Introduction
Electrical impedance (EI) or electrical resistance is a measure which represents the resistance to electric current flow through a tissue. EI is a property of all living tissues and depends on the structure and chemical composition of the tissue. Structural and/or chemical changes result in changes in the electrical resistance of the tissue. The application of EI in medical and dental diagnostics is based on this principle (1, 2). Diagnostic methods based on EI measurement are used in different fields of medicine. EI spectroscopy, which is the most widely used method, has a long history (Fere and Tarhanov 1888.g), and has been mostly used for the assessment of skin lesions (3-6). EI is also applied in the assessment of the severity of muscle ischaemia (2). Methods of EI tomography assist in the evaluation of ischemic heart disease and pulmonary edema (7, 8). In sports medicine and rehabilitation, EI measurements determine the increase of body mass due to accumulation of fat and water (9). Lately, these methods have been increasingly used in the detection of tumors in different tissues. Thus, EI spectroscopy can be used to assess different tumor lesions on the skin, breast and female reproductive organs (3, 4, 10, 11). The application of EI measurements has already been established in dentistry through the use of different apex locators for the root canal length determination (12, 13). However, the application of EI in the diagnostics of oral mucosal lesions has not been adequately evaluated so far. Nicader et al. (14) performed EI measurements of the oral mucosa in healthy subjects and established differences between keratinized, non-keratinized epithelium and specialized oral mucosa. The same authors (15) found statistically significant EI variations between the healthy oral mucosa and mucosa treated with a chemical irritant (sodium lauryl sulphate). Ching et al. (16) and Sun et al. (17) found statistically significant differences in EI between the healthy oral mucosa and early tongue cancer. Murdoch et al. (18) reported significantly lower values of EI in oral cancer and severely dysplastic lesions compared to mild dysplasia and healthy oral mucosa. Being non-invasive, methods based on EI have certain advantages over invasive methods: they are simpler to perform, more comfortable for the patient and pose a lesser problem regarding disinfection, sterilization and infection control (1, 16, 19). In order to use EI spectroscopy in the diagnostics of oral mucosal lesions, initial values for the comparison are needed i.e. it is necessary to determine the range of EI reference values on the healthy oral mucosa. The aims of this pilot study were: to determine the ranges of EI values on the healthy mucosa in healthy young subjects, to map EI spectra of different regions of the oral cavity, to establish frequencies which provide the most consistent measurements, to evaluate the effect of demographic and clinical parameters such as age, gender, the amount of saliva, or habits such as smoking on the EI measurement.
Materials and methods
Subjects
The study included 30 healthy subjects aged between 20 – 40 years. All subjects were informed about the aims of the study and signed the informed consent according to the declaration of Helsinki. The inclusion criteria were clinically normal oral mucosa, absence of systemic diseases and understanding the text of informed consent. The following patient data were recorded: age, gender, smoking and unstimulated salivary flow rate. Salivary flow was measured prior to each impedance measurement, between 8–10 AM, using the method by Wu Wang (20). One hour before the measurement, the subjects did not eat, drink, brush their teeth, or chew a chewing gum. During 5 min, participants expectorated saliva accumulated in the mouth into a sterile graded test tube. The total volume of saliva was divided by 5 to obtain the quantity in ml/min.
Methods
EI measurement was performed using a measuring set designed specifically for this purpose (21). The measuring set included an intraoral sensor, a measuring device and a laptop (Figure 1). The intraoral sensor consisted of three concentric rings made of high conductivity aluminum alloy, with a total diameter of 8 mm coated with an insolation layer of Teflon. In order to assure uniform pressure and contact with oral mucosa, the sensor was attached to a dental aspirator which produced a constant sub-pressure of 250 mbar to ensure stability of the sensor during measurements. The intraoral sensor was connected to the measuring device NI USB-6251 (National Instruments®, Austin SAD) by electric conductors. The device was attached to the laptop through a USB connection. The measurement program, based on LabView 8.5.1. software package (National Instruments®, Austin SAD), converted electrical impulses from the device into digital records and stored them in a database. Validation and calibration of the measuring device and the program were performed at the Department of Automation and Electronics of the Faculty of Engineering in Rijeka, using established resistance values in the standard replacement scheme. EI was measured at 7 reference locations in the oral cavity as follows:
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Upper labial mucosa - in the projection between the root apex of the maxillary second incisor and canine
Lower labial mucosa - in the projection between the root apex of the mandibular second incisor and canine
Hard palate mucosa - at the level of apex of the palatal root of the maxillary first molar
Buccal mucosa – at the projection of the first maxillary molar
Dorsum of the tongue – 1 cm towards the midline, on the imaginary line connecting the mesiobuccal cusps of the mandibular first molars
Ventral tongue - in the projection of the mandibular first molar
Sub-lingual mucosa - 1 cm from the mandibular ridge to the midline at the level of the mandibular first molar
The measurements were performed on the right and left side, making the total of 14 measuring points (Figure 2). Measurements were performed in such a way that the investigator placed the sensor on the selected point on the mucosa. When sub-pressure of 250 mbar was obtained, as verified by the manometer (Yuyao Yadong Plastic©, Huangzhou, China), the assistant started a computer program which recorded EI for each measuring point at nine frequencies (1, 2, 5, 7, 10, 20, 70 and 100 kHz). The same measuring procedure was repeated two more times after 7 and 14 days respectively, in order to observe intraindividual EI variations. Data analysis was performed using SPSS® software (IBM Inc, SAD). Normality of distribution was assessed by the Kolmogorov Smirnof test. To evaluate differences between groups, chi-square test, the Student's test and analysis of variance were used where appropriate. To assess differences between individual measurements for each subject, repeated measures analysis of variance was used. Correlations between variables were expressed by the Pearson correlation coefficient. P values with p<0.05 were considered statistically significant.
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Results
The study included 20 females and 10 males with the average age of 27.4 ± 5.9 years. Demographic and clinical characteristics of the subjects are shown in Table 1. No statistically significant differences in age, smoking and the salivary flow rate between females and males were found. Measurement summary data (minimum, maximum, mean value and standard deviation) for each region of the oral cavity are presented in Table 2. Measurements for the left and the right side of each region were unified. The lowest EI vales at all frequencies were measured on the dorsum of the tongue (3.62 + 1.19kΩ at the frequency of 1 kHz). The highest EI values were recorded on the hard palate at all frequencies (26 ± 19.3 kΩ at the frequency of 1 kHz). Statistically significant differences between measurements on the left and right side of any region were not found. Significantly higher EI values were measured in females on the upper labial mucosa (at frequencies 1kHz, 2kHz and 5 kHz), tongue dorsum (at all frequencies) and on the ventral surface of the tongue (at the frequency of 1kHz). Significantly higher EI values were determined in smokers in the sub-lingual mucosa (at frequencies 70 kHz and 100 kHz) and on the lower labial mucosa (at frequencies 50 kHz, 70 kHz and 100 kHz). A negative correlation between EI and the amount of saliva measured by sialometry was found on the upper labial mucosa (at frequencies 5 kHz and 7 kHz), hard palate (at all frequencies except 5 kHz), tongue dorsum (at frequencies 1 kHz, 2 kHz, 5 kHz and 7 kHz) and on the floor of the mouth (at 1 kHz, 2kHz, 7kHz). Individual measurements are shown in Figure 3. Statistically significant differences in EI between individual measurements were found on the hard palate (at frequencies 1 and 2 kHz), buccal mucosa (at frequencies 7, 10, 50, 70 and 100 kHz) and on the tongue dorsum (at 1, 2 and 20 kHz). Variability of measurements was higher at lower frequencies. Intraclass correlation coefficients for different regions were determined as follows: upper labial mucosa: 0.23 (1kHz)-0.30 (20 kHz), lower labial mucosa: 0.2 (1KHz)-0.42(20kHz), hard palate: 0.22 (1kHz)-0.57 (100kHz), buccal mucosa: 0.09(5kHz)-0.31(100kHz), dorsum of the tongue: 0.1 (1kHz)-0.40 (7 kHz), mucosa of the ventral surface of the tongue: 0.16 (20kHz)-0.24(7kHz), and sublingual mucosa: 0.23 (2kHZ)-0.41(100kHz).
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Discussion
The aim of this study was to determine EI reference values on the healthy oral mucosa. A similar investigation was conducted by Nicader et al. (14, 15) who analyzed EI measures on six different anatomical locations in the mouth of healthy subjects. The authors concluded that differences in EI values in the oral cavity among individuals were higher than EI values on the skin. Moreover, the authors claimed that the method of EI measurement allowed a more precise detection of experimentally caused mild allergic reaction than other methods such as inspection of the mucosa and histological tests (22). For the purpose of this pilot study, a group of subjects aged 20-40 years were selected. Although 66.6% of subjects were females, no statistically significant differences in age, the amount of saliva and smoking were found between females and males, thus suggesting that the study group was homogenous. The lowest EI measures were found on the tongue dorsum and the highest on the hard palate. This finding can be linked to a different structure of the mucosa at these locations. Factors such as vascular flow, humidity, amount of the underlying connective tissue and the thickness of the stratum corneum may affect the electric flow and its consequent resistance. The tongue dorsum has a rich vasculature, contains prominently less connective tissue and has thinner stratum corneum than the hard palate, so it is more likely to have lower electrical resistance. Saliva retained between filiform papillae can also lower resistance between poles of electrode and lead to decreased resistance. This finding complies with the results of our previous studies (23), and with results reported by Nicader et al. (14, 15) who also established the greatest differences in impedance between the hard palate and tongue dorsum. There were no statistically significant variations in EI values between the left and right side at any of the measuring points. This finding suggests that EI depends on histological and anatomical features of a specific region of the oral cavity regardless of the side. Absence of the difference between the left and right side in the same regions was also reported by Rantanen al. and Nicader et al. (15, 19, 22). Thus, there is a possibility that contralateral regions may be used as reference points for EI measurement of unilateral oral lesions. Gender is one of the factors the role of which should be clarified in determining EI values of oral mucosa. This study established significantly higher EI values in the females on the upper labial mucosa and on the ventral surface of the tongue at low frequencies (1, 2 and 5 kHz), and also on the dorsum of the tongue at all frequencies. This finding may result from the actual difference between genders, but it may occur due to a small number of subjects and/or a larger number of female subjects than male ones. These results need to be confirmed or rejected in studies with greater number of participants. There are no literature data available for comparison, while EI measurement on the skin did not show statistically significant differences between males and females (24). Smokers had somewhat higher EI values compared to non-smokers, but the differences were statistically significant only on the lower labial mucosa and on the floor of the mouth. It is known that tobacco smoke and elevated temperature can increase mucosal keratinization (25, 26), which may lead to increased resistance to the electric current flow. Whether this finding is actually an effect of smoking or it results from a small number of participants, remains to be determined by further studies. However, it is interesting to note that both the lower lip and the floor of the mouth are the sites which have the longest contact with a cigarette and ingredients of tobacco smoke. The EI values on the hard palate mucosa, (a localization which is also in intimate contact with tobacco smoke) did not differ between smokers and non-smokers. EI values on the hard palate were highly variable, and possible differences between smokers and non-smokers could therefore be undetected. Salivary flow may also affect the EI values. A negative correlation between impedance values and salivary flow was observed on the hard palate at all frequencies except at 5 kHz. Moreover, a negative correlation was also found between salivary flow and EI values on the upper labial mucosa, tongue dorsum and floor of the mouth at lower frequencies (1 kHZ to 7 kHz). Lower resistance is likely to be an effect of higher amount of saliva and better conductivity. There are no literature data to compare these findings with. Studies performed on the skin and oral mucosa suggest that the amount of tissue fluid has an impact on EI since inflammatory and allergic reactions of the skin and mucosa (characterized by increased extracellular fluid) have lower impedance values compared to the healthy mucosa/skin (1, 19, 22, 27). The results of EI measurement obtained in this study show intraindividual variability manifested by low intraclass correlation coefficients. Variability was more pronounced at lower frequencies. This finding showed that the impedance measures in each individual were not constant, but varied within a specific range. This range of intraindividual values is not too wide since in most cases the values of individual measurements did not differ significantly. No literature data on intraindividual variations in oral mucosal EI are available for comparison. Intraindividual variations may result from non-uniform pressure of the sensor on the mucosa (which we tried to eliminate with aspirator system) as well as the amount and quality of saliva during measurements. Circulatory changes caused by the vegetative nervous system may also lead to measurement variability since it is known that impedance values may depend on the amount of fluid in the tissue (1, 7, 19, 23, 27). The highest variability was noted on the hard palate where standard deviation was over 70% of the mean (0.74 at 1 kHz and 0.77 at 2 kHz), unlike other locations where standard deviations were 32-42%. This variability is most likely the effect of poor attachment of the sensor to the palatal mucosa. All of the abovementioned confirms the need for the determination of EI reference values on the healthy oral mucosa which could be used as the starting point for comparison with different mucosal lesions. This study has certain limitations which need to be addressed. First of all, there was a small number of participants. Although the obtained results suggest a specific pattern of influence of gender, smoking and salivary flow on the EI values, these results should be confirmed in studies with greater number of participants. Furthermore, it is necessary to standardize the measuring procedure in order to avoid artifacts and minimize non-homogeneity of the results. The standardization of the measuring procedure refers primarily to ensuring uniform pressure of the sensor on the mucosa. During the investigation, it was observed that the increase of sub-pressure exerted by the sensor contributed to lowering the intraindividual and interindividual differences. This investigation was conducted using sub-pressure of 250 mbar which created an adequate contact with the mucosa and was comfortable for the patients. Using higher sub-pressure, a more stable attachment of the sensor to the mucosa may be achieved and lead to more stable measurements. Based on the results of this pilot study, it can be concluded that the applied method may be used for determining the EI values of the oral mucosa in healthy subjects. EI values mostly depend on the degree of keratinization of the oral mucosa. Demographic and clinical factors such as gender, smoking and salivary flow probably affect their values, but they need to be clarified in further studies on a larger number of participants. The method shows a potential for developing into a diagnostic tool which may be used to determine the intensity and spread of oral mucosal lesions. Studies which assessed EI spectroscopy for the detection of malignant and potentially malignant lesions of the oral cavity speak in favor of the above claim (15-18). Before this is accomplished, factors that contribute to measurement deviations need to be eliminated, primarily the attachment of the sensor to oral mucosa and the amount of saliva in the measurement area.
