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Determinants of Myocardial Lactate Production During Acetylcholine Provocation Test in Patients With Coronary Spasm

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 5′‐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.

C oronary spasm plays an important role in the pathogenesis of acute and chronic coronary heart diseases. [1][2][3][4][5][6][7][8][9][10] Because coronary vasospastic angina (VSA) can be diagnosed in only %20% of the patients who present with spontaneous attack and ischemic electrocardiogram (ECG) changes, 11 the spasm-provocation test with acetylcholine (ACh) or ergonovine is widely used for the diagnosis of VSA. [12][13][14][15][16][17][18][19][20] While epicardial coronary spasm can be viewed angiographically during the ACh-provocation test, the test does not confirm the presence of myocardial ischemia. For this reason, measurement of myocardial lactate production in the coronary circulation is used as supporting diagnostic marker in the evaluation of coronary spasm-induced myocardial ischemia during ACh-provocation test in the left coronary artery (for simplicity, this is abbreviated in the text to "lactate production").
ACh-induced vasodilatation is thought to be mediated by nitric oxide (NO) released from the endothelium. 21,22 In patients with coronary spasm, the basal tone of the coronary arteries is increased, and the hyperreactivity to nitrovasodilators seen in patients with coronary spasm is consistent with decreased endothelial release of NO. 21,23 We have reported previously the presence of -786T/C polymorphism in the 5 0 -flanking region of the endothelial NO synthase (eNOS) gene and highlighted its association with coronary spasm. 24 We argued that eNOS gene mutation could reduce endothelial NO synthesis and predispose patients with the mutation to coronary spasm. 24 In another study, we also demonstrated the presence of a low lactate extraction ratio during the ACh-provocation test in patients with coronary spasm harboring -786T/C polymorphism in the eNOS gene. 25 To our knowledge, however, there is no information on the factors that contribute to lactate production (eg, eNOS gene polymorphism) in the general population. The purpose of the present study was to investigate the clinical features, predictive factors, and long-term prognosis of VSA patients with the lactate production.

Methods Study Population and ACh-Provocation Test
We analyzed retrospectively the angiographic coronary vasomotor response induced by ACh injection in 1877 consecutive patients who had typical and atypical angina-like chest pain and were admitted to Kumamoto University Hospital between January 1991 and December 2010. Among these, we excluded 117 patients for the following reasons: acute myocardial infarction (n=20), cardiomyopathy (n=75), Brugada syndrome (n=10), and other conditions (n=12). After the exclusion, data for 1760 patients who had undergone selective ACh-provocation test were analyzed. They included all 712 patients who also consented to genetic screening for eNOS gene -786T/C polymorphism.
The risk factors for coronary artery disease were defined as current smoking (smoking within 1 year), hypertension (>140/90 mm Hg or taking antihypertensive medications), dyslipidemia (high-density lipoprotein cholesterol <40 mg/ dL, low-density lipoprotein cholesterol ≥140 mg/dL, or triglycerides ≥150 mg/dL or taking medications for dyslipidemia), diabetes mellitus (symptoms of diabetes plus casual plasma glucose concentration ≥200 mg/dL, fasting plasma glucose concentration ≥126 mg/dL, 2-hour plasma glucose concentration ≥200 mg/dL during 75 g oral glucose tolerance test, or taking medications for diabetes mellitus), and family history of ischemic heart disease (IHD), including obstructive coronary artery disease, VSA, and myocardial infarction.
The ACh-provocation test was performed as described previously in "Guidelines for Diagnosis and Treatment of Patients With Vasospastic Angina" by the Japanese Circulation Society. 26 Coronary spasm was defined as total or subtotal obstruction within the borders of one isolated coronary segment as defined by the American Heart Association, 27  Lactate production was estimated by measuring serum lactate concentrations at the root of the aorta (AR) and coronary sinus (CS), sampled during myocardial ischemia induced by ACh-provocation (see Figure 1). The lactate production ratio was calculated by using the following formula: {[(lactate AR Àlactate CS )/lactate AR ]9100%}. The ratio is positive in healthy subjects, 28 while a negative ratio is a definite marker of myocardial ischemia. We usually examine coronary sinus lactate production during the ACh-provocation test only in the left coronary artery, because the great coronary sinus drains blood from the left coronary system but not from the right.

Screening for -786T/C Polymorphism in eNOS Gene
An allele-specific oligonucleotide method was used in screening for -786T/C polymorphism in the eNOS gene. Hybridization was accomplished with 32 P-radiolabeled oligonucleotides corresponding to the probe for either the -786T allele or the -786C allele. Details of the method have been published previously. 24 Briefly, the polymerase chain reaction fragments, 236 bp in length, including the -786T/C polymorphism site, were blotted in duplicate onto nylon membranes. Hybridization was accomplished with 32 P-radiolabeled oligonucleotides corresponding to either the -786T sequence (50-GGG TCA GCC AGC CAG GGAA-30; probe for the -786T sequence) or the -786C sequence (50-GGG TCA GCCGGC CAG GGAA-30; probe for the -786C sequence).  The study protocol was approved by the Human Ethics Review Committee of Kumamoto University, and a signed consent form was obtained from each subject.

Follow-up Data
Follow-up data were obtained directly from the patients, their families, or their family physicians, in addition to the information available on the medical records. Major adverse cardiac events (MACEs) were assessed by physicians blinded to the medical details available in the medical records. The primary end point was MACEs, defined as cardiac death, hospitalization for acute myocardial infarction, and unstable angina pectoris. The time frame in the survival analysis was defined as time from the date of diagnosis to the date of the first event or until December 2012. The secondary end point was all-cause mortality. Cardiac death was defined as sudden death or death associated with acute myocardial infarction. Acute myocardial infarction was defined by the presence of prolonged (>30 minutes) chest pain, associated with STsegment changes and elevated cardiac enzyme levels. Unstable angina pectoris represented recurrence or worsening of chest discomfort or pain, associated with ischemic ECG changes.

Statistical Analysis
Data for normally distributed continuous variables were expressed as meanAESD, whereas those with skewed distribution were expressed as median values (IQR). Continuous variables were analyzed by using the unpaired t test or Mann-Whitney U test, as appropriate. Categorical variables were presented by percentage values, and intergroup comparisons were analyzed by using the v 2 test or Fisher's exact test as appropriate. Age, sex, and the relationships between the results of ACh-provocation test and other significant parameters in simple logistic analysis were entered into multivariable logistic regression analysis by using the forced entry method, and the Hosmer-Lemeshow goodness-of-fit statistic was calculated to assess model calibration. The Breslow-Day test was carried out to confirm the relationship between lactate production and coronary spasm, after controlling for sex. Survival was analyzed by the Kaplan-Meier survival curve with the logrank test. The multivariable Cox hazard regression analysis was carried out for identification of predictors of outcome. Significant variables according to univariate analysis and found to be involved in VSA outcome were subjected to the forced entry method. A P value of <0.05 denoted statistical

Discussion
The present study described the clinical features, predictive factors, and long-term prognosis of VSA patients with lactate production in a large study population. The results showed that myocardial ischemia is more likely to be provoked by ACh in female diabetics with -786T/C eNOS polymorphism who present with VSA. The results also showed that despite these differences, the Kaplan-Meier survival analysis showed no difference in 5-year survival rates free from MACEs between lactate production-positive and -negative VSA patients. To the best of our knowledge, this is the first report that describes the relation between ACh-provoked myocardial ischemia and various predictors including sex and genetic factors in a large study population. Multivariable regression analysis identified female sex, diabetes mellitus, and spasm-positive findings as significant predictors for lactate production by using the data from 712 patients. Actually, 87% of lactate production-positive patients exhibited ACh-provoked coronary spasm in the left coronary artery, whereas 30% of lactate production-negative patients exhibited coronary spasm during the same test. These results suggest that lactate production could be a useful diagnostic tool for myocardial ischemia during the ACh-provocation test. It is not clear at present why angiographically evident coronary spasm did not induce lactate production in the remaining 137 of 356 VSA patients. It is possible that methodological (eg, gap in timing of blood sampling during ACh-provocation test) or physiopathological factors (eg, lowgrade ACh-provoked myocardial ischemia) were responsible for the lack of increase in serum lactate levels in the coronary circulation.
The present study identified female sex, diabetes mellitus, and -786T/C eNOS polymorphism to correlate with lactate production in VSA patients. In this regard, we identified previously -786T/C polymorphism in the eNOS gene and showed that this polymorphism was strongly associated with coronary spasm. 24 Further, the mutated -786T/C gene exhibited significant reduction in eNOS gene promoter activity based on luciferase reporter gene assays. 24 In the present study, the frequency of -786T/C eNOS mutation was higher in the VSA group compared with the no-spasm group (25% vs 15%, P=0.005, data not shown). Based on these findings, it is possible that -786T/C eNOS mutation could cause severe myocardial ischemia during ACh-provocation test, resulting in high serum lactate levels during coronary spasm. However, the presence of both -786T/C eNOS mutation and lactate production did not affect the incidence of MACEs in VSA patients (data not shown). In this retrospective study, VSA patients with eNOS mutation and lactate production were treated with antianginal drugs, such as calcium channel blockers and long-acting nitrates. Thus, one cannot rule out that such treatment reduced the cardiovascular events in those patients. Our data could not explain why lactate production in AChprovocation test was more common in female than in male VSA patients. Further studies are needed to examine this phenomenon. On the other hand, diabetes mellitus is known to be one of the major coronary risk factors and is closely associated with coronary endothelial dysfunction. [29][30][31][32][33][34] Previous studies reported the presence of endothelial dysfunction and impaired NO release in both type 2 diabetes 29,30 and insulin-dependent diabetes. 31,32 Patients with insulin resistance alone could also experience coronary endothelial dysfunction. 33,34 It is possible that in diabetic patients, impaired NO release induced by coronary endothelial dysfunction can cause coronary spasm and myocardial ischemia, as assessed by lactate production.
The present study has certain limitations. First, the number of patients was relatively small, mainly because some patients did not consent for the genetic screening test of eNOS gene -786T/C polymorphism. Second, we found that 33 (4.6%) unclassified IHD patients (of 712 patients) who underwent ACh-provocation test had lactate production without epicardial coronary spasm. Further analysis to define the diagnosis, clinical features, and treatment of those patients is needed. Third, because the study was retrospective in design, we could not examine the MACE rate in AChnegative patients. However, follow-up of the ACh-negative group will be clinically important to determine the prognosis and need for any medical treatment. Fourth, the outcome analysis had low statistical power because of the small number of events recorded during the follow-up period. A prospective multicenter study in a large study population with longer follow-up period is needed to investigate the clinical features, predictive factors, and prognoses of VSA patients who undergo the ACh-provocation test and measurement of lactate production.
In conclusion, myocardial lactate production in VSA patients during ACh-provocation test was closely associated with female sex, diabetes mellitus, and -786T/C eNOS polymorphism, although such production had no impact on the 5-year survival rate free from MACEs. Further large longterm studies are needed to determine the treatment strategy for VSA patients with lactate production during ACh-provocation test and eNOS gene mutation.

Sources of Funding
This study was supported in part by Grant-in-Aid for Scientific Research C24591062 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.