The peripheral blood samples were obtained from the brachial vein (4 mL) and were collected in Falcon tubes containing 0.025M EDTA. Then, the samples were centrifuged at 14000 rpm for five minutes, and the isolated serum was immediately frozen at -45 ^oC. Unstimulated saliva samples were collected in the morning (8 am to 10 am). 2 ml of saliva was collected in five minutes, while the subjects had not drunk any water, nor eaten any food and had not smoked about two hours before sampling. Before sampling, the subjects thoroughly washed their mouth, and after several minutes saliva was collected into Falcon tubes. Then, the samples were centrifuged at 20.000 rpm for 2-3 minutes, and the isolated transparent supernatants were immediately frozen at -45 ºC.

Firstly, serum and saliva samples were diluted (1:100) with 133 mM phosphate buffer. For the test tube, 1 mL of distilled water and 2 mL of diluted saliva or serum sample was mixed with 1 mL of 200 mM benzoylcholine chloride. In the blank tube, equal volumes of distilled water and serum or diluted saliva were added. The light absorbance at the wavelength of 240 nm at 25°C was measured after subtracting the absorbance of the blank tubes. Each sample was read five times in a five-minute period (every minute, one absorption). When benzoyl choline (200 nM) is hydrolyzed in a total volume of 4mL and the absorption changes are ∆A= 0.33, the serum and salivary BChE activity was calculated using the following formula:

The normal value of this enzyme is 0.6-1/Uk4/1 (at 25°C) and 1.08-1/Uk 4/2 (at 37^oC). The additional details (chemicals, kits, instruments, etc.) were reported in the studies of Shahmohamadnejad et al. 22 and Haghnazari et al. 23.

Firstly, 100 landa of whole blood was irrigated with saline buffer for three times in a glass tube. After irrigation, the sample was centrifuged, and the supernatant was removed, and saline buffer was added. When irrigation finished, the supernatant was removed totally, and 10 mL of 50 mM phosphate buffer was added. Then, in two cuvets (one for the test tube and one for the blank tube), 2 mL of DTNB was added. Then, 1 mL of cellular suspension was added to each cuvet and stored at 30C. After 3 minutes, 100 landa of the substrate was added to the cuvet containing the sample. At wavelength 412 nm, light absorbance of the sample and blank tube were measured. Test tubes were read in a 5-min interval (one reading for every minute). Using the following formula, salivary and serum AChE activity was calculated:

The normal value of the enzyme is 8-1/Uk 13.

Descriptive indices including frequency, percentage, mean and its standard deviation (SD) were used to express data. To determine the normal distribution of the data, the Kolmogorov-Smirnov (KS) test was used. To compare the enzyme values between different time points (i.e., immediately following MI, and hours 24 and 72 post-MI), repeated measures analysis of variance (ANOVA) was used. Whenever Mauchly’s test of sphericity was statistically significant, the Greenhouse-Geisser test was applied. For paired comparisons, the Bonferroni’s test was applied. For comparison of the variables between the MI and control groups, the independent sample T-test was used. To determine the correlation between serum and salivary enzyme measurements, the Pearson correlation test was used. The analyses were done by SPSS software (ver. 18.0). The significance level was set at less than 0.05.

There were 30 patients in the MI group and 30 subjects in the control group. In each group, there were 25 males (83.3%) and 5 females (16.7%). The mean (±SD) of ages in the MI and control groups were 52.37 (±6.42) and 50.73 (±7.36) years respectively; P= 0.364. The KS test showed that the variables had a normal distribution (P> 0.05).

The mean serum AChE levels immediately following MI, 24 hours and 72 hours post-MI were 12.65, 12.61, and 12.41, respectively (Table 1). There was no significant difference in AChE measurements at these three time points (P= 0.998) and also in each of three time points compared to the controls (P=0.713, P=0.650, and P=0.940, respectively). The serum levels have been shown based on sex which shows no significant difference in both male and female at the three time points, but there was a significant difference at each time compared to the controls.

As seen in Table 2, there is a significant difference in the salivary AChE level between the control and MI groups at the three time points (P<0.001 immediately, P<0.001 in 24 hours, and P<0.001 in 72 hours after MI). The mean salivary AChE levels immediately following MI, 24 hours and 72 hours post MI were 0.406, 0.434, and 0.429, respectively. The salivary levels have been shown based on sex which shows no significant difference in both male and female at the three time points, but there was a significant difference at each time point compared to the controls.

Table 2

	Healthy control	Myocardial infarction group (first hour)	Myocardial infarction group (after 24 hours)	Mmyocardial infarction group (after 72 hours)	P valuec	P valued	P valuee	P valuef
	Mean± SD	Mean± SD	Mean± SD	Mean± SD
Female	0.688±0.127	0.478±0.155	0.410±0.163	0.452±0.133	< 0.001	< 0.001	< 0.001	0.480
Male	0.596±0.241	0.392±0.185	0.439±0.178	0.425±0.191
Total	0.611±0.227	0.406±0.151	0.434±0.174	0.429±0.181

^cComparison between the control and myocardial infarction group (first hour)

There were significant differences in the serum BChE level between the control and MI groups immediately following MI, 24 hours and 72 hours post-MI (P=0.003, P=0.008, and P=0.002, respectively). Repeated measures ANOVA did not show significant changes regarding serum BChE at the three time points (P= 0.794); (Table 3). The serum levels have been shown based on sex which shows no significant difference in both male and female at the three time points, but there was a significant difference at each time point compared to the controls.

As seen in Table 4, There was no significant difference in the salivary BChE level measured at the three time points (P= 0.049) compared to the controls. In addition, the salivary levels have been shown based on sex which shows a significant difference in both male and female at the three time points, but there was no significant difference at each time point compared to the controls.

There was no significant correlation between the serum and salivary level of AChE immediately following MI (r= 0.08, P= 0.676), 24 hours after MI (r= 0.017, P= 0.929), and 72 hours after MI (r= 0.141, P= 0.457). No significant correlation existed between the salivary and serum level of AChE in the control group (r= 0.301, P= 0.106). Also, no significant correlation was found between the salivary and serum level of BChE immediately after MI (r= 0.233, P= 0.242), 24 hours after MI (r= 0.06, P= 0.771), and 72 hours after MI (r= 0.105, P= 0.642). No significant correlation existed between the salivary and serum level of BChE in the control group (r= 0.356, P= 0.054).

Acetylcholine is the main neurotransmitter of the parasympathetic system, and its level is regulated by AChE and BChE through hydrolysis 242526. This study investigated the activity of AChE and BChE enzymes in the saliva and serum in patients with MI in comparison to healthy subjects to identify markers to diagnose of MI. MI is one of the common cardiac conditions 27, and its main cause is atherosclerosis. One study 28 reported that serum AChE and BChE levels decreased following MI. The activity of these enzymes can be important in the development of MI and also be protective. Clinical findings and electrocardiography do not achieve the required sensitivity and specificity 28. The measurement of these enzymes can be useful in managing patients suspected to suffer MI. A limited number of studies has been performed regarding the activity of AChE and BChE in the MI patients 2927. One of the quick, cost-effective, and non-invasive methods for MI diagnosis could be the measurement of AChE and BChE activity in the serum and saliva. The previous studies have not been determined the changes in the activity of these enzymes in the saliva and serum of MI patients.

This study showed a significant decrease of AChE levels in the saliva of the MI patients compared to the control group immediately following MI, 24 hours and 72 hours post MI. We did not find any similar study in the literature assessing the salivary AChE and BChE levels in the MI patients. In a study by Mirzaii‐Dizgah et al. 30 including 30 salivary samples of patients with acute MI and 28 healthy subjects, salivary troponin level, as a marker for the diagnosis of MI, was higher in the MI patients than in the control group at 12 and 24 hours after MI. The observed findings in this study and increased salivary troponin levels in the mentioned study 30 showed increased cardiac-related enzymes following MI. Our results showed decreased serum BChE levels in the MI group immediately following MI, 24 hours and 72 hours post MI. In a previous study, it was reported that MI patients with reduced BChE levels experienced a higher mortality rate 29. As AChE and BChE enzymes are synthesized in cardiac muscle, their levels decrease after MI. This study is the first to investigate AChE and BChE serum and saliva levels in MI patients compared to the controls. During necrosis of myocardium, the AchE released by injury of cholinergic nerve and AchE can be a parameter for MI diagnosis.

Our findings showed a significant reduction in salivary AChE and serum BChE in the MI group in comparison to the control group. As the main objective of this study was to measure these two enzymes following MI, the results suggest that salivary AChE can be used as a marker the to diagnose MI in the future. As this study was done for the first time, we suggested that further studies with a larger number of salivary samples and for longer follow-up times be performed in the future. With such studies, the definitive role of AChE and BChE in diagnosing and managing MI patients will be validated.

Authors have no conflict of interest.

Neda Omidpanah: Design of the study and acquisition of data.

Negin Jalilian: Collection of data

Asad Vaisi-Raygani: Analysis and interpretation of data.

Masoud Sadeghi: Revision of the article and final approval of the version to be published.

Hamid Reza Mozaffari: Writing of the article.

This paper is issued from the thesis of Negin Jalilian and financial support was provided by Kermanshah University of Medical Sciences (Project code: 95336).

MI: Myocardial infarction

BChE: Butyrylcholinesterase

AChE: Acetylcholinesterase

CAD: Coronary artery disease

SD: Standard deviation

KS: Kolmogorov-Smirnov