Use of fecal metabolites as biomarkers in the preparation of a product for diagnosing the onset of a prostate hyperplasia disease

Abstract

The application discloses application of fecal metabolites as biomarkers in preparation of products for diagnosing occurrence of a prostatic hyperplasia disease, and with the support of omics research of a large sample population, three specific fecal metabolites, 3,4-Dihydroxybenzaldeh, 3-Hydroxybenzyl alcohol and 1-Methylhypoxanthine, are selected as BPH biomarkers, and the biomarker combination exhibits excellent diagnostic efficiency in preparation of products for diagnosing occurrence of the prostatic hyperplasia disease or evaluating the severity of the prostatic hyperplasia disease. The biomarker combination can accurately capture metabolic differences between BPH patients and healthy people, patients at different progression stages, and shows high accuracy in early diagnosis and progression evaluation of the disease. The biomarker combination not only effectively makes up for the defect of the existing diagnostic means in the evaluation of the severity of the disease, greatly reduces the risk of misdiagnosis and missed diagnosis, but also provides a key reference for clinicians to formulate individualized treatment schemes, helps to avoid improper treatment caused by inaccurate evaluation, and significantly improves the accuracy of diagnosis and treatment of the BPH.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of disease diagnosis, and particularly relates to application of fecal metabolites as biomarkers in preparation of products for diagnosing occurrence of benign prostatic hyperplasia. BACKGROUND

[0002] Benign prostatic hyperplasia (BPH) is a high-incidence disease in the middle-aged and elderly male population, and its incidence increases significantly with age, which has become an important problem affecting the health of the elderly male population. The current clinical diagnosis system for BPH has obvious limitations, mainly relying on prostate volume measurement and patient subjective symptom assessment (such as lower urinary tract symptoms, LUTS), but there is not a simple linear correlation between the two. This diagnosis mode relying on subjective symptoms and a single indicator, combined with the current situation that the development of diagnosis and treatment programs highly depends on the personal experience of physicians and lacks unified objective standards, is prone to lead to clinical misdiagnosis or missed diagnosis, and is difficult to meet the needs of precise diagnosis and treatment.

[0003] In the specific clinical diagnosis practice of BPH, the existing examination methods have significant deficiencies in accurately assessing the severity of the disease. Rectal examination as a traditional examination method has a large influence on the results by the experience and subjective judgment of the physician, and has poor consistency; although ultrasonic examination can provide morphological information such as prostate volume, it is difficult to effectively reflect the pathophysiological changes of prostate tissue and cannot reveal the nature of the disease progression; urinary flow dynamics examination can only assess the macroscopic performance of urination function and cannot directly reflect the degree of disease progression of BPH. The inherent defects of these examination methods make the clinical assessment of BPH lack comprehensiveness and depth.

[0004] The limitations of the existing diagnosis system directly lead to the predicament of BPH clinical diagnosis and treatment. Due to the lack of accurate grasp of the nature of the disease, it is difficult for the clinic to achieve precise diagnosis and individualized treatment in the true sense, and it is not uncommon for some patients with moderate to severe LUTS to have poor efficacy or difficulty tolerating adverse reactions despite receiving drug treatment. This situation highlights the urgent need to find new objective detection targets, therefore, finding an accurate and convenient biomarker for judging the occurrence and severity of BPH is of great significance for the precise diagnosis and individualized treatment of BPH. SUMMARY

[0005] The main purpose of the present application is to provide an application of a reagent for detecting fecal metabolites in the preparation of a product for diagnosing the occurrence of benign prostatic hyperplasia, aiming to solve the problem of insufficient assessment methods for the severity of BPH in the prior art, by selecting a specific combination of fecal metabolic markers, taking advantage of the significant difference in their expression in patients with benign prostatic hyperplasia, to achieve rapid diagnosis of the occurrence and severity of benign prostatic hyperplasia.

[0006] To achieve the above object, the present application provides a use of a reagent for detecting fecal metabolites in the preparation of a product for diagnosing the occurrence of a benign prostatic hyperplasia disease.

[0007] Preferably, the fecal metabolites include at least one of 3,4-Dihydroxybenzaldehy, 3-Hydroxybenzylalcohol and 1-Methylhypoxanthine.

[0008] Preferably, the fecal metabolites include a combination of 3-Hydroxybenzylalcohol and 1-Methylhypoxanthine.

[0009] Preferably, the fecal metabolites include a combination of 3,4-Dihydroxybenzaldehy and 1-Methylhypoxanthine.

[0010] Preferably, the fecal metabolites include a combination of 3,4-Dihydroxybenzaldehy and 3-Hydroxybenzylalcohol.

[0011] Preferably, the fecal metabolites include a combination of 3,4-Dihydroxybenzaldehy, 3-Hydroxybenzylalcohol and 1-Methylhypoxanthine.

[0012] Preferably, the diagnosing the benign prostatic hyperplasia disease includes distinguishing early benign prostatic hyperplasia subjects from healthy subjects.

[0013] The present application also provides a use of a reagent for detecting fecal metabolites in the preparation of a product for evaluating the severity of a benign prostatic hyperplasia disease.

[0014] Preferably, the fecal metabolites include a combination of 3,4-Dihydroxybenzaldehy and 3-Hydroxybenzylalcohol.

[0015] Preferably, the evaluating the severity of the benign prostatic hyperplasia disease includes distinguishing conservative treatment subjects, drug treatment subjects and surgical treatment subjects.

[0016] Compared with the prior art, the present application has the following advantages:

[0017] (1) With the support of large sample population omics research, the present application selects 3,4-Dihydroxybenzaldeh, 3-Hydroxybenzyl alcohol and 1-Methylhypoxanthine as three specific fecal metabolite combinations as BPH biomarkers, which exhibit excellent diagnostic efficiency in the preparation of products for diagnosing the occurrence or evaluating the severity of prostatic hyperplasia. The marker combination can accurately capture the metabolic differences between BPH patients and healthy people, patients at different stages of progression, and shows high accuracy in early diagnosis and progression evaluation of the disease. This not only effectively makes up for the defects of the existing diagnostic methods in evaluating the severity of the disease, greatly reduces the risk of misdiagnosis and missed diagnosis, but also provides a key reference for clinicians to develop personalized treatment plans, which helps to avoid improper treatment due to inaccurate evaluation, and significantly improves the accuracy of BPH diagnosis and treatment.

[0018] (2) The product based on the specific fecal metabolite marker combination has very high clinical application value and transformation potential. The detection sample is feces, which is simple and non-invasive to obtain, greatly improving the acceptance of patients, especially suitable for large-scale clinical screening and early diagnosis scenarios. The simple model only needs to input 3 metabolite indicators to quickly calculate the results, has the advantages of small calculation amount, low hardware requirement, stable results and easy interpretation, and is very suitable for large-scale popularization and application in primary medical units, physical examination centers and other scenarios. This not only helps to include the accurate screening of BPH into the routine health examination project, realizes the early detection and early intervention of the disease, but also can reduce the social and economic burden of late-stage treatment of the disease by expanding the coverage of screening, which has significant social significance and economic value. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0020] Figure 1Figure showing the fecal metabolites provided by the present application in different groups and their association with clinical symptoms; (A) Box plot showing the differential expression of 3,4-Dihydroxybenzaldehy, 3-Hydroxybenzyl alcohol and 1-Methylhypoxanthine metabolites between the benign prostatic hyperplasia patient group and the healthy control group; (B) Box plot showing the differential expression of 3,4-Dihydroxybenzaldehy, 3-Hydroxybenzyl alcohol and 1-Methylhypoxanthine metabolites between the BPH population requiring conservative treatment (mild-BPH) and the BPH patients requiring drug or surgical intervention (moderate-severe-BPH); ns, no significant meaning, *p<0.05; **p<0.01; ***p<0.001.

[0021] Figure 2 Figure showing the ROC curve of 3,4-Dihydroxybenzaldehy and 1-Methylhypoxanthine and their combination of two for the differential diagnosis between the benign prostatic hyperplasia patient group and the normal control group provided by the present application.

[0022] Figure 3 Figure showing the ROC curve of 3,4-Dihydroxybenzaldehy for benign prostatic hyperplasia progression provided by the present application.

[0023] Figure 4 Figure showing the ROC curve of 3-Hydroxybenzyl alcohol for benign prostatic hyperplasia progression provided by the present application.

[0024] Figure 5 Figure showing the ROC curve of 3,4-Dihydroxybenzaldehy and 3-Hydroxybenzyl alcohol and their combination of two for benign prostatic hyperplasia progression provided by the present application.

[0025] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0026] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not indicated, the conventional products that can be purchased in the market are adopted. In addition, the technical solutions in each embodiment can be combined with each other, but it should be considered that the combination of the technical solutions does not exist and is not within the protection scope of the present application when the combination of the technical solutions is contradictory or unachievable on the premise that the technical solutions can be realized by the ordinary skilled in the art. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without creative labor are within the protection scope of the present application.

[0027] The technical solutions of the present application will be further described in detail below in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present application and are not used to limit the present application.

[0028] Embodiment 1 Fecal sample collection, pretreatment and detection

[0029] 1. Fecal sample collection and pretreatment

[0030] Collection of samples: A total of 311 fecal samples of healthy people (N=93) and patients with benign prostatic hyperplasia (BPH, N=218) of different severity were collected. Among them, the training set was 220 cases, and the training set included healthy people (Healthy, N=66), BPH patients in conservative treatment (mild-BPH, N=46), and BPH patients who needed drug or surgical treatment (moderate to severe-BPH, N=108); the validation set was 91 cases, and the validation set included healthy people (Healthy, N=27), BPH patients in conservative treatment (mild-BPH, N=19), and BPH patients who needed drug or surgical treatment (moderate to severe-BPH, N=45). This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Ethical Number 2022173).

[0031] Pretreatment of samples: When collecting the fecal sample, first empty the bladder to prevent urine from contaminating the feces and discard the feces with blood. Prepare a bedpan and a feces container, wash your hands, and collect fresh fecal samples in a sterile sampling cup with gloves. Cut the middle section of the sample into a sterile 2.0 mL cryogenic tube (not more than 1 / 3 volume) with a sterile toothpick or fecal sampler, and take about a peanut-sized sample in each cryogenic tube. After aliquoting, immediately freeze in liquid nitrogen or directly store at -80°C.

[0032] 2. Fecal sample metabolite detection

[0033] Take 25 mg of fecal sample into a 2 mL grinding tube, add 800 μL of extraction solution (methanol: acetonitrile: water = 2:2:1, v:v:v, pre-cooled at -20°C) + 10 μL of internal standard, add two small steel balls, put into the tissue grinder for grinding (50 Hz, 5 min), 4°C water bath ultrasonic for 10 min, -20°C refrigerator for 1 h. 4°C, 25000g centrifugation for 15 min. After centrifugation, take 600 μL of supernatant, place it in a freeze vacuum concentrator to dry, then add 600 μL of redissolution solution (50% methanol) for redissolution, vortex for 1 min, 4°C water bath ultrasonic for 10 min, then centrifuge again at 4°C, 25000g for 15 min, take the supernatant and place it in a sample bottle. Take 50 μL of supernatant from each sample to mix into a QC quality control sample for evaluating the repeatability and stability of the LC-MS analysis process.

[0034] Chromatographic separation used a Waters BEH C18 column (1.7 μm, 2.1×100 mm), column temperature 45°C, flow rate 0.35 mL / min, injection volume 5 μL. The mobile phase was set as follows: 0.1% formic acid in water (A phase) and 0.1% formic acid in methanol (B phase) for positive ion mode; 10 mM ammonium formate in water (A phase) and 10 mM ammonium formate in 95% methanol (B phase) for negative ion mode, using gradient elution. Mass spectrometry detection used a Q Exactive HF mass spectrometer, ESI ion source, positive / negative ion mode spray voltage was 3.80 / 3.20 kV. First scan range m / z 70-1050, resolution 120,000; second scan selected Top3 parent ions, resolution 30,000, fragmentation energy 20 / 40 / 60 eV. Metabolite data obtained (see Figure 1 ).

[0035] 3. Data analysis and prediction performance

[0036] The mass spectrometry data metabolic peak area and metabolite identification results were analyzed using Compound Discoverer 3.3 (Thermo Fisher Scientific, USA) software combined with BMDB (BGI Metabolome Database), mzCloud database and ChemSpider online database. The data were normalized using a probabilistic quotient normalization method (PQN) to obtain the relative peak area of metabolites; QC-RLSC (Quality control-based robust LOESS signal correction) was used to correct batch effects; compounds with a relative peak area CV (Coefficient of Variation) greater than 30% in all QC samples were deleted.

[0037] Based on the pretreated data, R software package Ropls was used for dimensionality reduction analysis of sample data, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). According to the Wilcox test, the P-value was calculated, the fold change was calculated, the group difference was calculated, and the variable projection importance (VIP) was calculated by PLS-DA dimension reduction method. When p < 0.05, Fold Change ≧1.5 and VIP >1, the metabolite molecule was considered to have statistical significance. In the training set, possible screening and diagnostic metabolites were selected, and their performance was tested in the validation set. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the model, including the diagnostic accuracy of distinguishing between healthy people and BPH people, and in the BPH population, distinguishing between conservative treatment and drug / surgical treatment.

[0038] Example 2 Analysis of detection results

[0039] 1, 3, 4-dihydroxybenzaldehyde, 1-methylhypoxanthine and 3-hydroxybenzyl alcohol were significantly decreased in BPH and were significantly negatively correlated with clinical symptoms

[0040] Fecal metabolomics sequencing results showed that, compared with healthy individuals, the expression of 3,4-dihydroxybenzaldehyd, 1-methylhypoxanthine, and 3-hydroxybenzyl alcohol in the feces of patients with BPH was significantly reduced. Figure 1 A). Furthermore, in BPH patients, compared to those receiving conservative treatment, the levels of 3,4-dihydroxybenzaldehyd and 3-hydroxybenzyl alcohol were significantly lower in those requiring medication or surgery. Figure 1 B) suggests that 3,4-dihydroxybenzaldehyd can not only distinguish between healthy individuals and BPH patients, but also differentiate among BPH patients whether they require drug or surgical intervention.

[0041] 2. Receiver Operating Characteristic Curve Analysis for Early Diagnosis of Benign Prostatic Hyperplasia

[0042] The area under the receiver operating characteristic (AUC) curve was used to evaluate the accuracy of 3,4-Dihydroxybenzaldehy and 1-Methylhypoxanthine in the early diagnosis of benign prostatic hyperplasia. Results showed that the AUC of 3,4-Dihydroxybenzaldehy was 0.71, with a specificity of 81% and a sensitivity of 56%; the AUC of 1-Methylhypoxanthine was 0.62, with a specificity of 26% and a sensitivity of 97%. Figure 2 ).

[0043] Further combined use revealed that when 1-Methylhypoxanthine and 3,4-Dihydroxybenzaldehyde were used together for diagnosis, the AUC was 0.75, the specificity was 81%, and the sensitivity was 62%. The analytical results are shown in Table 1.

[0044] Table 1. Statistical analysis of ROC curves between BPH and Healthy populations.

[0045]

[0046] Note: AUC (Area under the curve); CI (Confidence interval); PPV (Positive predictive value); NPV (Negative predictive value).

[0047] 3. Receiver operating characteristic curve analysis of subjects with benign prostatic hyperplasia progression

[0048] The fecal metabolites can not only be used for early diagnosis of benign prostatic hyperplasia, but also be used for evaluation of benign prostatic hyperplasia progression. The area under the curve (AUC) of the receiver operating characteristic curve was used to evaluate the accuracy of 3,4-Dihydroxybenzaldehy and 3-Hydroxybenzyl alcohol in the diagnosis of benign prostatic hyperplasia progression. The results showed that the AUC of 3,4-Dihydroxybenzaldehy was 0.74, the specificity was 79%, and the sensitivity was 60%; the AUC of 3-Hydroxybenzyl alcohol was 0.77, the specificity was 74%, and the sensitivity was 71%; when the two were used together, the AUC was 0.79, the specificity was 84%, and the sensitivity was 64% (Table 2). Figure 3

[0049] Table 2. ROC curve statistical analysis of BPH conservative treatment population vs. drug or surgical treatment population

[0050]

[0051] Note: AUC (Area under the curve); CI (Confidence interval); PPV (Positive predictive value); NPV (Negative predictive value).

[0052] The above are only preferred embodiments of the present application, and do not limit the patent scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the patent protection scope of the present application.​

Claims

1. Use of a reagent for detecting a fecal metabolite in the manufacture of a product for diagnosing the occurrence of a benign prostatic hyperplasia disease, characterized in that, The fecal metabolites include a combination of 3,4-Dihydroxybenzaldehy and 1-Methylhypoxanthine.

2. Use of a reagent for detecting fecal metabolites including a combination of 3,4-Dihydroxybenzaldehy and 3-Hydroxybenzyl alcohol in the manufacture of a product for assessing the severity of benign prostatic hyperplasia, the assessment of the severity of benign prostatic hyperplasia comprising distinguishing between mild BPH - conservative treatment of subjects, moderate to severe BPH - drug treatment of subjects and subjects requiring surgical treatment.

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