Increased Plasma Level of Endothelin- 1 -Like Immunoreactivity During Coronary Spasm in Patients with Coronary Spastic Angina Kozaburo Matsuyama, MD, Hirofumi Yasue, MD, Ken Okumura, MD, Yoshihiko Saito, MD, Kazuwa Nakao, MD, Gotaro Shirakami, MD, and Hiroo Imura, MD

To examine the role of endothelin in coronary spasm, the plasma endothelin- 1 -like immunoreactivity in the coronary sinus and the aortic root was measured before and during spasm of the left coronary artery induced by injection of acetylcholine (20 to 100 erg) into the left coronary artery in 26 patients with coronary spastic angina. The plasma level of endothelin-l-like immunoreactivity was measured by the radioimmunoassay with a monoclonal antibody against endothelin-1. Plasma lactate levels in the coronary sinus and the aortic root were also measured to evaluate myocardial lactate metabolism during spasm. In 13 patients who had myocardial lactate production during spasm, the plasma level of endothelin-l-like immunoreactivity in the coronary sinus increased from 19.8 f 3.0 to 25.7 f 6.4 pg/ml (p 2 ENDOTHELIN IN CORONARY SPASM 991

hours before catheterization. The study was performed in the morning in the fasting state. A 6Fr GoodaleLubin catheter (USCI) for blood sampling was positioned in the coronary sinus through the right antecubital vein. The position of the catheter was confirmed by occasional injection of the contrast medium. A tripolar electrode catheter (USCI) was inserted into the right ventricular apex through the right femoral vein and was connectedto a temporary pacemakerset at a rate of 40 to 50 beats/min. Coronary arteriography was performed using the Sonestechnique. After the control left and right coronary arteriography, acetylcholine (20 to 100 pg) was injected into the left coronary artery for provocation of coronary spasmas reported previously.4,8 Left coronary arteriography was performed when STsegment changes on the electrocardiogram or chest pain, or both, developed,or 1.5 minutes after initiation of each injection. Coronary spasmwas defined as total or subtotal occlusion or diffuse severeconstriction of the coronary artery associatedwith chest pain or ST-segment changeson the electrocardiogram, or both. During the study, arterial blood pressureand 3 electrocardiographic leads (I, aVF and Vs or V4) were continuously monitored on an oscilloscope, and 6 electrocardiographic leads (I, II, aVF, Vi, V3 and Vs) were continuously recordedand 12 electrocardiographic leads were recorded at appropriate intervals. Wood sampling: Blood samplesfor measurementsof endothelin- 1-like immunoreactivity and lactate were obtained simultaneously from the coronary sinus and the aortic root during the study. In the coronary spastic angina group, blood sampleswere obtained just before the initial injection of acetylcholine and 20 to 30 secNS

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I Before

After (Spasm)

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FIGURE 1. Plots oi indiidwl and mean f standard deviation vahwe of plasma endotheftn-l-like immunweacW@ (ET-I-LI) kvols in the cerenary dnus (CS) and the aortic root (Ao) betere and after the injection of acetykhdine in the coronary spastic angina group as a whole. Left coronaryarlorialspasnl &as induc& at& the injection ot acetykholine in each patient. No signiticant changes in plasma Er-l-I.1 levels in both CS and Ao were observed. NS = not eignificant. 992

THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 68

onds after the arteriographic documentation of spasm. In the control group, blood sampleswere obtained just before the initial injection of acetylcholine and 20 to 30 secondsafter the coronary arteriography performed after injection of a maximum doseof acetylcholine. Each blood samplewas immediately withdrawn into a chilled siliconized glass tube that contained aprotinin (1,000 KIU/ml) and ethylenediaminetetraacetic acid (1 mg/ ml) and centrifuged at 3,000 g for 10 minutes at 4’C, separatedand stored at -80°C until analysis. Measurement reactivity level:

of plasma endothelin-l-like

immuno-

Plasma endothelin-1-like immunoreactivity level was measured in duplicate using the radioimmunoassay with a monoclonal antibody against endothelin-1 (KY-ET-l-I), which was validated in the previous studies.9J0The extraction of endothelin from plasma was performed using polystyrene beads coated with the monoclonal antibody. The value of 50% inhibitory concentration was 6 pg/tube and the minimal detectable amount was 0.5 pg/tube in this radioimmunoassay.Recoveriesof 25 and 50 pg endothelin-1 and 50 pg big endothelin added to 1 ml plasma were 52, 55 and 48%, respectively.9Inter- and intraassay variations in the radioimmunoassay were 6.8 f 2.8 and 7.3 f 2.2%, respectively.9 The radioimmunoassay showed cross-reactivitiesof 60% with endothelin-3 and 100% with endothelin-2 and human big endothelin.9 Measurement of plasma lactate level: The plasma concentration of lactate was measured in duplicate using an enzyme system assay kit, Determiner LA (Kyowa Medics Corp.), employing lactate oxidase combined with N-ethyl-N-( 3-methylphenyl)-N’-acetylethylenediamine.l l Myocardial lactate extraction ratio was calculated by the following formula: myocardial lactate extraction ratio (%) = [(LA~-L&/LAJ X 100 where LAGand Lcs representplasma lactate concentration (mg/dl) in the aortic root and in the coronary sinus, respectively. Statistical analysis: The 2-tailed paired Student’s t test was used in analysisof changesin endothelin-l-like immunoreactivity and lactate levels in each group. Comparisonsof endothelin-1-like immunoreactivity levels between the 2 groups were performed with the unpaired Student’s t test. All values were expressedas mean f standard deviation, and a p value