VOC markers in saliva for diagnosis of gastric cancer and gastric cancer diagnostic method using same

Abstract

Disclosed herein are a group of gastric cancer VOC markers in saliva and an application thereof in the preparation of a diagnostic reagent of gastric cancer. The markers are a combination of compounds selected from the group consisting of acetaldehyde, 2-methylbutyraldehyde, isopropanol, hexanal, n-butanol, cineole, nonanal, menthone, 2-ethylhexanol, menthol, anethole and dodecanol. The diagnostic reagent is used for detecting the contents of the marker in a saliva sample of a subject to perform the diagnosis of gastric cancer.

Claims

1. A method of diagnosing gastric cancer in a subject, the method comprising steps of: a) obtaining a saliva sample from the subject; b) detecting volatile organic compound (VOC) markers in the saliva sample by: concentrating the VOC markers in the saliva sample using a solid-phase microextraction device; and performing an analysis using a gas chromatography-mass spectrometry under a temperature program of 35° C. for 10 minutes (min), rise to 200° C. at a rate of 8° C./min, rise to 220° C. at a rate of 15° C./min and 220° C. for 12 min, wherein the VOC markers are compounds selected from the group consisting of acetaldehyde, isopropanol, hexanal, n-butanol, nonanal, menthone, menthol, anethole, dodecanol, and a combination thereof; and c) diagnosing the subject with gastric cancer when at least three of the compounds of acetaldehyde, isopropanol, hexanal, n-butanol and nonanal are present and at least three of the compounds of menthone, menthol, anethole and dodecanol are absent in the saliva sample.

2. A method of diagnosing gastric cancer in a subject, the method comprising steps of: a) obtaining a saliva sample from the subject; b) detecting volatile organic compound (VOC) markers in the saliva sample by: concentrating the VOC markers in the saliva sample using a solid-phase microextraction device; and performing an analysis using a gas chromatography-mass spectrometry under a temperature program of 35° C. for 10 min, rise to 200° C. at a rate of 8° C./min, rise to 220° C. at a rate of 15° C./min and 220° C. for 12 min, wherein the VOC markers are compounds selected from the group consisting of acetaldehyde, isopropanol, hexanal, n-butanol, nonanal, menthone, menthol, anethole, dodecanol, and a combination thereof; c) preliminarily diagnosing the subject with gastric cancer when significant increase in content of 2-methylbutyraldehyde, cineole and 2-ethylhexanol is detected; and d) diagnosing the subject with gastric cancer when at least three of the compounds of acetaldehyde, isopropanol, hexanal, n-butanol and nonanal are present and at least three of the compounds of menthone, menthol, anethole and dodecanol are absent in the saliva sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The FIGURE shows the GC-MS detection results of gastric cancer markers in saliva samples respectively from a patient with gastric cancer and a healthy control.

DETAILED DESCRIPTION OF EMBODIMENTS

(2) The invention will be described in detail below with reference to the accompanying drawings and embodiments.

Example 1 Collection of Saliva Samples

(3) Gastric cancer VOC markers in a saliva sample were screened by analyzing the saliva samples from the patients with gastric cancer and healthy people, where a total of 90 saliva samples were collected, including 36 samples from the patients with gastric cancer and 54 samples from healthy people.

(4) The participants all did not have other malignancies, blood diseases or metabolic system diseases.

(5) Those with gastric cancer were clinically diagnosed by biopsy, computed tomography (CT) and puncture, and did not receive chemotherapy or other treatment before the saliva collection.

(6) Before the collection, each participant was required to keep a natural and calm mood within 48 h and to avoid smoking and drinking alcohol within 24 h. Within 1 h before the sample collection, all participants were prohibited from ingesting any food to keep the mouth clean.

(7) All participants were required to gargle with pure water before the sample collection, and to collect 4-6 mL of a saliva sample 5 min after the gargling.

Example 2 Analysis of Saliva Samples

(8) 1 mL of the collected saliva sample was accurately pipetted, transferred to a vial and heated for about 20 min to 80° C. to achieve the chemical equilibrium of the vapor.

(9) The headspace sampling device was connected to the solid-phase microextraction (SPME) device. A 75 μm CAR/PDMS-coated SPME fiber was used to pre-concentrate VOCs in the saliva sample, where the pre-concentration lasted for 30-35 min.

(10) The GC-MS analysis was performed as follows. The SPME fiber was inserted into an injection port and heated at 280° C. for 1 min for desorption, and a splitless injection was chosen. 2 min later, a splitting valve was opened. A CD-1MS chromatographic column (1.4 μm×60 m×0.25 mm) was employed.

(11) The temperature was programmed as follows: 35° C. for 10 min; rise to 200° C. at 8° C./min; rise to 220° C. at 15° C./min; and 220° C. for 12 min.

(12) Full scanning range: 42-400 amu; Electron impact energy: 70 eV; Quadrupole mass spectrometer ion source temperature: 200° C.; Carrier gas: He; and flow rate: 44.2 cm/s.

(13) The detected volatile organic compounds were preliminarily identified according to the NIST08 library, and those with a similarity larger than 90% were further quantified according to the corresponding peak areas. The GC-MS analysis results of the saliva samples from the patients with gastric cancer and the healthy controls were shown in the FIGURE, and the corresponding gastric cancer VOC markers were presented in Table 1.

(14) TABLE-US-00001 TABLE 1 Gastric cancer markers in saliva samples from the patients with gastric cancer and the healthy controls Peak Compounds 1 Acetaldehyde 2 2-Methylbutyraldehyde 3 Isopropanol 4 Hexanal 5 n-Butanol 6 Cineole 7 Nonanal 8 Menthone 9 2-Ethylhexanol 10 Menthol 11 Anethole 12 Dodecanol

(15) It can be seen from the FIGURE and Table 1 that levels of nonanal, acetaldehyde, isopropanol, hexanal and n-butanol in the saliva samples of the patients with gastric cancer were significantly higher than those in the saliva samples of the healthy controls, while the levels of 2-methylbutanal, cineole and 2-ethylhexanol were relatively lower in the saliva samples of the patients with gastric cancer. It should be further noted that menthone, menthol, anethole and dodecanol were only present in the saliva of healthy people. Therefore, such 12 volatile organic compounds were suitable as markers to be applied to the diagnosis of gastric cancer, allowing for more accurate diagnosis.

Example 3

(16) The screened VOC markers were applied in the rapid diagnosis of gastric cancer for validation.

(17) 10 saliva samples from patients with gastric cancer and 10 saliva samples from healthy people were collected. When three compounds of acetaldehyde, isopropanol, hexanal, n-butanol and nonanal were present and at least three compounds of menthone, menthol, anethole and dodecanol were absent, the subject was diagnosed with gastric cancer; and when 2-methylbutanal, cineole and 2-ethylhexanol underwent significant decrease in content, the subject was preliminarily diagnosed with gastric cancer. Otherwise, the subject was considered to be healthy. The detection process was the same as that in Examples 1 and 2. The results showed that there were 8 gastric cancer samples and 9 normal samples that met the above criteria, indicating that the accurate rate of the diagnosis involving the use of such markers was greater than 80%.

(18) The above-mentioned embodiments are merely illustrative of the invention, and are not intended to limit the invention. Any change, replacement, modification and simplification made by those skilled in the art without departing from the spirit of the invention should still fall within the scope of the invention defined by the appended claims.