Study Design: This cross-sectional, analytical study was performed on 15 patients diagnosed with AD who were referred to the neurology ward of Imam Hossein Hospital and were on memantine therapy. Memantine is a drug that is used to treat the cognitive problems of Alzheimer’s patients, but it isn’t an AChE inhibitor [15]. Thus, any possible effects of enzyme inhibitors on the activity of AChE enzyme in saliva were prevented. The control group was randomly selected from 15 elderly non demented subjects without neurological or cognitive disease.

According to statistical sample size determination a minimum sample of 11 was required in each group, however 15 individuals were included to compensate for attrition.

Demographic information including age, sex and duration of disease was obtained by interviews.

The committee of medical ethics of Shahid Beheshti University of Medical Sciences approved the study procedure. The study was done between 2014 and 2015 year at the Department of Oral Medicine and Neurology, Shahid Beheshti University of Medical Sciences and Imam Hossein Hospital, Tehran, Iran. Informed consent was given by their caregivers before the onset of the study.

Salivary Collection: Subjects were advised to avoid from eating, drinking or smoking one hour prior to sampling. They were then asked to rinse their mouth with water. After five minutes, 2 ml of whole unstimulated saliva samples were collected from the subjects in the sitting and relaxing position by spitting into 15 ml Falcon tubes. All samples were collected between the hours of 9 am and 12 am [16]. Saliva samples were immediately transferred to the laboratory inside a chamber containing ice and were kept at the freezing temperature of -70°C.

On the day of testing, saliva samples were centrifuged for 10 minutes at 3000 rpm (Behdad, made in Iran) to separate any possible debris and squamous cells. The AChE activity levels in saliva were then determined using the Ellman colorimetric method [17]. About 50 μl from each sample was added to a buffer solution containing 0.1 mM dithionitrobenzene (Merck, made in Germany), 60 μg acetylthiocholine (Sigma, made in USA), and 10 mM phosphate buffer pH=8 at the total volume of 110 μl. The mixture was incubated for 15 minutes at 37°C [8].

The AChE in the reaction mixture broke into thiocholine and acetate due to the activity of the enzyme AChE. The obtained thiocholine reacted with the dithionitrobenzene producing 5-thio-2-nitro-benzoic acid anion, which produces a yellow color [14]. Catalytic activity levels were measured in the wavelength 412 nm by photometry using Cobas Mira auto analyzer (Roche, made in USA).

The protein content of saliva samples was measured by using the Bradford method, which is among the most accurate colorimetric methods [18]. A 25 μl from each sample was added to 250 ml Bradford containing 5% ethanol 97°, 0.01% Coomassie Brilliant Blue G-250 (Merck, made in Germany), 10% phosphoric acid 85%, glycerin and distilled water. As a result, the Coomassie Brilliant Blue G-250 dye binding to proteins caused maximum absorption to change from 465 nm (red color) to 595 nm (blue color) [19]. Finally, the degree of AChE enzyme activity in saliva samples was normalized with the protein content of the samples. Enzyme activity of saliva samples from AD and the non-Alzheimer’s group was compared and concluded.

Data analysis was performed using SPSS version 18.0. The p-value<0.05 was considered as level of significance. We used Mann Whitney U test to compare the average (median) of AChE activity between AD and controls. In order to adjust for possible confounding factors, partial correlation coefficient and multivariate linear regressions were used.

In this study, 15 patients with AD 9 (56.2%) males and 6 (42.9%) females aged 64 to 90 (average age 78.4 years) and 15 individuals who did not have AD 7 (43.8%) males and 8 (57.1%) females; aged 61 to 85 (average age 71 years) were tested. Mean duration of AD was 2.23±1.23 years and the average duration of disease was found to be longer in men than women.

According to the results of Mann Whitney test the overall salivary AchE in Alzheimer’s patients was lower than that of the control group which clinically may be important (138 in control group vs. 175 in Alzheimer patients) but we found no significant differences in the enzyme activity between two groups; p value=0.25 [Table/Fig-1], Enzyme activity was generally lower in males than in females. To evaluate the association between enzyme activity and age with respect to the variables of sex and disease, adjusted correlation coefficients were used and a positive, weak and no significant relationship was observed; partial correlation coefficient= -0.041; p-value=0.837 [Table/Fig-2].

There were no significant relationship between duration of AD and enzyme activity with respect to the age and sex; partial correlation coefficient = 0.192; p value=0.531 [Table/Fig-3].

In evaluation of enzyme activity with respect to the variables of disease, age and sex, no significant relationship was observed using multivariate linear regression models; regression coefficient β disease=0.256, β age=-0.044, β sex=0.033; p-value>0.05 [Table/Fig-4].

However, according to the average values (mean and median), the difference between the patient and healthy groups may be important in clinical terms.

Cholinergic dysfunction is a key biochemical event that is associated with the progression of AD. Currently, this is the target of most existing treatment plans [7]. The purpose of this study was to survey the activity of AChE in the saliva of patients with AD. A total of 30 eligible persons were selected, 15 patients with AD and 15 healthy subjects. After taking saliva samples from subjects, the AChE activity was evaluated by using Ellman colorimetric method and the enzyme activity of each sample was normalized with its protein content. This method had previously been used by other researchers such as Sayer R et al., and Boston PF et al., [8,20].

Based on the current study results, the reduction in salivary AChE enzyme activity in patients was not significant compared to the control group.

Moreover, there was no significant difference in AChE activity levels in saliva by age or gender, and enzyme activity did not change significantly with the duration of the disease. The results of the present study are similar to the Boston PF et al., study [20]. Although in some studies, salivary tau species has been proposed as diagnostic biomarker specially in the early stage of AD [13,14].

Boston PF et al., studied saliva AChE activity in three groups of 15 subjects with AD, 13 subjects with vascular dementia and 13 healthy subjects matched for age as control group. No significant difference in enzyme activity was observed between the diagnostic groups. This means that the rate of changes in enzyme activity was not significant with respect to Mini Mental Examination Test (MMSE) scores. In addition, gender did not cause significant changes in enzyme activity, which is similar to the present study [20].

Sayer S et al., found the relationship between salivary AChE and AD and the response to cholinesterase inhibitors. In their study, the patients with AD were treated with donepezil at a rate of 5 mg per day and cognitive assessment tests were performed on them [8]. Saliva samples were taken after classification of the subjects based on their response to therapy and also forming a control group. The AChE enzyme activity was determined by using the Ellman colorimetric method [17]. The protein content of the samples was also measured and enzyme activity was normalized with the protein content of the samples. In this study, a significant reduction in the catalytic activity of enzymes associated with age was observed in the control group. Additionally, the enzyme activity in Alzheimer’s patients who did not respond to treatment was significantly less than the control group matched for age. There was no difference between males and females in each group and changes in people with AD was not associated with age. There were also significant differences in enzyme activity between people who responded to treatment with AChE inhibitors and those who did not. The difference between the results of this study in comparison with our study could be due to differences in the study design. In this study, patients were treated with salivary AChE inhibitors and were classified based on whether they responded to treatment with inhibitors or not. Although, the inhibitor had been isolated from the saliva before sampling, it is possible that treatment with an inhibitor created the long term adaptive changes in the production of AchE. Alzheimer’s patients who participated in our study were on drug memantine, which is not an AChE inhibitor.

Overall, enzyme activity was lower in men than women in the present study, although this difference was not statistically significant.

Thus, it appears that although the enzyme activity in patients with AD was lower than the control group, the opinion of experts in this field is required in order to determine whether these differences are clinically important and can be used for diagnosis of AD, even if they are not statistically significant. In order to achieve more reliable results, it seems beneficial to design a study in which subjects are categorized according to the severity of their disease, so that enzyme activity in each group can be investigated separately.

In the present study, cooperation of Alzheimer’s patients was poor due to physical and mental disabilities. In future studies it is suggested to determine possible variables affecting the enzyme activity.

In addition, disease severity should be classified into mild, moderate and severe and the enzyme activity examined separately in each group.

We found that AChE activity levels in saliva vary in individuals with AD in comparison to healthy subjects. Considering confounding variables such as age and gender, the difference is not statistically significant, although it may be clinically important. Consequently, due to a lack of studies in this field, we cannot present a conclusion based on the evidence for the use of salivary AchE as a biomarker for diagnosing AD, moreover, AchE activity in the saliva of males and females does not change significantly with the duration of illness and age.