Study population and study protocol
The current study began in 2007 as a survey of the incidence of vascular events associated with lifestyle-related disease among the general population as a part of the Kyushu and Okinawa Population Study (KOPS) [19–22]. Our study was conducted with residents of Ishigaki City, Okinawa Prefecture who participated in a program of esopahagogastroduodenoscope screening for gastric cancer between October 2012 and January 2013. In the study protocol, after giving informed consent, the tongue was photographed. The photography was conducted in a shady controlled room. The subject’s face was fixed with a chin rest and a forehead rest. As the mouth cannot be opened when both chin and forehead are fixed, first the chin is placed on the chin rest and, after swallowing saliva, the mouth is opened and the tongue extended, following which the forehead is placed against the forehead rest. Each tongue extension is for 20s, and images are taken every 100 ms, for a total of 200 images. After that the operator visually confirmed the tongue color. Secondly, esophagogastroduodenoscopy was done, blood was taken to check for serum antibody to H.pylori (anti-H.pylori), and serum pepsinogen (PG) I /II and gastrin were measured. We then estimated the associations between tongue color, the endoscopic findings, H.pylori infection status, and serological atrophic gastritis. Tongue color was measured by the device-independent international commission on Illumination (CIE) 1976 L*a*b* color space standards at four points: (1) tongue edge, (2) tongue posterior, (3) tongue middle, and (4) tongue apex. The coating of the tongue does not grow on the edge of the tongue, and the color of that point can be considered the color of the tongue body. In contrast, at the other three points the color is a mixture of the coating and the body of the tongue.
From the point of view of Kampo medicine, it is said that the posterior of the tongue reflects kidney function (traditional medicine), the middle of the tongue reflects the stomach and pancreas (GI tract traditional medicine), and the apex of the tongue reflects the heart (traditional medicine). Therefore, we used these areas to assess the association of tongue color with endoscopic findings. Fig. 1. shows an example of tongue color imaging. The value for this patient with erosive gastritis (3a*, 3b*) is higher than that of the normal control.
In addition, we calculated the ratio of the tongue edge to the other three points measured to examine the association between the coating of the tongue and the endoscopic and laboratory findings. By calculating the ratio to the tongue edge we can confirm that the color of the tongue reflects the body of the tongue, not the coating.
Esophagogastroduodenoscopy was done for 919 residents, and the data of 896 residents from whom we were able to get consent for the tongue color and blood test (age range 28–86 years; mean age 57.7 years; 390 men and 506 women) was available for analysis.
To ensure the validity of the data, all physicians participating in the study were staff members of the Department of General Internal Medicine, Kyushu University Hospital or the Department of General Medicine and Emergency Care, Toho University School of Medicine Omori Hospital. All were trained with regard to the study protocol and the medical procedures necessary for the study. The study protocol was approved by the Ishigaki City Health Center and the Kyushu University Hospital Ethics Committee. Written informed consent was obtained from all participants prior to the examination. Consent to publish the images used was obtained from both of the patients featured. The study was conducted in accordance with the principles of the Helsinki Declaration of 1975, as revised in 2000.
Tongue image analyzing system (TIAS)
We previously reported on the functionality of the TIAS system, which is equipped with a diffused light source for recording the state of the tongue surface [17, 18, 23]. In brief, when doing photography using TIAS (film image was 1280 × 1024-pixels), calibration of the camera and light source is performed only once, when the power is turned on. When photographing the subject using TIAS, many tongue photographs are taken, from which one image is selected manually for tongue color analysis. We defined the position of the four points by a ratio calculated by manually specifying five points that define the shape of the tongue. The ratio used to determine the four measurement points is illustrated in Fig. 2 The size of the measured tongue area was two 5 mm diameter circles and the measurement of color value is calculated by the average of the two circles. The RGB values at each point are then converted to CIE1976 L*a*b* color space, which is device independent and is designed to be perceptually uniform. This means that a change of the same amount in the L*, a*, or b* value should produce a change of the same visual importance. A photograph taken with TIAS is shown in Fig. 3.
Esophagogastroduodenoscopy
Each participant underwent esophagastroduodenoscopy at the Ishigaki City Health Center by highly experienced endoscopists who performed each examination without knowledge of the serological data. The endoscopic examination was for esophageal hernia (EH), erosive esophagitis (EE), erosive gastritis (EG), superficial gastritis (SG), gastric ulcer (GU), gastric cancer (GC), erosive duodenitis (ED), duodenal ulcer (DU), and duodenal cancer (DC). EE was defined according to the Los Angeles classifications A-D [24]. GC was diagnosed based on the histological finding and the stage classification and was determined through an evaluation of the clinical examination.
Testing for antibody to H.pylori
The blood samples of all participants were separated and stored at −80 °C until testing.
The serum IgG level of HP was measured by a commercially available direct ELISA kit (“E Plate ‘Eiken’ HP Antibody” Eiken Kagaku). This ELISA kit was developed in Japan and uses antigen extracted from a domestic strain. It is commonly used in medical studies [25]. Positivity for HP infection was defined as an anti-HP IgG antibody level greater than 10 U/mL in serum.
Serum PG and gastrin measurement
Serum PG isozymes I and II were measured by a competitive-binding double-antibody radioimmunoassay (PGI/PGIIRIA-BEAD, Abbott Japan Co., Ltd., Tokyo, Japan). The serum gastrin level was measured using an RIA kit (Dinabot Co., Tokyo, Japan). Serological atrophic gastritis was defined based on the results of a serum PGIlevel ≤70 ng/ml and a PGI/IIratio ≤3.0. The assay has a sensitivity of 70.5 % and a specificity of 97.0 % for histological atrophic gastritis [26, 27]. Furthermore, we analyzed the serum PGIlevel as a marker gastric acid secretion [28].
Statistical analysis
Data are expressed as number (%), mean ± SD, or median with quartiles [25 % - 75 %]. Participant characteristics and the tongue color calculated from tongue photography were compared between participants with and without endoscopic findings, by H.pylori status, and by SAG using Fisher’s exact test for categorical variables and unpaired t test or Mann–Whitney test for continuous variables. Variables with a difference of P < 0.1 in the univariate analysis were used in multivariate analysis to determine independent, significant predictors. Odds ratios (OR) and 95 % Confident index (CI) were calculated from the multiple logistic regression model after adjustment with each variable. All statistical analyses were performed on a personal computer with the statistical package SPSS 18.0 for windows.