MARKER FOR HEALTH ASSESSMENT, AND APPLICATION THEREOF

Abstract

A marker for health assessment, and an application thereof, relating to the field of medical test. An application of a marker identification reagent in preparation of a reagent for measuring the health of a mammal, comprising: (1) measuring a marker in a test sample, wherein the marker is the level of 8-oxoGsn; and (2) comparing the marker with a reference, wherein the difference between the marker and the reference is used for assessing the health condition of the subject. According to the invention, comprehensive health condition assessment is performed on a subject by means of a simple clinical examination method, and the health condition and health laws of people can be obtained timely and quickly; compared with conventional medical test methods, the invention has remarkable advantages in detection and early warning of sub-health, disease treatment effect monitoring, and psychological emotional state detection in the field of health service management.

Claims

1. A use of a reagent for identifying a marker in manufacture of a reagent for detecting a health status of a mammal, comprising: (1) detecting a marker in a sample of a subject, wherein the marker is a ratio of 8-oxoGsn concentration to creatinine concentration; (2) comparing the marker with a reference, wherein a difference of the marker compared with the reference is used to evaluate the health status of the subject.

2. The use according to claim 1, wherein the sample is a body fluid.

3. The use according to claim 2, wherein the body fluid is selected from the group consisting of peripheral blood, serum, plasma, synovial fluid, aqueous humor, breast milk, semen, prostate fluid, sweat, tear, pleural effusion, ascites fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, vomitus, vaginal secretion, mucosal secretion, pancreatic juice, bronchopulmonary suction fluid, blastocyst cavity fluid, umbilical cord blood, urine, cerebrospinal fluid, saliva, lymph fluid and excreta.

4. The use according to claim 3, wherein the body fluid is urine, peripheral blood, serum, or plasma.

5. The use according to claim 1, wherein the reference is any one or both of the following: (1) an interval value of 8-oxoGsn level of a healthy mammal; (2) an interval value of the ratio of 8-oxoGsn concentration to creatinine concentration of a healthy mammal.

6. The use according to claim 5, wherein an interval value is 95% percentile.

7. The use according to claim 1, wherein the mammal is a human.

8. A use of a reagent for detecting 8-oxoGsn in manufacturing of a product for evaluating a health status of a mammal.

9. The use according to claim 8, wherein the reagent for detecting 8-oxoGsn is a reagent for determining 8-oxoGsn concentration.

10. A use of a reagent for detecting 8-oxoGsn concentration and creatinine concentration in manufacturing a product for evaluating a health status of a mammal.

11. The use according to claim 7, wherein the mammal is a human, and the product is a kit.

12. A kit for detecting a health status of a mammal, comprising a reagent for detecting 8-oxoGsn concentration and a reagent for detecting creatinine concentration.

13. The kit according to claim 12, wherein the mammal is a human.

14. The kit according to claim 12, wherein the kit comprises 8-oxoGsn standard, 10 mM ammonium acetate, methanol, 1 to 2000 pg/L isotope internal standard [.sup.13C,.sup.15N.sub.2]8-oxo-dGsn, and 1 to 2000 pg/L isotope internal standard [.sup.15N.sub.5]8-oxoGsn; preferably, the isotope internal standards have a concentration of 100 to 800 pg/L.

15. The kit according to claim 12[or 13], wherein the 10 mM ammonium acetate and methanol are 70% methanol+30% 10 mM ammonium acetate solution, and the solution after mixing has a pH of 3.5 to 4.0.

16. The use according to claim 6, wherein the mammal is a human.

17. The use according to claim 10, wherein the mammal is a human, and the product is a kit.

18. The kit according to claim 13, wherein the kit comprises 8-oxoGsn standard, 10 mM ammonium acetate, methanol, 1 to 2000 pg/L isotope internal standard [.sup.13C,.sup.15N.sub.2]8-oxo-dGsn, and 1 to 2000 pg/L isotope internal standard [.sup.15N.sub.5]8-oxoGsn; preferably, the isotope internal standards have a concentration of 100 to 800 pg/L.

19. The kit according to claim 13, wherein the 10 mM ammonium acetate and methanol are 70% methanol+30% 10 mM ammonium acetate solution, and the solution after mixing has a pH of 3.5 to 4.0.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1 shows the creatinine-corrected 8-oxoGsn/cre values in the urine samples of normal males and females aged 20-74 years.

[0044] FIG. 2A shows a comparison of the urine 8-oxoGsn/cre level of male patients with HIV with the upper limit of the 95th percentile of the reference interval for normal humans.

[0045] FIG. 2B shows a comparison of the urine 8-oxoGsn/cre level of male patients with hepatitis B with the upper limit of the 95th percentile of the reference interval for normal humans.

[0046] FIG. 2C shows a comparison of the urine 8-oxoGsn/cre level of female patients with hepatitis B with the upper limit of the 95th percentile of the reference interval for normal humans.

[0047] FIG. 2D shows a comparison of the urine 8-oxoGsn/cre level of male patients with hepatitis C with the upper limit of the 95th percentile of the reference interval for normal humans.

[0048] FIG. 2E shows a comparison of the urine 8-oxoGsn/cre level of female patients with hepatitis C with the upper limit of the 95th percentile of the reference interval for normal humans.

[0049] FIG. 2F shows a comparison of the urine 8-oxoGsn/cre level of male patients with syphilis with the upper limit of the 95th percentile of the reference interval for normal humans.

[0050] FIG. 2G shows a comparison of the urine 8-oxoGsn/cre level of female patients with syphilis with the upper limit of the 95th percentile of the reference interval for normal humans.

[0051] FIG. 2H shows a comparison of urine 8-oxoGsn/cre levels of male patients with ACS with the upper limit of the 95th percentile of the reference interval for normal humans.

[0052] FIG. 2I shows a comparison of the urine 8-oxoGsn/cre level of female patients with ACS with the upper limit of the 95th percentile of the reference interval for normal humans.

[0053] FIG. 2J shows a comparison of the urine 8-oxoGsn/cre level of male patients with coronary heart disease with the upper limit of the 95th percentile of the reference interval for normal humans.

[0054] FIG. 2K shows a comparison of the urine 8-oxoGsn/cre level of female patients with coronary heart disease with the upper limit of the 95th percentile of the reference interval for normal humans.

[0055] FIG. 2L shows a comparison of the urine 8-oxoGsn/cre level of male patients with liver cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

[0056] FIG. 2M shows a comparison of the urine 8-oxoGsn/cre level of female patients with liver cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

[0057] FIG. 2N shows a comparison of the urine 8-oxoGsn/cre level of male patients with kidney cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

[0058] FIG. 2O shows a comparison of the urine 8-oxoGsn/cre level of female patients with kidney cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

[0059] FIG. 2P shows a comparison of the urine 8-oxoGsn/cre level of male patients with bladder cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

[0060] FIG. 2Q shows a comparison of the urine 8-oxoGsn/cre levels of female patients with bladder cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

[0061] FIG. 2R shows a comparison of the urine 8-oxoGsn/cre level of male patients with prostate cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

[0062] FIG. 3A to 3H show comparisons the 8-oxoGsn/cre levels in 25 urine samples collected from 8 normal humans within 2 months with the upper limit of the 95th percentile of the reference interval for normal humans.

DETAILED DESCRIPTION OF THE INVENTION

Embodiment 1: Determination of 8-oxoGsn Content in Urine of Normal Humans Aged 20-74 Years

[0063] I. Reagents and Methods:

[0064] (1) Samples: 5179 urine samples from normal humans, including 2591 males and 2588 females.

[0065] (2) Reagents and instruments:

[0066] Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [.sup.13C, .sup.15N.sub.2]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [.sup.15N.sub.5]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

[0067] Chromatography column: Agilent SB-Aq, 3.0100 mm, 1.8 m

[0068] Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

[0069] (3) Determination method:

[0070] 3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at 80 C. until testing. The treatment of urine samples was as follows:

[0071] 1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4 C.

[0072] 2. 200 L of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 L of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

[0073] 3. 480 pg/L isotope internal standard [.sup.13C, .sup.15N.sub.2]8-oxo-dGsn and 480 pg/L isotope internal standard [.sup.15N.sub.5]8-oxoGsn, each 10 L, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

[0074] 4. After being placed in a 37 C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4 C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

[0075] 5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

[0076] 6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

[0077] 7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

[0078] II. Results

[0079] See FIG. 1 and Table 1 (creatinine-corrected mean values and percentiles of 8-oxoGsn in the reference population). A large sample study found that, for normal humans aged 20 to 74, regardless of gender, the 8-oxoGsn content had a positive correlation with the age, and could be used as an age-related indicator. Compared with normal humans, the level of 8-oxoGsn in patients with diseases was higher than that in normal humans, and the higher values, the more meaningful. Therefore, the 0-95 percentile of each age group was used as the reference interval for normal humans (see Table 1 for 0-95% reference interval for each age group).

Embodiment 2: Determination of 8-oxoGsn Content in Urine of Patients of Seven Diseases

[0080] I. Reagents and Methods:

[0081] (1) Samples: 111 urine samples from AIDS patients, 115 urine samples from hepatitis B patients, 104 urine samples from hepatitis C patients, 79 urine samples from syphilis patients, 90 urine samples from liver cancer patients, 119 urine samples from patients with coronary heart disease, and 98 urine samples from ACS patients were collected.

[0082] (2) Reagents and instruments:

[0083] Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [.sup.13C, .sup.15N.sub.2]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [.sup.15N.sub.5]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

[0084] Chromatography column: Agilent SB-Aq, 3.0100 mm, 1.8 m

[0085] Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

[0086] (3) Determination method:

[0087] 3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at 80 C. until testing. The treatment of urine samples was as follows:

[0088] 1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4 C.

[0089] 2. 200 L of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 L of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

[0090] 3. 480 pg/L isotope internal standard [.sup.13C, .sup.15N.sub.2]8-oxo-dGsn and 480 pg/L isotope internal standard [.sup.15N.sub.5]8-oxoGsn, each 10 L, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

[0091] 4. After being placed in a 37 C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4 C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

[0092] 5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

[0093] 6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

[0094] 7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

[0095] II. Results

[0096] The results were shown in FIG. 2 and Table 2 (the numbers and percentages of patients with various diseases that were of above the 95th percentile of the reference interval for normal humans), and for the 8-oxoGsn in urine samples of patients with seven diseases (AIDS, hepatitis B, hepatitis C, syphilis, liver cancer, coronary heart disease, acute coronary syndrome), a comparative study was performed between the results of the seven diseases and the reference interval of 8-oxoGsn for normal humans, and it was found that the levels of 8-oxoGsn in urine samples of patients with various diseases were significantly increased compared to that of the normal humans (see Table 2 and FIG. 2 for details), and at least 60% of the patients of each disease had higher values than the interval for normal humans. In particular, for the bacterial and viral infectious diseases, the proportion of number of patients having a value higher than the interval for normal humans was as high as about 90%, while chronic diseases such as syphilis, AIDS, hepatitis B, etc., come next, which proved that the increase of 8-oxoGsn was correlated with the diseases.

Embodiment 3: Determination of 8-oxoGsn Content in Urine of Healthy Volunteers

[0097] I. Reagents and Methods:

[0098] (I) Samples: collected from a total of 8 healthy volunteers, and urine was collected for 25 times from each of the volunteers.

[0099] (2) Reagents and instruments:

[0100] Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [.sup.13C, .sup.15N.sub.2]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [.sup.15N.sub.5]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

[0101] Chromatography column: Agilent SB-Aq, 3.0100 mm, 1.8 m

[0102] Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

[0103] (3) Determination method:

[0104] 3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at 80 C. until testing. The treatment of urine samples was as follows:

[0105] 1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4 C.

[0106] 2. 200 L of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 L of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

[0107] 3. 480 pg/L isotope internal standard [.sup.13C, .sup.15N.sub.2]8-oxo-dGsn and 480 pg/L isotope internal standard [.sup.15N.sub.5]8-oxoGsn, each 10 L, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

[0108] 4. After being placed in a 37 C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4 C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

[0109] 5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

[0110] 6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

[0111] 7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

[0112] II. Results

[0113] The results were shown in FIG. 3 and Table 3 (the physical discomfort records of the 8 healthy volunteers on the days for collecting 25 urine samples were assessed), and when the final test results of each volunteer's urine were compared with the corresponding upper limit of the 95th percentile of the reference interval for normal humans, it was found that most of the time points where the values were above the upper limit of the interval for normal humans corresponded to the special statuses of the day, such as illness, excessive fatigue, staying up late, strenuous exercise and the like. The above results fully reflected the high sensitivity of the level of 8-oxoGsn in urine, that was, as long as a normal healthy person felt an uncomfortable symptom, especially a state of disease, whose level would increase correspondingly, so that this could reflect a health status of a human to a certain extent. In addition, it was found in this experiment that the level of RNA oxidation in a human who exercised regularly was significantly lower than those who did not exercise, for example, as shown in FIG. 3, the healthy humans Nos. 201 and 202 basically maintained their 8-oxoGsn levels in urine below the upper limit of the 95th percentile of the reference interval for normal humans.

Embodiment 4

[0114] I. Reagents and Methods:

[0115] (I) Test population and sample size: plasma and urine samples were collected from a total of 146 patients with chronic kidney diseases, which included 30 patients with phase I chronic kidney disease, 30 patients with phase II chronic kidney disease, 31 patients with phase III chronic kidney disease, 30 patients with phase IV chronic kidney disease, and 25 patients with phase V chronic kidney disease.

[0116] (2) Reagents and instruments, comprising:

[0117] Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [.sup.13C, .sup.15N.sub.2]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [.sup.15N.sub.5]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

[0118] Chromatography column: Agilent SB-Aq, 3.0100 mm, 1.8 m

[0119] Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

[0120] (3) Determination method: urine and/or plasma samples could be selected for detection

[0121] Detection of urine sample: 3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at 80 C. until testing. The treatment of urine samples was as follows: 1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4 C.

[0122] 2. 200 L of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 L of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

[0123] 3. 480 pg/L isotope internal standard [.sup.13C, .sup.15N.sub.2]8-oxo-dGsn and 480 pg/L isotope internal standard [.sup.15N.sub.5]8-oxoGsn, each 10 L, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

[0124] 4. After being placed in a 37 C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4 C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

[0125] 5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

[0126] 6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

[0127] 7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

[0128] Determination of plasma samples: 3 ml of plasma was collected from a patient, evenly aliquoted and loaded into 1.5 ml EP tubes, and stored at 80 C. in a refrigerator until testing. The treatment of plasma samples was as follows:

[0129] 1. The collected plasma was taken from the 80 C. refrigerator and thawed on ice, mixed well and centrifuged at 14000 g and 4 C. for 10 minutes.

[0130] 2. 300 L was taken and added with two internal standards, 300 pg/L and 10 L for each. The internal standards were used to correct errors of the measuring instrument.

[0131] 3. After being mixed well with three times volume acetonitrile of 900 L, vortex was performed for approximately 1 minute, so that the proteins present in the plasma were precipitated with acetonitrile.

[0132] 4. After centrifugation at 14,000 g for 20 minutes at 4 C., a quantitative volume of 1 ml supernatant was taken and blow dried with nitrogen.

[0133] 5. After being redissolved with 100 L of mobile phase, centrifugation was carried out at 12000 g for 20 minutes at 4 C.

[0134] 6. 85 L was taken and placed into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

[0135] II. Results

[0136] The results were shown in Table 4 (plasma, urine creatinine and nucleic acid oxidation levels of patients with chronic kidney diseases). From Table 4, it could be found that the RNA oxidation level was positively correlated with the severity of the disease, so that the increased 8-oxoGsn ratio in plasma and urine could be a new indicator for advanced kidney diseases.

Embodiment 5: Reagent for Identifying Marker 8-oxoGsn and Use Thereof

[0137] I. Kit Composition

[0138] 1. 8-oxoGsn standard, used to prepare a standard curve of 8-oxoGsn concentration versus intensity.

[0139] 2. 10 mM ammonium acetate and pure methanol: used to prepare a working solution [70% methanol+30% 10 mM ammonium acetate (PH=3.7)]; which could also be a directly prepared 70% methanol+30% 10 mM ammonium acetate solution (pH was 3.5-4.0), that was, the working solution in this kit was prepared from methanol and 10 mM ammonium acetate solution in a volume ratio of 7:3.

[0140] 3. 500 pg/L isotope internal standard [.sup.13C, .sup.15N.sub.2]8-oxo-dGsn and 500 pg/L isotope internal standard [.sup.15N.sub.5]8-oxoGsn.

[0141] II. Use for Determining Health Status

[0142] 1. Detecting a marker in a sample of a subject, wherein the marker was level of 8-oxoGsn;

[0143] 2. The marker was compared with a reference (the 95% confidence interval value for normal humans obtained in Example 1), and the difference of the marker compared with the reference was used to evaluate the health status of the subject.

[0144] 3. The following explained how to compare the marker with the reference (the 95% reference interval value for normal humans obtained in Example 1) for evaluating the health status of the subject: among the 25 tests for the urine samples collected from the healthy volunteers, the marker concentrations obtained from the 25 urine tests were compared with the upper limit of the 95% reference interval for normal humans in the age group. Above 95% reference interval indicated that the subject was in a state of high oxidative stress. The concentrations of most or all of the 25 urine markers were higher than the 95% reference interval, indicating that the subject was in an unhealthy state with persistent high oxidative stress, and intervention was needed. Based on the physical discomfort records of the subjects (see Table 3), the effects of exogenous and endogenous pathogenic factors on 8-oxoGsn levels in human urine were identified, and the main factors that caused the increase of 8-oxoGsn level were screened, and real-time assessment of human health was performed to achieve the purpose of early warning of human sub-health and disease.

[0145] Although the present invention has been described in detail according to specific embodiments, it should be understood that those skilled in the art can readily conceive of changes, variations, or equivalents of these embodiments after gaining an understanding of the foregoing. Accordingly, the scope of the present invention should be evaluated as that of the appended claims and any equivalents thereof.

TABLE-US-00001 TABLE 1 Means and percentiles of 8-oxoGsn in reference population after creatinine correction Male Female Mean Mean Median standard Median standard Age group No. Creatinine 5th (IQR) 95th deviation No. Creatinine 5th (IQR) 95th deviation 20-24 121 19442(8878) 1.50 1.69(0.14) 1.87 1.7 0.11 121 15219(17118) 1.48 1.68(0.15) 1.82 1.67 0.1 25-29 125 18501(9586) 1.14 1.72(0.45) 2.38 1.7 0.32 124 12996(10642) 1.18 1.60(0.44) 2.00 1.6 0.25 30-34 130 15756(7010) 1.19 1.93(0.37) 2.43 1.89 0.34 130 12518(9591) 1.31 1.86(0.40) 2.29 1.84 0.28 35-39 120 14828(6971) 1.96 2.23(0.19) 2.45 2.23 0.14 131 12005(7572) 1.8 2.04(0.21) 2.32 2.03 0.14 40-44 125 16003(8118) 2.09 2.32(0.15) 2.55 2.33 0.13 122 10777(8401) 1.44 2.12(0.42) 2.55 2.06 0.33 45-49 132 15942(7864) 2.16 2.45(0.2) 2.61 2.43 0.14 133 11229(7000) 2.07 2.31(0.18) 2.56 2.31 0.13 50-54 133 15455(8087) 1.91 2.43(0.27) 2.74 2.38 0.25 127 10058(8086) 2.09 2.49(0.24) 2.61 2.44 0.17 55-59 128 14877(10014) 2.2 2.58(0.17) 2.83 2.55 0.16 137 9542(6640) 2.21 2.51(0.17) 2.70 2.5 0.13 60-64 129 12808(8747) 2.45 2.71(0.15) 2.89 2.68 0.12 120 10355(7945) 2.52 2.71(0.12) 2.90 2.71 0.09 65-69 132 12872(6817) 2.12 2.81(0.35) 3.26 2.75 0.32 125 9643(6428) 2.6 2.77(0.19) 2.94 2.78 0.11 70-74 121 12842(6522) 2.77 3.02(0.18) 3.28 3.02 0.16 128 8934(7056) 2.5 2.84(0.27) 3.11 2.83 0.17 Data were grouped by gender, and the concentration of creatinine was expressed as mol/L.

TABLE-US-00002 TABLE 2 Number and percentage of various diseases above the 95th percentile of the reference interval for normal humans 8-oxoGsn Total Disease Male Female Number number AIDS 90(90%) 90(90%) 100 Hepatitis B 43(85%) 43(85%) 86(86%) 100 Hepatitis C 37(74%) 45(90) 82(82%) 100 Syphilis 37(95%) 38(91%) 75 (93%) 81 Acute coronary syndrome 56(71%) 26(79%) 82(73%) 112 Coronary heart disease 38(75%) 26(52%) 64(64%) 100 Liver cancer 49 (69%) 17 (81%) 66(72%) 92 Kidney cancer 63(76%) 32(70%) 95(74%) 129 Bladder cancer 42(75%) 5 (45%) 47(70%) 67 Prostate 63(72%) 63(72%) 88

TABLE-US-00003 TABLE 3 Physical discomfort records on the days of 25 urine health assessments for 8 healthy volunteers Time of collecting Number of normal humans urine sample 201 202 203 205 206 209 211 214 1 Work hard Diarrhea and insomnia 2 Mood Slightly diarrhea, swings strenuous exercise 3 Cold Insomnia Stay-up late 4 Insomnia Insomnia 5 Diarrhea Stay-up late 6 Strenuous exercise 7 Cold and taking Diarrhea Stay-up late cold medicine 8 Cold and taking cold medicine 9 Cold and taking cold medicine 10 Diarrhea Cold and taking cold medicine 11 12 13 14 Insomnia Cold 15 Stay-up late 16 Stay-up late 17 Cold Stay-up late 18 Cold Stay-up late Cold and strenuous exercise 19 Stay-up late Cold 20 Stay-up late 21 Anxiety Stay-up late 22 Anxiety Stay-up late Anxiety and fatigue 23 Strenuous exercise Anxiety Stay-up late Anxiety and fatigue 24 Strenuous exercise Anxiety Stay-up late 25 Strenuous exercise Anxiety Stomach Stay-up late pain

TABLE-US-00004 TABLE 4 Plasma, urine creatinine, and nucleic acid oxidation levels in patients with chronic kidney disease CKD 1 CKD 2 CKD 3 CKD 4 CKD 5 Plasma creatinine 61.60 14.21 90.33 26.26 142.87 63.91 202.03 53.21 590.40 277.87 (umol/L) Urine creatinine 12494 6926.21 10270.07 6238.45 7359.17 3882.42 6447.93 4354.35 4268.64 5782.24 (umol/L) Glomerular 132.53 33.21 76.98 9.1 45.81 8.5 24.73 4.56 7.84 2.9 filtration rate Urine 1.87 0.87 1.92 1.01 1.84 0.81 1.47 0.89 1.60 1.59 8-oxo-dGsn/Cr (pmol/mol) Urine 8-oxo-Gsn/Cr (umol/mol) 3.07 1.07 3.42 1.34 3.72 1.47 3.90 1.93 3.75 2.26 Plasma 0.17 0.12 0.24 0.18 0.37 0.20 0.49 0.22 1.10 0.57 8-oxo-Gsn/Cr (pmol/mol) Plasma/urine 0.02 0.02 0.03 0.02 0.06 0.04 0.10 0.05 0.34 0.03 8-oxo-Gsn Data were expressed as mean SD; significant differences were considered when P < 0.05