Urate oxidation during percutaneous transluminal coronary angioplasty and thrombolysis in patients with coronary artery disease

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Urate oxidation during percutaneous transluminal

coronary angioplasty and thrombolysis in patients with

coronary artery disease

Sevgi YardVm-AkaydVn

a,

*, Mehmet KesVmer

b

, Ersin Imren

c

, Aylin Sepici

d

,

Bolkan S¸ims¸ek

a

, Meral Torun

a

a_{Gazi University, Faculty of Pharmacy, Department of Biochemistry, 06330, Etiler-Ankara, Turkey} b

Hacettepe University, Faculty of Medicine, Division of Metabolism and Nutrition, 06100, SVhhiye-Ankara, Turkey

c

Gazi University, Faculty of Medicine, Department of Cardiology, Bes¸evler-Ankara, Turkey

d

Ufuk University, Faculty of Medicine, Department of Biochemistry, Balgat, Ankara, Turkey Received 20 April 2005; received in revised form 3 June 2005; accepted 6 June 2005

Available online 1 July 2005

Abstract

Thrombolysis and percutaneous transluminal coronary angioplasty (PTCA) are kinds of procedures that can be used to restore the blood flow of previously ischemic myocardium that can be the result of excessive production of reactive oxygen and nitrogen species, such as superoxide and hydroxyl radical, hypochlorous acid and peroxynitrite. Reaction of urate with some of these potent oxidants results in allantoin production. In this study, we measured the serum allantoin levels, an oxidation product of urate, and bin vivoQ marker of free radical generation in reperfusion of ischemic myocardium.

After an overnight fasting state, blood samples were collected from 35 patients with coronary occlusive diseases (7 women and 28 men) and 31 healthy subjects (8 women and 23 men). Serum allantoin and urate levels were measured by a GC-MS method.

Serum allantoin levels of patients on PTCA therapy (meanFSD, 27.4 F 15.2 Amol/l) and thrombolytic therapy (24.6 F 8.6 A_{mol/l) were significantly higher than those of the patients without therapy (15.8 F 6.2 Amol/l, p b 0.05 with PTCA and} p b 0.006 with thrombolysis) and healthy controls (12.6 F 6.3 Amol/l, p b 0.002 with PTCA and p b 0.0001 with thrombo-lysis). Although serum urate levels in PTCA (380.1 F 72.6 Amol/l) and thrombolysis (359.5 F 60.0 Amol/l) were higher than those in the non-therapy patients (336.6 F 53.8 Amol/l) and controls (318.3 F 81.0 Amol/l), there were no significant differences among groups ( p N 0.05). The results of the study are consistent with others which have demonstrated, higher urate levels are associated with coronary occlusive diseases. Our data support the hypothesis that generation of ROS occurs

* Corresponding author. Gazi U¨ niversitesi, EczacVlVk Faku¨ltesi, Biyokimya ABD. 06330-Etiler Ankara Tu¨rkiye. Tel.: +90 312 215 44 68/ 1216; fax: +90 312 2235018.

E-mail address: sevgiy@gazi.edu.tr (S. YardVm-AkaydVn).

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during myocardial reperfusion. Increased allantoin levels may be used as an index of increased oxidative stress during reperfusion.

Keywords: Allantoin; Urate; Oxidative stress; Coronary occlusive disease; Reperfusion injury

1. Introduction

ROS-generated reperfusion injury may occur with reintroduction of oxygen to the ischemic myocardium after a critical period of coronary occlusion. Although the treatment of coronary occlusion by the use of thrombolytic agents (tissue plasminogen activator or streptokinase) or PTCA restores blood flow to the ischemic myocardium, reperfusion is still a major clinical problem due to myocardial damage [1–8]. During ischemia/reperfusion, a number of cell types, including cardiac myocytes, endothelial cells and neu-trophils, may have the ability of producing reactive oxygen species (ROS), such as superoxide radical (O2S), hydrogen peroxide (H2O2), hypochlorous

acid (HOCl), hydroxyl radical (HOS) and singlet ox-ygen (1O2), lipid peroxyl radical (ROOS) and

perox-ynitrite (ONOO )[3,5,9,10].

Several mechanisms have been proposed to explain the elevated urate levels that have been associated with coronary occlusive disease. Urate is continuously released from the cardiac tissue of humans, and its site of formation has been shown to be the micro vascular endothelium. It has been proposed that urate is an important antioxidant for humans. The antioxidant properties of urate have been attributed to its ability to chelate transition metal ions and to react with potent biological oxidants such as hydroxyl and per-oxyl radical, hypochlorous acid and peroxynitrite to produce relatively stable products. In recent studies, allantoin, which is the major product of urate oxida-tion, has been examined as a potential marker of oxidative stress in humans[11–13]. Several of these publications published methods capable of measuring low levels of allantoin that is present in human serum [14–17].

Since allantoin is formed by nonenzymatic oxida-tion of urate in humans, increasing number of studies have used it as a marker of free radical activity in several diseases as well as assessing oxidative stress [18–23].

The major aim of this study was to explore the clinical utility of allantoin as a marker of oxida-tive stress during reperfusion in coronary occlusive disease.

2. Experimental 2.1. Chemicals

Allantoin, urate, hydrochloric acid and N-(tert-butyl-dimethylsilyl)-N-methyltrifluoroacet-amide (MTBSTFA) were obtained from Merck (Darmstadt, Germany). Dimethylformamide (DMF) was purchased from Sigma (St. Louis, MO, USA). Acetonitrile was obtained from LabScan (Dublin, Ireland).

2.2. GC-MS instrumentation and conditions

HP 6890 Gas Chromatograph and HP 5972A mass spectrometer (Hewlett Packard, Germany) were used in the analysis. After isolation from aqueous standards or serum by extraction onto anion exchange columns (SPE Cartridges, 100 mg, 1 ml, Alltech, Illinois, US), the samples were applied onto HP-5MS capillary column (25 m 0.25 mm i.d., 0.33 Am film thickness) (Hewlett Packard) with helium as carrier gas (flow rate 1.2 ml/min). Injection port temperature was 280 8C and initial oven temperature was 150 8C rising to 270 8C at 20 8C/min for 6 min. Injections were made in the split mode (40 : 1). The ions at m/z 398 and 400 for allantoin and m/z 567 and 569 for urate were monitored under selective ion recording condi-tions (ionization energy 70 eV) with a 30 ms dwell time on each ion.

2.3. Study population and procedures

Thirty-one subjects without a history of hyperten-sion, hyperlipidemia and diabetes were selected as controls from healthy subjects that were attending

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the Clinic of Cardiology in Gazi University in Ankara. Coronary occlusive diseases (CODs) patients, 35, which had been hospitalized in the Coronary Intensive Care Unit of Gazi University Hospital were enrolled in the study group. Sixteen of 35 had documented as acute myocardial infarction (AMI), 8 chest pain (CP, had MI history), 7 unstable angina pectoris (UAP) and 4 stable angina pectoris (SAP). The most common associated diseases were hypertension (n = 6), hyper-lipidemia (n = 5) and diabetes (n = 3). The diagnosis of diseases for all patients was determined by ECG (echocardiography), cardiac enzymes (creatine kinase MB, troponin T and I, myoglobin) and angiography. Patients with acute coronary syndrome (n = 31) were on aspirin therapy with a dose range of 100– 300 mg/day with appropriate therapeutically manage-ment respect to specific considerations. Primary PTCA and stenting were the choice of therapy in 8 patients with AMI, CP and UAP. PTCA was performed accord-ing to a standard protocol. Thrombolytic therapy with tissue plasminogen activator or streptokinase was de-livered to the rest of the AMI patients. The streptoki-nase or the tissue-plasminogen activator doses of patients that were on thrombolytic therapy were 1.5 million units / 60 min or 100 mg / 90 min.

All the subjects, including controls, filled out a questionnaire giving the following information: age, gender, smoking habits, and taking vitamins or anti-oxidants as well subjects had not consumed vitamin or

other supplements in this study. Oral consent was obtained from subjects with study approval by the Hospital Ethics Committee.

2.4. Sample collection and storage

Early morning blood samples were taken from controls after the overnight fasting state to the vacu-tainer tube containing SSTR gel and clot activator for serum. The blood samples of subgroups were taken at the time of entrance to the coronary care unit. Blood samples of patients on therapy were collected within 4 h after reperfusion. After centrifugation at 2000 g for 10 min, serum samples were stored in the dark at

70 8C until the analysis.

2.5. Measurements of allantoin and urate

The levels of allantoin and urate in serum were determined by gas chromatography-mass spectrome-try (GC-MS) according to the method of Chen et al. [24] as described our previous study [22]. Shortly, after isolation from aqueous standards or serum by extraction onto an anion exchange column, allantoin and urate were derivatized with mixture of DMF: MTBSTFA. Derivatives were injected onto an HP-5MS column and analyzed using a Mass Selective Detector with Single Ion Monitoring at 398 and 399 m/z for allantoin and 567 and 568 m/z for urate.

Table 1

Baseline characteristics and laboratory values of the patients and controls

Variables Patients (n = 35) Controls (n = 31) p value Mean age (years) 58.8 F 12.0 57.3 F 8.6 Ns

Women included (%) 20.0 25.8

Number of smokers (%) 37.1 35.5

Diuretic therapy (n) 3 –

Aspirin therapy (n) 31 –

Allantoin (Amol/l) 21.5 F 10.7 12.6 F 6.3 0.0001 Uric Acid (Amol/l) 354.4 F 61.2 318.3 F 81.0 0.05 Diastolic blood pressure (mm Hg) 74.6 F 9.5 74.5 F 8.1 Ns Systolic blood pressure (mm Hg) 121.4 F 11.4 120.3 F 10.2 Ns Glucose (mg/dl) 108.4 F 16.1 103.2 F 18.0 Ns Total cholesterol (mg/dl) 209.6 F 38.7 210.7 F 33.2 Ns Triglycerides (mg/dl) 188.3 F 71.6 150.9 F 68.0 0.05 HDL-Cholesterol (mg/dl) 38.3 F 8.8 41.6 F 9.0 Ns LDL-Cholesterol (mg/dl) 128.5 F 39.6 138.9 F 28.3 Ns VLDL-Cholesterol (mg/dl) 37.7 F 14.3 30.2 F 13.6 0.05 Creatinine (mg/dl) 1.1 F 0.3 0.9 F 0.1 0.005 GFR (ml/min/1.73 m2₎ _{75.43 F 26.23} _{86.48 F 15.15} _0.05

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2.6. Statistical analysis

Data were expressed as the mean F SD, and statis-tical analyses were performed by Student’s t-test and Mann–Whitney U test (for groups of small numbers). Pearson and Spearman correlation coefficients were calculated for the relationship between allantoin and urate. Multiple regression analysis was made using uric acid and allantoin as the dependent variables and gender, age, creatinine, and GFR as independent vari-ables. Statistical analyses were performed with a SPSS 10.0 Package (SPSS Inc., U.S.A.).

3. Results

Table 1 presents baseline characteristics and labo-ratory values of all patients and controls. The serum allantoin and urate levels were markedly elevated in the patients than in the controls, as shown in Table 2. Statistically significant relations were found between the serum allantoin and urate levels of the patient and

control groups ( p = 0.0001 and p = 0.05, respectively)

(Table 1). All patients on therapy (both PTCA and

thrombolytic) have increased allantoin levels (26.5 F 10.8 Amol/l) than those without therapy (15.8 F 6.2 A_{mol/l) and controls (12.6 F 6.3 Amol/l) ( p b 0.005} and p b 0.0001, respectively). The levels of urate of all patients on therapy (367.7 F 64.3 Amol/l) were higher than patients without therapy (336.6 F 53.8 Amol/l) and controls (318.3 F 81.0 Amol/l), differences were statistically significant in controls ( p b 0.03).

When we evaluated the effects of PTCA therapy on our parameters, the levels of allantoin were higher in patients on PTCA therapy than those without therapy and healthy controls ( p b 0.05 and p b 0.002, respec-tively). Although the serum urate levels in PTCA were higher than in both patients without therapy and healthy controls, the differences were not statistically significant ( p N 0.05) (Table 2).

The serum allantoin levels of patients on thrombo-lytic therapy were higher than those of the patients without therapy and controls ( p b 0.006 and p b 0.0001, respectively). There were increased urate

Table 2

Mean levels (F SD) of serum allantoin and uric acid in patients and controls

Mean levels F SD (95% confidence interval)

Allantoin (Amol/l) Uric acid (Amol/l) Patients on PTCA therapy (n = 8) 27.4 F 15.2a, b_{(14.7–40.2)} _{380.1 F 72.6 (319.4–440.8)}

Patients on thrombolytic therapy (n = 12) 24.6 F 8.6c, d _{(19.2–30.1)} _{359.5 F 60.0 (321.4–397.6)}

Patients without therapy (n = 15) 15.8 F 6.2 (12.3–19.3) 336.6 F 53.8 (306.8–366.4) Healthy controls (n = 31) 12.6 F 6.3 (10.3–14.9) 318.3 F 81.0 (288.6–348.0)

a

p b 0.05. PTCA with non-therapy.

b

p b 0.002. PTCA with healthy controls.

c

p b 0.006. Thrombolysis with non-therapy.

d

p b 0.0001. Thrombolysis with healthy controls.

Table 3

Multiple regression analyses with three different sets of variables

Model 1 Model 2 Model 3

Stand. b p-value Stand. b p-value Stand. b p-value Gender 0.326 0.008 0.303 0.016 0.266 0.098 Age 0.101 0.399 0.075 0.542 0.093 0.486 Creatinine 0.106 0.405 0.171 0.434 GFR 0.085 0.713 R 0.346 0.360 0.363 R square 0.120 0.130 0.132 Adj. R square 0.092 0.088 0.075

Dependent variable: uric acid. St. b: standardized beta-coefficient. Adj. R square: Adjusted R square.

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levels in patients on thrombolysis than in the patients without therapy and controls, but differences were not statistically significant ( p N 0.05) (Table 2).

It is known that babnormalQ creatinine levels and GFR affect kidney functions and also urate levels. GFR of the subjects was assessed by MDRD formula[25]. Creatinine levels and glomerular flow rate (GFR) were higher in cases than in controls ( p b 0.0001 and p b 0.05, respectively) (Table 1). Focusing on the con-tribution of the biochemical parameters, different sets of independent variables were considered in three com-plementary models: (1) age and gender; (2) age, gender and creatinine; (3) age, gender, creatinine and GFR. Gender was found to be independently associated with uric acid (Table 3). Creatinine levels weakly affected the allantoin levels (Standardized b coefficient = 0.229 and p = 0.082 in Model 2).

4. Discussion

Reperfusion of ischemic myocardium is accepted as a clinical problem which is called breperfusion injuryQ[3,5–7]. As mentioned above, several reactive oxygen and nitrogen species produced in ischemia/ reperfusion.

In the current study, we found increased urate levels in PTCA and thrombolysis groups than in patients without therapy, and healthy controls al-though the differences were not statistically signifi-cant. Hyperuricemia was seen in several hypoxic conditions, such as interruption of limb arterial flow, coronary artery bypass and coronary angioplas-ty, reflecting oxidative stress [26–30]. An increased urate levels in ischemia may be a result of increased XO activity. During myocardial ischemia, xanthine dehydrogenase is converted to xanthine oxidase, an enzyme that produces O2S and urate from

hypoxan-thine or xanhypoxan-thine and molecular oxygen [1,2,9,31]. However, it has been suggested that hyperuricemia may reflect a response to increased production of free radicals. The antioxidant properties of urate have been attributed to its ability to react with potent biological oxidants to produce relatively stable pro-ducts. It has been shown that some of the ROS which are produced during inflammation can react rapidly with urate to form allantoin [13,16,32–34]. Since urate oxidation by ONOO has been shown in

many studies, it is essentially accepted a natural scavenger of this reactive nitrogen species [35–38]. In consistent with these, we found slightly high allantoin levels in non-therapy group when compared to controls and significant rise on allantoin levels in PTCA or thrombolytic therapy groups than in both non-therapy and control groups.

Doehner et al. investigated serum urate and allan-toin levels before and after allopurinol therapy in chronic heart failure[21]. After allopurinol treatment, serum urate levels were decreased by almost 60% due to reduction of XO activity. Interestingly, they also observed a decreasing, approximately 20%, in allan-toin levels of patients on allopurinol therapy and only placebo therapy. We were not able to compare their results with ours, as our study did not include the measurement of initial serum allantoin and urate levels of patients on therapy. When increased reactive species in reperfusion were taken into consideration, high allantoin levels in reperfusion are more logical than low levels. In another study, Kock et al. investi-gated xanthine, hypoxanthine, allantoin and uric acid levels in acute myocardial infarction and other ische-mic diseases [39]. They obtained slightly increased allantoin levels, but not statistically significant, in patients with angina pectoris and MI than those in healthy controls. In their study, they also had higher levels of xanthine/hypoxanthine due to increased ac-tivity of xanthine oxidoreductase in myocardial infarc-tion. As we and they mentioned, the activity of this enzyme results from the production of O2S that is

converted to more reactive species. As a result of this, an increase in allontoin levels is expected. They explained these results by other radical scavenging mechanisms. In addition, it was indicated that urate also could prevent extracellular superoxide dismutase degradation, thus increasing superoxide dismutation to hydrogen peroxide, and decreasing the availability of superoxide[40]. Although we found similar results in our other subgroups with theirs, we obtained dif-ferent allantoin and urate levels in our AMI group and controls. These differences between our and their results may depend on other radical scavenging mechanisms.

According to the results of multiple regression analyses, we found independently effect of gender on uric acid levels, but not allantoin. Normally, it is known that serum uric acid levels are higher in males

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than in females although their levels in females are slightly increased during or after menopause. It is suggested that estrogen may enhance excretion of uric acid via urine [41]. We can not expect altered levels of allantoin by gender as it is only a product of oxidative stress.

A large body of studies have provided evidence that post-ischemic myocardial dysfunction may be mediat-ed in part by the generation of reactive oxygen and nitrogen species [42–47]. In these studies, however, results on the levels of several markers of oxidative stress in reperfusion are controversial. As serum urate levels already increase by XO activity during ischemia and easily react with reactive species in reperfusion, the measurements of allantoin levels in serum, or urine, represent oxidative stress in reperfusion.

In conclusion, since there is not any known enzy-matic mechanism for synthesis of allantoin, oxidation of urate in humans can be implied by the presence of allantoin. Our results suggest that the measurement of serum allantoin levels may be prior to lipid peroxida-tion products as a marker of increased oxidative stress in reperfusion. Our data also support findings about the possible involvement of free radicals in COD. Our animal studies on allantoin levels during ischemia/ reperfusion are continuing. Further studies are needed to support our hypothesis.

Acknowledgements

This study was supported by Gazi University Re-search Fund. We want to thank to Nezir Ko¨se for his helpful assistance in statistical evaluation and Elvan Laleli-Sahin for her helpful assistance in grammar.

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