2.1 Patients and control subjects: selection
The investigation compared patients with known CAS, either due to underlying large vessel spasm (PA; n = 39) or to the CSFP (n = 24). In all cases this diagnosis was made at coronary angiography, which showed the absence of haemodynamically significant stenoses in epicardial coronary arteries plus either:-
(a) Inducible coronary artery spasm with intracoronary injection of 25 to 100 µg of ACh, with a positive test being defined on the basis of a > 75% reduction in luminal diameter (either focal or diffuse) together with onset of chest pain (Ong et al. , 2012)
or (b) Presence of CSFP, defined as TIMI-2 flow in at least one major epicardial vessel (Beltrame et al. , 2002; Beltrame et al. , 2003).
All patients (n = 63) were subjected to elective peripheral venous blood sampling during chronic treatment of their angina. However, in 16 cases, sampling was also performed during presentation to hospital with prolonged pain, implying the onset of an acute exacerbation. In such cases, blood samples were taken before initiation of emergency treatment measures, 2-12 hours thereafter, and 2 -6 weeks post resolution of symptoms, in order to compare these 3 phases of the disease process.
Treatment during the acute phase of the disease consisted of the intravenous infusion of GTN at the low rate of 2.5µg/min, utilizing non-adsorptive tubing, together with N-acetylcysteine (NAC, 10 g per 24 hours), which has been shown to potentiate the vasodilator (Horowitz et al. , 1983) and anti-aggregatory (Loscalzo, 1985) effects of NTG, and which has been shown to suppress cyclical coronary vasoconstriction and associated phasic platelet aggregation in a canine model of coronary artery spasm (Foltset al. , 1991).
Healthy control subjects (n= 31) were recruited by advertisement. Clinical characteristics of patients and control subjects are summarized in the Table.
In patients undergoing coronary angiography, blood sample collection was carried out at least 20 minutes post contrast injection. Blood was taken by venesection into plastic tubes containing1:10 volume of citrate anticoagulant (2 parts of 0.1 M citric acid to 3 parts of 0.1 M trisodium citrate, pH 5) for platelet aggregation studies, and into EDTA Vacutainer tubes for plasma syndecan-1 and tryptase assays.
All human studies were performed in accordance with the Declaration of Helsinki. The study was approved by the Institutional Human Research and Ethics Committee, and informed consent was obtained from all subjects.