed that the Ht-HOCl reaction was triphasic. The first two phases were HOCl concentration dependent and attributable to HOCl binding to the monomeric and dimeric forms. The third phase was HOCl concentration independent and attributed to Ht destruction with the release of free iron. HPLC and LC-ESI-MS analyses of the Ht-HOCl reaction revealed the formation of a number of degradation products, resulting from the cleavage or modification of one or more carbon-methene bridges of the porphyrin ring. Similar studies with PPIX showed that HOCl also mediated tetrapyrrole ring destruction. Collectively, this work demonstrates the ability of HOCl to modulate destruction of heme, through a process that occurs independent of the iron molecule that resides in the porphyrin center. This phenomenon may play a role in HOCl-mediated oxidative injury in pathological conditions.

Effect of zinc supplementation on respiratory tract infections in children with cystic fibrosis

Zinc (Zn) has significant anti‐oxidant and anti‐inflammatory activity. Zn deficiency can occur in subsets of patients with cystic fibrosis (CF) especially those with malabsorption and impaired growth. Although supplemental Zn has significantly reduced infections in various disorders, its efficacy has not been thoroughly investigated in CF. We performed a double blind placebo controlled pilot study to investigate the effect of daily 30 mg elemental Zn for 1 year on the rate of respiratory tract infections (RTIs), use of antibiotics and plasma cytokines in 26 children with CF (ages 7–18 years). Plasma Zn, Cu, inflammatory cytokines and ex vivo generation of IL‐2 were measured at baseline and at the end of the study. The number of days of oral antibiotics was lower in Zn treated patients compared to placebo (P = 0.05). However, compared to placebo, the effect of Zn was greater in patients who exhibited low plasma Zn at baseline (P = 0.02) than those who had plasma Zn levels identical to normal subjects (P = 0.55). Zn supplementation was marginally effective in reducing percentage increase in plasma IL‐6 and IL‐8 while increasing the percentage change in ex vivo generation of IL‐2 in isolated mononuclear cell. In conclusion, oral intake of 30 mg/day of Zn reduced the number of days of oral antibiotics used to treat RTIs in children with CF. A higher daily Zn dose may be needed to decrease RTIs and modify immune responses. Pediatr Pulmonol. 2008; 43:281–287.

Analysis of the mechanism by which tryptophan analogs inhibit human myeloperoxidase.

Myeloperoxidase (MPO) catalyzes the formation of oxidants that have been implicated in the pathogenesis of various diseases, including cardiovascular and pulmonary diseases and cancer. Inhibition of MPO oxidant-generating activity represents an attractive target for preventing the progression of inflammatory conditions. Peroxidation and chlorination catalytic activity were utilized to screen for the most effective tryptophan analog that inhibits MPO. Rapid kinetic measurements were performed to determine the mechanisms through which these compounds inhibit the catalytic activity of MPO. Substituents on the amino and carboxyl termini of tryptophan enhance its affinity toward MPO compared to a substituent on the indole ring. Hydrogen-bond donor capabilities and a positive charge on the amino group are not required for MPO inhibition. Hydroxyl-containing indoles did not inhibit MPO H(2)O(2)-consumption activity. Elimination of the negative charge from the carboxyl terminus by introducing a hydrophobic character significantly enhanced tryptophan analog affinity for MPO and improved its inhibitory properties. Further mechanistic studies indicated that indole compounds inhibited MPO activity through the accumulation of compound II, an inactive MPO intermediate. Our results show that specific structural features of tryptophan analogs are involved in increasing the affinity for MPO and providing a significantly greater inhibition of MPOs catalytic activities.

Potential role of tryptophan and chloride in the inhibition of human myeloperoxidase

Myeloperoxidase (MPO) binds H2O2 in the absence and presence of chloride (Cl-) and catalyzes the formation of potent oxidants through 1e(-) and 2e(-) oxidation pathways. These potent oxidants have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Thus, inhibition of MPO and its by-products may have a wide application in biological systems. Using direct rapid kinetic measurements and H2O2-selective electrodes, we show that tryptophan (Trp), an essential amino acid, is linked kinetically to the inhibition of MPO catalysis under physiological conditions. Trp inactivated MPO in the absence and presence of plasma levels of Cl(-), to various degrees, through binding to MPO, forming the inactive complexes Trp-MPO and Trp-MPO-Cl, and accelerating formation of MPO Compound II, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO Compound II without any sign of Compound I accumulation. This behavior indicates that Trp binding modulates the formation of MPO intermediates and their decay rates. Importantly, Trp is a poor substrate for MPO Compound II and has no role in destabilizing complex formation. Thus, the overall MPO catalytic activity will be limited by: (1) the dissociation of Trp from Trp-MPO and Trp-MPO-Cl complexes, (2) the affinity of MPO Compound I toward Cl(-) versus Trp, and (3) the slow conversion of MPO Compound II to MPO-Fe(III). Importantly, Trp-dependent inhibition of MPO occurred at a wide range of concentrations that span various physiological and supplemental ranges.

Myeloperoxidase interaction with peroxynitrite: chloride deficiency and heme depletion

Myeloperoxidase (MPO) is a hemoprotein involved in the leukocyte-mediated defense mechanism and uses hydrogen peroxide (H2O2) and chloride (Cl(-)) to produce hypochlorous acid. In human saliva and in hypochloremic alkalosis syndrome occurring in breast-fed infants, the MPO-H2O2 system functions in a lower Cl(-) concentration (10-70 mM) compared to plasma levels (100 mM) as part of the antibacterial defense system. The impact of low Cl(-) concentration and exposure to high peroxynitrite (ONOO(-)) synthesized from cigarette smoke or oxidative stress on MPO function is still unexplored. Rapid mixing of ONOO(-) and MPO caused immediate formation of a transient intermediate MPO Compound II, which then decayed to MPO-Fe(III). Double mixing of MPO with ONOO(-) followed by H2O2 caused immediate formation of Compound II, followed by MPO heme depletion, a process that occurred independent of ONOO(-) concentration. Peroxynitrite/H2O2-mediated MPO heme depletion was confirmed by HPLC analysis, and in-gel heme staining showing 60-70% less heme content compared to the control. A nonreducing denaturing SDS-PAGE showed no fragmentation or degradation of protein. Myeloperoxidase heme loss was completely prevented by preincubation of MPO with saturating amounts of Cl(-). Chloride binding to the active site of MPO constrains ONOO(-) binding by filling the space directly above the heme moiety or by causing a protein conformational change that constricts the distal heme pocket, thus preventing ONOO(-) from binding to MPO heme iron. Peroxynitrite interaction with MPO may serve as a novel mechanism for modulating MPO catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Quality of life as a treatment outcome in patients with cystic fibrosis

We attempted to determine the responsiveness and validity of the Quality of Well‐Being (QWB) scale in 20 consecutive children and adolescents with cystic fibrosis. The QWB score was determined for 6‐day periods immediately before and after hospital admission, and at 6‐ and 12‐month follow‐up. With the instruments scale of zero‐1, responsiveness was indicated by significant changes in QWB score (0.09), physical (0.019), social (0.021), and symptom‐problem complexes (0.04) domains, and all pulmonary function tests from before to after treatment of an acute exacerbation. Only the symptom‐problem complex domain significantly changed from after treatment to 6‐ and 12‐month follow‐up. Validity was shown by significant correlations between before and after QWB scores and forced vital capacity (r=0.476), residual volume total lung capacity ratio (r=0.452), forced expiratory volume in 1 second (r=0.358), and forced expiratory flow between 25% and 75% of vital capacity (r=0.35).