WO2011140800A1

WO2011140800A1 - Kit for detecting 27-nor-5β-cholestane-3, 7, 12, 24, 25 pentol glucuronide in serum and using method thereof

Info

Publication number: WO2011140800A1
Application number: PCT/CN2010/079202
Authority: WO
Inventors: 许国旺; 陈静; 徐丛剑; 曹锐; 路鑫; 张晓燕; 王宜生; 黄以萍
Original assignee: 中国科学院大连化学物理研究所; 复旦大学附属妇产科医院; 大连市妇产医院
Priority date: 2010-05-14
Filing date: 2010-11-26
Publication date: 2011-11-17
Also published as: CN102243216A

Abstract

The present invention discloses a kit for early diagnosis of ovarian cancer by detecting 27-nor-5β-cholestane-3, 7, 12, 24, 25 pentol glucuronide in serum and using method thereof. Said kit is composed of leucine enkephalin as internal standard, aqueous solution (containing 0.1% formic acid, 2% acetonitrile) and acetonitrile. 27-nor-5β-cholestane-3, 7, 12, 24, 25 pentol glucuronide is detected by Liquid Chromatography-Mass Spectrometry (LC-MS) platform.

Description

Detection of serum 27-5β-norcholestane-3, 7, 12, 24, 25-pentaol glucose

Kit for aldehyde acid and method of using same

The invention relates to the field of analytical chemistry and medicine, and is based on the detection of 27-5β-cholestaxin-3, 7, 12, 24, 25-pentitol glucuronic acid conjugate (27-nor-5P-cholestane- in serum). 3, 7, 12, 24, 25 pentol glucuronide) High-throughput HPLC analysis of human serum by ultra-high performance liquid chromatography-mass spectrometry for rapid determination of 27-5β-cholestane-3,7 in serum 12, 24, 25-penta-glycol glucuronide conjugate (27-nor-5p-cholestane-3, 7, 12, 24, 25 pentol glucuronide) content for early diagnosis of ovarian cancer. Background technique

Ovarian cancer, endometrial cancer and cervical cancer are also called "gynecological three cancers". The incidence of primary ovarian cancer in China is on the rise. The ovary is located deep in the pelvic cavity and is concealed. It is difficult to find its presence in the early stage of malignant tumor formation. General physical examination and imaging examinations have limited effects. Once they can be found and clearly diagnosed, they often reach the middle and late stages and lack early warning value.

Ovarian cancer is mostly epithelial ovarian cancer, and the early symptoms are not obvious. Once the symptoms of ovarian cancer appear, the cancer tissue begins to increase or spread, indicating that most of the patients have entered the advanced stage. At present, the five-year survival rate of advanced ovarian cancer is low, and the five-year survival rate of early ovarian cancer is generally higher. Therefore, the development of new kits is important for the early diagnosis of ovarian cancer.

Tumor markers and imaging examination are the main methods for the diagnosis of ovarian tumors, and the detection of serum markers is particularly important. Cancer antigen 125 (CA125) is currently the most commonly used serum marker for serous ovarian cancer, but its specificity is not satisfactory. There is also a significant increase in malignancies including inflammation, tuberculosis, benign tumors, endometriosis and other organs. (Document 1: Bast RC, Xu FJ, Yu YH, et al. Int J Biol Markers 13: 179-187, 1998 Document 2: An HJ, Miyamoto S, Lancaster KS, et al. J Proteome Res 5: 1626-1635 , 2006). Serum levels of CA125 are less sensitive and specific in the diagnosis of ovarian cancer, and sensitivity to early diagnosis of ovarian cancer is often less than 25% (Reference 3: Rosen DG, Wang L, Atkinson JN, et al. Gynecol Oncol 99) :267-277, 2005 ). Therefore, in order to improve the sensitivity and specificity of clinical diagnosis, it is imperative to develop new early detection methods.

We used metabolomics research to separate and detect small molecules of metabolites in the serum of healthy women, benign ovarian tumors and ovarian cancer patients based on ultra-high performance liquid chromatography-mass spectrometry. The statistical analysis was used to analyze the metabolic differences between the three groups. , found that 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide in serum The content is related to ovarian cancer. 27-5β-norcholin-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5p-cholestane-3, 7, 12, 24 , 25 pentol glucuronide) is a bile glycol glucuronic acid conjugate found in human blood and urine. 27-5β-cholestane-3, 7, 12, 24, 25-pentaol (27-nor -5P-cholestane-3, 7, 12, 24, 25 The precursor of pentol) is cholesterol. According to the current high incidence of ovarian cancer in western developed countries, it is generally believed that a large intake of cholesterol in the diet is one of the causes of ovarian cancer. Therefore, the 27-5β-cholestyr-3,7,12,24,25-pentanol glucuronic acid conjugate in serum was determined (27-nor-5p-cholestane-3, 7, 12, 24, 25 pentol The change in the content of glucuronide) helps us understand the metabolic changes of ovarian cancer.

There are only a few reports on 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugates (27-nor-5P-cholestane-3, 7, 12, 24 , 25 pentol glucuronide), mainly by gas chromatography and GC/MS (Document 4: Karlaganis G, Alme B, Karlaganis V, et al. J Steroid Biochem 14:341-345, 1981 Document 5: Une M, Takenaka S, Kuramoto T, at al. J Lipid Res 41: 1562-1567, 2000). The pretreatment process of gas chromatography and mass spectrometry is complicated and needs to be derivatized, which brings unpredictable errors to the detection process. At present, there is no fast and efficient method for detecting 27-5β-cholestane-3, 7, 12, 24, 25-penta-glucuronate conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide), and no one has yet linked it to ovarian cancer. The present invention provides a method for rapid detection of 27-5β-cholestane-3, 7, 12, 24, 25-pentitol glucuronic acid conjugate in serum (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide) A kit for the diagnosis and early detection of ovarian cancer. The kit has simple pretreatment, high throughput, low cost and good repeatability. Summary of the invention

The object of the present invention is to develop a method for detecting 27-5β-cholestrazine-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide) A kit for the diagnosis and early detection of ovarian cancer. This kit was used to detect 27-5β-cholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate in serum (27-nor-5P-cholestane-3, 7, 12, 24, The method of 25 pentol glucuronide) has the characteristics of low detection cost, good repeatability, high stability and high flux. It takes only 10 minutes to measure an actual sample, which is suitable for clinical application.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

The kit consists of three parts: (1) an internal standard-leucine enkephalin standard; (2) a weak eluent-water (containing a volume concentration of 0.1% formic acid, a volume concentration of 2% acetonitrile); (3) Strong eluent - acetonitrile or methanol.

The kit uses leucine enkephalin as an internal standard to enhance the detection accuracy. The mobile phase can be quickly separated and analyzed by using water (containing 0.1% formic acid, 2% acetonitrile in volume) and acetonitrile system. The purpose of cholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide). The present invention has also been developed based on the use conditions of the test kit and has been applied in normal control, benign ovarian tumor, and ovarian cancer samples.

Specific steps are as follows:

1) Optimize the optimal composition of the kit: The internal standard is selected as leucine enkephalin, which can effectively enhance the detection accuracy; the weak eluent selects water (containing volume concentration 0.1% formic acid, volume concentration 2% acetonitrile); De-liquid selection of acetonitrile.

2) Kit use conditions: The chromatographic instrument is Waters ultra performance liquid chromatography, the column is C18 column, column The temperature is 35 degrees Celsius, the flow rate is 0.4 ml/min, and the mass spectrometer is a single quadrupole mass spectrometer.

3) Take the isolated serum of healthy pregnant women, benign ovarian tumors and ovarian cancer in the morning under the same conditions. After collection, they should be allowed to stand at 4 ° C for half an hour, centrifuged for 15 minutes, and the supernatant (serum) is stored immediately. Stand by in a refrigerator at 80 °C.

4) Pretreatment of serum samples: samples should be thawed at room temperature, 180 μl serum, 720 μl acetonitrile (containing 0.625 μg/ml leucine enkephalin), shaken for 30 seconds, 4°C 13000g After centrifugation for 10 minutes, the supernatant was lyophilized, reconstituted with 150 μl of a mixed solution of acetonitrile in a volume ratio of 1: 4, and directly loaded for analysis. The ion chromatogram was extracted from m/z 556 and m/z 403 to calculate leucine. Enkephalin, 27-5β-cholesterol-3, 7, 12, 24, 25-penta-glycol glucuronide conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide The peak area and the ratio of the two to determine 27-5β-norcholest-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7 , 12, 24, 25 pentol glucuronide) content.

5) Analytical methods were characterized, single-shot injection repeatability and intra-day precision were in compliance with the US Food and Drug Administration (FDA) test standards. The three groups of serum samples were subjected to ultra-high performance liquid chromatography (HPLC) mass spectrometry in random order.

6) In the serum sample test, 27-5β-norformin-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24 , 25 pentol glucuronide) was significantly elevated in the ovarian cancer group relative to the healthy group and benign ovarian tumor group. 27-5β-cholinergic-3, 7, 12, 24, 25-pentanol glucuronide conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide) for ovarian cancer The sensitivity was 72.6% and the specificity was 68.9%. The sensitivity of CA125 for ovarian cancer was 64.2% and the specificity was 45.1%. (Document 6: Chi DS, Zivanovic 0, Palayekar MJ, at al. Gynecol Oncol 112:6- 10, 2009). This indicates that 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronide conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide) has Better ovarian cancer screening value.

The method of the present invention has the following effects: leucine enkephalin and 27-5β-cholinergic-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3) , 7, 12, 24, 25 pentol glucuronide) retention time is similar, and leucine enkephalin is relatively stable and easy to obtain. Based on the above advantages, leucine enkephalin is used as an internal standard, and the detection result is accurate and highly reliable. 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate based on the detection method (27-nor-5P-cholestane-3, 7, 12, 24, 25 The pentol glucuronide) content is accurate and reliable for the diagnosis of ovarian cancer. The mobile phase selects water (containing 0.1% formic acid, 2% acetonitrile) and acetonitrile system to obtain the best separation effect. The method based on kit development has the following characteristics: The sample is collected and stored using standardized operating procedures to avoid introducing human error; the pretreatment is simple, and the analysis process uses ultra-efficient liquid chromatography to reduce the matrix to the analyte. Interference, and greatly improved the analytical throughput. In a serum sample test, 27-5β-norcholestane-3, 7, 12, 24, 25-pentitol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 Pentol glucuronide) can assist or even replace CA125 for the diagnosis of primary ovarian cancer. Its sensitivity and specificity for early diagnosis are superior to CA125. The method for diagnosing ovarian cancer established by this method has high throughput, high sensitivity and specificity. Features, suitable for screening and assisted diagnosis of high-risk populations of ovarian cancer. DRAWINGS

Figure 1 Results of human serum LC/MS analysis, 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, Extracted ion chromatograms of 12, 24, 25 pentol glucuronide) and leucine enkephalin.

Figure 2 (A) Serum 27-5β-norcholin-3, 7, 12, 24, 25-penta-glycol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 Pentol glucuronide) Changes in the content of healthy, benign ovarian tumors, and ovarian cancer serum samples (mean + standard deviation).

Figure 2 (B) Serum 27-5β-norcholin-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 Changes in the content of pentol glucuronide in serum samples from healthy, benign ovarian tumors, and early ovarian cancer (stage I) (mean + standard deviation).

Figure 3 (A) 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate in serum samples from non-ovarian cancer (healthy + benign ovarian tumors) and ovarian cancer groups (27) ROC plot of -nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide): AUC value of 0.74.

Figure 3 (B) Non-ovarian cancer (healthy + benign ovarian tumor) and early ovarian cancer (stage I) serum samples 27-5β-norcholestane-3, 7, 12, 24, 25-pentitol aldol ROC plot of the acid conjugate (27-nor-5p-cholestane-3, 7, 12, 24, 25 pentol glucuronide): AUC value of 0.743. detailed description

Example 1

1 . Serum sample collection

Before the collection, volunteers were included to sign the informed consent form.

The serum of 43 healthy women, 92 benign ovarian tumors diagnosed, and 84 ovarian cancer patients (including 45 cases of stage I ovarian cancer) were collected under the same conditions. After collection, they were allowed to stand at 4 ° C for half an hour and centrifuged for 15 minutes. Immediately after storage, store in a refrigerator at -80 °C for use.

2. Analysis method

2. 1 serum sample pretreatment

The sample was thawed at room temperature, 180 μl serum, 720 μl acetonitrile (containing 0.625 μg/ml leucine enkephalin), shaken for 30 seconds, centrifuged at 4V 13000g for 10 minutes, and then the supernatant was frozen. Dry, reconstitute with 150 μl of a mixed solution of acetonitrile in a volume ratio of 1:4 and directly load the sample.

2. 2 high performance liquid chromatography mass spectrometry high temperature high speed analysis

(1) The liquid phase conditions are: the chromatographic instrument is Waters ultra performance liquid chromatography, the column is Waters ACQUITY UPLCTM BEH C18 column, the mobile phase A is water (containing volume concentration 0.1% formic acid, volume concentration 2% acetonitrile), B is Acetonitrile; Gradient elution conditions: 0~0.5 min for volume concentration 2% B phase, 0.5~7 min linear change To volume concentration 100% B phase, maintain volume concentration 100% phase B at 7 min-8min, decrease to volume concentration 2% phase B at 8.1min, and keep for 2 min; flow rate 0.4 mL/min, column temperature 35 °C, injection The amount of 3 L, the effluent after the column is directly into the mass spectrometry without splitting.

(2) Mass spectrometry conditions: Mass spectrometer is Waters single quadrupole mass spectrometer, detected by electrospray ion source positive ion mode; desolvation gas and cone gas flow are divided into 500 L/h and 50 L/h, desolvation gas Both the cone and the gas are high purity nitrogen; the temperature of the desolvation gas is 300. C, ion source temperature is 120 °C; capillary voltage and cone voltage are 3350V and 33V respectively; detection by selective ion monitoring mode: detection ion is m/z 556 (internal standard), m/z 403 (27- 5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate). Data is collected every 0.05 seconds (Figure 1).

2. 3 data analysis

The ionic flow diagram was extracted from m/z 556 and m/z 403 to calculate leucine enkephalin, 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor -5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide), the peak area, find the ratio of the two to determine 27-5β-cholinergic-3, 7, 12, 24, 25-pentaol The content of glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide).

Analysis of serum test results and auxiliary diagnostic potential

In the serum sample test, 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24, 25 Pentol glucuronide) The contents of 43 healthy women, 92 benign ovarian tumors, and 84 ovarian cancer patients (including 45 stage I ovarian cancer) are summarized in Table 1.

Table 1: 27-5 β-norcholin-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5 P-cholestane-3, 7, 12, 24, 25 pentol glucuronide ) in 43 healthy women, 92 benign ovarian tumors and 84 ovarian cancer serum

Number ^a a, number a, number a

A001 0. 00000 B001 0. 00054 C001 0. 00239

A002 0. 00038 B002 0. 00058 C002 0. 00222

A003 0. 00051 B003 0. 00058 C003 0. 00255

A004 0. 00061 B004 0. 00063 C004 0. 00239

A005 0. 00063 B005 0. 00066 C005 0. 00208

A006 0. 00072 B006 0. 00071 C006 0. 00130

A007 0. 00096 B007 0. 00074 C007 0. 00162

A008 0. 00098 B008 0. 00080 C008 0. 00224

A009 0. 00102 B009 0. 00084 C009 0. 00420

A010 0. 00109 B010 0. 00084 C010 0. 00224

A011 0. 00114 B011 0. 00109 C011 0. 00283

A012 0. 00114 B012 0. 00125 C012 0. 00184

§000 ζ> . /s/u/ Ososld 0080ΗΠ0ΖAV

§00 z 2∞g B083 0. 00291 C083 0. 00247

B084 0. 00449 C084 0. 00245

B085 0. 00459

B086 0. 00171

B087 0. 00183

B088 0. 00110

B089 0. 00127

B090 0. 00159

B091 0. 00184

B092 0. 00680

a: A stands for healthy women, B stands for benign ovarian tumors, and C stands for ovarian cancer. Sample C001-C045 is stage I of ovarian cancer.

27-5β-cholestrazine-3, 7, 12, 24, 25-pentitol glucuronic acid conjugate (27-nor-5P-cholestane-3) in healthy women, benign ovarian tumors, and ovarian cancer samples 7, 12, 24, 25 pentol glucuronide) content average and standard deviation, found 27-5β-norane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor- 5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide) was significantly elevated in the ovarian cancer group (Fig. 2A). The ROC curve was obtained, and the ROC curve AUC value was 0.74 (Fig. 3A). Diagnosing with 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronide conjugate (27-nor-5p-cholestane-3, 7, 12, 24, 25 pentol glucuronide) In ovarian cancer, the sensitivity was 72.6% and the specificity was 68.9% based on the sum of sensitivity and specificity, which was superior to the reported value of CA125. Based on the obtained sensitivity and specificity data, the threshold was found to be 0.00188.

27-5β-norcholestane-3, 7, 12, 24, 25-pentitol glucuronic acid conjugate (27-nor-5P-cholestane- in healthy women, benign ovarian tumors, and stage I ovarian cancer samples) The content of 3, 7, 12, 24, 25 pentol glucuronide) is averaged and standard deviation. 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5P-cholestane-3, 7, 12, 24 found even in early stage ovarian cancer , 25 pentol glucuronide) was also significantly higher in serum than healthy women and benign ovarian tumors (Fig. 2B). Corresponding ROC curve AUC value is 0.75

(Figure 3B). Similarly, with a threshold of 0.00188 and a specificity of 68.9%, the sensitivity was 75.5%.

Therefore, 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate

(27-nor-5P-cholestane-3, 7, 12, 24, 25 pentol glucuronide) is especially suitable for the development of early diagnosis kits for ovarian cancer.

For unknown female serum samples, the 27-5β-cholesteryl-3,7,12,24,25-pentanol glucuronic acid conjugate (27-nor-5p-) was obtained using the above operating conditions described in this patent. The content of cholestane-3, 7, 12, 24, 25 pentol glucuronide). Comparing this content to a threshold of 0.00188, it can be determined whether the unknown serum is from a woman with ovarian cancer.

Claims

Claim

A kit for early diagnosis of ovarian cancer by detecting metabolites in serum, characterized in that the kit consists of three parts: (1) an internal standard-leucine enkephalin; (2) a weak eluate- Water (containing a volume concentration of 0.1% formic acid, a volume concentration of 2% acetonitrile or methanol); (3) A strong eluent, acetonitrile or methanol.

A process according to claim 1, wherein the serum detected by the kit of claim 1 is collected fasting serum.

3. The use process according to claim 2, characterized in that the detection process is by liquid chromatography-mass spectrometry for the detection of 27-5?-norcholestane-3, 7, 12, 24, 25-f in serum. Alcohol glucuronic acid conjugate

(27-nor-5 P-cholestane-3, 7, 12, 24, 25 pentol glucuronide);

Positive ion of 27-5β-norcholestane-3, 7, 12, 24, 25-pentanol glucuronic acid conjugate (27-nor-5p-cholestane-3, 7, 12, 24, 25 pentol glucuronide) The characteristic mass-to-charge ratio in the mode is m/z 403. The leucine enkephalin is selected as the internal standard, which can enhance the detection accuracy. The characteristic mass-to-charge ratio in positive ion mode is m/z 556.

4. The use process according to claim 3, characterized in that the serum sample is thawed at room temperature when tested, taking 100-200 μl of serum, and adding 3-5 volumes of acetonitrile (containing 0.6-0.65 μg/ml leucine) Enkephalin), shaking for 20-40 seconds, centrifugation for 10-15 minutes at 4-10 °C 10000-15000g, then lyophilizing the supernatant, using a 1:1 volume of acetonitrile water mixed solution 5/6 serum Load analysis after volume reconstitution.

5. The use process according to claim 3, characterized in that the chromatographic analysis employs a C18 column having an inner diameter of 2.1 mm and a length of 30-100 mm.

6. The use process according to claim 3, characterized in that the gradient elution conditions are: 0~0.5 min is a volume concentration of 2% strong eluent, 0.5~7 min linearly changes to a volume concentration of 100% strong eluent Maintain a volumetric concentration of 100% strong eluent at 7 min-8 min, 8. Imin reduced to a volumetric concentration of 2% strong eluent for 2 min, flow rate 0.2-0.5 mL/min, column temperature 30-40 °C, The injection volume is 1-5 μΙ^.

7. The use process according to claim 3, characterized in that the electrospray ion source positive ion mode is used for detecting; the desolvation gas and the cone gas flow are divided into 400-600 L/h and 30-60 L/h, Both solvent gas and cone gas are high purity nitrogen; desolvation gas temperature is 280-320 V, ion source temperature is 110-130 °C; capillary voltage and cone voltage are 3000-3500V and 30-35 V respectively; Data is collected once in 0.03-0.1 seconds.