CN117630234A - Benign prostatic hyperplasia diagnosis related biomarker and application thereof - Google Patents
Benign prostatic hyperplasia diagnosis related biomarker and application thereof Download PDFInfo
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- CN117630234A CN117630234A CN202311559964.5A CN202311559964A CN117630234A CN 117630234 A CN117630234 A CN 117630234A CN 202311559964 A CN202311559964 A CN 202311559964A CN 117630234 A CN117630234 A CN 117630234A
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- prostatic hyperplasia
- benign prostatic
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- 206010004446 Benign prostatic hyperplasia Diseases 0.000 title claims abstract description 71
- 208000004403 Prostatic Hyperplasia Diseases 0.000 title claims abstract description 71
- 238000003745 diagnosis Methods 0.000 title claims abstract description 48
- 239000000090 biomarker Substances 0.000 title claims abstract description 40
- DTERQYGMUDWYAZ-ZETCQYMHSA-N N(6)-acetyl-L-lysine Chemical compound CC(=O)NCCCC[C@H]([NH3+])C([O-])=O DTERQYGMUDWYAZ-ZETCQYMHSA-N 0.000 claims abstract description 36
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims abstract description 34
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 claims abstract description 34
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 claims abstract description 33
- 235000019393 L-cystine Nutrition 0.000 claims abstract description 31
- 239000004158 L-cystine Substances 0.000 claims abstract description 31
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- 238000001514 detection method Methods 0.000 claims abstract description 8
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a benign prostatic hyperplasia diagnosis related biomarker and application thereof. The biomarker is selected from at least one of N6-acetyl-L-lysine, L-cystine and L-homoarginine; the study shows that the content of N6-acetyl-L-lysine, L-cystine and L-homoarginine in serum of benign prostatic hyperplasia patients is obviously higher than that of healthy controls, ROC curve analysis is further carried out on the results to evaluate the diagnostic efficacy of indexes, and the results show that the 3 metabolites can be used as biomarkers for diagnosing benign prostatic hyperplasia and have better diagnostic performance. Further, the sensitivity and the specificity of the detection can be further improved when more than two combinations are used for diagnosing benign prostatic hyperplasia, and the diagnosis effect is better. The invention provides a benign prostatic hyperplasia diagnosis related biomarker which has the advantages of easily available samples, no wound, simplicity, convenience and rapidness when being used for diagnosing benign prostatic hyperplasia.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a benign prostatic hyperplasia diagnosis related biomarker and application thereof.
Background
Benign prostatic hyperplasia (Benign prostatic hyperplasia, BPH) is commonly known as "prostatic hypertrophy", and is one of the common diseases of older men, which is caused by a substantial change in the proliferation of the middle and lateral lobes of the prostate. The pathogenesis of benign prostatic hyperplasia is quite well studied, but the etiology has not yet been elucidated. May interact with androgens and their interactions with estrogens, interactions of the prostate stroma with glandular epithelial cells, growth factors, inflammatory cells, neurotransmitters, genetic factors, and the like.
Benign prostatic hyperplasia is hidden and slow, and most patients cannot recall the exact onset time; the diagnosis is definitely carried out when the patient is in a visit due to acute urinary retention, obvious urinary flow slowing and other reasons; or prostatic hyperplasia is found at routine physical examination. Common symptoms can be divided into two major categories: obstructive symptoms such as fine urine flow, unclean urination, dribbling in the later stage of urination, intermittent urine column, and the like. The irritative symptoms include frequent urination (urination is not performed for two hours), urgent urination (urgent urination is even suffocated), nocturia, etc. Since urination is often not clear, bacterial cystitis, vesical calculus, urinary retention and the like are easy to occur, abdominal hernia, internal hemorrhoids and the like are caused, and hydronephrosis and even renal insufficiency can be caused by serious patients. In addition, as the prostate gland increases, its blood flow increases relatively, and painless hematuria may occur in patients.
The current diagnosis technology for the prostatic hyperplasia mainly comprises digital rectal examination, transrectal ultrasound, pathological tissue detection and the like. The rectal examination refers to palpating the prostate by the physician's finger through the anus, and indicates the approximate size, whether there is a lesion such as a hard mass or a abscess. Meanwhile, prostate massage can be used to obtain the prostate pressing liquid for further examination by doctors. The length, width, height, volume and other parameters of the prostate can be accurately measured by transrectal ultrasonic energy, and the abnormal focus can be punctured to take out tissues for pathological examination. Existing diagnostic techniques are further to be improved in terms of operational procedures and patient compliance.
Biomarkers (biomarks) refer to biochemical markers that can mark alterations or possible changes in system, organ, tissue, cell and subcellular structures or functions, and have a very wide range of uses in diagnosis and treatment of diseases. A biomarker specific to the disease is found, and the biomarker can play a role in the identification, early diagnosis, prevention and monitoring in the treatment process of the disease. Thus, the search for and discovery of valuable biomarkers associated with disease has become an important hotspot in current research.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a benign prostatic hyperplasia diagnosis related biomarker and application thereof. The benign prostatic hyperplasia diagnosis related biomarker has good sensitivity and specificity when used for benign prostatic hyperplasia diagnosis, can be used for preparing a reagent for diagnosing benign prostatic hyperplasia, and has the advantages of easily available samples, no wound, simplicity, convenience and rapidness.
The aim of the invention is achieved by the following technical scheme:
use of a benign prostatic hyperplasia diagnosis-related biomarker selected from at least one of N6-acetyl-L-lysine, L-cystine, and L-homoarginine in the manufacture of a product for diagnosis of benign prostatic hyperplasia.
Further, the benign prostatic hyperplasia diagnosis-related biomarker is N6-acetyl-L-lysine.
Further, the benign prostatic hyperplasia diagnosis related biomarker is L-cystine.
Further, the benign prostatic hyperplasia diagnosis-related biomarker is L-homoarginine.
Further, the biomarkers related to benign prostatic hyperplasia diagnosis are N6-acetyl-L-lysine and L-cystine.
Further, the biomarkers related to benign prostatic hyperplasia diagnosis are N6-acetyl-L-lysine and L-homoarginine.
Further, the biomarkers related to benign prostatic hyperplasia diagnosis are L-cystine and L-homoarginine.
Further, the biomarkers related to benign prostatic hyperplasia diagnosis are N6-acetyl-L-lysine, L-cystine and L-homoarginine.
Further, the product comprises a reagent for detecting the content of a benign prostatic hyperplasia diagnosis related biomarker;
still further, the product comprises a reagent for detecting the content of the benign prostatic hyperplasia diagnosis related biomarker by using an ultra performance liquid chromatography-tandem mass spectrometry technology.
Further, the detection sample of the product is serum; further, the sample is venous blood serum.
The invention also provides a biomarker related to benign prostatic hyperplasia diagnosis, wherein the biomarker related to benign prostatic hyperplasia diagnosis is selected from at least one of N6-acetyl-L-lysine, L-cystine and L-homoarginine.
The invention also provides a kit for benign prostatic hyperplasia diagnosis, which comprises a reagent for detecting the content of benign prostatic hyperplasia diagnosis related biomarker in a sample, wherein the benign prostatic hyperplasia diagnosis related biomarker is at least one of N6-acetyl-L-lysine, L-cystine and L-homoarginine.
Further, the kit further comprises a standard for the benign prostatic hyperplasia diagnosis-related biomarker.
Further, the kit further comprises an internal standard; still further, the internal standard is [2H2] -L-threonine.
Further, the sample is serum; further, the sample is venous blood serum.
Further, the kit also comprises a sample pretreatment reagent, wherein the sample pretreatment reagent is 20% acetonitrile methanol extract.
Compared with the prior art, the invention has the following advantages and effects:
according to the invention, the content of N6-acetyl-L-lysine, L-cystine and L-homoarginine in serum of benign prostatic hyperplasia patients is obviously higher than that of healthy control people, ROC curve analysis is further carried out on detection results to evaluate the diagnostic efficacy of indexes, and the results show that the N6-acetyl-L-lysine and AUC=0.808; l-cystine, auc=0.755; l-homoarginine, auc=0.688. The results show that the N6-acetyl-L-lysine, L-cystine and L-homoarginine can be used as biomarkers for diagnosing benign prostatic hyperplasia, and have better diagnostic performance, especially N6-acetyl-L-lysine and higher diagnostic efficiency. Further, more than two of N6-acetyl-L-lysine, L-cystine and L-homoarginine are combined for diagnosing benign prostatic hyperplasia, and the results show that the AUC is more than 0.7, wherein the AUC is as high as 0.866 when the three are combined for diagnosis, which shows that the combination of the 3 metabolites can be used as a high-efficiency biomarker for diagnosing benign prostatic hyperplasia; the invention provides a novel benign prostatic hyperplasia diagnosis related biomarker which has the advantages of easily available samples, no wound, simplicity, convenience and rapidness when being used for benign prostatic hyperplasia diagnosis.
Drawings
FIG. 1 is a graph showing the results of comparison of the content of N6-acetyl-L-lysine in serum of benign prostatic hyperplasia patients with healthy controls (B) and ROC curve analysis (A).
FIG. 2 is a graph showing the results of comparison of L-cystine content in serum (B) and ROC curve analysis (A) of benign prostatic hyperplasia patients and healthy control serum.
FIG. 3 is a graph showing the results of comparison of L-homoarginine content in serum of benign prostatic hyperplasia patients and healthy controls (B) and ROC curve analysis (A).
FIG. 4 is a graph of ROC for N6-acetyl-L-lysine in combination with L-cystine for diagnosis of benign prostatic hyperplasia.
FIG. 5 is a graph of ROC for N6-acetyl-L-lysine in combination with L-homoarginine for diagnosing benign prostatic hyperplasia.
FIG. 6 is a graph of ROC for the diagnosis of benign prostatic hyperplasia in combination of L-cystine and L-homoarginine.
FIG. 7 is a graph of ROC for N6-acetyl-L-lysine, L-cystine, and L-homoarginine in combination for diagnosing benign prostatic hyperplasia.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The structural formulas of the N6-Acetyl-L-Lysine (CAS number 692-04-6), L-Cystine (L-Cystine, CAS number 56-89-3) and L-Homoarginine (homoL-arginine or L-Homoarginine, CAS number 156-86-5) are respectively shown as the formula (I), the formula (II) and the formula (III):
example 1
The method for detecting the content of N6-acetyl-L-lysine, L-cystine and L-homoarginine in venous blood serum of benign prostatic hyperplasia patients and healthy controls comprises the following steps of:
1. subject inclusion criteria
Healthy controls (Control group) inclusion criteria: 1) Transrectal digital examination and prostate B-ultrasonic examination are normal; 2) Eliminating urinary system diseases, various benign and malignant tumors and various chronic diseases;
benign prostatic hyperplasia (BPH group) patients were included as standard: 1) Digital rectal examination and ultrasonic diagnosis of the prostate gland B indicate the increase of the volume of the prostate gland; 2) The patients have the symptoms of unsmooth urination, frequent urination, nocturia, difficult progressive urination, partial patients have the medical history of urinary retention, and the medical history of other malignant tumors is not combined; 3) All patients underwent urethral prostatectomy, and post-operative pathological tissue diagnosis proved to be benign prostatic hyperplasia.
All subjects signed patient informed consent, and the study was approved by the central hospital ethical committee of the canthus, inc.
Information about healthy controls and benign prostatic hyperplasia patients are shown in table 1:
TABLE 1 information about healthy controls and benign prostatic hyperplasia patients
2. Specimen collection
All subjects were preoperatively drawn 3ml of early morning fasting venous blood (inspection procedures such as digital rectal examination, massage, puncture, etc. were prohibited 1 week before blood collection) and serum was collected and stored at-80 degrees celsius until analysis.
3. Sample detection
The data acquisition instrument system mainly comprises ultra-high performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) (ExionLC) TM AD，https://sciex.com.cn/) And tandem mass spectrometry (Tandem Mass Spectrometry, MS/MS)6500+，https://sciex.com.cn/)。
3.1 serum sample treatment
(1) After the sample is thawed, vortex for 10s and mix well;
(2) Taking 50 mu L of a sample (the sampled sample is put back into a refrigerator at the temperature of minus 80 ℃ as soon as possible) and added into a corresponding numbered 1.5mL centrifuge tube, and 250 mu L of 20% v/v acetonitrile methanol extract is added;
(3) Centrifuging after swirling for 3min, centrifuging for 12000r/min, and centrifuging for 10min at 4 ℃;
(4) Sucking 250 mu L of supernatant into another 1.5mL centrifuge tube with the same number after centrifugation, and standing in a refrigerator at-20 ℃ for 30min;
(5) 12000r/min, and centrifuging at 4deg.C for 10min;
(6) Taking 180 mu L of supernatant after centrifugation, passing through a protein precipitation plate, and then performing on-machine analysis, and placing on-machine liquid at the temperature of minus 20 ℃ for preservation.
3.2 liquid phase conditions mainly include:
1) Chromatographic column: ACQUITY BEH Amide column (1.7 μm,100mm×2.1mm i.d.);
2) Mobile phase: phase a, ultrapure water (containing 2mM ammonium acetate, 0.04% formic acid); phase B, acetonitrile (containing 2mM ammonium acetate, 0.04% formic acid);
3) Gradient elution procedure: 0-1.2min A/B is 10:90 (V/V), 9min A/B is 40:60 (V/V), 10-11min A/B is 60:40 (V/V), 11.01-15min A/B is 10:90 (V/V);
4) The flow rate is 0.4mL/min; column temperature 40 ℃; the sample injection amount was 2. Mu.L.
3.3 Mass Spectrometry conditions mainly include:
electrospray ion source (Electrospray Ionization, ESI) temperature 550 ℃, mass spectral voltage 5500V in positive ion mode, mass spectral voltage-4500V in negative ion mode, and Gas Curtain (curtaingas, CUR) 35psi. In Q-Trap 6500+, each ion pair is scan detected based on an optimized declustering voltage (declustering potential, DP) and Collision Energy (CE).
4. Principle of qualitative and quantitative determination
And constructing a MWDB (Metware Database) database based on the standard substance, and carrying out qualitative analysis on the data of the mass spectrum detection.
Quantification was done using a multiple reaction monitoring mode (Multiple Reaction Monitoring, MRM) analysis of triple quadrupole mass spectrometry. In the MRM mode, the quaternary rod firstly screens precursor ions (parent ions) of target substances, and eliminates ions corresponding to other molecular weight substances to primarily eliminate interference; the precursor ions are ionized by the collision chamber in an induction way to be broken to form a plurality of fragment ions, and the fragment ions are filtered by the triple quaternary rod to select the required characteristic fragment ions, so that the interference of non-target ions is eliminated, the quantification is more accurate, and the repeatability is better. After obtaining mass spectrometry data of different samples, the chromatographic peaks of all the targets are integrated, and quantitative analysis is carried out through a standard curve.
Preparing 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000ng/mL and other standard substance (N6-acetyl-L-lysine, L-cystine or L-homoarginine) solutions with different concentrations, and obtaining mass spectrum peak intensity data corresponding to the standard substances with different concentrations; the internal standard is [2H2] -L-threonine (product number: IR-22713, CAS number: 1202936-45-5, on-line concentration 1000 ng/mL); and drawing standard curves of different substances by taking the concentration Ratio (Concentration Ratio) of the external standard and the internal standard as an abscissa and the peak Area Ratio (Area Ratio) of the external standard and the internal standard as an ordinate. The standard curve linear equation and the correlation coefficient of the substances detected in the project are shown in Table 2. The contents of N6-acetyl-L-lysine, L-cystine and L-homoarginine in the samples were calculated according to the regression equation in Table 2 and the ratio of the integrated peak areas corresponding to the respective substances.
TABLE 2 Linear equation form
Note that: RT represents a retention time; LLOQ represents the lowest limit of quantification in ng/mL; ULOQ represents the highest limit of quantification in ng/mL.
5. Statistical analysis
And performing Principal Component Analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) dimension reduction analysis on the sample data by adopting an R software package Ropls. Calculating P-value according to T test, calculating variable projection importance (VIP) by PLS-DA dimension reduction method, calculating component difference multiple by foldchange, measuring influence intensity and interpretation ability of each metabolite component content on sample classification discrimination, and assisting in screening marker metabolites. Metabolite molecules are considered statistically significant when P-value <0.05 and VIP value > 1. The area under the subject working characteristic curve (AUC), which is plotted on the ordinate with the true positive rate (sensitivity) and the false positive rate (1-specificity) on the abscissa, was used to evaluate the model for identifying the diagnostic accuracy of the patient in each comparison. AUC <0.5 indicates diagnostic nonsensical; auc=0.5 to 0.7, indicating lower diagnostic accuracy; auc=0.7 to 0.9, indicating moderate diagnostic accuracy; AUC >0.9 indicates high diagnostic accuracy.
6. Results
Analysis of the serum levels of N6-acetyl-L-lysine and ROC curves of benign prostatic hyperplasia patients and healthy controls is shown in FIG. 1: the content of N6-acetyl-L-lysine in serum of benign prostatic hyperplasia patients is obviously higher than that of healthy controls, the AUC is 0.808, the sensitivity is 65.3%, and the specificity is 82.8%.
As shown in fig. 2: the content of L-cystine in serum of benign prostatic hyperplasia patient is obviously higher than that of healthy control, AUC is 0.755, sensitivity is 59.3%, and specificity is 78.9%.
As shown in fig. 3: the content of L-homoarginine in serum of benign prostatic hyperplasia patients is obviously lower than that of healthy controls, the AUC is 0.688, the sensitivity is 54.5 percent, and the specificity is 78.90 percent.
The results of the ROC curve analysis of the three are shown in Table 3, in which threshold is the optimal cut-off value of the ROC curve.
TABLE 3 ROC Curve analysis results
The results show that the N6-acetyl-L-lysine, L-cystine and L-homoarginine can be used as biomarkers for diagnosing benign prostatic hyperplasia, and have better diagnostic performance, in particular N6-acetyl-L-lysine.
Further analysis, when the combination of more than two of N6-acetyl-L-lysine, L-cystine and L-homoarginine is used for diagnosing benign prostatic hyperplasia, the sensitivity and the specificity of detection can be further improved, and the diagnosis effect is better:
the AUC was 0.843, sensitivity was 82.6% and specificity was 69.7% for the combined diagnosis of N6-acetyl-L-lysine and L-cystine (fig. 4);
N6-acetyl-L-lysine was diagnosed with L-homoarginine with an AUC of 0.841, a sensitivity of 74.3% and a specificity of 84.2% (FIG. 5);
when L-cystine was diagnosed in combination with L-homoarginine, AUC was 0.789, sensitivity was 51.5%, and specificity was 92.1% (FIG. 6);
N6-acetyl-L-lysine, L-cystine and L-homoarginine were diagnosed with an AUC of 0.866, a sensitivity of 79.0% and a specificity of 80.3% (FIG. 7);
the ROC curve analysis results of the combination of two or more of the three are specifically shown in table 4, in which threshold is the optimal cut-off value of the ROC curve.
TABLE 4 ROC Curve analysis results
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. Use of a biomarker associated with diagnosis of benign prostatic hyperplasia in the manufacture of a product for diagnosis of benign prostatic hyperplasia, characterized by: the benign prostatic hyperplasia diagnosis-related biomarker is selected from at least one of N6-acetyl-L-lysine, L-cystine and L-homoarginine.
2. The use according to claim 1, characterized in that:
the benign prostatic hyperplasia diagnosis related biomarker is N6-acetyl-L-lysine, L-cystine or L-homoarginine.
3. The use according to claim 1, characterized in that: the biomarkers related to benign prostatic hyperplasia diagnosis are N6-acetyl-L-lysine and L-cystine; N6-acetyl-L-lysine and L-homoarginine; l-cystine and L-homoarginine; alternatively, N6-acetyl-L-lysine, L-cystine, and L-homoarginine.
4. A use according to any one of claims 1 to 3, characterized in that: the product comprises reagents for detecting the content of biomarkers associated with benign prostatic hyperplasia diagnosis.
5. The use according to claim 4, characterized in that: the product comprises a reagent for detecting the content of the benign prostatic hyperplasia diagnosis related biomarker by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology.
6. A use according to any one of claims 1 to 3, characterized in that: the detection sample of the product is serum.
7. A kit for diagnosis of benign prostatic hyperplasia, characterized in that: the kit comprises reagents for detecting the content of the benign prostatic hyperplasia diagnosis-related biomarker according to any one of claims 1 to 3 in a sample.
8. The kit of claim 7, wherein: the kit further comprises a standard for the benign prostatic hyperplasia diagnosis-related biomarker.
9. The kit of claim 7 or 8, wherein: the sample is serum.
10. The kit of claim 7 or 8, wherein: the kit further comprises a sample pretreatment reagent, wherein the sample pretreatment reagent is 20% v/v acetonitrile methanol extract.
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