This is a cross-sectional study. Patients who were over 20 years old and under 80 years old who were treated at a single medical center and who were diagnosed with oral cancer by stomatologist met the study entry criteria. The exclusion criteria included the following: 1) liver (hepatitis B or C), kidney or gastrointestinal diseases; 2) the use of antioxidant supplements; and 3) pregnant or lactating women. The study was approved by the Institutional Review Board of Taichung Veterans General Hospital, Taiwan. The subjects participated in the research after signing the informed consent form.
Characteristics and anthropometrics
A questionnaire was used to understand the characteristics, including sex, age, lifestyle habits, and family medical history, of the subjects. The definitions for lifestyle habits as follows: smokers: individuals regularly smoking one or more cigarettes per day; for alcohol use: individuals regularly consuming one or more drink per day; betel nut use: individuals regularly consuming one or more betel nut per day. exercise: individuals regularly exercising at least 3 times per week. The height, weight, and waist circumference of each subject were measured, and then the body mass index (BMI) was calculated. Central obesity was defined by a waist circumference ≥ 90 cm for male; ³ 80 cm for female. Blood pressure was measured by an electronic sphygmomanometer, and a systolic blood pressure (SBP) ≥ 130 mmHg or a diastolic blood pressure (DBP) ≥ 85 mmHg was defined as high blood pressure.
Biochemical measurements and pathological diagnosis
Subjects who were hospitalized and asked to fast for at least 8 hours before surgical therapy, blood samples were collected in vacutainers containing K3-EDTA anticoagulant, sodium fluoride, or in the tube without anticoagulant. Then, these samples were centrifuged for 15 minutes to separate plasma, red blood cells (RBCs), and serum and stored at - 80ºC until analysis. After tumor resection surgery, the pathologist determined the pathological staging of the tumor in patients with oral cancer.
Fasting glucose was analyzed by an automated chemistry analyzer (Labospect, Tokyo, Japan), and a level ≥ 5.55 mmol/L was defined as hyperglycemia. Lipid profiles, including triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were analyzed by an automated chemistry analyzer (Hitachi 7070 & 7600, Tokyo, Japan), and hyperlipidemia was defined by TG ≥ 1.70 mmol/L, LDL-C ≥ 2.59 mmol/L, or HDL-C ≤ 1.04 mmol/L for male and < 1.30 mmol/L for female. High sensitivity C-reactive protein was measured by latex immunoassay. The level of IL-6 was analyzed by an enzyme-linked immunosorbent assay using commercial kits (R&D Systems Inc., Minneapolis, USA). The definition of high blood pressure, central obesity, hyperglycemia, and hyperlipidemia were according to the guidelines of the Administration of Health Promotion, Ministry of Health and Welfare, Taiwan (2007). The diagnostic criteria for metabolic syndrome in Taiwan are in accordance with the National Cholesterol Education Program Adult Treatment Panel III (ATP III, 2001), International Diabetes Federation (IDF, 2005), and the American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI, 2005) criteria [21-23].
Oxidative stress and antioxidant enzymes measurements
The concentration of malondialdehyde was analyzed using the thiobarbituric acid colorimetric method . The antioxidant enzyme activity of RBCs, including catalase, glutathione peroxidase, and superoxide dismutase, was determined by calculating the changes in the optical density of the enzyme activity reaction over one minute [25-27], and the data are shown as units/mg protein. RBC protein was analyzed using the bicinchoninic acid protein assay reagent kit (Thermo Scientific, Rockford, IL, USA).
Antioxidant vitamins measurements
Antioxidant vitamins status was measured by high-performance liquid chromatography (HPLC). The measurement of ubiquinone was performed according to the method used by Littarru et al. . The analysis column used was a LiChroCART®RP-18 (Merck, Germany), and the ultraviolet detector was set at 275 nm. The measurement of vitamins A and E was performed according to the method used by Karpińska et al. . The analysis column for vitamin A and vitamin E analyses was a LiChrospher® 100 RP-18 (Merck, Germany), and the ultraviolet detector was set at 325 nm and 292 nm, respectively. The measurement of β-carotene was based on the method used by Kand’ár et al. . The column for β-carotene analysis was a Purospher® STAR RP-18 (Merck, Germany), and the ultraviolet detector was set at 450 nm.
All statistical tests in the study were conducted using SigmaPlot software (version 12.0, Systat, San Jose, California, USA). Descriptive statistics are presented as the mean ± standard deviation (median) or percentages. The normality of the distribution of the data was examined using the Shapiro-Wilk test. One-way ANOVA or Kruskal-Wallis test was used to examine the differences in antioxidant vitamins, metabolic parameters, oxidative stress, antioxidant enzymes activity, and inflammation according to the stage of the oral cancer; post hoc tests were further used to compare the differences among the stages. A chi-square test or Fisher’s exact test was used to compare the differences in categorical variables. Spearman’s rank order correlation coefficient was used to determine the correlations between antioxidant vitamins status and metabolic parameters in patients with oral cancer. The logistical regression analyses were used to examine the correlations between antioxidant vitamins and metabolic syndrome (the diagnostic criteria for metabolic syndrome were based on the guidelines of the Administration of Health Promotion, Ministry of Health and Welfare, Taiwan). The significance of level was set at a p value < 0.05.