ORIGINAL RESEARCH Determinants of Myocardial Lactate Production During Acetylcholine Provocation Test in Patients With Coronary Spasm Koichi Kaikita, MD, PhD; Masanobu Ishii, MD; Koji Sato, MD, PhD; Masafumi Nakayama, MD, PhD; Yuichiro Arima, MD, PhD; Tomoko Tanaka, MD, PhD; Koichi Sugamura, MD, PhD; Kenji Sakamoto, MD, PhD; Yasuhiro Izumiya, MD, PhD; Eiichiro Yamamoto, MD, PhD; Kenichi Tsujita, MD, PhD; Megumi Yamamuro, MD, PhD; Sunao Kojima, MD, PhD; Hirofumi Soejima, MD, PhD; Seiji Hokimoto, MD, PhD; Kunihiko Matsui, MD, MPH; Hisao Ogawa, MD, PhD Background-—Myocardial lactate production in the coronary circulation during acetylcholine (ACh)-provocation test (abbreviated as lactate production) provides supporting evidence for coronary spasm–induced myocardial ischemia. The purpose of this study was to examine the clinical features, predictive factors, and prognosis of patients with coronary vasospastic angina (VSA) and lactate production. Methods and Results-—We examined all 712 patients who underwent both myocardial lactate measurement during ACh- provocation test in the left coronary artery and genetic screening test of a –786T/C polymorphism in the 50-flanking region of the endothelial nitric oxide synthase (eNOS) gene between January 1991 and December 2010. Lactate production was observed in 252 of the 712 patients and in 219 of 356 VSA patients diagnosed by ACh-provocation test. Compared with lactate production– negative VSA patients, the lactate production–positive counterparts were more likely to be nonsmoker female diabetics with – 786T/C eNOS polymorphism (61% vs 31%, P<0.001, 62% vs 34%, P<0.001, 24% vs 14%, P=0.016, and 25% vs 15%, P=0.018, respectively). Multivariable logistic regression analysis identified female sex, diabetes mellitus, and –786T/C eNOS polymorphism to correlate with lactate production (odds ratio 3.51, 95% CI 2.16 to 5.70, P<0.001; odds ratio 2.53, 95% CI 1.38 to 4.65, P=0.003; and odds ratio 1.85, 95% CI 1.02 to 3.35, P=0.044, respectively). Kaplan–Meier survival curve showed no difference in 5-year survival rate free from major adverse cardiac events between lactate production–positive and –negative VSA patients (P=0.319). Conclusions-—The results indicated that female sex, diabetes, and mutation in –786T/C eNOS gene correlate with ACh-provoked myocardial ischemia in patients with coronary spasm. ( J Am Heart Assoc. 2015;4:e002387 doi: 10.1161/JAHA.115.002387) Key Words: acetylcholine-provocation test • coronary spasm • endothelial nitric oxide synthase polymorphism • myocardial lactate production oronary spasm plays an important role in the pathogen- is widely used for the diagnosis of VSA.12–20 While C esis of acute and chronic coronary heart diseases.1–10 epicardial coronary spasm can be viewed angiographically Because coronary vasospastic angina (VSA) can be diagnosed during the ACh-provocation test, the test does not confirm in only 20% of the patients who present with spontaneous the presence of myocardial ischemia. For this reason, attack and ischemic electrocardiogram (ECG) changes,11 the measurement of myocardial lactate production in the spasm-provocation test with acetylcholine (ACh) or ergonovine coronary circulation is used as supporting diagnostic marker in the evaluation of coronary spasm–induced myocardial ischemia during ACh-provocation test in the left From the Department of Cardiovascular Medicine, Graduate School of Medical coronary artery (for simplicity, this is abbreviated in the text Sciences, Kumamoto University, Kumamoto, Japan (K.K., M.I., K. Sato, M.N., to “lactate production”). Y.A., T.T., K. Sugamura, K. Sakamoto, Y.I., E.Y., K.T., M.Y., S.K., H.S., S.H., H.O.); Department of Community Medicine, Kumamoto University Hospital, ACh-induced vasodilatation is thought to be mediated by Kumamoto, Japan (K.M.). nitric oxide (NO) released from the endothelium.21,22 In Correspondence to: Koichi Kaikita, MD, PhD, Department of Cardiovascular patients with coronary spasm, the basal tone of the coronary Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, arteries is increased, and the hyperreactivity to nitrovasodila- Honjo, Chuo-ku, Kumamoto 860-8556, Japan. E-mail: kaikitak@kumamoto-u. ac.jp tors seen in patients with coronary spasm is consistent with 21,23 Received July 9, 2015; accepted October 23, 2015. decreased endothelial release of NO. We have reported ª 2015 The Authors. Published on behalf of the American Heart Association, previously the presence of –786T/C polymorphism in the Inc., by Wiley Blackwell. This is an open access article under the terms of the 50-flanking region of the endothelial NO synthase (eNOS) gene Creative Commons Attribution-NonCommercial License, which permits use, 24 distribution and reproduction in any medium, provided the original work is and highlighted its association with coronary spasm. We properly cited and is not used for commercial purposes. argued that eNOS gene mutation could reduce endothelial NO DOI: 10.1161/JAHA.115.002387 Journal of the American Heart Association 1 Myocardial Lactate Production in ACh Test Kaikita et al ORIGINAL RESEARCH synthesis and predispose patients with the mutation to exclusion, data for 1760 patients who had undergone coronary spasm.24 In another study, we also demonstrated selective ACh-provocation test were analyzed. They included the presence of a low lactate extraction ratio during the all 712 patients who also consented to genetic screening for ACh-provocation test in patients with coronary spasm eNOS gene –786T/C polymorphism. harboring –786T/C polymorphism in the eNOS gene.25 To The risk factors for coronary artery disease were defined our knowledge, however, there is no information on the as current smoking (smoking within 1 year), hypertension factors that contribute to lactate production (eg, eNOS gene (>140/90 mm Hg or taking antihypertensive medications), polymorphism) in the general population. The purpose of the dyslipidemia (high-density lipoprotein cholesterol <40 mg/ present study was to investigate the clinical features, dL, low-density lipoprotein cholesterol ≥140 mg/dL, or predictive factors, and long-term prognosis of VSA patients triglycerides ≥150 mg/dL or taking medications for dyslipi- with the lactate production. demia), diabetes mellitus (symptoms of diabetes plus casual plasma glucose concentration ≥200 mg/dL, fasting plasma glucose concentration ≥126 mg/dL, 2-hour plasma glucose Methods concentration ≥200 mg/dL during 75 g oral glucose toler- ance test, or taking medications for diabetes mellitus), and Study Population and ACh-Provocation Test family history of ischemic heart disease (IHD), including We analyzed retrospectively the angiographic coronary vaso- obstructive coronary artery disease, VSA, and myocardial motor response induced by ACh injection in 1877 consecutive infarction. patients who had typical and atypical angina-like chest pain The ACh-provocation test was performed as described and were admitted to Kumamoto University Hospital between previously in “Guidelines for Diagnosis and Treatment of January 1991 and December 2010. Among these, we Patients With Vasospastic Angina” by the Japanese Circulation excluded 117 patients for the following reasons: acute Society.26 Coronary spasm was defined as total or subtotal myocardial infarction (n=20), cardiomyopathy (n=75), Brugada obstruction within the borders of one isolated coronary syndrome (n=10), and other conditions (n=12). After the segment as defined by the American Heart Association,27 or Control ACh 100 μg ISDN ABC AR AR CS PC CS RAO35 DE F AR AR PC CS CS LAO55 CRA25 Figure 1. ACh-induced coronary spasm and sampling source for serum lactate at the AR and CS in the coronary circulation. A through C, CAG at RAO35°. D through F, CAG at LAO55° RAO35°. B and E, Injection of 100 lg ACh into the left coronary artery induced diffuse spasm in the entire left coronary artery. ACh indicates acetylcholine; AR, aortic root; CS, coronary sinus; PC, pacing catheter; ISDN, isosorbide dinitrate. DOI: 10.1161/JAHA.115.002387 Journal of the American Heart Association 2 Myocardial Lactate Production in ACh Test Kaikita et al ORIGINAL RESEARCH All patients who underwent ACh- provocation test in left coronary artery between Jan 1991 and Dec 2010 n=1877 (male/female: 966/911) Excluded n=117 (65/52) Acute myocardial infarction (n=20) Cardiomyopathy (n=75) Brugada syndrome (n=10) n=1,760 (903/857) Others (n=12) Patients who underwent both lactate <No> measurement and screening for n=1048 (539/509) -786T/C eNOS gene polymorphism <Yes> n=712 (364/348) Lactate production No lactate production n=252 (100/152) n=460 (264/196) Presence of coronary spasm <No> <No> No spasm no Unclassified IHD lactate production n=33 (15/18) n=323 (170/153) <Yes> <Yes> Spasm & lactate production Spasm no lactate production n=219 (85/134) n=137 (94/43) Figure 2. Flow chart of the study recruitment process. ACh indicates acetylcholine; eNOS, endothelial nitric oxide synthase; IHD, ischemic heart disease. severe diffuse vasoconstriction observed in >2 adjacent coronary sinus drains blood from the left coronary system but coronary segments of epicardial coronary arteries associated not from the right. with transient myocardial ischemia, as evidenced by ischemic ST-segment changes on the ECG. In this study, ischemic ST- – eNOS segment changes were defined as ST-segment elevation Screening for 786T/C Polymorphism in (>0.1 mV), ST-segment depression (>0.1 mV) from baseline Gene level occurring at 60 to 80 ms after the J point in ≥2 An allele-specific oligonucleotide method was used in screen- contiguous leads on the 12-lead ECG, or appearance of a new ing for –786T/C polymorphism in the eNOS gene. Hybridiza- negative U-wave on ECG. tion was accomplished with 32P-radiolabeled oligonucleotides Lactate production was estimated by measuring serum corresponding to the probe for either the –786T allele or the – lactate concentrations at the root of the aorta (AR) and 786C allele. Details of the method have been published coronary sinus (CS), sampled during myocardial ischemia previously.24 Briefly, the polymerase chain reaction frag- induced by ACh-provocation (see Figure 1).