Author + information
- Received October 24, 2001
- Revision received May 20, 2002
- Accepted June 27, 2002
- Published online October 2, 2002.
- Yasuko Shimizu, MD*,
- Shinya Minatoguchi, MD*,
- Kazuaki Hashimoto, MD*,
- Yoshihiro Uno, MD*,
- Masazumi Arai, MD*,
- Ningyuan Wang, MD*,
- Xuehai Chen, MD*,
- Chuanjian Lu, MD*,
- Genzou Takemura, MD*,
- Masaaki Shimomura, MSc†,
- Takako Fujiwara, MD† and
- Hisayoshi Fujiwara, MD*,* ()
- ↵*Reprint requests and correspondence:
Dr. Hisayoshi Fujiwara, Second Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa Machi, Gifu 500, Japan.
Objectives We aimed to clarify the relation between sarpogrelate (SG), a 5-hydroxytryptamine (5-HT)-2 receptor blocker, and myocardial interstitial serotonin or infarct size during ischemia and reperfusion.
Background In cardiac tissues serotonin is rich in vascular platelets, mast cells, sympathetic nerve endings, and the receptors are present in platelets and cardiomyocytes.
Methods The myocardial interstitial serotonin levels were measured using a microdialysis technique during 30-min ischemia with and without SG in in vivo as well as isolated rabbit hearts. Other rabbits underwent 30 min of ischemia and 48 h of reperfusion, and the effect of SG on the infarct size was investigated in the absence and presence of a selective protein kinase C (PKC) inhibitor, chelerythrine (5 mg/kg, intravenously), or a mitochondrial adenosine triphosphate sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate (5-HD) (5 mg/kg, intravenously). In another series, the effect of SG on PKC isoforms in cytosol and membrane fraction was assessed after a 20-min global ischemia in isolated rabbit hearts.
Results Interstitial serotonin levels were markedly increased during 30-min ischemia in in vivo and isolated hearts, and the increases were inhibited by SG in each. The infarct size was reduced by SG (27 ± 2% vs. 40 ± 3% of control). This effect was blocked by chelerythrine and 5-HD, respectively. Sarpogrelate further enhanced the ischemia-induced translocation of PKC-ϵ to the membrane fraction.
Conclusions Sarpogrelate reduces the myocardial infarct size by inhibiting the serotonin release followed by enhancement of PKC-ϵ translocation and opening of the mitochondrial KATP channel in ischemic myocytes.
In general, platelets in the vascular beds are rich in serotonin (5-hydroxytryptamine [5-HT]) in the cytoplasm (1). The 5-HT2A receptors are observed on the cell membranes of platelets and smooth muscle cells (2,3), and the binding with released serotonin can induce release of serotonin from platelet cytoplasm, platelet aggregation, and contraction of smooth muscle cells (4). These effects are inhibited by 5-HT2 receptor blockers. That is, 5-HT2 receptor blockers are also inhibitors of serotonin release (5). On the other hand, 5-HT1 receptors are present on the cell membrane of endothelial cells, stimulation of which releases endothelial-derived relaxing factor followed by relaxation of smooth muscle cells (1). In the cardiac tissues, serotonin has been identified in platelets in the vascular beds (6), mast cells (7), and sympathetic nerve endings (8). The 5-HT2 receptors are not found in the mast cells or sympathetic nerve endings but are found in the vascular platelets (1). It is postulated that 5-HT receptors are present in cardiomyocytes, although the function is unclear.
The transcardiac plasma serotonin concentration is increased in some patients with angina pectoris (9). It was reported that blockade of 5-HT2 receptors by ketanserine and cinanserine can protect the isolated rat heart against ischemia in terms of cardiac function (10) and that cinanserine has a protective effect on pacing-induced ischemia in an in vivo model of dogs (11). Sarpogrelate (SG), a selective 5-HT2 receptor blocker, is the only drug, among many 5-HT2 receptor blockers, that is currently clinically used for patients with arteriosclerotic obliterance. Sarpogrelate may be protective against human angina pectoris through an increase in collateral circulation (12). However, the role of serotonin and 5-HT2 receptor blockers in myocardial ischemic cellular damage is unclear. Our hypothesis is as follows. First, serotonin is released from platelets in the vascular beds, mast cells, and sympathetic nerve endings at the area at risk during ischemia and reperfusion, as indicated by the increase in myocardial interstitial serotonin. Second, SG can inhibit the serotonin release and the bindings between serotonin and the 5-HT2 receptor of cardiomyocytes, as indicated by the disappearance of the elevated myocardial interstitial serotonin levels after treatment with SG, and, thus, can reduce the infarct size. Therefore, the aim of the present study was to clarify in rabbit hearts with minimum collateral circulation: 1) whether the myocardial interstitial serotonin level is increased during ischemia and reperfusion, and, if the increase is blocked by SG; 2) whether SG can reduce the myocardial infarct size, and if translocation of protein kinase C (PKC) and opening of the adenosine triphosphate sensitive potassium (KATP) channels, which are the main pathways of the protective effect in ischemic preconditioning (13), are associated with the infarct size-reducing effect.
Protocol 1: myocardial interstitial serotonin in in vivo and isolated beating hearts
In vivo study
Surgical preparation was as previously reported (14). Briefly, rabbits were anesthetized with sodium pentobarbital (30 to 40 mg/kg, intravenous), and additional doses were administered when required throughout the experiment. They were intubated and ventilated with room air supplemented with low-flow oxygen using a mechanical ventilator (tidal volume, 20 to 30 ml, respiratory rate, 20/min to 30/min; Model SN-480-5, Shinano, Tokyo, Japan). Serial blood gas analysis was performed, and ventilatory conditions were adjusted to keep the arterial blood gas within the physiologic range. The left carotid artery and jugular vein were cannulated to monitor peripheral arterial pressure and to administer drugs or saline. The rabbits were then systematically heparinized (500 U/kg). A thoracotomy was performed at the third intercostal space, and the heart was exposed after excising the pericardium. A 4-0 silk suture on a small curved needle was passed through the myocardium beneath a large marginal branch of the left circumflex coronary artery, which supplies much of the anterolateral and apical walls of the left ventricle (LV). A small vinyl tube was passed into both ends of the suture to make the snare.
A microdialysis probe (PNF 1700, 20 mm length, 0.31 mm outside diameter, 0.2 mm inside diameter, transverse type, 50,000 MW cut-off) for dialysate sampling was implanted in the risk region of the myocardium, which was served by the anterolateral coronary artery along the axis of the ventricular fibers and reached from the epicardial outer layer to the endocardial inner layer of the myocardium. The probe placement was confirmed at autopsy. The microdialysis probe was perfused with Ringer’s solution at a rate of 10 μl/min. After a 60-min rest following the completion of the instrumentation, the dialysate was sampled during a 30-min period before ischemia and during a 30-min period of ischemia and during two consecutive 30-min periods of reperfusion. Ischemia was induced by occluding the coronary branch by pulling the snare, which was then fixed by clamping the tube with a mosquito hemostat. Myocardial ischemia was confirmed by regional cyanosis and electrocardiographic change. Reperfusion was confirmed by the myocardial blush over the risk area after releasing the snare. After the initial preparation but before the coronary occlusion, the animals were assigned to one of the two groups described in the following text.
A total of 14 rabbits underwent 30-min ischemia and 60-min reperfusion in the presence of saline (control group, n = 7) or the 5-HT2 receptor blocker SG (SG group, n = 7; 10 mg/kg/h, intravenous, starting 10 min before ischemia until 20 min after reperfusion). In the preliminary study using three rabbits, the plasma concentration of SG in arterial blood assessed by high-performance liquid chromatography (HPLC) was 588 ± 169 ng/ml and 688 ± 308 ng/ml at 10 min and 60 min, respectively, after the start of venous infusion of SG at a rate of 10 mg/kg/h. The concentration was reported to be adequate to block the 5-HT2 receptors (5). Therefore, the dose of 10 mg/kg/h was selected in the present study.
Isolated heart study
The 12 rabbits were anesthetized and ventilated as described above. The hearts were excised, immediately arrested in ice-cold buffer, mounted, and then perfused at a constant pressure of 100 cm H2O with Krebs-Henseleit solution gassed with 95% O2 and 5% CO2 at 37°C. After a 20-min stabilization period, the hearts were perfused with buffer for 30 min and then global ischemia was induced by stopping the flow of perfusate for 30 min, and then reperfused for 30 min without ischemia. Sarpogrelate was administered at a dose of 10 mg/kg/h for 10 min before global ischemia in six hearts. Another six hearts without SG were used as controls. A microdialysis probe for dialysate sampling was implanted in the myocardium along the axis of the ventricular fibers and reached from the epicardial outer layer to the endocardial inner layer of the myocardium. The microdialysis probe was perfused with Ringer’s solution at a rate of 10 μl/min. After a 20-min rest following the completion of the instrumentation, the dialysate was sampled during a 30-min period before ischemia, and during a 30-min period of ischemia, and during a 30-min period of reperfusion.
The dialysate samples obtained from both in vivo and isolated hearts were frozen at −83°C until further analysis. The dialysate serotonin concentrations were measured using a reversed-phase HPLC system (MCM column, MC Medical, Tokyo, Japan) coupled with electrochemical detection (Coulochem II, ESA, Chemsford, Massachusetts).
Protocol 2: infarct size
A total of 110 rabbits underwent a surgical preparation similar to that in protocol 1 except for the insertion of the microdialysis probe (Table 1). Two types of studies (pre-treatment and post-treatment) were performed. All rabbits underwent 30 min of ischemia followed by 48-h reperfusion, and the rabbits were randomly assigned to each treatment group by lot. In the control group (n = 15), 1 ml of placebo saline (0.9% NaCl, pH 7.4) was injected 10 min before ischemia. In the SG pretreated groups, each rabbit was intravenously infused with SG at a rate of 5 or 10 mg/kg/h (n = 13 and n = 12, respectively), starting 10 min before ischemia until 20 min after reperfusion for 60 min. In the SG post-treated group (n = 14), SG (10 mg/kg/h) was intravenously infused starting 10 min before reperfusion until 50 min after reperfusion for 60 min. In the SG + chelerythrine group (n = 15), chelerythrine (a selective PKC inhibitor, 5 mg/kg) was intravenously administered 10 min before SG infusion (10 mg/kg/h × 60 min) starting 10 min before ischemia. In the SG + 5-hydroxydecanoate (5-HD) group (n = 14), 5-HD (a mitochondrial KATP channel blocker, 5 mg/kg) was intravenously administered 10 min before SG infusion (10 mg/kg/h × 60 min) starting 10 min before ischemia. The chelerythrine group (n = 13) or 5-HD group (n = 14) was intravenously injected with chelerythrine (5 mg/kg) or 5-HD (5 mg/kg) 20 min before ischemia. After the experiment the chest was closed, and the rabbits were allowed to recover from anesthesia for two days.
The postmortem examination was as previously reported (14). At the end of the study, the rabbits were heparinized (500 U/kg) and killed by an overdose of pentobarbital. The heart was excised and mounted on a Langendorff apparatus. The coronary branch was reoccluded, and monastral blue dye (4%, Sigma Chemical Co., St. Louis, Missouri) was injected from the aorta at 80 mm Hg. The LV was sectioned into seven slices parallel to the atrioventricular ring. Each slice was weighed, incubated in a 1% solution of triphenyl tetrazolium chloride at 37°C to visualize the infarct area (15), and photographed. The area of the ischemic region and the infarcted myocardium were traced on each LV slice and multiplied by the slice’s weight, then the infarct size was expressed as a fraction of the risk region of LV for each heart. The measurement of the infarct size was performed by two persons blinded to the treatment.
Protocol 3: assessment of the subcellular distribution of PKC-ϵ
The 20 rabbits were anesthetized and ventilated as described in the preceding text. The hearts were excised, immediately arrested in ice-cold buffer, mounted, and perfused at a constant pressure of 100 cm H2O with Krebs-Henseleit solution gassed with 95% O2 and 5% CO2 at 37°C. After a 20-min stabilization period, the hearts were perfused with buffer containing either SG of 10 mg/kg/h (n = 10) or an equivalent volume of saline (n = 10) for 10 min. Then, 20-min global ischemia was induced in a half of each group. Another half of each group (nonischemia groups) was perfused with buffer for 20 min. We selected 20 min of ischemia because we previously demonstrated that PKC-ϵ is maximally translocated at 20 min of ischemia in rabbit hearts (16). At the end of the 20-min ischemia, the ventricles were quickly separated, frozen in liquid nitrogen, and stored at −83°C.
Frozen samples were weighed and homogenized in five volumes of buffer containing 300 mM sucrose, 4 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 2 mM ethyleneglycoltetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 20 mM leupeptin using a Polytron homogenizer at the maximum speed in five 5-s bursts. The homogenates were centrifuged at 1,000 × g for 10 min, after which the supernatant was referred to as the cytosolic fraction. The pellet (membrane fraction) was dispersed in a buffer containing 20 mM 2-amino-2-hydroxymethyl-1,3-propanedial-HCl (Tris-HCl), 1 mM EGTA, 1 mM PMSF, 20 μM leupeptin, and 0.5% Triton X-100. The protein concentration in samples was determined by the Bradford method (Bio-Rad protein assay kit, California).
The PKC-ϵ present in the cytosolic and membrane fractions was assessed using a standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Western immunoblotting technique. The proteins (30 mg) present in each sample fraction were separated by SDS-PAGE on 8.0% gels and then electrophoretically transferred to 0.45 μm polyvinilidene difluoride membranes using a Semi-Dry transfer cell (Bio-Rad). The transfer buffer contained 25 mM Tris-HCl and 192 mM glycine in 20% methanol. The blots were initially blocked overnight with 5% milk in buffer containing 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 0.05% Tween-20, and then incubated for 1.5 h with anti-PKC-ϵ antibody (1:100 dilution). The primary antibody was monoclonal raised against peptides corresponding to amino acids 1 to 175 as mapped to the carboxyterminus of human PKC-ϵ. After washing, the blots were incubated for 1 h at room temperature with a peroxidase-linked, goat anti-mouse secondary antibody (1:5,000 dilution). The bound antibody was then visualized using an enhanced chemiluminescence kit (Amersham). Protein kinase C was quantified densitometrically using suitable autoradiographs.
Values are expressed as group means ± SEM. To compare the group means of the infarct sizes and quantitation of PKC-ϵ, one-way analysis of variance (ANOVA) was performed, and if the ANOVA results were significant, a Scheffé’s post-hoc test was performed. To compare the group means of the hemodynamic parameters, interstitial serotonin levels, a two-way repeated measures ANOVA followed by a Scheffé’s post-hoc test was performed. A p value of <0.05 was considered to be statistically significant.
Mortality and animal exclusion
A total of 110 rabbits were initially enrolled in the infarct size study. There was no significant difference in the number of animals assigned to each of the eight groups or in the incidence and time of ventricular fibrillation and mortality (Table 1). Among these animals seven were excluded because of technical problems, six had ventricular fibrillation during coronary occlusion, and four had ventricular fibrillation during reperfusion; all of these rabbits died. Six rabbits died after the first day of the experiment. Thus, the experiments were completed in the remaining 88 rabbits (11 in each of the eight groups), and the findings from these animals were used for the analysis.
There were no significant differences in the basal systolic and diastolic blood pressures and basal heart rate among any group in protocol 1 (Table 2) and protocol 2 (Table 3). Throughout the experiment the blood pressure and heart rate were similar among all groups.
Myocardial interstitial serotonin levels
As shown in Figure 1A, the myocardial interstitial serotonin level in the in vivo rabbit model significantly increased during 30-min ischemia and during the first 30-min reperfusion (41.6 ± 6.7 ng/ml and 47.2 ± 16.6 ng/ml vs. 16.3 ± 3.4 ng/ml at control), and decreased to below the control level during the second 30-min reperfusion (7.8 ± 1.9 ng/ml). This increase in interstitial serotonin during 30-min ischemia and the first 30-min reperfusion was blocked by the pretreatment with the 5-HT2 blocker SG.
In the isolated heart model, as shown in Figure 1B, the myocardial interstitial serotonin level before ischemia (0.052 ± 0.018 ng/ml) was quite low compared with that of the in vivo model (16.3 ± 3.4 ng/ml), but was significantly increased during ischemia (1.155 ± 0.427 ng/ml). The increase disappeared during early reperfusion (0.243 ± 0.103 ng/ml). The serotonin increases were inhibited by SG.
Myocardial infarct size
The mean percentages of the area at risk (percentage of LV) were: 34 ± 1%, 30 ± 3%, 31 ± 1%, 31 ± 2%, 34 ± 2%, 35 ± 2%, 37 ± 3%, and 37 ± 2% in the control, SG pre-5 group, SG pre-10 group, SG post group, SG + chelerythrine group, chelerythrine group, SG + 5-HD group, and the 5-HD group, respectively. No significant difference was seen among these groups.
As shown in Figure 2, the infarct size as a percentage of the area at risk was 45 ± 3% in the control group. The SG pre-5 and SG pre-10 groups showed a significant reduction in infarct size (31 ± 3% and 27 ± 2%, respectively, p < 0.05 vs. the control group). The SG post group did not show a significant reduction in infarct size (36 ± 2%). Treatment with chelerythrine and 5-HD blocked the infarct size-reducing effect of SG, respectively (38 ± 3% and 39 ± 3%, respectively). Chelerythrine or 5-HD alone did not affect the infarct size (38 ± 2% and 41 ± 3%, respectively).
Subcellular translocation of PKC isoform
Protein kinase C-ϵ was detected as a single band with a molecular mass of 98 kDa. The quantitative densitometric analyses illustrated in Figure 3 showed that 20 min of ischemia caused translocation of PKC-ϵ from the cytosolic to the membrane fraction and that SG enhanced the ischemia-induced increase in PKC-ϵ in the membrane fraction. Sarpogrelate did not cause any translocation of PKC-ϵ in the nonischemic heart tissue.
Release of serotonin into the myocardial interstitial space during ischemia and the blocking effect by SG
Although microdialysis technique has been used for the measurements of adenosine and noradrenaline in the myocardial interstitium during ischemia (16,17,18), no study is published regarding serotonin, to our knowledge.
The present study revealed an increase in serotonin in myocardial interstitial tissues during ischemia using microdialysis. In in vivo cardiac tissues, serotonin is rich in platelet cytoplasm of the vascular beds, although mast cells and sympathetic nerve endings also have some serotonin (Fig. 4). Ischemia, indicating the lack of blood flow, induces platelet aggregation and release of serotonin initially. As platelets have 5-HT2A receptors, their binding with released serotonin induces a large amount of serotonin release from platelet cytoplasm and more platelet aggregation. The presence of 5-HT2 receptors has not been reported in mast cells or sympathetic nerve endings. The interstitial serotonin increases were almost completely blocked by SG, a selective 5-HT2 receptor blocker. These findings suggest that an increase in myocardial interstitial serotonin during ischemia is merely due to serotonin release from platelet cytoplasm via the vicious cycle between platelet 5-HT2A receptor and serotonin rather than the release from mast cells and sympathetic nerve endings.
In the present study, isolated hearts were perfused with Krebs-Henseleit solution, which did not include blood or platelets. The myocardial interstitial serotonin level at the baseline was quite low, compared with that of the in vivo hearts. This finding could be explained by platelet washout due to perfusion with Krebs-Henseleit solution before ischemia. The myocardial interstitial serotonin level of isolated hearts was clearly increased during ischemia, although the increase was small compared with that of the in vivo hearts. One of the possible mechanisms is the release of serotonin from mast cells and/or sympathetic nerve endings. However, small amounts of platelets with serotonin would remain in a part of myocardial capillary beds despite the washout effect by Krebs-Henseleit solution. The findings of the present study showed that the increase in myocardial interstitial serotonin was abolished by SG. These findings suggest that the serotonin release from the remaining platelet via the vicious cycle plays an important role in the increase of myocardial interstitial serotonin in isolated hearts during ischemia.
In in vivo hearts, the increase in interstitial serotonin was observed during early reperfusion and disappeared thereafter. On the other hand, the increase was not seen even during early reperfusion in isolated hearts. This suggests that the increase during early reperfusion in in vivo hearts depends on blood reperfusion with platelets. That is, aggregation of new reperfused platelets and serotonin release from these may be important.
Translocation of myocardial PKC-ϵ and myocardial interstitial serotonin in isolated hearts
Protein kinase C consists of various subtypes and is present in various types of cells, especially rich in blood cells. Furthermore, rapid freezing in liquid nitrogen is needed for the precise measurement. Therefore, we used isolated hearts perfused with Krebs-Henseleit solution, but not in vivo hearts, for the measurement of myocardial PKC, as previously reported (19). The findings of this study in isolated hearts that PKC-ϵ was translocated from the cytosolic fraction to the membrane fraction during ischemia confirmed the previous observation (19). In the present study we found that SG, a 5-HT2 receptor blocker, enhanced translocation of myocardial PKC-ϵ and inhibited the increase in myocardial interstitial serotonin during ischemia in isolated hearts. This suggests that increased translocation of myocardial PKC-ϵ may be associated with inhibition of myocardial interstitial serotonin (Fig. 4).
Possible mechanisms of the infarct size-reducing effect by SG
Sarpogrelate significantly reduced the myocardial infarct size in in vivo rabbit hearts. Because SG had no effect on heart rate or blood pressure, the infarct-size-reducing effect of SG was not due to the decrease in the double product, an indicator of oxygen demand. In addition, the infarct size-reducing effect of SG was independent of the regional blood flow because the collateral circulation in the rabbit heart was minimal (20).
Cardiomyocytes are protected against ischemic cellular damage via translocation of PKC-ϵ in isolated rabbit hearts, followed by opening of the mitochondrial KATP channel (13,21), which was confirmed in this study. The findings of this study showed an increase in myocardial interstitial serotonin during ischemia and reperfusion, which was inhibited by SG, a selective 5-HT2 receptor blocker, as discussed previously. Sarpogrelate enhanced further translocation of PKC-ϵ and reduced infarct size. The infarct-size-reducing effect of SG disappeared after treatment with chelerythrine, a selective PKC inhibitor, and with 5-HD, a selective inhibitor of the mitochondrial KATP channel. These findings suggest that increased myocardial interstitial serotonin can inhibit translocation of PKC-ϵ followed by opening of the mitochondrial KATP channel, and SG reduces infarct size by inhibiting an increase in serotonin release during ischemia and reperfusion (Fig. 4).
Other possible mechanisms of the infarct size-reducing effect by SG are as follows. Stimulation of 5-HT2 receptors by serotonin accumulates inositol phosphates through phospholipase C-mediated hydrolysis of phosphoinositides (22), which can release calcium from intracellular pools, and may cause calcium overload and damage the myocytes (23). Serotonin induces vasoconstriction and thrombus formation in intramyocardial small vessels, and may cause the so-called no-reflow phenomenon (24). The calcium overload and so-called no-reflow phenomenon are associated with early reperfusion injury. In this study serotonin was increased not only during ischemia, but also during early reperfusion, and the increases were inhibited by SG. Therefore, the inhibition of calcium overload and the so-called no-reflow phenomenon may be included in the protective mechanism of SG.
Sarpogrelate is currently used for the treatment of patients with arteriosclerosis obliterance because of its vasodilating and antiplatelet actions. It has been reported that SG is beneficial for human angina pectoris through an increase in the collateral circulation (12). In this study we found a cardioprotective effect against infarction, which was independent of collateral flow or double products. The doses of SG used in this study were 5 or 10 mg/kg/h, and plasma concentrations of SG 10 and 60 min after infusion of 10 mg/kg/h of SG was 588 and 688 ng/ml, respectively. The clinical dose of SG is 100 mg × 3 times/day, and the maximum plasma concentration of SG after oral administration of 100 mg of SG was reported to be 540 ng/ml in healthy volunteers (25), which is very similar to that of the present study. In the present study SG reduced myocardial infarct size only when administered before ischemia. Therefore, SG may be useful for reducing infarct size when acute myocardial infarction occurs in the patients with angina pectoris and atherosclerosis obliterance that have previously been being treated with SG. Further clinical investigations are warranted.
☆ Supported, in part, by a Grant-in-Aid for Scientific Research (C) (10670639) from the Japan Society for the Promotion of Science.
- analysis of variance
- ethyleneglycol tetraacetic acid
- high-performance liquid chromatography
- adenosine triphosphate sensitive potassium
- left ventricle
- protein kinase C
- phenylmethylsulfonyl fluoride
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Received October 24, 2001.
- Revision received May 20, 2002.
- Accepted June 27, 2002.
- American College of Cardiology Foundation
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