Public trials registry number: Clinicaltrials.gov (NCT 01248338).Correspondence: Martin Serg, MD, Department of Cardiology, University of Tartu, 8 Puusepa Street, Tartu 51014, Estonia. Tel: � 372 7318 455. Fax: � 372 7318 404. E-mail: Martin.Serg@kliinikum.ee

(Received 8 January 2012; accepted 27 April 2012)

ORIGINAL ARTICLE

Nebivolol and metoprolol: long-term effects on infl ammation and oxidative stress in essential hypertension

MARTIN SERG 1,2 , PRIIT KAMPUS 1,2,3 , JAAK KALS 2,4 , MAKSIM ZAGURA 1,2 , MIHKEL ZILMER 2 , KERSTI ZILMER 2 , TIIU KULLISAAR 2 & JAAN EHA 1,3

1 Department of Cardiology, University of Tartu, Tartu, Estonia, 2 Department of Biochemistry, Centre of Excellence for Translational Medicine, University of Tartu, Tartu, Estonia, 3 Heart Clinic of Tartu University Hospital, Tartu, Estonia, and 4 Department of Vascular Surgery, Tartu University Hospital, Tartu, Estonia

Abstract Arterial hypertension is characterised by increased oxidative stress and infl ammation, which are associated with further cardiovascular risk. The aim of our study was to investigate the long-term effects of nebivolol and metoprolol succinate on oxidative stress, and on infl ammatory and pro-infl ammatory markers in patients with hypertension. Eighty patients with never-treated mild-to-moderate essential hypertension, aged 30 – 65 years, were randomised to a 5 mg daily dose of nebivolol or a 50 – 100 mg daily dose of metoprolol succinate. Brachial blood pressure, plasma oxidized LDL (oxLDL), interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP), fi brinogen, intercellular adhesion molecule-1 (ICAM-1), asymmetric dimethylarginine (ADMA), and urine 8-isoprostane levels were measured before and after 1 year of treatment. Nebivolol and metoprolol reduced equally signifi cantly brachial blood pressure. The oxLDL was signifi cantly reduced in both groups ( p � 0.01 and for both drugs), but only nebivolol reduced 8-isoprostanes ( p � 0.01). In the metoprolol group, change in oxLDL levels correlated with change in systolic blood pressure (r � 0.45; p � 0.01) and pulse pressure (r � 0.47; p � 0.01). Both metoprolol and nebivolol reduced ICAM-1 ( p � 0.01). There was no change in IL-6, hsCRP, fi brinogen, or ADMA levels in either group. These data suggest that in long-term antihypertensive treatment both the cardioselective beta blocker metoprolol succinate and the vasodilating beta blocker nebivolol have infl ammation-related effects but only nebivolol has a favourable blood pressure-independent effect on oxidative stress.

Key Words: Adrenergic beta-antagonists , isoprostanes , oxidized low density lipoprotein , cell adhesion molecules

Introduction

Beta blockers have been widely used in the treatment of essential hypertension. However, a large meta-analysis has shown that beta blockers are inferior to other antihypertensives in preventing cardiovascular events [1]. The studies included in the meta-analysis mainly involved atenolol which is shown to have no effect on oxidative stress, pro-infl ammation, infl am-mation, and central aortic blood pressure [2 – 4].

Oxidative stress and infl ammation, independent predictors of cardiovascular events, are increased in essential hypertension [5 – 7]. It has been demonstrated previously that elevated oxidative stress and infl am-mation are associated with vascular dysfunction and progression of atherosclerosis in hypertension [8,9].

Oxidized low density lipoprotein (oxLDL) and isoprostanes have emerged as useful markers of oxi-dative stress in various pathological states [10]. It has been shown that oxLDL but not native LDL is rec-ognised by scavenger receptors and is taken up by macrophages – a pivotal process in the development of atherosclerosis [11]. The oxLDL is a prognostic marker of subclinical atherosclerosis and is related to severity of the acute coronary syndrome [12,13]. The more recently introduced group of markers, iso-prostanes, are chemically stable lipid peroxidation products of arachidonic acid. Currently, isoprostanes are considered the most sensitive markers of lipid peroxidation and the most reliable markers of sys-temic oxidative stress in humans [14].

Scandinavian Journal of Clinical & Laboratory Investigation, 2012; 72: 427–432

ISSN 0036-5513 print/ISSN 1502-7686 online © 2012 Informa HealthcareDOI: 10.3109/00365513.2012.691991

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We have recently shown that nebivolol, a highly selective beta1-receptor antagonist with vasodilating properties, reduces central aortic blood pressure in long-term antihypertensive therapy [15]. In addition, nebivolol has been demonstrated to reduce aortic pulse wave velocity [16] and to reverse endothelial dysfunction [17], which are the characteristics infl u-enced largely by oxidative stress and infl ammation [18,19]. To date, only short- and mid-term studies have dealt with the effects of nebivolol and meto-prolol succinate, the latter being a widely used beta blocker in Eastern and Northern Europe, on oxida-tive stress and infl ammation. These studies have shown that nebivolol decreases markers of oxidative stress and infl ammation, while metoprolol has no effect on these markers [2,20].

The aim of our study was to investigate the long-term (1 year) effects of nebivolol and metoprolol suc-cinate on oxidative stress and infl ammation.

Methods

Study design

A randomised, double-blind, active controlled parallel-group trial (NCT 01248338) was conducted in the Department of Cardiology, University of Tartu. Patients, aged 30 – 65 years, with never-treated mild-to-moderate essential hypertension, were recruited from a hypertension outpatient clinic or from local general practices. Eighty patients were randomised to a 5 mg daily dose of nebivolol (Nebilet ® , dl-nebivolol hydrochloride, Berlin-Chemie) or a 50 mg daily dose of metoprolol (BetalocZOC ® , metoprolol succinate, Astra Zeneca). Randomisation was performed in blocks of four. All patients who met the inclusion and exclu-sion criteria were assigned a patient number in the ascending order. This number determined whether treatment was to be performed with nebivolol or metoprolol. The allocation of patients into the treat-ment arms remained unknown for all patients throughout the study. The duration of the study for each patient was 52 weeks (one year) plus the screen-ing period of 2 weeks. The patients were scheduled for follow-up visits at weeks 2, 4, 12, 24, 40, and 52. Brachial blood pressure was measured at all visits, and blood and morning urinary samples were taken at baseline and after 12 months of therapy. If blood pressure � 140/90 mmHg was not achieved 2 weeks after the randomisation, the dosage in the metoprolol group was raised to 100 mg daily. After 4 weeks of randomisation we allowed the addition of 12.5 – 25 mg of hydrochlorothiazide daily in both groups if a blood pressure decrease to � 140/90 mmHg was not achieved.

Mild or moderate hypertension was defi ned as sys-tolic blood pressure 140 – 179 mmHg and/or diastolic blood pressure 90 – 109 mmHg on at least two occa-sions separated by 1 month. Patients with diabetes

mellitus, obesity, ischaemic heart disease, heart failure, arrhythmias, secondary hypertension, acute or chronic infl ammatory disease, and hypercholes-terolaemia were excluded during the screening period. Detailed exclusion criteria have been reported previously [15].

The subjects were studied between 8:00 and 10:00 a.m. after an overnight fast and abstinence from tobacco, alcohol, tea, or coffee. After 15 min of rest, blood pressure was measured at each visit whereas laboratory tests were performed at baseline and at week 52. Body mass index was calculated using the standard formula.

This investigator-initiated study was carried out in accordance with the Declaration of Helsinki and was approved by the local Research Ethics Committee. Written informed consent was given by all subjects.

Measurement of haemodynamics

Brachial blood pressure and heart rate were mea-sured in a sitting position in the non-dominant arm as a mean of three consecutive measurements at 5 min intervals using a validated oscillometric tech-nique (OMRON M4-I; Omron Healthcare Europe BV, Hoofddorp, The Netherlands). The mean of the two closest blood pressure readings was used in fur-ther analysis. Pulse pressure was calculated as the dif-ference between systolic blood pressure and diastolic blood pressure. Mean arterial pressure (MAP) was calculated as described previously [19].

Biochemical analysis

Centrifuged blood samples and urine samples were divided into aliquots and stored at � 70 ° C until anal-ysis. The oxLDL levels were measured using an ELISA kit (Catalogue no. 10 - 1143-01 Mercodia AB, Upp-sala, Sweden). The urinary content of 8-isoprostanes was analysed by a competitive ELISA (Cayman Chemical Company ® , Ann Arbor, MI, USA). The urinary concentrations of 8-isoprostanes were cor-rected by urinary creatinine concentrations to account for differences in renal function. The plasma level of asymmetric dimethylarginine (ADMA) was deter-mined by a competitive ELISA using a commercially available kit (DLD Diagnostika ® , Hamburg, Ger-many). The plasma level of high-sensitivity C-reactive protein (hsCRP) was determined by using a validated latex particle-enhanced immunoturbidimetric assay (CRP (Latex) HS; Roche Diagnostics Gmbh ® , Man-nheim, Germany). Soluble intercellular adhesion molecule-1 (ICAM-1) was measured by ELISA using a commercially available kit (Human soluble ICAM-1 Immunoassay, R&D Systems ® , Minneapolis, MN, USA). Interleukin-6 (IL-6) was analysed by the quan-titative sandwich enzyme immunoassay technique (R&D Systems ® , Minneapolis, MN, USA). Fibrino-gen was measured by the clotting method after Clauss

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Beta blockers and oxidative stress 429

using the Stago Compact analyser (Diagnostica Stago ® , Asni è res, France). White blood cell count (WBC), plasma glucose, total cholesterol, LDL cho-lesterol, HDL cholesterol, triglycerides, and urine creatinine were determined by standard laboratory methods, using certifi ed assays, in a local clinical laboratory.

Statistical analysis

Statistical analysis was performed with the Statistica software (version 8; Statsoft ® , Tulsa, OK, USA). All data were tested for normality using the Kolmogo-rov-Smirnov test. Normally distributed data are pre-sented as mean � standard deviation; non-normally distributed data are presented as the median with the interquartile range. For categorical variables, contingency tables were composed and the chi-square test was used to compare the distributions for the two randomised groups. Changes from the baseline to the endpoint were tested for the differ-ence from zero using the paired t-test; for continu-ous variables, which were not normally distributed � 1 group, the Wilcoxon rank-sum test was employed. Additionally, oxidative stress and infl ammatory markers, adjusted to MAP, were analysed as well. The independent t-test or the Mann-Whitney U-test were used to test for the difference between the groups in baseline values. Correlations between the variables were examined using univariate linear regression analysis. Intention-to-treat analysis was employed in statistics. Signifi cance was defi ned as two-sided p � 0.05.

Results

A total of 40 patients (50%) were enrolled in the nebivolol arm and 40 (50%) were enrolled in the metoprolol arm. The baseline demographic and clinical characteristics of the untreated hypertensive subjects are presented in Table I. Before the randomisation there were no statistically signifi cant differences in the demographic and clinical characteristics between the treatment groups.

Out of the 80 patients enrolled, 17 were with-drawn from the study for different reasons: seven patients for lack of compliance (four in the nebivolol group and three in the metoprolol group), four patients for dizziness (two patients in each group), two patients for being non-respondent (both in the nebivolol group), two patients for bradycardia (one patient in each group), one patient for hyperglycae-mia (in the metoprolol group) and one patient for anxiety (in the nebivolol group). Up-titration of metoprolol to 100 mg was performed for 13 patients (32%). During the treatment period, 30% of the patients (12 subjects) in the nebivolol group and 22.5% of the patients (9 subjects) in the metoprolol

Table I. Demographic and clinical characteristics of the patients.

Nebivolol Metoprolol p

Age (years) 49 � 11 44 � 9 0.07Sex (M), n (%) 20 (50) 21 (53) 0.82BMI (kg/m 2 ) 26.6 � 2.7 26.8 � 2.4 0.82Smokers, n (%) 5 (12.5) 5 (12.5) 1.00SBP (mmHg)

Before 146 � 12 145 � 11 0.57After 129 � 8 134 � 5 p � 0.01 � 0.01

DBP (mmHg)Before 90 � 8 91 � 7 0.85After 78 � 8 80 � 8 p � 0.01 � 0.01

PP (mmHg)Before 56 � 11 54 � 10 0.34After 51 � 7 55 � 12 p � 0.05 0.67

Heart rate (bpm)Before 68 � 9 70 � 9 0.2After 60 � 7 65 � 10 p � 0.01 � 0.01

Values are mean � SD or n (%). BMI, body mass index; DBP, diastolic blood pressure; PP, pulse pressure; SBP, systolic blood pressure.

group ( p � 0.5) received additionally 12.5 – 25 mg hydrochlorothiazide.

The haemodynamic results of this study have been published previously [15]. Briefl y, after 1 year of treat-ment, nebivolol and metoprolol signifi cantly reduced brachial systolic and diastolic blood pressure ( p � 0.01 versus baseline) (Table I). Both drugs signifi cantly reduced heart rate ( p � 0.01 versus baseline).

The unadjusted and MAP-adjusted data of oxida-tive stress and infl ammatory markers are presented in Table II. At baseline there were no differences in oxi-dative stress or infl ammatory markers between the treatment arms. Both drugs signifi cantly reduced oxLDL levels ( p � 0.01 for both groups). Only nebiv-olol signifi cantly reduced 8-isoprostane levels ( p � 0.01). Both metoprolol and nebivolol signifi cantly reduced ICAM-1 levels ( p � 0.01 for both drugs). There were no changes in WBC, hsCRP, IL-6, fi brin-ogen, or ADMA levels in either group. Adjustment to MAP did not change the statistical signifi cance of the effects of the drugs on oxidative stress and infl amma-tory markers. In the nebivolol group, changes in oxLDL and 8-isoprostane levels were not correlated with change in brachial systolic, diastolic or mean arterial pressure. In the metoprolol group, change in oxLDL levels was correlated with systolic blood pres-sure change (r � 0.45; p � 0.01) and pulse pressure change (r � 0.47; p � 0.01). There were no correlations between ICAM-1 level change and change in systolic, diastolic, or mean arterial pressure in either group.

Total cholesterol, triglycerides, and glucose did not change signifi cantly in either treatment group, but both drugs signifi cantly reduced HDL choles-terol (Table III). An increase in LDL cholesterol

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suggests a low probability of the presence of plaques in the vasculature as a source of oxLDL. It is plau-sible that increased shear stress induces oxidation of LDL cholesterol. Thus, reducing blood pressure per se might reduce oxLDL.

In hypertension, several pro-infl ammatory factors activate endothelial cells to express adhesion mole-cules (i.e. ICAM-1) which initiate recruitment of cir-culating lymphocytes to the blood vessel wall. Several studies have shown that renin-angiotensin system

level was observed only in the metoprolol group ( p � 0.02).

Discussion

The main fi nding of this 1-year study was that both nebivolol and metoprolol reduced oxLDL and ICAM-1 levels in essential hypertension patients while only nebivolol reduced 8-isoprostane levels. Furthermore, the effect of metoprolol on oxLDL was associated with blood pressure change while nebiv-olol decreased oxLDL and 8-isoprostane levels inde-pendently of blood pressure reduction. To our knowledge, this is the fi rst study to assess the long-term effects of nebivolol and metoprolol on oxidative stress and infl ammatory markers.

Harmful oxidative stress results from the imbal-ance between the generation of reactive oxygen spe-cies and the antioxidant defence systems. It has been shown that such oxidative stress reduces the bioavail-ability of nitric oxide, leading to endothelial dysfunc-tion and atherosclerosis [21]. Thus, reversal of elevated oxidative stress could represent an adjunc-tive target for antihypertensive treatment [22]. S á ez et al. have previously shown that antihypertensive treatment reduces oxidative stress markers in a time-dependent manner [23]. This evidence emphasises the need for long-term studies and careful selection of markers to investigate the antioxidative properties of antihypertensive drugs.

Urine isoprostanes were recently accepted by the European Food Safety Authority as a new biochem-ical marker for oxidative stress [24]. Isoprostanes are stereoisomers of prostaglandins that are formed pri-marily through the non-enzymatic peroxidation of arachidonic acid by reactive oxygen species [25]. To our knowledge, only one study has investigated the long-term effect of antihypertensive drugs on iso-prostane levels. The study showed that zofenopril is superior to enalapril in reducing plasma isoprostane levels [26]. However, plasma values of isoprostanes may overestimate isoprostane levels because of the auto-oxidation of lipids [25]. Our study showed that nebivolol decreased urinary 8-isoprostane levels independently of blood pressure reduction in two respects. Firstly, the reduction in 8-isoprostane levels was not correlated with reduction in blood pressure. Secondly, nebivolol reduced 8-isoprostane levels even when 8-isoprostane levels were adjusted for blood pressure. Our results suggest that in long-term antihypertensive therapy nebivolol possesses blood pressure-independent effects on systemic oxidative stress.

Plasma levels of oxLDL are mainly infl uenced by degree of local oxidative stress in the arterial wall and by the susceptibility of LDL to oxidation [27]. The patients included in our study had been recently diagnosed with hypertension; they were relatively young and were at low cardiovascular risk, which

Table II. Markers of infl ammation and oxidative stress at baseline and after 12 months of treatment.

Nebivolol Metoprolol p

Oxidized LDL (U/L)Before 85.5 � 33.8 88.6 � 31.9 0.67After 62.0 � 18.7 65.5 � 29.1 p � 0.01 � 0.01 p ∗ � 0.01 � 0.01

8-isoprostanes (ng/mmol creatinine)Before 43.0 � 28.4 43.2 � 34.9 0.66After 21.8 � 18.6 34.4 � 25.7 p 0.01 0.37 p ∗ 0.01 0.11

ICAM-1 (ng/mL)Before 235.7 � 57.1 234.0 � 57.2 0.94After 200.8 � 55.9 213.1 � 45.5 p � 0.01 � 0.01 p ∗ � 0.01 � 0.01

ADMA ( μ mol/L)Before 0.6 � 0.2 0.6 � 0.2 0.52After 0.7 � 0.2 0.6 � 0.1 p 0.42 0.3 p ∗ 0.8 0.31

IL-6 (pg/mL)Before 1.6 � 0.7 1.6 � 0.9 0.86After 1.7 � 1 2 � 1.7 p 0.77 0.8 p ∗ 0.7 0.66

hsCRP (mg/L)Before 0.95 (0.42; 1.72) 0.85 (0.53;2.56) 0.29After 1.16 (0.67; 2.13) 1 (0.52; 3.3) p 0.98 0.86 p ∗ 0.8 0.71

Fibrinogen (g/L)Before 3.1 � 0.5 2.9 � 0.5 0.11After 3.1 � 0.7 2.9 � 0.8 p 0.95 0.73 p ∗ 0.8 0.43

White cell blood count ( � 10 9 /L)Before 6.1 � 1.4 5.6 � 1.6 0.15After 6.2 � 1.3 5.9 � 1.4 p 0.21 0.2 p ∗ 0.2 0.2

Values are mean � SD or median (interquartile range). ADMA, asymmetric dimethylarginine; hsCRP, high-sensitivity C-reactive protein; ICAM, intercellular adhesion molecule; IL, interleukin; LDL, low density lipoprotein. p ∗ , p adjusted for treatment mean arterial pressure.

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Beta blockers and oxidative stress 431

side effects of the drugs. Secondly, as the patients who remained hypertensive after week 4 received additionally hydrochlorothiazide, it is possible that the combination of a beta blocker and the thiazide diuretic could have infl uenced their effect on oxida-tive stress. However, hydrochlorothiazide was added to almost the same number of patients. Furthermore, it has been shown that thiazide diuretics have no effect on oxidative stress [34].

In conclusion, our study suggests that in the long-term treatment of essential hypertension the vasodilating beta blocker nebivolol is superior to the conventional cardioselective beta blocker meto-prolol succinate regarding their systemic antioxidant properties. Whether these effects implicate cardio-vascular outcome in hypertensive patients remains to be determined. Our study further emphasises the heterogeneity of beta blockers in antihypertensive treatment.

Acknowledgements

The authors would like to thank all patients who participated in the study. We are grateful to the study physicians Drs Kristina Lotam õ is and Ingmar Lind-str ö m, the study nurse Eva-Brit M ö lder and the tech-nician Annelii Sikk for their invaluable assistance. We thank Ester Jaigma for the linguistic revision of the manuscript and Mart Kals for his assistance with the statistical analysis.

Funding Sources: This study was supported by the Berlin-Chemie Menarini, Estonian Science Founda-tion grants Nos. 7480, 8273, and 9094 and by target fi nancing Nos. 0180105s08 and SF0180001s07.

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.

References

Lindholm LH, Carlberg B, Samuelsson O. Should beta [1] blockers remain fi rst choice in the treatment of primary hypertension? A meta-analysis. Lancet 2005;366:1545 – 53. Fratta Pasini A, Garbin U, Nava MC, Stranieri C, [2] Davoli A, Sawamura T, Lo Cascio V, Cominacini L. Nebivolol decreases oxidative stress in essential hypertensive patients and increases nitric oxide by reducing its oxidative inactivation. J Hypertens 2005;23:589 – 96. Williams B, Lacy PS, Thom SM, Cruickshank K, Stanton [3] A, Collier D, Hughes AD, Thurston H, O ’ Rourke M; CAFE Investigators; Anglo-Scandinavian Cardiac Outcomes Trial Investigators; CAFE Steering Committee and Writing Com-mittee. Differential impact of blood pressure-lowering drugs on central aortic pressure and clinical outcomes: principal results of the Conduit Artery Function Evaluation (CAFE) study. Circulation 2006;113:1213 – 25. Touyz RM, Savoia C, He Y, Endemann D, Pu Q, [4] Ko EA, Deciuceis C, Montezano A, Schiffrin EL. Increased

inhibitors and calcium antagonists effectively decrease ICAM-1 levels in hypertension [28,29]. Our study is the fi rst one to assess the effect of metoprolol or nebivolol on ICAM-1 levels in hypertensive patients. It can be suggested that both drugs potentially inhibit endothelial activation in vessel walls.

There was no effect of metoprolol or nebivolol on ADMA levels in long-term antihypertensive therapy. In addition to beta-1 antagonist characteristics, nebivolol has beta-3 agonist properties. There is evi-dence that the beta-3 agonist properties contribute to nitric oxide release while beta-1 antagonism has converse effects [30]. These two pharmacological effects of nebivolol may account for the fi nding that, in some studies, nebivolol decreases ADMA levels, while in ours and other studies ADMA concentration did not change [31]. Neither drug had an effect on infl ammatory markers (hsCRP, WBC, fi brinogen, and IL-6). It could be speculated that the studied patients were indeed at a relatively low cardiovascu-lar risk according to the hsCRP (0.85 – 0.95 mg/L) risk categories [32]. Furthermore, hsCRP values have been somewhat higher in a similar study (1.94 mg/L) [33].

There are some limitations to our study. Firstly, there is a possible shortage of power because of the small number of study patients. When the current study was designed in 2005, there were no relevant publications providing data about the effect of anti-hypertensive drugs on oxLDL and on isoprostane levels to undertake formal power calculation. Quite a high proportion of patients (17 of 80) were withdrawn from the study. We studied relatively young patients with newly diagnosed uncomplicated essential hyper-tension, which might explain the low tolerance to the

Table III. Comparison of the basic biochemical parameters between treatment groups.

Nebivolol Metoprolol p

Total cholesterol (mmol/L)Before 5.3 � 0.8 5.2 � 0.8 0.58After 5.3 � 1.0 5.4 � 1.0 p 0.88 0.24

LDL cholesterol (mmol/L)Before 3.4 � 0.8 3.5 � 0.8 0.92After 3.5 � 1.0 3.8 � 1.0 p 0.91 0.02

HDL cholesterol (mmol/L)Before 1.7 � 0.4 1.6 � 0.4 0.22After 1.5 � 0.4 1.4 � 0.4 p � 0.01 � 0.01

Triglycerides (mmol/L)Before 1.2 � 0.8 1.4 � 1.1 0.41After 1.4 � 1.2 1.3 � 0.7 p 0.08 0.37

Glucose (mmol/L)Before 5.2 � 0.5 5.2 � 0.8 0.87After 5.3 � 0.6 5.3 � 0.8 p 0.23 0.42

Values are mean � SD. HDL, high density lipoprotein; LDL, low density lipoprotein.

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