CN114813997A - Bile acid marker composition, application and serum detection kit - Google Patents
Bile acid marker composition, application and serum detection kit Download PDFInfo
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- CN114813997A CN114813997A CN202210292592.3A CN202210292592A CN114813997A CN 114813997 A CN114813997 A CN 114813997A CN 202210292592 A CN202210292592 A CN 202210292592A CN 114813997 A CN114813997 A CN 114813997A
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- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
Abstract
The invention relates to a new application of a serum detection kit for gastric cancer/early gastric cancer auxiliary screening, which is prepared by taking bile acid micromolecule metabolites, namely taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid as marker combinations. The invention also relates to a serum detection kit for auxiliary screening of gastric cancer/early gastric cancer, which detects the serum concentrations of the two bile acid metabolites of a testee by a liquid chromatogram tandem mass spectrometry method, substitutes the serum concentrations into a binary logistic regression model to calculate the positive predicted value of the gastric cancer/early gastric cancer of the marker combination, and then compares the positive predicted value with the optimal cutoff value of a diagnosis model, thereby judging whether the testee is a positive case of gastric cancer/early gastric cancer, and accordingly, accurate layering of whether high-risk people need to receive gastroscope fine screening can be realized. The combined application of the two bile acid marker combinations has good auxiliary effects on screening of gastric cancer and early gastric cancer of high risk people. The detection kit has the characteristics of high diagnosis specificity and sensitivity, simple combination, capability of determination by a single method, low detection cost, good compliance of a subject, no wound and the like, and is beneficial to improving the screening popularization rate and accuracy of gastric cancer/early gastric cancer.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to a bile acid marker combination and a serum detection kit for assisting in screening gastric cancer/early gastric cancer of high-risk people, belonging to the field of inspection medicine, clinical chemistry and clinical medicine.
[ background of the invention ]
Gastric Cancer is a highly invasive and fatal malignant digestive tract tumor, and according to the authoritative report of clinical Journal of tumor (CA) in 2015, the incidence rate of gastric Cancer is high in the fourth place of male tumor and the mortality rate is high in the third place of male tumor [ Torre, L.A.et al.Global Cancer statistics,2012, CA.cancer J.Clin.65, 87-108 (2015) ], all around the world. At present, surgery and chemotherapy are the main clinical means for treating gastric cancer, the diagnosis and treatment time and pathological grading of gastric cancer determine the prognosis of patients, early treatment is found early, the surgical trauma is small, the complications are few, and the prognosis is better. However, about 90% of new cases of Gastric Cancer in our country currently belong to the advanced stage, the 5-year-after-operation survival rate is less than 30%, and according to the research reports from Japan, the 5-year-after-operation survival rate of early Gastric Cancer can exceed 90% [ Katai, H.et al.five-year overview analysis of surgery-responded Cancer cases in Japan: a retroactive analysis of more than 100,000Patients from the national convention of the Japanese patient health of the Japanese patient Cancer Association (2001) 2007. Gastric Cancer of J.Int.gastric Cancer Association. Therefore, the screening and diagnosis of gastric cancer/early gastric cancer in high-risk population has very important clinical value for the prevention, control and treatment of gastric cancer.
According to the current clinical industry consensus in China, the high-risk group with gastric cancer refers to a natural group aged 40 years and older and having the past history of gastric diseases, or helicobacter pylori infection, or from a region with high gastric cancer incidence, or exposed to risk factors such as smoking, drinking, being addicted to pickled food and the like. Currently, in clinic, detection of endoscopic and serum tumor markers is a main means for screening and diagnosing gastric cancer. Gastroscopy and tissue biopsy are the golden standards for diagnosing gastric cancer and are suitable for screening gastric cancer in the advanced stage. The gastroscope screening can improve the detection rate of early gastric cancer, but the implementation of the gastroscope screening depends on special and fine gastroscope equipment such as electronic dyeing, high-definition electronic amplification and the like and the super-high operating skills of skilled endoscopists, and the two are not enough, which means that medical resources for carrying out gastric cancer screening by utilizing the gastroscope screening are very limited. Meanwhile, gastroscopy is also traumatic, painful and anxiety-phobic [ Ho, s.w.t. & Tan, p.diagnosis of gastric cancer pathogenesis for precision on-line. cancer sci.110, 3405-3414 (2019) ], and thus results in poor patient compliance. However, serum tumor markers commonly used in clinical practice, such as Carcinoembryonic Antigen (CAE), carbohydrate antigen CA199, CA125, CA242, etc., although having advantages of non-invasive property and high patient compliance, are not ideal in sensitivity and specificity (the positive rate of gastric cancer at the advanced stage is about 20-30%, and the positive rate of early gastric cancer is less than 10%), so that the methods have not been suggested as detection means for screening gastric cancer/early gastric cancer in industry consensus published by institutions 2018 such as national digestive system disease clinical medicine research center [ national digestive system disease clinical medicine research center, health management of chinese medical society, gastroenterology of physicians' society, digestive endoscopy professional committee of chinese medical society, digestive endoscopy health management and physical examination professional committee of chinese society & national digestive endoscopy quality control center ], early gastric cancer screening process expert consensus opinion (draft) (2017, shanghai) gastroenterology 23, (2018). On the other hand, the technical route of assisting gastric cancer screening by simultaneously combining various means such as serum pepsinogen ratio (PGR), pepsin (G17) and helicobacter pylori antibody detection still faces the challenges of high detection cost caused by multi-index superposition, need of detection technology participation depending on various different systems, still less popularization of clinical application and the like. Therefore, the serum detection kit which is simple, convenient and effective to develop, measurable by a single method, low in cost, noninvasive and good in patient compliance is used for auxiliary screening of the gastric cancer/early gastric cancer, the defects of endoscopic screening and serum tumor marker value detection are overcome, accurate layering of whether a subject needs to receive endoscopic scrutiny (including biopsy, pathological section and the like) is realized, the popularization and accuracy of developing the gastric cancer/early gastric cancer screening in high-risk groups are improved, and certain practical significance is realized for improving the severe gastric cancer prevention and control current situation in China.
Metabonomic technology is an important omics approach to find tumor markers and study disease mechanisms [ Faubert, b., Solmonson, a. & debarkini, r.j. metabolic profiling and cancer progression. science 368, eaaw5473 (2020) ], and numerous studies have confirmed that the onset of gastric cancer is associated with metabolic regulation abnormalities. For example, it has been reported That metabolic abnormalities in energy, amino acids and lipids are associated with Gastric Cancer progression [ Huang, S.et al.identification and differentiation of Plasma Metabolic Signatures in Precancerous gases threshold progression to Cancer. JAMA Net. open 4, e2114186(2021) ], peritoneal recurrence of Gastric Cancer with unique metabolic characteristics [ Kaji, S.et al.Metabolic profiling of Gastric Cancer tissue samples concentration. Gastric Cancer official. J.J.internal.gastric Cancer analyte analysis. Jpn. Gastric Cancer Strand nucleic acid application. 23, 874-3 ]. In a multicenter clinical trial, Bile reflux has been shown to be An independent risk factor for precancerous lesions and gastric cancer development [ Zhang, l.y.et al. Bile reflux is An independent risk factor for precancerous lesions and gastric cancer development: An organic cross-sectional study.j.di.dis.22, 282-290 (2021) ], while Bile acids are the major components in Bile, which play An important regulatory role in the maintenance of digestive tract function and gastrointestinal immunity, and more studies have revealed a diverse role for Bile acid metabolism involved in the molecular regulation of the development of digestive tract carcinogenesis [ Jia, w.xie., g. & je, g. & gt, w.bile acid-microbial cross in gastric metastasis growth hormone production. natural gene.20115. natural cancer. 7. 11. 7. and 7. natural cancer. Therefore, the bile acid micromolecular metabolite has practical potential and development value in screening and diagnosing gastric cancer. The invention utilizes a deep-coverage serum bile acid targeted metabonomics technology to detect 258 gastric cancer/early gastric cancer and serum samples of healthy control group subjects, measures derivative indexes such as 49 serum bile acid concentration values, 28 concentration ratio values, the sum of subclass concentrations and the like, and performs metabolite optimization and combination research on the basis, wherein the process is shown in figure 1. Firstly, calculating the contribution degree of metabolites in a model to sample classification by utilizing orthogonal partial least square-discriminant analysis, and selecting 34 valuable candidate indexes by combining T test and multiple test correction significance level; subsequently, 34 candidate indices were compressed to 6 reduced indices using LASSO regression (LASSO) (table 1); then gradually reducing indexes one by one, and utilizing different bile acid markers to combine and construct a diagnosis model, and evaluating the diagnosis performance (table 1); and after factors such as convenience of kit engineering implementation, readability of a diagnosis model, detection cost and the like are comprehensively considered, two bile acid metabolites are finally selected and used as a combined marker for auxiliary screening of gastric cancer/early gastric cancer by adopting a binary logistic regression model. Our studies also confirm that the bile acid has significant correlation with a plurality of clinical indexes of gastric cancer and tumor proliferation related proteins such as HER-2, p53, Ki-67 and the like in cancerated tissues. Among the known bile acid molecules, taurine-conjugated lithocholic acid is the strongest agonist of bile acid receptor TGR5, and can promote the occurrence of protocarcinoma. 3-glucuronic acid conjugated deoxycholic acid is a product formed by transferring a glycosyl donor, guanosine diphosphate glucuronic acid, to deoxycholic acid by glucuronyl transferase UGT, the biological function of the product is not clear at present, and the mechanism of the participation of the product in gastric cancer canceration is to be further researched. At present, no research report for combining the two bile acid metabolites for auxiliary screening of gastric cancer/early gastric cancer exists.
[ summary of the invention ]
Aiming at the problems that the existing clinical gastric cancer/early gastric cancer screening is carried out in high risk population, such as poor patient compliance caused by invasive endoscopy, poor accuracy of the existing serum tumor marker, limited clinical resources for gastroscope precise examination, low popularity and the like, the invention provides a novel marker combination and a serum detection kit based on quantitative detection of two serum bile acids, which are applied to gastric cancer/early gastric cancer screening and/or precise layering of high risk population. The marker combination and the detection kit of the invention are characterized as follows:
the bile acid marker combination comprises two bile acid micromolecular metabolites, namely: taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid.
The serum detection kit comprises a quantitative liquid, an extracting solution, a microporous plate and a reference sample. The quantitative liquid contains quantitative external standard taurine conjugated lithocholic acid monosodium salt and 3-glucuronic acid conjugated deoxycholic acid, and the external standard is used for quantitative and/or qualitative identification of the concentration of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in serum to be detected of a subject. The extracting solution is a methanol-acetonitrile mixed solution (the volume ratio is 1:1, the concentrations of the internal standard substances are both 50ng/mL) containing internal standard substance deuterated taurine conjugated lithocholic acid-d 4 monosodium salt and deuterated n-ursodeoxycholic acid-d 5, and the methanol-acetonitrile mixed solution is used for preprocessing a serum sample to be detected of a subject and extracting bile acid, and the internal standard substances are also respectively used for quantifying taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid. The kit comprises 3 micro-porous plates, wherein one plate is a deep-hole plate with the capacity of 750 microliters in a single hole and is used for pretreatment of serum to be detected and extraction of bile acid of a subject; the second plate is a shallow plate with a single-well capacity of 450. mu.l, and is used for drying the extract of the serum to be tested of the subject transferred from the first plate; the third plate is the same as the second plate, the extract in the second plate is redissolved after being dried, and the redissolved solution is transferred to the third plate to be used for the computer detection of the liquid chromatography tandem mass spectrometer.
The work flow of the serum detection kit provided by the invention is as follows: (1) after a subject takes blood, 80-100 mu L of serum sample to be detected is transferred to a first plate micropore one by one, 320-400 mu L of extracting solution is added, vortex oscillation is carried out for 3 minutes at the rotating speed of 1500rpm, the first plate is placed into a centrifuge, and the first plate is centrifuged for 20 minutes at the rotating speed of 5000rpm, so as to obtain bile acid extract; (2) absorbing 260-320 mu L of bile acid extract from each sample to be detected, transferring the bile acid extract to a second-plate micropore, drying by nitrogen blowing or vacuum reduced pressure drying, re-dissolving the dried extract by using 150-200 mu L of 15% methanol-water mixed solution (the volume ratio of methanol can be 5-100%, but the high proportion of methanol can influence the chromatographic peak shape of the detected bile acid, and the low proportion of methanol can influence the dissolution of the bile acid during re-dissolving), so as to obtain a sample re-solution to be detected, and re-dissolving the reference sample by the same method; (3) centrifuging the complex solution at the rotating speed of 5000rpm for 20 minutes, sucking 140-190 mu L of complex solution of each sample to be detected and each reference sample, transferring the complex solution into a third plate micropore, and performing on-machine detection on a liquid chromatography tandem mass spectrometer; (4) and respectively detecting the quantitative liquid, the treated blood serum sample to be detected of the subject and the reference sample by liquid chromatography tandem mass spectrometry, comparing the retention time of the chromatographic peak recorded by mass spectrometry of the external standard taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid and the blood serum to be detected of the subject in the quantitative liquid (any concentration point between 20 and 200 ng/mL), and judging that the chromatographic peak is the chromatographic peak of two markers detected in the blood serum to be detected of the subject within +/-0.05 minutes, thereby realizing the qualitative identification of the two bile acid markers in the blood serum to be detected of the subject. If no chromatographic peak exists in the retention time window with the difference of +/-0.05 minutes, judging that the contents of the two bile acid markers in the serum to be detected of the subject are not detected due to the fact that the contents of the two bile acid markers are lower than the detection limit, and setting the peak area value to be zero; and comparing the retention time of the chromatographic peaks recorded by mass spectrum scanning of the internal standard substance deuterated taurine conjugated lithocholic acid-d 4 and deuterated n-ursodeoxycholic acid-d 5 in the quantitative liquid at any concentration point with the serum to be detected of the subject, and judging that the chromatographic peaks are chromatographic peaks of the internal standard substance corresponding to the two markers respectively within +/-0.05 minutes of the retention time, thereby realizing the qualitative identification of the internal standard substance in the serum to be detected of the subject. And respectively recording and calculating the peak area values and the ratio of the peak area values to the d4 of the taurine conjugated lithocholic acid and the internal standard substance deuterated taurine conjugated lithocholic acid in the quantitative liquid, the serum to be detected of the tested object and the reference sample, and the peak area values and the ratio of the peak area values to the d5 of the 3-glucuronic acid conjugated deoxycholic acid and the internal standard substance deuterated ursodeoxycholic acid. And respectively drawing quantitative working curves of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid by taking the concentration value of the external standard substance in the quantitative liquid as X and the corresponding ratio as Y, and establishing a quantitative working equation. And substituting the peak area ratios recorded and calculated in the serum sample to be detected and the reference sample of the subject into an equation to obtain mass concentration, and calculating the serum concentration values of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid of the subject in unit nmol/L through sample pretreatment volume conversion and molar quantity conversion. And determining that the serum concentration measurement result of the reference sample is within 75-125% of the specification calibration value, and determining that the quantitative inspection quality of the serum bile acid is qualified. (5) The method comprises the steps of taking blood serum concentration values of two bile acids of a training set subject for constructing a diagnosis model as independent variables, taking classification information of the subject as response values (gastric cancer component class information is set to be 1, and health control component class information is set to be 0), conducting binary logistic regression analysis to combine the two bile acid metabolites into the diagnosis model, recording intercept of the model and coefficients of the two bile acids, and transforming mathematical equations of the binary logistic regression model to obtain a calculation formula of a gastric cancer positive predictive value (GC-PPV). In the formula, CTLCA and CDCA-3G are serum concentration values of taurine conjugated lithocholic acid and 3-glucuronic acid respectively, and e is a natural constant. Substituting the bile acid serum concentration of the subject into a formula to calculate the GC-PPV value of the subject.
The formula:
(6) the present invention determines that the optimal cut-off value (cut-off value) for gastric cancer screening based on the two bile acid marker combinations is 0.829, and a subject with a GC-PPV value higher than 0.829 is judged as a gastric cancer positive case, and vice versa is negative. The method for setting the cutoff value is to draw ROC for short according to GC-PPV values and classification information of all training set testers, wherein the X axis represents 100% -specificity% of the diagnostic model, and the Y axis represents sensitivity% of the diagnostic model. The optimal cut-off is the GC-PPV value corresponding to the maximum sum of sensitivity and specificity in the ROC curve. The serum detection kit using unit can determine a new binary logistic regression model according to a new training set and field experimental results, and set a new optimal cutoff value.
The two bile acids have obvious concentration difference between the gastric cancer group and the healthy control group, and have good effect on assisting gastric cancer screening. After the two bile acids are used as the marker combination, the specificity of the diagnostic model is improved to 99.3%, the area under the curve (AUC) of the ROC curve is improved to 0.992, and the sensitivity is still maintained at an excellent level of 98.2%. Accordingly, the good diagnostic performance exhibited by the preferred two bile acid marker combinations of the present invention makes them of practical value for assisting in screening for gastric cancer. Meanwhile, the bile acid marker combination has satisfactory performance in assisting early gastric cancer screening and also has clinical practical value.
The serum detection kit has the characteristics of high diagnostic sensitivity and specificity, simple combination, single method measurement, low cost, no wound, good patient compliance and the like. The combination of the two bile acid markers can realize the aim of accurately layering screening on whether a subject needs to receive endoscopic scrutiny (comprising biopsy, pathological section and the like).
The invention relates to a new application of a serum detection kit for gastric cancer/early gastric cancer auxiliary screening, which is prepared by taking bile acid micromolecule metabolites, namely taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid as marker combinations. The invention also relates to a serum detection kit for the auxiliary screening of gastric cancer/early gastric cancer, which detects the serum concentrations of the two bile acid metabolites of a testee by a liquid chromatography tandem mass spectrometry method, substitutes the serum concentrations into a binary logistic regression model to calculate the gastric cancer/early gastric cancer positive prediction value of the marker combination, and compares the gastric cancer/early gastric cancer positive prediction value with the optimal cutoff value of a diagnosis model, thereby judging whether the testee is a gastric cancer/early gastric cancer positive case, and accordingly, accurate layering of whether a high-risk group needs to receive gastroscope fine screening can be realized. The combined application of the two bile acid marker combinations has good auxiliary effects on screening of gastric cancer and early gastric cancer of high risk people. The detection kit has the characteristics of high diagnosis specificity and sensitivity, simple combination, capability of determination by a single method, low detection cost, good compliance of a subject, no wound and the like, and is beneficial to improving the screening popularization rate and accuracy of gastric cancer/early gastric cancer.
[ description of the drawings ]
FIG. 1 is a schematic representation of a preferred bile acid metabolite combination study procedure according to the invention with markers.
FIG. 2 is a bar graph of 6 bile acid markers under combined study with markers in examples 1-5 in the gastric cancer group and healthy control group. (A) Taurine conjugated lithocholic acid serum concentration, (B) 3-glucuronic acid conjugated deoxycholic acid serum concentration, (C) n-cholic acid serum concentration, (D) swine cholic acid serum concentration, (E) 3-sulfated taurine conjugated lithocholic acid serum concentration, and (F) ratio of swine deoxycholic acid to lithocholic acid serum concentration
FIG. 3 is a ROC curve chart of the independent use of 6 bile acid indexes including the combination study of markers in examples 1-5 for assisting gastric cancer screening diagnosis. (A) Taurine conjugated lithocholic acid, (B) 3-glucuronic acid conjugated deoxycholic acid, (C) orthocholic acid, (D) hyocholic acid, (E) 3-sulfated taurine conjugated lithocholic acid, and (F) the serum concentration ratio of hyodeoxycholic acid/lithocholic acid. The information of sensitivity, specificity, AUC and AUC 95% confidence interval is labeled by the figure.
FIG. 4 is a ROC curve chart of different marker combinations in examples 1-6 for assisting gastric cancer/early gastric cancer screening diagnosis. (A) Example 1: the combination of two bile acid markers is used for assisting the ROC curve of gastric cancer screening (taurine conjugated lithocholic acid + 3-glucuronic acid conjugated deoxycholic acid); (B) example 2: the combination of three bile acids is used for assisting the ROC curve of gastric cancer screening (taurine conjugated lithocholic acid + 3-glucuronic acid conjugated deoxycholic acid + orthocholic acid); (C) example 3: the combination of four bile acids is used for assisting the ROC curve of gastric cancer screening (taurine conjugated lithocholic acid + 3-glucuronic acid conjugated deoxycholic acid + orthocholic acid + hyocholic acid); (D) example 4: the combination of five bile acids is used for assisting the ROC curve of gastric cancer screening (taurine conjugated lithocholic acid + 3-glucuronic acid conjugated deoxycholic acid + n-cholic acid + hyocholic acid + 3-sulfated taurine conjugated lithocholic acid); (E) example 5: the combination of six bile acid indexes is used for assisting the ROC curve of gastric cancer screening (the ratio of taurine-conjugated lithocholic acid + 3-glucuronic acid-conjugated deoxycholic acid + orthocholic acid + hyocholic acid + 3-sulfated taurine-conjugated lithocholic acid + hyodeoxycholic acid/lithocholic acid, and five of the ratios are serum concentration values). (F) Example 6: two bile acid markers were combined to assist the ROC curve for early gastric cancer screening (taurine conjugated lithocholic acid + 3-glucuronic acid conjugated deoxycholic acid). The information of sensitivity, specificity, AUC and AUC 95% confidence interval is labeled by the figure.
Fig. 5 (a) example 1: a distribution scatter diagram of the gastric cancer positive prediction rate (GC-PPV) of the tested person calculated based on the combination of the two bile acid markers in the invention between the gastric cancer group and the healthy control group, wherein a red horizontal line represents the optimal cutoff value of the model to be 0.829; (B) the distribution scatter diagram of the early gastric cancer positive prediction rate (EGC-PPV) of the tested person calculated based on the combination of the two bile acid markers provided by the invention in the early gastric cancer group and the healthy control group shows that the red horizontal line represents the optimal cutoff value of the model of 0.715. (C) Example 7: 168 blind samples (80 gastric cancers versus 88 healthy controls) were tested.
[ detailed description ] embodiments
Example 1 gastric cancer-assisted screening based on combination of two bile acid markers
1. And (3) designing a clinical test: the study was approved by the medical symposium and subjects signed an informed consent prior to inclusion in the clinical trial. The training set used to construct the gastric cancer screening diagnostic model included 145 healthy control group subjects (age 60.31 ± 12.15 years, male 108, female 37) and 113 gastric cancer group subjects (age 63.33 ± 10.59 years, male 79, female 34), of which 68 were advanced gastric cancer (cancer tissue infiltration beyond the gastric wall submucosa and invaded the gastric wall muscularis under pathological section), and 45 were early gastric cancer (cancer tissue did not invade the gastric wall muscularis under pathological section, limited to the gastric wall mucosa and submucosa). According to the cancer staging system established by the eighth edition of the united states joint committee for cancer (AJCC), 47 patients were stage I, 10 were stage II, 34 were stage III, and 22 were stage IV.
2. Serum collection of subjects: blood serum of a subject is collected according to the routine operation of venous blood collection in the clinical laboratory of a hospital. The blood is collected by using a red tube cap vacuum negative pressure blood collection tube specified in the national people's republic of China sanitary industry standard WS/T661-2020, standing for 30 minutes at room temperature after blood collection, centrifugally separating serum, and storing in a refrigerator at-20 ℃ for detection.
3. Pretreatment of serum to be detected of a subject: the serum sample to be detected of a subject is unfrozen at room temperature, 80 mu L of the sample is respectively transferred into the micropores of a first microporous plate in portions, respectively adding 320 μ L of extractive solution (methanol-acetonitrile mixed solution with volume ratio of 1: 1) containing internal standard substance deuterated taurine conjugated lithocholic acid-d 4 monosodium salt and deuterated n-ursodeoxycholic acid-d 5 (concentration is 50ng/mL) by using an electric dispenser, vortex oscillating at 1500rpm × 3min, centrifuging at 5000rpm × 20min to remove protein precipitate, transferring 260 μ L bile acid extract to micropores of the second plate, vacuum drying under reduced pressure for 3h, adding 150 μ L15% methanol-water mixture hole by hole to redissolve the dried sample, vortex oscillating at 1500rpm × 3min, centrifuging at 5000rpm × 20min for removing protein precipitate again, transferring 120 μ L of redissolved solution to the micropores of the third plate one by one respectively, and performing detection on the three-dimensional plate by using a liquid chromatography tandem mass spectrometry machine.
Preparation and treatment of reference samples: 9.900mL of Ultra-Low Hormons & Steroids model artificial serum produced by Golden West Diagnostics is added into a test tube with a plug, 50 μ L of a 15% methanol-water solution (V/V, the same below) containing taurine conjugated lithocholic acid with the concentration of 2nmol/mL and 50 μ L of a 15% methanol-water solution containing 3-glucuronic acid conjugated deoxycholic acid with the concentration of 60nmol/mL are respectively added, and the mixture is shaken on an electric shaking table for 10min and fully mixed to obtain reference serum samples containing the taurine conjugated lithocholic acid and the 3-glucuronic acid conjugated deoxycholic acid with the serum concentrations of 10nmol/L and 300nmol/L respectively. Respectively transferring 96 parts of the bile acid extract into micropores of a microporous plate with the same model as the first plate according to the use volume of 80 mu L of each part, adding 320 mu L of the extract (the composition and the concentration of the extract solvent and the internal standard are the same as those of the first plate) hole by using an electric separator, carrying out vortex oscillation at 1500rpm multiplied by 3min, centrifuging at 5000rpm multiplied by 20min to remove protein precipitates, respectively transferring 260 mu L of the bile acid extract from each hole, placing the bile acid extract into another test tube with a plug, fully and uniformly mixing the bile acid extract by shaking on an electric shaking table for 10min, respectively transferring 90 parts of the bile acid extract (each 260 mu L) into 90 spiral cover tubes, and carrying out vacuum reduced pressure drying for 3 hours to obtain 90 parts of reference sample. Adding 150 mu L of 15% methanol-water mixed solution into any reference sample for redissolving, after vortex oscillation at 1500rpm multiplied by 3min, centrifuging at 5000rpm multiplied by 20min for removing protein precipitate again, and transferring 120 mu L of reference sample redissolution to a micropore of a third plate for detection by a liquid chromatography tandem mass spectrometry machine.
4. Liquid chromatography tandem mass spectrometry detection: bile acid detection was performed by using Shimadzu LC-20ADXR type liquid chromatograph of Shimadzu corporation, Japan, in tandem with SCIEX 5500+ triple quadrupole mass spectrometer of Aibotisi, USA, scanning using a selective reaction monitoring mode (SRM), and quantifying by an internal standard method.
4.1 liquid chromatography method: bile acid isolation was performed using an ACE Excel PFP C18 chromatography column (packing size 2 μm, inner diameter 2.1mM, column length 100mM), 2mM ammonium acetate aqueous solution (A) and acetonitrile (B) as binary elution mobile phases, flow rate 0.4mL/min, column temperature 45 ℃ and injection volume 3 μ L. The chromatographic elution gradient was set as: and the volume of the phase B is 17 percent of the initial proportion, the phase B is maintained for 0.5 minute, the linear increase is carried out to 30 percent in 0.5-10 minutes, the linear increase is carried out to 55 percent in 10-13 minutes, the linear increase is carried out to 55 percent in 13-14 minutes, the linear decrease is carried out to 17 percent of the initial proportion in 14.1 minutes, and the linear increase is carried out to 16 minutes, thus completing the preparation.
4.2 Mass Spectrometry method: the negative ion ionization voltage of the Turbo V electrospray ion source is-4.5 kV, the temperature of the ion source is 550 ℃, the pressure of air curtain gas is 35 psi, the pressure of the spray gas is 50 psi, and the pressure of auxiliary heating gas is 50 psi. Scanning by using a selective reaction monitoring mode, wherein a taurine-conjugated lithocholic acid scanning ion pair is a parent ion 482.3 → a child ion 80, the declustering voltage and the collision voltage are-130V, the scanning ion pair corresponding to an internal standard substance deuterated taurine-conjugated lithocholic acid-d 4 is a parent ion 486.3 → a child ion 80, and the declustering voltage and the collision voltage are-130V and-127V respectively; the 3-glucuronic acid conjugated deoxycholic acid scanning ion pair is a parent ion 567.3 → a daughter ion 505.3, the declustering voltage and the collision voltage are-120V and-50V respectively, the scanning ion pair corresponding to the internal standard substance deuterated n-ursodeoxycholic acid-d 5 is a parent ion 382.3 → a daughter ion 382.3 (the parent ion and the daughter ion of the SRM scanning are the same because the structure of deuterated n-ursodeoxycholic acid-d 5 is difficult to generate secondary mass spectrum fragmentation), and the declustering voltage and the collision voltage are-160V and-20V respectively.
5. Quantifying the working curve and quantifying the bile acid: the quantification solution was 12 parts methanol-water solution (methanol volume ratio of 15%) containing external standard (12 concentration points) and internal standard of the same concentration, and was used to establish the internal standard method quantification working curve. The external standard substances are taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid, and the concentrations of the two external standard substances in 12 parts of quantitative working curve solution are as follows from low to high in sequence: 0.2271, 0.4541, 0.9083, 1.8165, 3.6329, 7.2657, 14.5313, 29.0625, 58.1250, 116.2500, 232.5000 and 465.0000ng/mL, which are abbreviated as L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11 and L12. The internal standard substances corresponding to the two external standard substances are respectively deuterated taurine conjugated lithocholic acid-d 4 and deuterated n-ursodeoxycholic acid-d 5, and the concentration of the internal standard substances is 70 ng/mL. And respectively carrying out liquid chromatography tandem mass spectrometry on the quantitative working curve solution, the treated serum sample of the subject and the reference sample, and comparing the retention time of the chromatographic peak recorded by mass spectrometry of the external standard taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid and the serum to be detected of the subject in the quantitative solution (58.1250ng/mL concentration point), wherein the retention time is within +/-0.05 minute, namely judging that the chromatographic peak is the chromatographic peak of two markers detected in the serum to be detected of the subject, and realizing the qualitative identification of the two bile acid markers in the serum to be detected of the subject. If no chromatographic peak exists in the retention time window with the difference of +/-0.05 minutes, judging that the contents of the two bile acid markers in the serum to be detected of the subject are not detected due to the fact that the contents of the two bile acid markers are lower than the detection limit, and setting the peak area value to be zero; comparing the retention time of the chromatographic peaks recorded by mass spectrum scanning of the internal standard substance deuterated taurine conjugated lithocholic acid-d 4 and deuterated n-ursodeoxycholic acid-d 5 in the quantitative liquid at any concentration point with the serum to be detected of the subject, and judging that the chromatographic peaks are chromatographic peaks of the internal standard substance corresponding to the two markers respectively within +/-0.05 minutes of the retention time, thereby realizing the qualitative identification of the internal standard substance in the serum to be detected of the subject;
respectively recording and calculating peak area values and ratios of the quantitative liquid, serum to be detected of a subject, and taurine conjugated lithocholic acid and an internal standard substance deuterated taurine conjugated lithocholic acid-d 4 in a reference sample, and peak area values and ratios of the 3-glucuronic acid conjugated deoxycholic acid and the internal standard substance deuterated ursodeoxycholic acid-d 5 (table 2: peak areas of the quantitative liquid, the serum to be detected of the subject, and the external standard substance in the reference sample, and table 3: peak areas of corresponding internal standard substances in the quantitative liquid, the serum to be detected of the subject, and the reference sample). And respectively drawing quantitative working curves and establishing a quantitative working equation by taking the concentration of the external standard substance in the corresponding quantitative liquid as X and the corresponding ratio as Y as follows. Quantitative working curve equation of taurine conjugated lithocholic acid: Y0.01296X-0.000692
Quantitative working curve equation of 3-glucuronic acid conjugated deoxycholic acid: Y0.00134X +4.914E-4
Substituting the peak area ratios calculated by the blood serum sample to be detected of the subject and the reference sample into an equation to calculate mass concentration values (in ng/mL) of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in the blood serum to be detected of the subject and the reference sample, multiplying the mass concentration values by a volume conversion coefficient 2884.6154 (from the numerical value described in the section of 3. blood serum pretreatment of the subject, the calculation formula is 1000 x [ (A + B) ÷ A ] × [ C ÷ D ], 1000 represents unit conversion between ng/mL and ng/L, A represents the use volume of the blood serum to be detected, B represents the use volume of an extracting solution, C represents the use volume of a 15% methanol-water mixed solution added into a second plate for redissolving the dried extract, D represents the volume of the bile acid extract transferred from the first plate to the second plate), and the two bile acid metabolites are divided by the molar masses of 483.70424g/mol and 612.65978g/mol respectively to finally obtain the serum concentrations of the two bile acid metabolites with the unit of nmol/L (table 4). The measured values of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in the reference sample are respectively 112.4% and 110.1% of the calibrated value, and the quantitative detection quality of the serum bile acid in the example 1 is qualified. The serum concentrations of taurine conjugated lithocholic acid in the gastric cancer group and the healthy control group were respectively (mean ± sd) 13.22 ± 2.64nmol/L and 5.30 ± 2.65nmol/L (fig. 2A), and the serum concentrations of 3-glucuronic acid conjugated deoxycholic acid in the gastric cancer group and the healthy control group were respectively 2153.0 ± 207.9nmol/L and 1651.0 ± 718.7nmol (fig. 2B). The serum concentrations of the two bile acid metabolites in the gastric cancer group and the healthy control group are subjected to t test by using SPSS statistical software, and the results show that the two groups have significant difference (p <0.0001), and the serum concentration of the gastric cancer group is higher than that of the healthy control group.
6. Single metabolite based diagnostic models and diagnostic performance evaluation: ROC curves (fig. 3B) of taurine-conjugated lithocholic acid (fig. 3A) and 3-glucuronic acid-conjugated deoxycholic acid were plotted using GraphPad Prism software, where the X-axis is 100% -specificity of the diagnostic model and the Y-axis is sensitivity of the diagnostic model, based on the serum concentration values and classification information (divided into gastric cancer group and healthy control group) of taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid, respectively, of all subjects. The diagnosis sensitivity and specificity of the gastric cancer screening assisted by the taurine conjugated lithocholic acid are respectively 99.1% and 97.2%, and the diagnosis sensitivity and specificity of the gastric cancer screening assisted by the 3-glucuronic acid conjugated deoxycholic acid are respectively 98.2% and 50.3%.
7. Marker combination based diagnostic model and positive predictive value calculation: combining the two bile acids into a diagnosis model by using GraphPad Prism software to perform binary logistic regression analysis by taking the serum concentration values of the two bile acids of all the subjects as independent variables and the classification information of the subjects as response values, wherein the classification information of the gastric cancer component is set as 1, the classification information of the healthy control component is set as 0, the intercept of the result model is equal to 18.18, the coefficients of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid are respectively-1.223, -0.003013, and the mathematical equation of the binary logistic regression model is as follows: ln ((1- [ GC-PPV ])/[ GC-PPV ]) 18.18-1.223 × CTLCA-0.003013 × CDCA-3G, ln represents the logarithm with the natural constant as the base. Converting the concentration values to obtain a formula 1, respectively substituting the bile acid serum concentration values of each sample of the subjects in the gastric cancer group and the healthy control group into the formula 1, and calculating the gastric cancer positive predictive value (GC-PPV) of the subjects. ROC curves for gastric cancer screening diagnostic models were plotted using GraphPad Prism software (fig. 4A) based on GC-PPV values and classification information (divided by gastric cancer group and healthy control group) of all subjects, where X-axis is 100% -specificity and Y-axis is sensitivity of the diagnostic model.
Equation 1:
in the formula, CTLCA and CDCA-3G are serum concentration values of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid respectively, and e is a natural constant.
The ROC curve shows that the sensitivity of the diagnostic model of the two marker combinations is 98.2%, the specificity is 99.3%, the area under the curve AUC is 0.991, the AUC 95% confidence interval is 0.980-1.000, in other words, the probability of 95% of AUC occurring in the interval indicates that the two bile acid marker combinations have good diagnostic effect on auxiliary gastric cancer screening. Meanwhile, compared with the single metabolite of taurine conjugated lithocholic acid or 3-glucuronic acid conjugated deoxycholic acid, the combined application of the two bile acid metabolites improves the specificity of a diagnosis model (single optimal 99.1% and combined 98.2%) on the premise of maintaining excellent sensitivity and accuracy (single optimal 97.2% and combined 99.3%), reduces the false positive rate of misdiagnosis of a negative subject to be positive, and is particularly important for gastric cancer screening (table 1). The combined application of the two bile acid markers enables the sensitivity and specificity of a diagnosis model to be more balanced, and the diagnosis performance of assisting gastric cancer screening to be more excellent.
The optimal cut-off value for the gastric cancer screening diagnostic model was determined to be 0.829, based on the principle that the sum of sensitivity and specificity is the greatest, i.e., at the corner of the upper left corner of the ROC curve. In this study example, 111 of the 113 gastric cancer groups had a GC-PPV higher than the cutoff value, and were judged to be positive for gastric cancer with an accuracy of 98.23% and a false negative rate of 1.77%. Among the 145 healthy control groups, 144 healthy control groups were judged to be negative for gastric cancer with a GC-PPV lower than the cutoff value, an accuracy of 99.31% and a false positive rate of 0.69% (table 5, fig. 5A).
Example 2 gastric cancer-assisted screening based on three bile acid marker combinations
1. And (3) designing a clinical test: same as example 1 (i.e. using the same subject as example 1).
2. Serum collection of subjects: the corresponding procedures and conditions were the same as those in example 1.
3. Pretreatment of serum to be detected of a subject: based on example 1, the extract was supplemented with deuterated Beta-murine cholic acid-d 5 at a concentration of 50ng/mL, and the corresponding procedures and conditions were otherwise the same as those in example 1.
4. Liquid chromatography tandem mass spectrometry detection: the corresponding procedures and conditions were the same as those in example 1.
4.1 liquid chromatography method: the corresponding procedures and conditions were the same as those in example 1.
4.2 Mass Spectrometry method: the detection of the normal cholic acid is increased on the basis of the embodiment 1, and the rest is the same as the embodiment 1. The scanning ion pair of the positive cholic acid is a parent ion 393.3 → a daughter ion 393.3, the declustering voltage and the collision voltage are-160V and-20V respectively, the scanning ion pair corresponding to the internal standard substance deuterated Beta-murichoic acid-d 5 is a parent ion 412.3 → a daughter ion 412.3, and the declustering voltage and the collision voltage are-160V and-20V respectively.
5. Quantifying the working curve and quantifying the bile acid: on the basis of example 1, a positive cholic acid was newly added to the quantitative external standard, 12 concentration points were set to the taurine conjugated lithocholic acid in example 1, a deuterated Beta-murine cholic acid-d 5 was newly added to the internal standard, the concentration was the same as that of the deuterated taurine conjugated lithocholic acid-d 4 in example 1, and the corresponding procedures and conditions were otherwise the same as those in example 1. Respectively recording and calculating peak area values and ratios of the quantitative liquid, serum to be detected of a subject, taurine conjugated lithocholic acid and an internal standard substance deuterated taurine conjugated lithocholic acid-d 4 in a reference sample, peak area values and ratios of 3-glucuronic acid conjugated deoxycholic acid and an internal standard substance deuterated n-ursodeoxycholic acid-d 5, peak area values and ratios of n-cholic acid and an internal standard substance deuterated Beta-murine cholic acid-d 5 (table 2: peak areas of the detected bile acid and an external standard substance in the quantitative liquid, the serum to be detected of the subject and the reference sample, and table 3: peak areas of the corresponding internal standard substance in the quantitative liquid, the serum to be detected of the subject and the reference sample). And respectively drawing quantitative working curves and establishing a quantitative working equation by taking the concentration of the external standard substance in the corresponding quantitative liquid as X and the corresponding ratio as Y.
Quantitative working curve equation of taurine conjugated lithocholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of 3-glucuronic acid conjugated deoxycholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of n-cholic acid: Y-0.01243X-2.63752 e-5
Substituting the peak area ratios calculated by the sample record of the serum to be detected of the subject into an equation, so as to calculate mass concentration values and orthocholic acid (unit ng/mL) of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in the serum to be detected of the subject, multiplying the mass concentration values and the orthocholic acid by a volume conversion coefficient 2884.6154 introduced in a sample pretreatment link, dividing the mass values by the molar masses of external three bile acid markers, namely 483.70424g/mol, 612.65978g/mol and 394.54482g/mol, and finally obtaining serum concentration measurement results of the three bile acid metabolites, wherein the unit is nmol/L (table 4). Serum concentrations of taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid were the same as in example 1. Serum concentrations of n-cholic acid in the gastric cancer group and healthy control group were 31.54. + -. 17.37nmol/L and 17.94. + -. 10.32 nmol/L, respectively (FIG. 2C). The SPSS statistical software was used to perform a t-test on the serum concentrations of the three bile acid metabolites between the gastric cancer group and the healthy control group, and the results showed significant differences between the two groups (p <0.0001), and the serum concentration of the gastric cancer group was higher than that of the healthy control group.
6. Single metabolite based diagnostic models and diagnostic performance evaluation: ROC curves (fig. 3C) were plotted for orthocholic acid using GraphPad Prism software based on the serum concentration values and classification information (divided by gastric cancer group versus healthy control group) of all subjects, where the X-axis is 100% -specificity and the Y-axis is sensitivity of the diagnostic model. The diagnostic sensitivity and specificity of the screening for gastric cancer assisted by the use of orthocholic acid alone were 64.6% and 82.8%, respectively, which are far from the diagnostic performance of the marker combination of taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid in example 1.
7. Marker combination based diagnostic model and positive predictive value calculation: combining the three bile acid metabolites into a diagnostic model by using GraphPad Prism software to perform binary logistic regression analysis by using the serum concentration values of the three bile acid metabolites of all the subjects as independent variables and the classification information of the subjects as response values, wherein the classification information of the gastric cancer component is set as 1 and the classification information of the healthy control component is set as 0, the intercept of the result model is equal to 18.84, the coefficients of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid and n-cholic acid are-1.151, -0.003054 and-0.05935 respectively, and the mathematical equation of the binary logistic regression model is as follows: ln ((1- [ GC-PPV ])/[ GC-PPV ]) 18.84-1.151 × CTLCA-0.003054 × CDCA-3G-0.05935 × CNorCA, ln represents a logarithm based on a natural constant. And (3) converting the concentration value to obtain a formula 2, substituting the bile acid serum concentration value of each sample of the testee into the formula 2, and calculating the gastric cancer positive predictive value (GC-PPV) of the testee. ROC curves for screening diagnostic models for gastric cancer were plotted using GraphPad Prism software (fig. 4B) based on GC-PPV values and classification information for all subjects, where the X-axis is 100% -specificity and the Y-axis is sensitivity of the diagnostic model.
Equation 2:
in the formula, CTLCA and CDCA-3G, CNorCA are serum concentration values of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid and orthocholic acid respectively, and e is a natural constant.
The ROC curve shows that the sensitivity of the three marker combination diagnostic model is 98.2%, the specificity is 99.3%, the area under the curve AUC is 0.992, the AUC 95% confidence interval is 0.982-1.000, in other words, the AUC has a probability of 95% to appear in the interval. The optimal cutoff value for the gastric cancer screening diagnostic model was determined to be 0.768 according to the principle that the sum of sensitivity and specificity is the largest, i.e., at the corner of the upper left corner of the ROC curve. In this study example, 111 of the 113 gastric cancer groups had a GC-PPV higher than the cutoff value, and were judged to be positive for gastric cancer with an accuracy of 98.23% and a false negative rate of 1.77%. Among the 145 healthy control groups, 144 healthy control groups were judged to be negative for gastric cancer with a GC-PPV lower than the cut-off value, an accuracy of 99.31% and a false positive rate of 0.69% (Table 5). From the above data, the diagnostic performance of the three marker combination in example 2 was not different from the diagnostic performance of the two marker combination in example 1.
Example 3 gastric cancer-assisted screening based on four bile acid marker combinations
1. And (3) designing a clinical test: same as example 1 (i.e. using the same subject as example 1).
2. Serum collection of subjects: the corresponding procedures and conditions were the same as those in example 1.
3. Pretreatment of serum to be detected of a subject: based on example 1, the extract solution added with deuterated Beta-murcholic acid-d 5 and deuterated cholic acid-d 4 at a concentration of 50ng/mL, and the corresponding procedures and conditions are the same as those in example 1.
4. Liquid chromatography tandem mass spectrometry detection: the corresponding procedures and conditions were the same as those in example 1.
4.1 liquid chromatography method: the corresponding procedures and conditions were the same as those in example 1.
4.2 Mass Spectrometry method: the detection of the normal cholic acid and the hyocholic acid is increased on the basis of the embodiment 1, and the rest is the same as the embodiment 1. Mass spectrum scanning conditions of the n-cholic acid and the internal standard substance deuterated Beta-mouse-cholic acid-d 5 are the same as those of example 2. The scanning ion pair of the hyocholic acid is a parent ion 407.3 → a child ion 407.3, the declustering voltage and the collision voltage are-160V and-20V, the scanning ion pair corresponding to the internal standard substance deuterated cholic acid-d 4 is a parent ion 411.3 → a child ion 411.3, and the declustering voltage and the collision voltage are-160V and-20V respectively.
5. Quantifying the working curve and quantifying the bile acid: on the basis of example 1, orthocholic acid and hyocholic acid were added to the quantitative external liquid standard, and taurine-conjugated lithocholic acid as in example 1 was set at 12 concentration points. The internal standard substance is newly added with deuterated Beta-mouse cholic acid-d 5 and deuterated cholic acid-d 4, the concentration of the internal standard substance is the same as that of the deuterated taurine conjugated lithocholic acid-d 4 in the example 1, and the rest is the same as that in the example 1. The quantitative liquid, the serum to be detected of a subject, the peak area value and the ratio of the peak area value of taurine conjugated lithocholic acid and an internal standard substance deuterated taurine conjugated lithocholic acid-d 4 in a reference sample, the peak area value and the ratio of the peak area value of 3-glucuronic acid conjugated deoxycholic acid and an internal standard substance deuterated ursodeoxycholic acid-d 5, the peak area value and the ratio of the peak area value of hyocholic acid and an internal standard substance deuterated cholic acid-d 4 in a reference sample are recorded and calculated respectively (Table 2: the peak areas of the quantitative liquid, the serum to be detected of the subject and the external standard substance in the reference sample, and Table 3: the peak areas of the corresponding internal standard substance in the quantitative liquid, the serum to be detected of the subject and the reference sample). And respectively drawing quantitative working curves and establishing a quantitative working equation by taking the concentration of the external standard substance in the corresponding quantitative liquid as X and the corresponding ratio as Y.
Quantitative working curve equation of taurine conjugated lithocholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of 3-glucuronic acid conjugated deoxycholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of n-cholic acid: the corresponding procedures and conditions were the same as those in example 2.
Quantitative working curve equation of hyocholic acid: Y-0.01310X +8.86370e-4
Substituting the peak area ratios calculated by the sample record of the serum to be detected of the subject into an equation, so as to calculate mass concentration values (unit ng/mL) of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, orthocholic acid and hyocholic acid in the serum to be detected of the subject, multiplying the mass concentration values by a volume conversion coefficient 2884.6154 introduced in a sample pretreatment link (the calculation formula is the same as that in example 1), dividing the mass values by the molar masses 483.70424g/mol, 612.65978g/mol, 394.54482g/mol and 408.57140g/mol of three bile acid external standard substances, and finally obtaining the serum concentration measurement results of the four bile acid metabolites with the unit of nmol/L (Table 4). The serum concentrations of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid were the same as in example 1, the serum concentration of n-cholic acid was the same as in example 2, and the serum concentrations of hyocholic acid in the gastric cancer group and the healthy control group were 38.47 ± 43.88nmol/L and 21.92 ± 21.59nmol/L, respectively (fig. 2D). The SPSS statistical software was used to perform a t-test on the serum concentrations of the four bile acid metabolites between the gastric cancer group and the healthy control group, and the results showed significant differences between the two groups (p <0.0001), and the serum concentration of the gastric cancer group was higher than that of the healthy control group.
6. Single metabolite based diagnostic models and diagnostic performance evaluation: ROC curves of hyocholic acid were plotted using GraphPad Prism software (fig. 3D) according to the serum concentration values and classification information of all subjects hyocholic acid (divided by gastric cancer group and healthy control group), in which the X-axis is 100% -specificity of the diagnostic model and the Y-axis is sensitivity of the diagnostic model. The diagnostic sensitivity and specificity of screening for gastric cancer assisted by using hyocholic acid alone were 69.0% and 58.6%, respectively, which are far from the diagnostic performance of the marker combination of taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid in example 1.
7. Marker combination based diagnostic model and positive predictive value calculation: taking the serum concentration values of the four bile acid metabolites of all the subjects as independent variables, and taking classification information of the subjects as response values, wherein the classification information of the gastric cancer component is set as 1, the classification information of the healthy control component is set as 0, and the four bile acid metabolites are combined into a diagnosis model by using GraphPad Prism software to carry out binary logistic regression analysis, the intercept of the result model is equal to 18.88, the coefficients of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, n-cholic acid and n-cholic acid hyocholic acid are respectively-1.124, -0.002846, -0.05061, -0.03613, and the mathematical equation of the binary logistic regression model is as follows: ln ((1- [ GC-PPV ])/[ GC-PPV ]) 18.88-1.124 × CTLCA-0.002846 × CDCA-3G-0.05061 × CNorCA-0.03613 × CHCA, ln represents a logarithm with a natural constant as the base. And (3) converting the concentration values to obtain a formula 3, substituting the bile acid serum concentration values of all the test sample into the formula 3, and calculating the gastric cancer positive predictive value (GC-PPV) of the test sample. ROC curves for screening diagnostic models for gastric cancer were plotted using GraphPad Prism software (fig. 4C) based on GC-PPV values and classification information for all subjects, where the X-axis is 100% -specificity and the Y-axis is sensitivity of the diagnostic model.
Equation 3:
in the formula, CTLCA, CDCA-3G, CNorcA and CHCA are serum concentration values of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, orthocholic acid and hyocholic acid respectively, and e is a natural constant.
The ROC curve shows that the sensitivity of the four marker combination diagnostic model is 98.2%, the specificity is 99.3%, the area under the curve AUC is 0.992, the AUC 95% confidence interval is 0.984-1.000, in other words, the AUC has a 95% probability of appearing in the interval. The optimal cut-off value for the gastric cancer screening diagnostic model was determined to be 0.643 on the principle that the sum of sensitivity and specificity is the largest, i.e., at the corner of the upper left corner of the ROC curve. In this study example, 111 of the 113 gastric cancer groups had a GC-PPV higher than the cutoff value, and were judged to be positive for gastric cancer with an accuracy of 98.23% and a false negative rate of 1.77%. Among the 145 healthy control groups, 144 healthy control groups were judged to be negative for gastric cancer with a GC-PPV lower than the cut-off value, an accuracy of 99.31% and a false positive rate of 0.69% (Table 5). From the above data, the diagnostic performance of the four marker combination in example 3 was not different from the diagnostic performance of the two marker combination in example 1.
Example 4 gastric cancer-assisted screening based on five bile acid marker combinations
1. And (3) designing a clinical test: same as example 1 (i.e. using the same subject as example 1). .
2. Serum collection of subjects: the corresponding procedures and conditions were the same as those in example 1.
3. Pretreatment of serum to be detected of a subject: based on the example 1, the extract is added with deuterated Beta-murrocic acid-d 5, deuterated cholic acid-d 4 and deuterated 3-sulfated taurine conjugated lithocholic acid-d 4 monosodium salt, the concentration is 50ng/mL, and the rest is the same as the example 1.
4. Liquid chromatography tandem mass spectrometry detection: the corresponding procedures and conditions were the same as those in example 1.
4.1 liquid chromatography method: the corresponding procedures and conditions were the same as those in example 1.
4.2 Mass Spectrometry method: the detection of the n-cholic acid, the hyocholic acid and the 3-sulfated taurine conjugated lithocholic acid is added on the basis of the example 1, and the rest is the same as the example 1. Mass spectrum scanning parameters of the n-cholic acid and the internal standard substance deuterated Beta-mouse cholic acid-d 5 are the same as in example 2, and the mass spectrum scanning conditions of the hyocholic acid and the internal standard substance deuterated cholic acid-d 4 are the same as in example 3. The scanning ion pair of the 3-sulfated taurine conjugated lithocholic acid is a parent ion 562.3 → a child ion 482.3, the declustering voltage and the collision voltage are-120V and-45V, the scanning ion pair corresponding to the internal standard substance deuterated 3-sulfated taurine conjugated lithocholic acid-d 4 is a parent ion 566.3 → a child ion 486.3, and the declustering voltage and the collision voltage are-120V and-40V respectively.
5. Quantifying the working curve and quantifying the bile acid: on the basis of example 1, orthocholic acid, hyocholic acid and 3-sulfated taurine-conjugated lithocholic acid were added to the liquid external standard, and the same taurine-conjugated lithocholic acid as in example 1 was set at 12 concentration points. The internal standard substance is newly added with deuterated Beta-murcholic acid-d 5, deuterated cholic acid-d 4 and deuterated 3-sulfated-taurine conjugated lithocholic acid-d 4 monosodium salt, the concentration of the internal standard substance is the same as that of the deuterated taurine conjugated lithocholic acid-d 4 in example 1, and the rest is the same as that of example 1. The quantitative liquid, the serum to be detected of a subject, the peak area value and the ratio of the peak area value of taurine conjugated lithocholic acid and internal standard substance deuterated taurine conjugated lithocholic acid-d 4 in a reference sample, the peak area value and the ratio of the peak area value of 3-glucuronic acid conjugated deoxycholic acid and internal standard substance deuterated n-ursodeoxycholic acid-d 5, the peak area value and the ratio of the peak area value of orthocholic acid and internal standard substance deuterated Beta-murine cholic acid-d 5, the peak area value and the ratio of the peak area value of hyocholic acid and internal standard substance deuterated cholic acid-d 4, the peak area value and the ratio of the peak area value of 3-sulfated taurine conjugated lithocholic acid and internal standard substance 3-sulfated taurine conjugated lithocholic acid-d 4 in a reference sample are respectively recorded and calculated (Table 2: the peak areas of the quantitative liquid, the serum to be detected of the subject, and the external standard substance, table 3: the peak areas of corresponding internal standard substances in the quantitative liquid, the serum to be detected of the subject and the reference sample). And respectively drawing quantitative working curves and establishing a quantitative working equation by taking the concentration of the external standard substance in the corresponding quantitative liquid as X and the corresponding ratio as Y.
Quantitative working curve equation of taurine conjugated lithocholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of 3-glucuronic acid conjugated deoxycholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of n-cholic acid: the corresponding procedures and conditions were the same as those in example 2.
Quantitative working curve equation of hyocholic acid: the corresponding procedures and conditions were the same as those in example 3.
3-sulfated taurine-conjugated lithocholic acid: y is 0.01160X +0.00119
Substituting the peak area ratios calculated by the sample records of the serum to be detected of the subject into an equation, so as to calculate mass concentration values (unit ng/mL) of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, orthocholic acid, hyocholic acid and 3-sulfated taurine conjugated lithocholic acid in the serum to be detected of the subject, multiplying the mass concentration values by a volume conversion coefficient 2884.6154 introduced in a sample pretreatment link, and dividing the mass values by the molar masses 483.70424g/mol, 612.65978g/mol, 394.54482g/mol, 408.57140g/mol and 607.73110g/mol of five bile acid external standard substances respectively, so as to finally obtain the serum concentration measurement results of the five bile acid metabolites, wherein the unit is nmol/L (Table 4). Serum concentrations of taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid were the same as in example 1, serum concentrations of orthocholic acid were the same as in example 2, and serum concentrations of hyocholic acid were 90.17 ± 103.10nmol/L and 56.59 ± 65.17nmol/L in gastric cancer group and healthy control group, respectively, as in example 3, 3-sulfated taurine-conjugated lithocholic acid (fig. 2E). The serum concentrations of the five bile acid metabolites in the gastric cancer group and the healthy control group are subjected to t test by using SPSS statistical software, and the results show that the serum concentrations of the gastric cancer group and the healthy control group are significantly different (p < 0.0001).
6. Single metabolite based diagnostic models and diagnostic performance evaluation: ROC curves (fig. 3E) were drawn for 3-sulfated taurine-conjugated lithocholic acid using GraphPad Prism software based on the serum concentration values and classification information (divided by gastric cancer group and healthy control group) of 3-sulfated taurine-conjugated lithocholic acid of all subjects, where the X-axis is 100% -specificity% and the Y-axis is sensitivity of the diagnostic model. The diagnostic sensitivity and specificity of screening for gastric cancer assisted by the use of 3-sulfated taurine-conjugated lithocholic acid alone were 66.4% and 55.2%, respectively, and the diagnostic performance of the marker combination of taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid in example 1 was far from the same.
7. Marker combination based diagnostic model and positive predictive value calculation: using the serum concentration values of the five bile acid metabolites of all the subjects as independent variables, using classification information of the subjects as response values, wherein the classification information of the gastric cancer component is set to 1, the classification information of the healthy control component is set to 0, and using GraphPad Prism software to perform binary logistic regression analysis to combine the five bile acid metabolites into a diagnostic model, the intercept of the resulting model is equal to 17.48, and the coefficients of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, orthocholic acid, hyocholic acid, and 3-sulfated taurine conjugated lithocholic acid are-1.221, -0.002058, -0.05879, -0.03128, and 0.01251, respectively, and the mathematical equation of the binary logistic regression model is: ln ((1- [ GC-PPV ])/[ GC-PPV ]) 17.48-1.221 × CTLCA-0.002058 × CDCA-3G-0.05879 × CNorCA-0.03128 × CHCA +0.01251 × CTLCA-3S, ln represents a logarithm with a natural constant as the base. And (3) converting the concentration values to obtain a formula 4, and substituting the bile acid serum concentration values of each sample of the subjects in the gastric cancer group and the healthy control group into the formula 4 respectively to calculate the gastric cancer positive predictive value (GC-PPV) of the subjects. ROC curves for screening diagnostic models for gastric cancer were plotted using GraphPad Prism software (fig. 4D) based on GC-PPV values and classification information for all subjects, where the X-axis is 100% -specificity% and the Y-axis is sensitivity of the diagnostic model.
Equation 4:
in the formula, CTLCA, CDCA-3G, CNorcA, CHCA and CTLCA-3S are serum concentration values of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, orthocholic acid, hyocholic acid and 3-sulfated taurine conjugated lithocholic acid respectively, and e is a natural constant.
The ROC curve shows that the sensitivity of the five marker combination diagnostic model is 98.2%, the specificity is 99.3%, the area under the curve AUC is 0.994, the AUC 95% confidence interval is 0.985-1.000, in other words, the AUC has a probability of 95% to appear in the interval. According to the principle that the sum of sensitivity and specificity is the largest, namely the corner point at the upper left corner of the ROC curve, the optimal cutoff value for the gastric cancer screening and diagnosing model is determined to be 0.597. In this study example, 111 of the 113 gastric cancer groups had a GC-PPV higher than the cutoff value, and were judged to be positive for gastric cancer with an accuracy of 98.23% and a false negative rate of 1.77%. Among the 145 healthy control groups, 144 healthy control groups were judged to be negative for gastric cancer with a GC-PPV lower than the cut-off value, an accuracy of 99.31% and a false positive rate of 0.69% (Table 5). From the above data, the diagnostic performance of the five marker combination in example 4 was not different from the diagnostic performance of the two marker combinations in example 1.
Example 5 gastric cancer-assisted screening based on six bile acid marker combinations
1. And (3) designing a clinical test: same as example 1 (i.e. using the same subject as example 1).
2. Serum collection of subjects: the corresponding procedures and conditions were the same as those in example 1.
3. Pretreatment of serum to be detected of a subject: based on the example 1, the extract was supplemented with deuterated Beta-mucic acid-d 5, deuterated cholic acid-d 4, deuterated 3-sulfated taurine-conjugated lithocholic acid-d 4 monosodium salt, deuterated ursodeoxycholic acid-d 4 and deuterated lithocholic acid-d 4, the concentrations of which were all 50ng/mL, and the corresponding procedures and conditions were otherwise the same as those in example 1.
4. Liquid chromatography tandem mass spectrometry detection: the corresponding procedures and conditions were the same as those in example 1.
4.1 liquid chromatography method: the corresponding procedures and conditions were the same as those in example 1.
4.2 Mass Spectrometry method: the detection of n-cholic acid, hyocholic acid, 3-sulfated taurine-conjugated lithocholic acid, hyodeoxycholic acid, and lithocholic acid was performed in addition to example 1, and the rest was the same as example 1. Mass spectrum scanning conditions of n-cholic acid and internal standard deuterated Beta-mouse-cholic acid-d 5 are the same as in example 2. The scanning parameters of the mass spectrum of the hyocholic acid and the internal standard deuterated cholic acid-d 4 are the same as those of example 3. The scanning parameters of the mass spectrum of the 3-sulfated-taurine conjugated lithocholic acid and the internal standard 3-sulfated-taurine conjugated lithocholic acid-d 4 are the same as those of example 4. The porcine deoxycholic acid scanning ion pair is a parent ion 391.3 → a daughter ion 391.3, the declustering voltage and the collision voltage are-160V and-20V, the scanning ion pair corresponding to the internal standard substance deuterated ursodeoxycholic acid-d 4 is a parent ion 395.3 → a daughter ion 395.3, and the declustering voltage and the collision voltage are-180V and-20V respectively. The lithocholic acid scanning ion pair is a parent ion 375.3 → a child ion 375.3, the declustering voltage and the collision voltage are-160V and-25V, the scanning ion pair corresponding to the internal standard substance deuterated lithocholic acid-d 4 is a parent ion 379.3 → a child ion 379.3, and the declustering voltage and the collision voltage are-160V and-20V respectively.
5. Quantifying the working curve and quantifying the bile acid: on the basis of example 1, the external standard substance of the liquid is quantitatively added with ortho-cholic acid, hyocholic acid, 3-sulfated taurine conjugated lithocholic acid, hyodeoxycholic acid and lithocholic acid, and the setting of 12 concentration points is the same as that of the taurine conjugated lithocholic acid in example 1. The concentrations of the internal standard substance newly added deuterated Beta-murcholic acid-d 5, deuterated cholic acid-d 4, deuterated 3-sulfated-taurine-conjugated lithocholic acid-d 4 monosodium salt, deuterated ursodeoxycholic acid-d 4 monosodium salt and deuterated lithocholic acid-d 4 are the same as that of deuterated taurine-conjugated lithocholic acid-d 4 in example 1, and the rest is the same as that in example 1. Quantitative liquid, serum to be detected by a subject, respective peak area values and ratios of two in taurine conjugated lithocholic acid and internal standard substance deuterated taurine conjugated lithocholic acid-d 4 in a reference sample, respective peak area values and ratios of two in 3-glucuronic acid conjugated deoxycholic acid and internal standard substance deuterated n-ursodeoxycholic acid-d 5, respective peak area values and ratios of two in orthocholic acid and internal standard substance deuterated Beta-murine cholic acid-d 5, respective peak area values and ratios of two in hyocholic acid and internal standard substance deuterated cholic acid-d 4, respective peak area values and ratios of two in 3-sulfated taurine conjugated lithocholic acid and internal standard substance 3-sulfated taurine conjugated lithocholic acid-d 4, respective peak area values and ratios of two in hyodeoxycholic acid and internal standard substance deuterated ursodeoxycholic acid-d 4, respective peak area values and ratios of two in lithocholic acid and internal standard substance deuterated lithocholic acid-d 4 And (4) performing record calculation (table 2: peak areas of bile acid and an external standard substance detected in the quantitative liquid, the serum to be detected of the subject and the reference sample, and table 3: peak areas of corresponding internal standard substances in the quantitative liquid, the serum to be detected of the subject and the reference sample). And respectively drawing quantitative working curves and establishing a quantitative working equation by taking the concentration of the external standard substance in the corresponding quantitative liquid as X and the corresponding ratio as Y.
Quantitative working curve equation of taurine conjugated lithocholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of 3-glucuronic acid conjugated deoxycholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of n-cholic acid: the corresponding procedures and conditions were the same as those in example 2.
Quantitative working curve equation of hyocholic acid: the corresponding procedures and conditions were the same as those in example 3.
The quantitative working curve equation of the 3-sulfated taurine conjugated lithocholic acid: the corresponding procedures and conditions are the same as those in example 4.
Quantitative working curve equation of hyodeoxycholic acid: 0.01955X +0.01491
Quantitative working curve equation of lithocholic acid: 0.01851X +0.01377
Respectively substituting the peak area ratios calculated by the sample records of the serum to be detected of the testee into an equation, calculating the mass concentration values (unit ng/mL) of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, orthocholic acid, hyocholic acid, 3-sulfated taurine conjugated lithocholic acid, hyodeoxycholic acid and lithocholic acid in the serum to be detected of the testee, respectively multiplying the mass concentration values by a volume conversion coefficient 2884.6154 introduced in the sample pretreatment link, and respectively dividing the mass concentration values by the molar mass 483.70424g/mol of seven external bile acid markers, 612.65978g/mol, 394.54482g/mol, 408.57140g/mol, 607.73110g/mol, 392.572g/mol and 376.5726g/mol, obtaining the serum concentration measurement results of seven bile acids, wherein the unit is nmol/L (table 4), and finally calculating the concentration ratio of the hyodeoxycholic acid to the lithocholic acid. The serum concentrations of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid were the same as in example 1, the serum concentration of n-cholic acid was the same as in example 2, and the serum concentration of hyocholic acid was the same as in example 3, and the serum concentration of 3-sulfated taurine conjugated lithocholic acid was the same as in example 4. The concentration ratio of hyodeoxycholic acid/lithocholic acid was 1.039 + -1.927 and 0.551 + -0.782 in the gastric cancer group and the healthy control group, respectively (FIG. 2F). The SPSS statistical software was used to perform a t-test on the two bile acid concentrations compared to the serum concentration between the gastric cancer group and the healthy control group, and the results showed significant differences between the two groups (p <0.0001), with the hyodeoxycholic acid/lithocholic acid concentration ratio being higher in the gastric cancer group than in the healthy control group.
6. Single metabolite based diagnostic models and diagnostic performance evaluation: ROC curves (fig. 3F) were plotted using GraphPad Prism software for the hyodeoxycholic acid/lithocholic acid concentration ratio according to the hyodeoxycholic acid/lithocholic acid concentration ratio and classification information (divided by gastric cancer group versus healthy control group) for all subjects, where the X-axis is 100% -specificity% and the Y-axis is sensitivity of the diagnostic model. The diagnostic sensitivity and specificity of screening for gastric cancer assisted by the concentration ratio of hyodeoxycholic acid/lithocholic acid alone were 53.1% and 69.7%, respectively, which are far from the diagnostic performance of the marker combination of taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid in example 1.
7. Diagnostic model and positive predictive value calculation based on marker combinations: using the five bile acid metabolite serum concentration values and the hyodeoxycholic acid/lithocholic acid concentration ratio of all subjects, totaling 6 indexes as independent variables, and using subject classification information as response values, wherein the gastric cancer component classification information is set to be 1, and the health control component classification information is set to be 0, using GraphPad Prism software to perform binary logistic regression analysis to combine the six indexes into a diagnosis model, and the intercept of the result model is equal to 17.06, and the coefficients of the concentration ratios of taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, orthocholic acid, hyocholic acid, 3-sulfated taurine conjugated lithocholic acid, and hyodeoxycholic acid/lithocholic acid are-1.233, -0.001818, -0.05152, -0.02882, 0.01354, -0.3342 respectively, and the mathematical equations of the binary logistic regression model are: ln ((1- [ GC-PPV)])/[GC-PPV])＝17.06-1.233×C TLCA -0.001818×C DCA-3G -0.05152×C NorCA -0.02882×C HCA +0.01354×C TLCA-3S -0.3342×R HDCA/LCA And ln represents a logarithm based on a natural constant. And converting the concentration values to obtain a formula 5, and substituting the bile acid serum concentration value and the hyodeoxycholic acid/lithocholic acid concentration ratio of each sample of the testee of the gastric cancer group and the healthy control group into the formula 5 respectively to calculate the gastric cancer positive predictive value (GC-PPV) of the testee. ROC curves for screening diagnostic models for gastric cancer were plotted using GraphPad Prism software (fig. 4E) based on GC-PPV values and classification information for all subjects, where the X-axis is 100% -specificity% and the Y-axis is sensitivity of the diagnostic model.
Equation 5:
in the formula C TLCA 、C DCA-3G 、C NorCA 、C HCA 、C TLCA-3S Serum concentration values, R, of taurine-conjugated lithocholic acid, 3-glucuronic acid-conjugated deoxycholic acid, orthocholic acid, hyocholic acid, and 3-sulfated taurine-conjugated lithocholic acid HDCA/LCA Is the ratio of the serum concentration between hyodeoxycholic acid and lithocholic acid, and e is a natural constant.
The ROC curve shows that the sensitivity of the 6-index combined diagnostic model is 98.2%, the specificity is 98.6%, the area under the curve, AUC, is 0.993, and the confidence interval of AUC 95% is 0.985-1.000, namely that the AUC has the probability of 95% in the interval. The optimal cutoff value for the gastric cancer screening diagnostic model was determined to be 0.539 on the principle that the sum of sensitivity and specificity is the greatest, i.e., at the corner of the upper left corner of the ROC curve. In this study example, 111 of the 113 gastric cancer groups had a GC-PPV higher than the cutoff value, and were judged to be positive for gastric cancer with an accuracy of 98.23% and a false negative rate of 1.77%. Among the 145 healthy control groups, 143 healthy control groups were judged to be negative for gastric cancer with a GC-PPV lower than the cut-off value, with an accuracy of 98.62% and a false positive rate of 1.38% (Table 5). From the above data, the six marker combinations in example 5 were less specific than the two marker combinations in example 1.
Based on the research results of examples 1, 2, 3, 4, and 5, the diagnosis effect of two bile acid marker combinations (taurine-conjugated lithocholic acid + 3-glucuronic acid-conjugated deoxycholic acid) for assisting gastric cancer screening is combined with three bile acid combinations (taurine-conjugated lithocholic acid + 3-glucuronic acid-conjugated deoxycholic acid + orthocholic acid), four bile acid combinations (taurine-conjugated lithocholic acid + 3-glucuronic acid-conjugated deoxycholic acid + orthocholic acid + hyocholic acid), five bile acid combinations (taurine-conjugated lithocholic acid + 3-glucuronic acid-conjugated deoxycholic acid + orthocholic acid + hyocholic acid + 3-sulfated taurine-conjugated lithocholic acid), and six bile acid index combinations (taurine-conjugated lithocholic acid + 3-glucuronic acid-deoxycholic acid + orthocholic acid + 3-sulfated taurine-conjugated lithocholic acid + hyodeoxycholic acid- Lithocholic acid ratio) (sensitivity 98.2% and specificity 99.3% respectively). Therefore, from the viewpoints of convenience in implementation of kit engineering, readability of a diagnostic model, cost of test, and the like, the marker combination in which taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid are used in combination is preferably used in the present invention, and the present invention has balanced sensitivity and specificity for assisting gastric cancer screening and excellent diagnostic performance (tables 1 and 5).
Example 6 assisted screening for early gastric cancer
1. And (3) designing a clinical test: the study was approved by the medical symposium and subjects signed an informed consent prior to inclusion in the clinical trial. The cohort training set for the early gastric cancer screening study included 145 healthy control subjects and 45 early gastric cancer subjects. According to the cancer staging system established by the eighth edition of the united states joint committee for cancer (AJCC), 45 patients were all stage I gastric cancer.
2. Serum collection of subjects: the corresponding procedures and conditions were the same as those in example 1.
3. Pretreatment of serum to be detected of a subject: the corresponding procedures and conditions were the same as those in example 1.
4. Liquid chromatography tandem mass spectrometry detection: the corresponding procedures and conditions of example 1 were followed.
4.1 liquid chromatography method: the corresponding procedures and conditions were the same as those in example 1.
4.2 Mass Spectrometry method: the corresponding procedures and conditions were the same as those in example 1.
5. Quantifying the working curve and quantifying the bile acid: the same as in example 1. Respectively recording and calculating peak area values and ratios of the quantitative liquid, serum to be detected of a subject, and taurine conjugated lithocholic acid and an internal standard substance deuterated taurine conjugated lithocholic acid-d 4 in a reference sample, and peak area values and ratios of the 3-glucuronic acid conjugated deoxycholic acid and the internal standard substance deuterated ursodeoxycholic acid-d 5 (table 2: peak areas of the quantitative liquid, the serum to be detected of the subject, and the external standard substance in the reference sample, and table 3: peak areas of corresponding internal standard substances in the quantitative liquid, the serum to be detected of the subject, and the reference sample). And respectively drawing quantitative working curves and establishing a quantitative working equation by taking the concentration of the external standard substance in the corresponding quantitative liquid as X and the corresponding ratio as Y. The rest of the procedure was the same as in example 1.
Quantitative working curve equation of taurine conjugated lithocholic acid: the corresponding procedures and conditions were the same as those in example 1.
Quantitative working curve equation of 3-glucuronic acid conjugated deoxycholic acid: the corresponding procedures and conditions were the same as those in example 1.
The measured values of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in the reference sample are respectively 112.4% and 110.1% of the calibrated value, and the quantitative detection quality of the serum bile acid in the example 1 is qualified. The serum concentrations of taurine-conjugated lithocholic acid in the early gastric cancer group and the healthy control group were (mean. + -. standard deviation) 12.85. + -. 1.93nmol/L and 5.30. + -. 2.65nmol/L, respectively, and the serum concentrations of 3-glucuronic acid-conjugated deoxycholic acid in the gastric cancer group and the healthy control group were 2162.0. + -. 106.7nmol/L and 1651.0. + -. 718.7nmol/L, respectively (Table 4). The serum concentrations of the two bile acid metabolites in the early gastric cancer group and the healthy control group are subjected to t test by using SPSS statistical software, and the results show that the two groups have significant difference (p <0.0001), and the serum concentration of the early gastric cancer group is higher than that of the healthy control group.
6. And (3) calculating a diagnosis model and a positive predictive value based on the marker combination: taking serum concentration values of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid of all subjects as independent variables, taking classification information of the subjects as response values, wherein classification information of an early gastric cancer group is set as 1, classification information of a healthy control group is set as 0, performing binary logistic regression analysis by using GraphPad Prism software to combine the two indexes into a diagnostic model, and the intercept of the result model is equal to 27.13, coefficients of the taurine conjugated lithocholic acid and the 3-glucuronic acid conjugated deoxycholic acid are respectively-1.323 and-0.006491, and the mathematical equation of the binary logistic regression model is as follows: ln ((1- [ EGC-PPV ])/[ EGC-PPV ]) 27.13-1.323 × CTLCA-0.006491 × CDCA-3G, ln represents a logarithm with a natural constant as a base. And converting to obtain a formula 6, substituting the bile acid serum concentration value of each sample of the subjects in the early gastric cancer group and the healthy control group into the formula 6, and calculating the positive predictive value (EGC-PPV) of the subjects in the early gastric cancer. ROC curves for the early gastric cancer screening diagnostic model were plotted using GraphPad Prism software (fig. 4F) based on EGC-PPV values and classification information (divided by early gastric cancer group and healthy control group) for all subjects, where the X-axis is 100% -specificity and the Y-axis is sensitivity of the diagnostic model.
Equation 6:
the sensitivity of the ROC curve is 97.8%, the specificity is 100.0%, the AUC of the area under the curve is 0.991, the confidence interval of the AUC 95% is 0.973-1.000, in other words, the probability that the AUC appears in the interval of 95%, and the results show that the two bile acid marker combinations also have good diagnosis effect on auxiliary early gastric cancer screening.
The optimal cutoff value for the early gastric cancer screening diagnostic model was determined to be 0.715 based on the principle that the sum of sensitivity and specificity is the greatest, i.e., at the corner of the upper left corner of the ROC curve. In this study example, 44 cases of the 45 early gastric cancer groups had EGC-PPV higher than the cutoff value, and were judged as positive for early gastric cancer with an accuracy of 97.78% and a false negative rate of 2.22%. EGC-PPV in 145 healthy control groups was below the cut-off value, and was judged as negative for gastric cancer with an accuracy of 100% and a false positive rate of 0% (table 5, fig. 5B). This shows that the two bile acid marker combinations involved in the invention also have good discrimination effect and high application value for the auxiliary screening of early gastric cancer.
Example 7 Blind sample test
1. Clinical verification experiment design: the study was approved by the medical symposium and subjects signed an informed consent prior to inclusion in the clinical trial. The blind validation set included 88 healthy control subjects and 80 gastric cancer subjects. According to the cancer staging system established by the eighth edition of the united states joint committee for cancer (AJCC), 25 patients were stage I, 29 were stage II, 16 were stage III, and 10 were stage IV. Blind sample treatment is carried out on a sample of a subject before testing, and mass spectrum inspectors, data analyzers and process recheckers do not know the actual classification of the blind sample in the testing process. And (4) after the test is finished, blind uncovering, comparing the difference between the diagnosis model judgment result and the actual clinical diagnosis, and calculating the screening accuracy.
2. Serum collection of subjects: the corresponding procedures and conditions were the same as those in example 1.
3. Pretreatment of serum to be detected of a subject: the corresponding procedures and conditions were the same as those in example 1.
4. Liquid chromatography tandem mass spectrometry detection: the corresponding procedures and conditions were the same as those in example 1.
4.1 liquid chromatography method: the corresponding procedures and conditions were the same as those in example 1.
4.2 Mass Spectrometry method: the corresponding procedures and conditions were the same as those in example 1.
5. Quantifying the working curve and quantifying the bile acid: the corresponding procedures and conditions were the same as those in example 1. And respectively recording and calculating the peak area values and the ratio of the peak area values to the d4 of the taurine conjugated lithocholic acid and the internal standard substance deuterated taurine conjugated lithocholic acid in the quantitative liquid, the serum to be detected in the blind sample and the reference sample, and the peak area values and the ratio of the peak area values to the d5 of the 3-glucuronic acid conjugated deoxycholic acid and the internal standard substance deuterated ursodeoxycholic acid (Table 6). And respectively drawing quantitative working curves and establishing a quantitative working equation by taking the concentration of the external standard substance in the corresponding quantitative liquid as X and the corresponding ratio as Y.
Quantitative working curve equation of taurine conjugated lithocholic acid: Y-0.01745X-0.01616.
Quantitative working curve equation of 3-glucuronic acid conjugated deoxycholic acid: Y0.00161X + 1.34173E-4.
Substituting the peak area ratios recorded and calculated by the blind sample serum sample to be detected and the reference sample into an equation, calculating mass concentration values of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in the blind sample serum to be detected and the reference sample, performing volume conversion and molar quantity conversion (the volume conversion formula and the molar mass are the same as in example 1) according to the operation described in example 1, and finally obtaining the serum concentrations of two bile acid metabolites with the unit of nmol/L (Table 6). In this example, the measured values of the reference sample taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid were 76.6% and 93.7% of the standard values, respectively, and the quantitative assay quality of the serum bile acid in example 7 was determined to be acceptable.
6. Calculating the positive predictive value of the gastric cancer and uncovering the blind: substituting the bile acid serum concentration value of each blind sample into formula 1 to calculate the positive predictive value (GC-PPV) of the gastric cancer of the subject. If the GC-PPV value is higher than the optimal cutoff value of 0.829, the blind sample is judged to be positive for gastric cancer, otherwise, the blind sample is judged to be negative. In example 7, 79 blind samples were screened positive for gastric cancer and 89 were negative for gastric cancer. After blindness is uncovered, 80 patients with actual clinical diagnosis of gastric cancer have the positive screening accuracy rate of 95.0 percent and the false positive rate of 5.0 percent; there were 88 healthy subjects who were not clinically diagnosed with gastric cancer, with a negative screening accuracy of 96.6% and a false negative rate of 3.4% (table 7). The bile acid marker combination and the serum detection kit show good auxiliary screening effect of gastric cancer in blind sample verification test.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
TABLE 5
TABLE 6
TABLE 7
Claims (8)
1. A bile acid marker composition comprises taurine conjugated lithocholic acid (Taurolithocholic acid), 3-glucuronic acid conjugated Deoxycholic acid (Deoxycholic acid 3-glucuronide).
2. The application of the bile acid marker composition in preparing a serum detection kit for auxiliary screening or diagnosis of gastric cancer and/or early gastric cancer, or the application of the bile acid marker composition in preparing a serum detection kit for auxiliary screening or diagnosis of gastric cancer and/or early gastric cancer.
3. A serum detection kit for assisted screening or diagnosis of gastric cancer/early gastric cancer, the kit comprising:
1) quantitative liquid: the method is used for establishing an internal standard method quantitative working curve, is a series of methanol-water solutions or methanol solutions which simultaneously contain external standards (when establishing an internal standard method quantitative working curve, different external standards respectively and independently select 3-15 different concentration points within the range of 0.1-1000 ng/mL) and internal standards (when establishing an internal standard method quantitative working curve, different internal standards respectively and independently select one concentration point within the range of 5-500 ng/mL), and adopts a solvent with the volume ratio of methanol of 5-100%, wherein the external standard is taurine conjugated lithocholic acid, 3-glucuronic acid conjugated deoxycholic acid, the external standard is respectively used for quantitatively and/or qualitatively identifying the concentration of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in serum to be detected of a subject; the internal standard substances are deuterated taurine conjugated lithocholic acid-d 4 and deuterated n-ursodeoxycholic acid-d 5, the concentrations of the internal standard substances are respectively 5-500 ng/mL, and the internal standard substances are respectively used for quantifying the concentrations of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in serum to be detected of a subject;
2) extracting solution: and (2) methanol and/or acetonitrile solutions containing internal standard substance deuterated taurine conjugated lithocholic acid-d 4 and deuterated n-ursodeoxycholic acid-d 5 (the volume ratio of methanol in the solvent adopted by the solution is 0-100%), wherein the concentrations of the internal standard substances are respectively 5-500 ng/mL, and the internal standard substances are used for pretreatment of a serum sample to be detected of a subject and bile acid extraction.
4. The serum detection kit according to claim 3, further comprising three microplates: the first microporous plate, the second microporous plate and the third microporous plate are respectively, wherein the first microporous plate is a 24-96-hole microporous plate used for pretreatment of a serum sample to be detected of a subject and extraction of bile acid, and the capacity of each hole is 500-2000 muL; the second microporous plate is a 24-96-hole microporous plate for drying and redissolving bile acid extract of a serum sample to be detected of a subject, wherein the capacity of each hole is 100-1000 mu L; the third microporous plate is a 24-96-hole microporous plate for machine detection after the redissolution of a serum sample to be detected, wherein the capacity of each hole is 100-1000 mu L.
5. The serum test kit according to claim 3 or 4, wherein the extract is used by a method comprising:
1) transferring 50-500 mu L of serum of a subject to be detected to 1 or more than 2 parts of frozen serum samples to be detected (if the frozen serum samples to be detected are firstly thawed at room temperature) of different micropores of a first microporous plate one by one, adding 200-2000 mu L of extracting solution into each pore, and after vortex oscillation at 1200-2000 rpm for 2-5 min (rpm represents a rotating speed unit: rpm), centrifuging at 4000-6000 rpm for 10-60 min to remove protein precipitates;
2) respectively transferring 150-1500 mu L of bile acid extract to 1 or more than 2 different micropores of a second plate one by one, blowing dry with nitrogen or drying under vacuum and reduced pressure for 2-5 h, adding 100-1000 mu L of 15-100% methanol-water mixed solution (V/V, the volume ratio of methanol in the solvent is 15-100%) into each pore to redissolve the dried sample, and centrifuging at 1200-2000 rpm for 2-5 min after vortex oscillation at 4000-6000 rpm for 10-60 min to remove protein precipitate again;
3) transferring 80-800 mu L of the complex solution to 1 or more than 2 different micropores of a third plate respectively one by one for detection on a machine by liquid chromatography tandem mass spectrometry.
6. The serum detection kit according to claim 3 or 5, further comprising a reference sample, wherein the reference sample is an artificial serum (such as an Ultra-Low Hormones & Steroids model artificial serum manufactured by Golden West Diagnostics), the freeze-dried substance is obtained by adding an external standard (the final concentration of taurine conjugated lithocholic acid in the reference sample is 1-1000 nmol/L, and the final concentration of 3-glucuronic acid conjugated deoxycholic acid is 5-5000 nmol/L) and then performing the treatment by the method processes described in the steps 1) and 2) of claim 5, the concentration values of the taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid are determined by using a liquid chromatography tandem mass spectrometry method, and the reference sample is calibrated in the kit specification for reference value comparison and test quality control of the concentration determination result of the bile acid marker of the serum sample to be detected;
before the reference sample is used, 100-1000 mu L of 15-100% methanol-water mixed liquor (V/V, the volume ratio of methanol in a solvent is 15-100%) is used for redissolving to the required volume, then the concentration of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid is measured by using a liquid chromatography tandem mass spectrometry method, and the quantitative detection quality of serum bile acid is qualified when the measured concentration value is within 75-125% of the specification value.
7. The serum detection kit according to claim 3 or 6, wherein the quantitative liquid, the treated serum sample to be detected of the subject and the reference sample are respectively detected by liquid chromatography tandem mass spectrometry, retention time of chromatographic peaks recorded by mass spectrometry of external standard taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in the quantitative liquid (any concentration point between 20 ng/mL and 200 ng/mL) and the serum to be detected of the subject is compared, and the retention time is within +/-0.05 minutes, so that the chromatographic peaks are judged to be chromatographic peaks of two markers detected in the serum to be detected of the subject, and qualitative identification of the two bile acid markers in the serum to be detected of the subject is realized; comparing retention time of chromatographic peaks recorded by mass spectrum scanning of the internal standard substance deuterated taurine conjugated lithocholic acid-d 4 and deuterated ursodeoxycholic acid-d 5 in the quantitative liquid at any concentration point (any concentration point between 20 ng/mL and 200 ng/mL) and the serum to be detected of the subject, and judging that the chromatographic peaks are chromatographic peaks of two markers respectively corresponding to the internal standard substance within +/-0.05 minute of retention time, thereby realizing qualitative identification of the internal standard substance in the serum to be detected of the subject;
respectively recording and calculating peak area values and ratios of the quantitative liquid, serum to be detected of a subject and reference sample taurine conjugated lithocholic acid and internal standard substance deuterated taurine conjugated lithocholic acid-d 4, and peak area values and ratios of 3-glucuronic acid conjugated deoxycholic acid and internal standard substance deuterated n-ursodeoxycholic acid-d 5, respectively drawing quantitative working curves of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid and establishing respective quantitative working equations with the concentration value of the external standard substance in the quantitative liquid as X and the corresponding ratio as Y;
substituting the peak area ratios recorded and calculated in a blood serum sample to be detected and a reference sample of a subject into an equation to obtain mass concentration, and calculating the blood serum concentration values of taurine conjugated lithocholic acid and 3-glucuronic acid conjugated deoxycholic acid in the blood serum to be detected of the subject in unit nmol/L through sample pretreatment volume conversion and molar weight conversion;
and determining that the serum concentration measurement result of the reference sample is within 75-125% of the specification calibration value, and determining that the quantitative inspection quality of the serum bile acid is qualified.
8. The serum test kit according to claim 3 or 7, wherein a binary logistic regression analysis is performed to combine the two bile acid metabolites into a diagnostic model by using the serum concentration values of the taurine-conjugated lithocholic acid and 3-glucuronic acid-conjugated deoxycholic acid as independent variables and training set subject classification information for diagnostic model construction as response values (gastric cancer group is set to 1, healthy control group is set to 0, and each of the two groups of subjects is not less than 100 in the case of gastric cancer screening, thereby forming a training set; early gastric cancer group is set to 1, healthy control group is set to 0, early gastric cancer group is not less than 40, healthy control group is not less than 100 in the case of gastric cancer screening, thereby forming a training set); recording the intercept value of a binary logic mathematical model and the coefficients of the two bile acids, converting the mathematical equation of the binary logic regression model from natural logarithm to natural number power form to obtain a stomach cancer or early stage stomach cancer positive predictive value calculation formula, respectively substituting the bile acid serum concentration value of each training set subject sample into the formula to calculate the stomach cancer or early stage stomach cancer Positive Predictive Value (PPV) of each subject, and drawing a receiver operating characteristic curve (ROC curve) according to the classification information of the training set subjects (classified according to a stomach cancer group and a health control group when screening the stomach cancer and classified according to an early stage stomach cancer group and a health control group when screening the stomach cancer) and the PPV value obtained by the calculation, determining the optimal cutoff value (Cut-off) of a gastric cancer or early gastric cancer diagnosis model according to the principle that the sum of diagnosis specificity and sensitivity in the ROC curve is maximum;
substituting the serum concentrations of the two bile acid markers measured by the tested human subjects to be tested into the formula, respectively calculating the PPV (gastric cancer) value or early gastric cancer value of each tested human subject to be tested, comparing the PPV value with the optimal cutoff value, and judging the test to be positive if the PPV value is higher than the cutoff value, wherein the tested human subject needs to rapidly adopt one or more than two clinical diagnosis measures such as gastroscopy, tissue biopsy, pathological section and the like; on the other hand, a PPV value lower than the cutoff value is a negative result.
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| CN115326953A (en) * | 2022-08-02 | 2022-11-11 | 云谱康(大连)生物科技有限公司 | Metabolite combination and application, detection kit and application |
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