Plasma Concentrations of Myeloperoxidase Predict Mortality After Myocardial Infarction

ObjectivesThis study investigated relationships between plasma myeloperoxidase (MPO), protein oxidation markers, and clinical outcome retrospectively in patients after acute myocardial infarction (MI).BackgroundReactive oxidants are implicated in cardiovascular disease, and elevated plasma MPO is reported to predict adverse outcome in acute coronary syndromes.MethodsDetailed demographic information, radionuclide ventriculography, neurohormone measurements, and clinical history were obtained for 512 acute MI patients at hospital admission. Plasma levels of MPO and protein carbonyls were measured in patients and 156 heart-healthy control subjects. 3-Chlorotyrosine was measured in selected patients. Patient mortality was followed for 5 years.ResultsPlasma MPO and protein carbonyl concentrations were higher in MI patients 24 h to 96 h after admission than in control subjects (medians: MPO 55 ng/ml vs. 39 ng/ml, and protein carbonyls 48 pmol/mg vs. 17 pmol/mg protein, p < 0.001 for each). Both markers were significantly correlated with each other and with cardiovascular hormone levels. Chlorotyrosine was not elevated in patients with high MPO or carbonyl levels. Above-median levels of MPO but not protein carbonyls were independently predictive of mortality (odds ratio 1.8, 95% confidence interval 1.0 to 3.0, p = 0.034). Patients with above-median MPO levels in combination with above-median plasma amino-terminal pro-brain natriuretic peptide (NT-proBNP) or below-median left ventricular ejection fraction (LVEF) had significantly greater mortality compared with other patients.ConclusionsMyeloperoxidase and protein carbonyl levels are elevated in plasma after acute MI, apparently via independent mechanisms. High MPO is a risk factor for long-term mortality and adds prognostic value to LVEF and plasma NT-proBNP measurements

A myeloperoxidase precursor, promyeloperoxidase, is present in human plasma and elevated in cardiovascular disease patients

Myeloperoxidase (MPO)-derived oxidants have emerged as a key contributor to tissue damage in inflammatory conditions such as cardiovascular disease. Pro-myeloperoxidase (pro-MPO), an enzymatically active precursor of myeloperoxidase (MPO), is known to be secreted from cultured bone marrow and promyelocytic leukemia cells, but evidence for the presence of pro-MPO in circulation is lacking. In the present study, we used a LC-MS/MS in addition to immunoblot analyses to show that pro-MPO is present in human blood plasma. Furthermore, we found that pro-MPO was more frequently detected in plasma from patients with myocardial infarction compared to plasma from control donors. Our study suggests that in addition to mature MPO, circulating pro-MPO may cause oxidative modifications of proteins thereby contributing to cardiovascular disease

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nM

Increased detection of pro-MPO in plasma from patients with myocardial infarction (MI) compared to healthy controls.

<p>Plasma was purified by affinity chromatography with MPO-specific antibodies, subjected to digestion with trypsin and analyzed for the presence of pro-MPO and mature MPO-specific peptides by SRM-based LC-MS/MS. Samples that showed a peak are denoted by “+”, those that don’t by “-”.</p

Halogenation activity of mature MPO and recombinant pro-MPO.

<p>Initial rates of NADH oxidation were determined with various concentrations of A) bromide or B) chloride, 20 nM mature MPO or recombinant pro-MPO and 100 μM NADH in 50 mM phosphate buffer (pH 7.4) at 21°C. The reaction was started by adding 50 μM H₂O₂. The kinetics of the reaction of myeloperoxidase-generated hypobromous and hypochlorous acids with NADH were monitored by measuring the bromohydrin and chlorohydrin product at 275 nm. Data are representative of two or more experiments.</p

LC-MS/MS analysis of mutual, pro-MPO- and MPO- specific peptides.

<p><b>A)</b> Location of tryptic peptides used for LC-MS/MS analysis within the MPO sequence. The <i>N-</i>terminal signal peptide is shown in dark grey, the pro-peptide in bold light grey and the sequence of mature MPO in bold black. Highlighted by the black and grey boxes are tryptic peptides specific to pro-MPO or present in both mature MPO and pro-MPO, respectively. Twenty-five μg of recombinant pro-MPO or mature MPO were digested with trypsin and analyzed by LC-MS/MS. <b>B), D) +F)</b> Extracted ion chromatograms for SRM transitions specific for the mutual, pro-MPO and mature MPO-specific peptides (MS1/MS2 551.8/272.2, 1000.9/1033.5 and 648.8/935.5, respectively, MS1 = m/z for the doubly charged precursor species and MS2 = m/z for the singly charged y–ion fragment, c–carbamidomethyl-cysteine). <b>C), E) +G)</b> CID-MS/MS spectra confirming the sequence of the respective peptide. Representative chromatograms and spectra are shown.</p

Detection of mature MPO and pro-MPO in plasma using affinity-purification and immunblot analysis.

<p><b>A)</b> Plasma was spiked with MPO standard (10 nM) and HL60 cell lysate (containing 10 nM MPO) and subjected to affinity purification. Purified samples along with MPO, pro-MPO standards and HL-60 lysates were separated by 10% SDS/PAGE, transferred to PVDF and probed with MPO-specific antibody. <b>B)</b> Neutrophils (5x10⁶ cells/ml) were added back into plasma and then stimulated with CytB and FMLP for 30 min at 37°C. Neutrophils were centrifuged and cell free plasma MPO was subjected to affinity purification and analyzed as described in A. After the ECL fluorescence of blots was developed, a photograph of the blot showing the molecular weight markers was taken and aligned with the fluorescence image as indicated by the black line.</p

LC-MS/MS detection of pro-MPO in affinity-purified plasma separated by SDS/PAGE.

<p>Plasma was purified by affinity chromatography with an MPO-specific antibody and subjected to separation by SDS/PAGE. Bands with the molecular weight of pro-MPO (90 kDa) were subjected to in-gel tryptic digestion and analyzed by LC-MS/MS using SRM for a pro-MPO-specific peptide (1000.9->1033.5). <b>(A)</b> Extracted ion chromatogram for the SRM transition for the pro-MPO specific peptide. <b>(B)</b> CID-MS/MS spectrum confirming the sequence for the pro-MPO-specific peptide SSGcAYQDVGVTcPEQDK. Representative chromatograms and spectra are shown.</p