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Peptides 43 (2013) 121–125

Contents lists available at SciVerse ScienceDirect

Peptides

j ourna l h o mepa ge: www.elsev ier .com/ locate /pept ides

lasma intermedin levels in patients with acute myocardial infarction

hengbing Lva,1, Kai Wub,1, Xiaoping Chena,∗, Xin Zhanga, Biying Honga

Department of Cardiology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, ChinaDepartment of Cardiology, 903 Hospital, Jiangyou, Sichuan 621700, China

r t i c l e i n f o

rticle history:eceived 18 February 2013eceived in revised form 7 March 2013ccepted 7 March 2013vailable online 15 March 2013

eywords:ntermedin

a b s t r a c t

It has been shown that adrenomedullin (ADM) may function as a cardiovascular-regulatory peptide inhumans. Intermedin (IMD) is a newly discovered peptide related to ADM and has a greater range of bio-logical effects on the cardiovascular in animal experiments. The purpose of the study was to investigatethe pathophysiological role of IMD in patients with acute myocardial infarction (AMI). The present studyincluded twenty patients with acute ST-segment elevation myocardial infarction (STEMI), thirty-threewith stable coronary heart disease (SCHD), and eighteen healthy controls. Plasma levels of IMD, mal-onaldehyde (MDA), and superoxide dismutase (SOD) and cardiac biomarkers were determined at one,

cute myocardial infarctionxidative stress

two, four and seven days following AMI. Plasma IMD levels were significantly increased on day 1 in AMIpatients when compared with SCHD subjects (P = 0.014), and reached a peak of 181.88 ± 9.47 pg/ml at96 h. Plasma IMD concentrations were correlated with MDA and SOD. Furthermore, patients with severelesions in their coronary arteries tended to have higher plasma IMD levels (P < 0.05) in AMI patients. Asignificant increase in plasma IMD following AMI may be associated with oxidative stress, and could beused as a marker to reflect the severity of the coronary stenosis.

. Introduction

Cardiovascular diseases are a leading cause of mortality andorbidity according to estimates by the World Health Organization

WHO) [13]; however, the mechanisms of these diseases remainnclear. Vascular endothelial cell damage, insulin resistance andtherosclerosis induced by adipose infiltration, inflammation andxidative stress play critical roles in the pathogenesis of coronaryeart disease and its complications [14,16,23].

Intermedin (IMD), also called adrenomedullin 2 (ADM2), is newly discovered peptide related to calcitonin gene-relatedeptide (CGRP) and ADM. Many studies have shown that concen-rations of ADM in cardiovascular tissue, plasma and urine werelevated in patients with acute coronary syndrome (ACS) [3,10,15].ontinuous administration of ADM had beneficial effects on remod-ling of left ventricle and hemodynamics in rates with myocardialnfarction, suggesting the possibility that ADM could be a usefulherapeutic tool for acute myocardial infarction (AMI) [18] throughounteracting renin–angiotensin–aldosterone system (RAAS) and

xidative stress [12,22]. It may also be an independent predic-or of prognosis in patients with AMI [19]. IMD has significantasodilator and cardiac protective actions similar to the related

∗ Corresponding author. Tel.: +86 028 85423352; fax: +86 028 85422175.E-mail address: xiaopingc12@yahoo.cn (X. Chen).

1 These authors contributed equally to this work.

196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.peptides.2013.03.007

© 2013 Elsevier Inc. All rights reserved.

peptide ADM [21,24]. It exists extensively in the heart, kidney,hypothalamus, and plasma, and has a greater range of biologicaleffects on the cardiovascular, respiratory and central nervous sys-tems [17,24]. It dilates coronary arteries and enhances myocardialcontraction [26] and reduces reactive oxygen species formation invivo. The pathophysiological role of upregulated gene expressionof AM2/IMD in the hearts of congestive heart failure (CHF) rats andincreased plasma IMD levels in ischemia/reperfusion injury in iso-lated rat hearts seems to be related to its cardioprotective effects[8,27]. Recently, we discovered that exogenous administration ofIMD could prevent the progression of atherosclerotic plaque [29].Qin et al. [20] also found that the levels of IMD increased in patientswith ACS. However, the dynamic tendency of IMD in patients withAMI is not clear, and whether it is a sensitive marker for the earlydiagnosis of coronary heart disease (CHD) remains unknown. Thepresent study was designed to investigate the time course of plasmaIMD levels and the potential role of IMD in AMI patients.

2. Materials and methods

2.1. Patients and study protocol

Twenty consecutive patients (75% men and 25% female; mean

age 61.75 ± 13.521) were initially examined by ST-segment ele-vation myocardial infarction (STEMI) from the Intensive CareUnit (ICU) of West China Hospital Sichuan University within sixhours following the onset of chest pain. We included a group of

1 des 43 (2013) 121–125

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Table 1Baseline characteristics of the study patients.

Risk factors Groups

Control (n = 18) SCHD (n = 33) AMI (n = 20)

Age (years) 60.70 ± 2.21 61.20 ± 5.03 61.75 ± 13.52Sex (male%) 60% 77.4% 75.0%Hypertension 0% 70.9% 35.0%DM history 0% 22.6% 20.0%Smoking 10% 41.9% 40.0%SBP (mmHg) 118.20 ± 10.40 122.80 ± 13.80 124.00 ± 17.49DBP (mmHg) 69.50 ± 10.80 73.60 ± 7.09 73.30 ± 12.25HR (bpm) 74.00 ± 6.00 74.10 ± 11.51 73.90 ± 12.99BMI (kg/cm2) 22.84 ± 1.87 23.03 ± 2.66 24.01 ± 2.90Cr (�mol/L) 73.44 ± 16.23 81.96 ± 22.08 88.51 ± 16.93UA (�mol/L) 300.67 ± 39.75 369.91 ± 138.89 372.67 ± 88.46FBG (mmol/L) 4.41 ± 0.56 5.72 ± 1.20* 7.40 ± 1.99*

TG (mmol/L) 1.64 ± 0.42 2.07 ± 0.63 1.91 ± 0.96TC (mmol/L) 4.04 ± 0.94 3.63 ± 0.80* 4.07 ± 1.04*

HDL (mmol/L) 1.65 ± 0.29 1.21 ± 0.36* 1.26 ± 0.33*

* *

22 Z. Lv et al. / Pepti

hirty-three outpatients (77.4% men and 22.6% female; mean age1.20 ± 5.029) with stable coronary heart disease (SHCD) from ourospital, and a group of eighteen control subjects from a commu-ity (age- and sex-matched), who had no previous diagnosis ofypertension, diabetes mellitus, other chronic diseases, and hadot received any pharmacological therapy during the month prioro the study. AMI diagnosis was made using the following criteria15]: (1) typical chest pain with a duration >30 min; (2) electro-ardiographic ST-segment elevation ≥0.1 mV in either two or moreimb leads, or ≥0.2 mV in either two or more precordial leads; (3)nd an elevation of serum creatine phosphokinase more than twoimes the upper limit of the normal range. All subjects providedritten informed consent to participate in the study. The currentrotocol was approved by the Human Ethics Committee of the Westhina Hospital Sichuan University. Patients with renal failure (eGFRestimated glomerular filtration rate) < 30 ml/min/1.73 m2), malig-ancy, valvular heart disease, inflammatory diseases, and bloodoagulation system disorders were excluded from the presenttudy.

Peripheral venous blood samples for the measurements of IMD,alonaldehyde (MDA), superoxide dismutase (SOD) and cardiac

iomarkers (myoglobin (Mb), creatine kinase MB (CK-MB) and car-iac troponin T (CTn-T)) were obtained from each patient with AMIt 12–24 h (day 1), 48 h (day 2), 96 h (day 4) and 168 h (day 7)ollowing AMI, respectively. Blood samples from both SCHD andontrol subjects were collected for further assay. Plasma was sep-rated using centrifugation at 3000 × g for 15 min at 4 ◦C and wastored at −80 ◦C until extracted.

.2. Peptide extraction and radioimmunoassay of plasma levels ofMD

Plasma IMD levels were assayed according to the method of Ryoorimoto et al. [17]. Briefly, plasma samples were extracted with

ep-Pak C18 cartridges (Waters, Milford, MA, USA) and assayedy use of a radioimmunoassay kit (Phoenix Pharmaceuticals Inc.,elmont, CA, USA). Human AM2/IMD1–47 (Peptide Institute) wassed as a standard. [125I] AM2/IMD1–47 (Phoenix) was used as aadioligand. The assay could detect changes of 3.3 ± 1.2 fmol/tubemean ± SD; n = 5) from zero at 95% confidence with duplicateubes. The crossreactivities were 100% with human IMD and lesshan 0.001% with other peptides including human ADM and CGRP.ntra- and interassay coefficients of variation were less than 5% and0%, respectively.

.3. Measurement of other variables

Fasting plasma glucose, serum lipids, brain natriuretic peptideBNP) and myocardial markers were determined using standardaboratory measurements in our hospital laboratories. Lipid perox-dation was estimated by measuring TBARS according to a modified

ethod of Jentzsch et al. [9]. Superoxide dismutase levels wereeasured using an ELISA assay, as previously described [1].

.4. Documentation of coronary artery disease severity

During hospitalization, all patients with AMI underwentoronary angiography. The angiograms were assessed by two car-iologists who were unaware that the patients were to be included

n the study. The coronary angiography score [25] was used to esti-ate the degree of coronary atherosclerosis. It was defined as the

umber of coronary branches (right, left anterior descending, andeft circumflex) with more than 75% stenosis, with scores rangingrom 0 to 3. We also excluded patients who only showed signifi-ance (more than 50% luminal obstruction) left of the main disease.

LDL (mmol/L) 2.92 ± 0.78 2.46 ± 0.55 3.23 ± 0.82

* P < 0.05 versus control subjects.

2.5. Statistical analyses

Data are expressed as mean ± standard error of the mean, and allother variables are shown as a percentage of the control patients.Statistical significance of differences among groups was evaluatedusing ANOVA. The time course of plasma IMD, MDA, SOD and car-diac biomarkers was evaluated by analysis of variance for repeatedmeasures. When the F value was found to be significant, the datawere compared using a Dunnett’s multiple comparison tests. Dif-ferences between the two groups were analyzed using either a �2

test or an unpaired Student’s t-test. Differences were consideredsignificant if P < 0.05.

3. Results

3.1. Patient characteristics

Baseline characteristics, including age, sex, body mass index(BMI), blood pressure, blood biochemical data, history of hyper-tension and diabetes mellitus among the patients with AMI, SCHDand the control subjects are listed in Table 1. We observed a largenumber of men with AMI (male:female = 3:1). The three groups didnot differ significantly among age, gender, blood pressure, creati-nine and uric acid, but the ANOVA showed significant differencesin FBG, TC, HDL and LDL among the three groups. Statistical dif-ferences were also determined using pairwise comparison. TC andLDL were significantly lower in patients with SCHD than in con-trols (P < 0.05) and the AMI group (P < 0.05); patients with AMI hadthe highest FBG among the three groups (P < 0.05), and the SCHDpatients had the lowest HLD (P < 0.05).

3.2. Levels of IMD, SOD, MDA, BNP and cardiac biomarkers inSCHD and control groups and the time course of these parametersin AMI patients

Plasma IMD, MDA and BNP levels in SCHD patients were signif-icantly higher than in controls (P < 0.05), with a decrease in SODactivity in AMI patients (P < 0.001) when compared with the con-trols (Table 2). The time course of IMD, MDA, SOD and cardiacbiomarkers is shown in Table 2, which reveals the variations inthese indicators from the time of admission to the hospital to the

seventh day following MI. At the time of admission to the hospi-tal, patients with AMI exhibited elevated CTn-T levels comparedto SCHD subjects (P < 0.001). The dynamic tendencies of CTn-T and

Z. Lv et al. / Peptides 43 (2013) 121–125 123

Table 2Changes in data in the patients with AMI.

Varies Control SCHD Time after AMI

12–24Yh (day 1) 48Yh (day 2) 96Yh (day 4) 168Yh (day 7)

IMD (pg/ml) 125.92 ± 9.24 148.80 ± 6.04 a,** 158.94 ± 11.96b,* 174.08 ± 11.15 181.88 ± 9.47 159.17 ± 8.40b,*

MDA (nmol/ml) 3.78 ± 3.38 5.39 ± 0.40a,** 6.99 ± 0.50b,** 8.47 ± 0.46 9.36 ± 0.22 7.73 ± 0.36b,**

SOD (u/ml) 111.59 ± 2.91 90.85 ± 4.49a,** 72.68 ± 4.98 b,** 60.51 ± 7.12 53.19 ± 7.00 72.14 ± 5.72b,**

CK-MB (ng/ml) – – 76.02 ± 71.42 78.69 ± 96.74 14.58 ± 26.99 2.94 ± 1.35CTn-T (ng/L) – 9.80 ± 2.49 773.46 ± 960.71b,** 2983.40 ± 2726.63 2503.10 ± 2310.0 1158.52 ± 1017.42b,**

BNP (pg/L) 52.70 ± 15.14 196.90 ± 50.34a,** 1940.00 ± 2220.81b,** – – 1895.50 ± 1439.15b,**

a P < 0.05 versus control subjects.b P < 0.05 versus SHCD subjects.* P < 0.05.

** P < 0.01.

Table 3Correlation coefficients between plasma IMD levels and other related parameters at various time points following the onset of AMI.

12–24 h 48 h 96 h 168 h

r P r P r P r P

MDA 0.480 0.032 0.392 0.087 0.586 0.008 0.015 0.951SOD −0.733 <0.001 −0.734 <0.001 −0.734 <0.001 −0.55 0.015Mb −0.309 0.185 −0.073 0.767 −0.158 0.545 −0.34 0.343CK-MB −0.011 0.962 −0.093 0.705 −0.047 0.858 0.326 0.357

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CTn-T 0.324 0.163 −0.156

BNP 0.174 0.464

: Pearson correlation.

K-MB measured in the current study were similar to those previ-usly described [2].

Plasma IMD levels on day 1 were higher than SCHD subjects158.94 ± 11.96 versus 148.80 ± 6.04 pg/ml, P = 0.014) and controls158.94 ± 11.96 versus 125.92 ± 9.24 pg/ml, P < 0.001), and con-inued to increase throughout the four-day observation periodP < 0.001). Peak values of mean plasma IMD in all patients with AMI181.88 ± 9.47) occurred 96 h following the onset of AMI. The levelas then gradually decreased, but was higher on the seventh day

han the SCHD subjects (159.17 ± 8.39 versus 148.80 ± 6.04 pg/ml, = 0.002) and controls (159.17 ± 8.39 versus 125.92 ± 9.24 pg/ml, < 0.001; Fig. 1).

Correlations between plasma IMD and the other related param-ters at each time point following the onset of AMI are shown inable 3. Plasma IMD concentrations were correlated with MDA

ig. 1. The levels of IMD in the patients with AMI and SHCD and in controls. The IMDevels were significantly higher on day 1 after the onset of AMI as compared withhe SHCD and control subjects (P < 0.05). The IMD levels were significantly decreasednd returned to the first day range on day 7 (P = 0.628). The error bars indicate SEM.P < 0.05 versus control subjects; †P < 0.05 versus SHCD subjects.

−0.174 0.49 −0.39 0.2720.034 0.917

(r = 0.48, P = 0.032; r = 0.586, P = 0.008, respectively) at 12–24 and96 h (Fig. 2A) post-infarction, and IMD and SOD were nega-tively correlated at each time point post-MI (r = −0.733, P < 0.001;r = −0.734, P < 0.001; r = −0.734, P < 0.001 (Fig. 2B); r = −0.55,P = 0.015, respectively). There was no correlation between IMD andBNP (Fig. 2C), CK-MB, and CTn-T.

3.3. Severity of coronary artery disease and IMD

We subdivided the patients with AMI. Eight patients had oneinvolved coronary branch (right, left anterior descending, or leftcircumflex) with ≥75% stenosis (group A), nine subjects had twoinvolved coronary branches with ≥75% stenosis (group B), and onlythree individuals had ≥75% stenotic lesions in all three coronarybranches (group C) (Fig. 3). IMD levels were increased in group Ccompared with groups A and B; however, there were no significantdifferences between groups A and B.

4. Discussion

The present study showed that plasma IMD levels were higher inpatients with AMI than in patients with SCHD and healthy controls.The time course of plasma IMD was similar to the change in plasmaMDA, and was opposite that of SOD. In the present study, we foundthat plasma IMD levels in AMI patients were positively correlatedwith degree of coronary stenosis.

There is accumulating evidence suggesting that IMD has simi-lar or even wider cardio-protective effects than ADM and CGRP [7].The expression of the counter-regulatory peptide in animal mod-els, such as hypertrophied cardiomyocytes [5], ischemic injury [28]and heart failure [8] suggests possible pathophysiological roles ofIMD in cardiovascular system. However, few studies have showedthe role of IMD in patients. Qin et al. [20] has recently investigatedplasma IMD levels in patients with ACS, and they found that plasma

IMD levels were markedly higher in ACS patients than that in con-trols (P < 0.05) and positively correlated with degree of coronarystenosis (r = 0.758, P < 0.05). In the present study, we also foundthat plasma levels of IMD were significantly increased in patients

124 Z. Lv et al. / Peptides 43 (2013) 121–125

Fig. 2. (A) Correlation between plasma levels of IMD and MDA in AMI patients at 96 h afand SOD in AMI patients at 96 h after onset of AMI. r = −0.734, P < 0.001; (C) there was no

Fig. 3. Mean values of IMD (mean ± SD) in different severity degrees of coronarystenosis at 12–24 h, 48 h, 96 h and 168 h. *P < 0.05 versus group A and group B.

ter onset of AMI. r = 0.586, P = 0.008; (B) correlation between plasma levels of IMDcorrelation between IMD and BNP at 12–24 h after onset of AMI. r = 0.174, P = 0.464.

with AMI. Therefore, IMD may be a new biomarker for diagnosis ofACS/AMI.

In the current study, we found that plasma IMD levels on day 1were elevated in patients with AMI. The peak value of mean plasmaIMD in all patients with AMI occurred 96 h following the onsetof AMI. Analyses indicated that plasma IMD concentrations werecorrelated with MDA at 12–24 and 96 h post-infarction. Moreover,there were good correlations between IMD and SOD, which showeda negative correlation at each time point. These results suggest thatthe increase in IMD may be associated with oxidative stress, andmay have beneficial effects on antioxidant activity. Studies havefound that IMD ameliorates vascular and renal injury by inhibi-tion of oxidative stress in rats [6] and has the ability to protectthe heart muscle against the deleterious effects of oxidative stressassociated with ischemia–reperfusion injury in rat [11]. David Bellet al. [4] found that the augmented expression of IMD/receptoractivity-modifying protein 1 (RAMP1) in cardiomyocytes from ratssubjected to chronic NO-deficiency can be prevented by concurrentadministration of exogenous anti-oxidants (Vitamin C). These datasupport a specific causal association between increased oxidative

stress and induction of this cardio-protective peptide, IMD, actingvia calcitonin receptor-like receptor (CRLR)/RAMP1.

Furthermore, patients with more severe lesions of the coro-nary arteries had higher plasma IMD levels at various measuring

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oints. Plasma IMD levels may be correlated with degree of coro-ary stenosis. We have recently found that IMD could prevent therogression of atherosclerotic plaque [29]. This suggests that IMDay protect the myocardium in patients with AMI and might be a

ew biomarker for risk stratification in AMI patientsHowever, our study found no relationship between IMD and

yocardial markers (CK-MB, CTn-T) and BNP. The increase of IMDollowing an AMI was presented later than myocardial markers;herefore, IMD may not be a sensitive marker for the early diagno-is of myocardial injury. The present study has some limitationsuch as a relatively low number of patients and the correlationetween IMD and the severity of CAD may be doubted by the limitedase number, and we also cannot get the cutoff of IDM levels foriagnosing AMI

In conclusion, our study showed plasma levels of IMD werencreased in patients with AMI. Increased plasma IMD levels maye associated with oxidative stress, and are potentially correlatedith the severity of coronary stenosis. The detailed mechanism

emains unknown, nevertheless, it may provide a new potentialor the diagnosis and treatment of CHD.

cknowledgement

We thank Medjaden Bioscience Limited for assisting in thereparation of this manuscript.

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