Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders

Summary

Intracoronary acetylcholine testing has been established for the assessment of epicardial coronary spasm more than 30 years ago. Recently, the focus has shifted towards the microcirculation and it has been shown that microvascular spasm can be detected using ACH-testing. This article describes the ACH-test and its implementation in daily routine.

Abstract

Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the catheterization laboratory which was introduced more than 30 years ago. Due to the short half-life of acetylcholine it can only be applied directly into the coronary arteries. Several studies have demonstrated the safety and clinical usefulness of this test. However, acetylcholine testing is only rarely applied in the U.S. or Europe. Nevertheless, it has been shown that 62% of Caucasian patients with stable angina and unobstructed coronary arteries on coronary angiography suffer from coronary vasomotor disorders that can be diagnosed with acetylcholine testing. In recent years it has been appreciated that the ACH-test not only assesses the presence of epicardial spasm but that it can also be useful for the detection of coronary microvascular spam. In such cases no epicardial spasm is seen after injection of acetylcholine but ischemic ECG shifts are present together with a reproduction of the patient's symptoms during the test. This article describes the experience with the ACH-test and its implementation in daily clinical routine.

Introduction

Angina pectoris is the hallmark of coronary artery disease and the concept of an epicardial stenosis causing myocardial ischemia and exercise-induced angina has been established for many years. However, many patients with angina pectoris do not have the typical triad of retrosternal pain, onset during exercise and relief by nitroglycerine or rest. Frequently patients report angina pectoris at rest or a combination of exertional and resting angina as well as shortness of breath upon exertion as a possible angina equivalent. In 1959 Prinzmetal was the first to introduce the concept of transient spasm of the coronary arteries causing angina at rest associated with ST-segment elevation on the electrocardiogram (ECG) but with preserved exercise capacity¹. This hypothesis was confirmed later using coronary angiography and it appeared that coronary spasm can be present in patients with epicardial stenoses or normal coronary arteries². In the 1980ies intracoronary acetylcholine provocation testing (ACH-test) for the detection of coronary artery spasm was established in Japan and subsequently the interested in clinical research for coronary spasm increased³.

However, after introduction of percutaneous coronary intervention in 1977 and the first stent implantation in 1986 the interest in coronary vasomotor disorders declined considerably at least in Europe and the United States. This may be also due to the invasive nature of the ACH-test (due to the short half-life of ACH it can only be administered into the coronary arteries for the assessment of coronary spasm). Still, many patients with signs and symptoms of myocardial ischemia do not have any relevant epicardial stenosis on coronary angiography^4,5,6. In such patients intracoronary acetylcholine provocation testing is useful to detect a clinically relevant coronary vasomotor disorder and institute appropriate medical therapy⁷.

Acetylcholine is a neurotransmitter in the parasympathetic nervous system. It acts via nicotinergic as well as muscarinergic (mAChR) receptors. The latter are important for vascular homeostasis and acetylcholine binds on mAChR as a non-selective agonist. Activation of these receptors at the endothelial level leads to nitric oxide mediated vasodilatation whereas activation of mAChR on the vascular smooth muscle cells leads to vasoconstriction⁸. Depending on the integrity of the endothelium and the reactivity of the smooth muscle cells the net effect of intracoronary acetylcholine administration is vasodilatation or vasoconstriction. The physiologic response of coronary arteries in response to acetylcholine in human beings is not fully understood but it has been reported that vasodilatation as well as vasoconstriction of up to 25% of the vessel diameter may be physiologic as shown in patients with normal coronary arteries and no angina pectoris⁹.

Intracoronary acetylcholine provocation testing has been recommended by the European Society of Cardiology guidelines¹⁰ as well as the Japanese Circulation Society guidelines¹¹ for assessment of epicardial and/or microvascular spasm in patients with angina pectoris and unobstructed coronary arteries. Intracoronary acetylcholine provocation testing has been established in daily clinical routine in the catheterization laboratory of our institution in 2003. Since 2006 a standardized protocol has been followed¹². ACH-testing is generally performed in all patients with signs and symptoms of myocardial ischemia yet no relevant epicardial stenosis (<50%) on coronary angiography. ACH-testing is performed immediately after diagnostic coronary angiography according to the protocol described below. Ergonovine is another agent that is used for spasm provocation testing with a different mechanism of action. Detailed information on ergonovine testing can be found elsewhere¹².

Protocol

NOTE: Intracoronary acetylcholine testing has been approved by the local ethics committee and the protocol follows the guidelines of our institution for human research.

1. Preparation of the Acetylcholine Solutions (See Materials Table)

1. Mix the 20 mg acetylcholine with the 2 ml solvent provided with the package.
2. Add the 2 ml ACH solution to 98 ml of NaCl 0.9 %. This corresponds to a dose of 0.2 mg/ml and is called stock solution 1. Add 9 ml of stock solution 1 to 91 ml of NaCl 0.9%, This corresponds to a dose of 18 µg/ml and is called stock solution 2.
3. Prepare 3 perfusion syringes (each of 50 ml) labeled as "high", "medium" and "low". Fill the perfusion syringe 1 labeled "high" with 40 ml of stock solution 2.
4. Fill the perfusion syringe 2 (labeled "medium") with 8 ml of the perfusion syringe 1 (high) and add 32 ml of NaCl 0.9%. This corresponds to a dose of 3.6 µg/ml.
5. Fill the perfusion syringe 3 (labeled "low") with 4 ml of the perfusion syringe 2 (medium) and add 36 ml of NaCl 0.9%. This corresponds to a dose of 0.36 µg/ml.

2. Preparation of the Syringes for Intracoronary Injection of Acetylcholine

1. Prepare 5 syringes for the intracoronary injection (4 syringes of 5 ml [#1, #2, #3, #4] and 1 syringe of 10 ml).
2. Fill the 5 ml syringe #1 with 6 ml from the perfusion syringe labeled "low" (see step 1.5). This corresponds to a dose of approximately 2 µg.
3. Fill the 5 ml syringe #2 with 6 ml from the perfusion syringe labeled "medium". This corresponds to a dose of approximately 20 µg.
4. Fill the 5 ml syringe #3 with 5.5 ml from the perfusion syringe labeled "high". This corresponds to a dose of approximately 100 µg.
5. Fill the 10 ml syringe with 11 ml from the perfusion syringe labeled "high". This corresponds to a dose of approximately 200 µg.
6. Fill the 5 ml syringe #4 with 4.5 ml from the perfusion syringe labeled "high". This corresponds to a dose of approximately 80 µg. Put this syringe aside and only use it for assessment of the right coronary artery.

3. Diagnostic Coronary Angiography

1. Inject local anaesthesia either in proximity to the right radial artery (usually 2 ml of mepivacain) or in proximity to the right femoral artery (usually 15 ml of mepivacain).
2. Confirm the success of local anesthesia is by pricking the anesthetized-skin with the needle and asking the patient if pain is still present.
3. Puncture the artery according to the Seldinger technique¹³ with a cannula, then insert the wire through the cannula and remove it. Insert the sheath (usually 5F) over the wire. Perform coronary angiography under sterile conditions.
4. Advance the wire through the sheath to the ascending aorta and place the diagnostic catheter above the aortic valve. Then remove the wire and connect the catheter with the contrast syringe.
   NOTE: The contrast agent contains Iomeprol, Trometamol, hydrochloric acid and water.
5. Place the diagnostic catheter for the left coronary artery in the left main stem by slightly pulling and turning the catheter. Confirm correct positioning of the catheter by injecting 2 ml of contrast agent.
6. Perform coronary angiography according to the Judkins technique¹⁴ with manual injections of approximately 10 ml of contrast to visualize the coronary arteries in different views.
   NOTE: Usually LAO 40° and RAO 35° are used for the right coronary artery and LAO 45°/CRAN 25°, RAO 30°/CRAN 30° and RAO 20°/CAUD 30° are used for the left coronary artery.
7. Start acetylcholine testing after exclusion of any relevant epicardial stenosis (≥50%) by visual assessment.

4. Intracoronary Injection of Acetylcholine

1. Inject 6 ml of the 2 µg syringe #1 into the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient's symptoms (e.g. chest pain, shortness of breath, dizziness). Perform coronary angiography of the left coronary artery (usually a RAO 20°/CAUD 30° projection is best projection) after injection of the 6 ml.
2. Perform coronary angiography by injecting 10 ml of contrast manually via the contrast syringe into the catheter. The 12-lead-ECG should be recorded and printed after every dose of acetylcholine. A pause of 1 min should lie between every dose.
3. Inject 6 ml of the 20 µg syringe #2 into the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient's symptoms. Perform coronary angiography of the left coronary artery after injection of the 6 ml as mentioned above.
4. Inject 5.5 ml of the 100 µg syringe #3 into the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient's symptoms. Perform coronary angiography of the left coronary artery after injection of the 5.5 ml as mentioned above.
   NOTE: Most patients report some symptoms, show ECG changes or epicardial vasoconstriction at this dose. Sometimes the speed of the manual injection needs to be slowed down. As mentioned below different protocols for ACH-testing are in use with a different speed of injection. A slower injection over a period of 3 min compared to the injection within 20 sec may also be feasible.
5. If no spasm (i.e. >90% vasoconstriction compared to the relaxed state of the vessel after 200 µg nitroglyercine injection) occurs at the 100 µg dose continue with the 200 µg (10 ml syringe) ACH dose. Inject the 11 ml within 20 sec with continuous monitoring of the ECG and the patient's symptoms.
6. Perform coronary angiography of the left coronary artery after injection of the 11 ml as mentioned above (section 3).
   NOTE: Although the 200 µg dose has been applied in many patients it should be mentioned that appropriate doses of intracoronary acetylcholine remain to be fully defined. Some authors suggest that 100 µg should be the maximum dose for the left coronary artery.
   NOTE: Often bradycardia occurs and the speed of the injection needs to be slowed down.
7. Inject 80 µg ACH (4.5 ml, syringe #4) into the right coronary artery if no abnormal findings are seen during testing of the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient's symptoms. Often bradycardia occurs and the speed of the injection needs to be slowed down to prevent prolonged bradycardia and/or asystole.
8. Perform coronary angiography of the right coronary artery after injection of the 4.5 ml (usually a LAO 40°/0° projection is best) as mentioned above (section 3).
9. Inject intracoronary nitroglycerine with a dose of 200 µg into each tested artery after the test or when severe symptoms (i.e. severe chest pain or severe shortness of breath), ischemic ECG shifts or epicardial spasm occurs.
10. Image the vessel as mentioned in step 4.2 after one minute to document reversion of spasm.

Results

Interpretation of the acetylcholine test is based on three criteria. First, the patient is asked throughout the test whether or not symptoms occur. Frequently, patients report a reproduction of their usual symptoms such as chest pain, shortness of breath or other symptoms. This represents an important point for the overall interpretation of the test. Second, a 12-lead-ECG registration is continuously performed throughout the test with a special emphasis on ischemic ECG shifts such as ST-s...

Discussion

It is feasible to implement the acetylcholine test in daily clinical routine in the catheterisation laboratory. Apart from the preparation of the ACH solutions there are several technical issues that have to be resolved before starting the test including radiolucent ECG leads for continuous 12-lead ECG registration. This is essential to be able to detect transient ischemic ECG changes during the test. Moreover, it is important to know that the ACH solutions can only be used for 2 hr. After that they should be newly prepa...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vial of 20 mg acetylcholine chloride powder and 1 Ampoule of 2 ml diluent	Bausch & Lomb	NDC 24 208-539-20
3x 100 ml NaCl 0.9 %	BBraun	3200950
3x syringe 50 ml each	BBraun	4187903
1x 2 ml syringe	BBraun	4606027V
1x 10 ml syringe	BBraun	4606108V
2x cannula 20 G 70 mm	BBraun	4665791
5x 5 ml syringe	BBraun	4606051V
Contrast agent Imeron 350 with a 10 ml syringe for contrast injection	Bracco Imaging	30699.03.00
Coronary angiography suite (AXIOM Artis MP eco )	Siemens	n/a