Use of aromatic l-amino acid decarboxylase as marker in detection product for hepatic encephalopathy

Abstract

Use of an aromatic L-amino acid decarboxylase as a marker in a detection product for hepatic encephalopathy. It has been confirmed that the abundance of aromatic L-amino acid decarboxylase genes encoded by gut microbiota, as well as the product of aromatic L-amino acid decarboxylase, phenylethylamine, are increased in patients with hepatic encephalopathy; and compared with blood ammonia, hepatic encephalopathy can be more accurately diagnosed by means of phenylethylamine. This suggests that the occurrence of hepatic encephalopathy and the degree of progression of the disease can be quickly confirmed by detecting the patients' feces and serum, which increases the demand for clinical detection of hepatic encephalopathy. It has been confirmed that the baseline serum phenylethylamine level can effectively predict the incidence of hepatic encephalopathy at 3 months after TIPS surgery, which provides a detection means for early warning of hepatic encephalopathy.

Description

Application of aromatic amino acid decarboxylases as biomarkers in detection products for hepatic encephalopathy

[0001] This application claims priority to Chinese Patent Application No. 2024106666804, filed on May 27, 2024, entitled "Application of Aromatic Amino Acid Decarboxylase as a Marker in a Detection Product for Hepatic Encephalopathy", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of biodetection technology, and in particular to the application of an aromatic amino acid decarboxylase as a biomarker in a detection product for hepatic encephalopathy. Background Technology

[0003] Hepatic encephalopathy (HE) is a serious complication of end-stage liver disease, a major cause of hospitalization for patients with end-stage liver disease, and has an extremely high mortality rate and very poor short-term prognosis. Timely prediction and diagnosis of hepatic encephalopathy can help with preventative or early medication, which are effective measures to reduce hospitalization and mortality rates among liver disease patients.

[0004] The inventors recognized that currently used clinical neuropsychological scoring scales are complex to operate and highly subjective, failing to meet the requirements for early warning. While blood ammonia testing is widely used clinically, it lacks a significant correlation with the occurrence, severity, and prognosis of hepatic encephalopathy, and is not the optimal choice as an early warning biomarker. Discovering new and more precise early warning biomarkers for hepatic encephalopathy would be crucial for early warning and appropriate prevention in high-risk patients, significantly reducing hospitalization and mortality rates in patients with cirrhosis, and thus represents a significant clinical need. Summary of the Invention

[0005] The purpose of this application is to provide an application of aromatic amino acid decarboxylase (AADC) as a biomarker in a detection product for hepatic encephalopathy, targeting bacterial aromatic amino acid decarboxylase (AADC) and its product phenylethylamine.

[0006] To achieve the above technical objectives, the technical solution adopted in this application is as follows:

[0007] Application of aromatic amino acid decarboxylases as biomarkers in detection products for hepatic encephalopathy.

[0008] Alternatively, the aromatic amino acid decarboxylase is used as a marker, and the detection items include at least one of the following:

[0009] The abundance of genes encoding aromatic amino acid decarboxylases in the sample was detected.

[0010] The microbial species and their abundance that produce aromatic amino acid decarboxylases in the samples were detected.

[0011] The concentration of the metabolites of aromatic amino acid decarboxylases in the sample was measured.

[0012] Alternatively, the sample may be derived from feces and / or serum.

[0013] Specifically, the sample processing procedure for feces is as follows:

[0014] Genomic DNA was extracted from the sample, and its concentration and purity were detected.

[0015] A DNA library was prepared based on the extracted genomic DNA;

[0016] The abundance of genes encoding aromatic amino acid decarboxylases was quantitatively analyzed using the DNA library described above.

[0017] The abundance of microorganisms producing aromatic amino acid decarboxylases was quantitatively analyzed using the DNA library.

[0018] The DNA library was used to identify the species of microorganisms that produce aromatic amino acid decarboxylases.

[0019] Furthermore, compared with the control sample, the abundance of genes encoding aromatic amino acid decarboxylases was increased in the test sample.

[0020] Furthermore, compared with the control sample, the microorganism with the highest abundance of aromatic amino acid decarboxylases in the test sample was Ruminococcus gnavus.

[0021] More specifically, the sample processing procedure for serum-derived samples is as follows:

[0022] Purify the target substance in the serum sample;

[0023] The concentration of the target substance was confirmed using a mass spectrometry-chromatography system;

[0024] The target substance is a metabolite of aromatic amino acid decarboxylase.

[0025] Specifically, the target substance is phenylethylamine.

[0026] Furthermore, compared with the control sample, the concentration of the target substance in the test sample increased.

[0027] Furthermore, the abundance of genes encoding aromatic amino acid decarboxylases in the samples, the microbial species that produce aromatic amino acid decarboxylases and their corresponding abundances are significantly positively correlated with the concentrations of the metabolites of aromatic amino acid decarboxylases.

[0028] Alternatively, the concentration difference of the metabolites of aromatic amino acid decarboxylases can be used to differentiate between cirrhosis with hepatic encephalopathy and cirrhosis without hepatic encephalopathy.

[0029] This application confirms the abundance of the AADC gene encoded by gut microbiota in patients with hepatic encephalopathy and the increased presence of the AADC product phenylethylamine in these patients. Furthermore, phenylethylamine is more accurate in diagnosing hepatic encephalopathy than blood ammonia, suggesting that the presence and progression of hepatic encephalopathy can be rapidly confirmed by testing patients' stool and serum, thereby increasing the demand for clinical testing of hepatic encephalopathy.

[0030] This application also confirms that baseline serum phenylethylamine levels can effectively predict the incidence of hepatic encephalopathy 3 months after Tips surgery, providing a detection method for early warning of hepatic encephalopathy. Attached Figure Description

[0031] Figure 1 shows the results of metagenomic analysis of fecal samples using this application.

[0032] Figure 2 shows the results of metabolomics analysis of serum samples using this application.

[0033] Figure 3 shows the ROC curves of monoamines and ammonia in blood using the method described in this application.

[0034] Figure 4 shows the serum phenylethylamine concentration in patients who developed hepatic encephalopathy after TIPS compared with that in patients who did not, using the method described in this application.

[0035] Figure 5 shows the ROC curve for predicting the occurrence of hepatic encephalopathy one month postoperatively using the baseline serum phenylethylamine level of this application. Detailed Implementation

[0036] The present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] The relevant technical terms in this application are defined as follows:

[0038] Hepatic encephalopathy (HE): Hepatic encephalopathy (HE), also known as hepatic coma, is a syndrome of central nervous system dysfunction caused by severe liver disease and based on metabolic disorders. Its main clinical manifestations are altered consciousness, behavioral abnormalities, and coma.

[0039] Gene abundance: In metagenomic analysis, the ratio of a specific gene or a specific type of gene to all genes in gut bacteria.

[0040] Monoaminergic neurotransmitters: neurotransmitters and neuromodulators containing one amino group, linked to an aromatic ring via a double carbon chain (such as -CH2-CH2-), including serotonin, phenylethylamine, etc.

[0041] Phenylacetamine, molecular formula as follows:

[0042] This application initially included 122 cases for cross-sectional comparison: cirrhosis without hepatic encephalopathy (n=41); cirrhosis with hepatic encephalopathy (n=40); and healthy controls (n=44). The specific inclusion and exclusion criteria for cirrhosis were as follows: 1) Patients diagnosed with cirrhosis based on clinicopathological features—including a history of liver disease, endoscopic findings, or radiological examination results (the clinical diagnosis of cirrhosis was determined by two independent hepatologists in a blinded manner; both experts must agree on the diagnosis of clinical cirrhosis for inclusion); 2) Patients had at least 6 months of clinical evidence of chronic liver disease, the etiology of which may include: alcoholic liver disease (history of alcohol consumption: more than 2 ounces of ethanol per day for men, or 1 ounce of ethanol per day for women (for more than 10 years) without other known causes of liver disease), non-alcoholic fatty liver disease, and viral hepatitis B or C (via HCV RNA or HBV). DNA testing or autoimmune liver disease (based on the International Autoimmune Score). Hepatic encephalopathy is carefully evaluated by two independent hepatologists, with the specific criteria being: exclusion of sudden changes in mental status due to other causes. Inclusion criteria for healthy subjects are as follows: 1) Subjects from a health checkup center, matched for age and sex with patients with cirrhosis; 2) No clinically diagnosed chronic disease. Exclusion criteria include: 1) Received antibiotic, probiotic, or proton pump inhibitor treatment within one month prior to sample collection; 2) Underwent gastrointestinal surgery or colonoscopy within six months prior to sample collection; 3) Existing infection.

[0043] Stool (cirrhosis without hepatic encephalopathy, n=26; cirrhosis with hepatic encephalopathy, n=21; healthy controls, n=10) and serum (cirrhosis without hepatic encephalopathy, n=37; cirrhosis with hepatic encephalopathy, n=40; ​​healthy controls, n=44) samples were collected from the above subjects and subjected to metagenomic and metabolomics analysis.

[0044] The methods for metagenomic sample detection and analysis of fecal samples are as follows:

[0045] 1) Genomic DNA was extracted, and its integrity and purity were then assessed using a 1% agarose gel. DNA concentration and purity were simultaneously measured using a Qubit 3.0 (Thermo Fisher Scientific, Waltham, USA) and a Nanodrop One (Thermo Fisher Scientific, Waltham, USA).

[0046] 2) DNA Library Preparation and Sequencing: Sequencing libraries were generated using the ALFA-SEQ DNA Library Prep Kit according to the manufacturer's instructions, and index codes were added. Library quality was assessed using a Qubit 4.0 fluorometer (Life Technologies, Grand Island, NY) and a Qsep400 high-throughput nucleic acid and protein analysis system (Houze Biological Technology Co., Hangzhou, China). Finally, the library was sequenced on an Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads.

[0047] 3) Data Quality Control and Analysis: Kneaddata was used to remove low-quality bases, host DNA (human DNA), and qPCR repeats; FastQC was used to check sequence quality; ShortBred (version 0.9.5) was used to quantify AADC gene abundance: First, AADC sequences were clustered with 95% similarity, and then the UniRef90 database was used to identify short marker sequences. Finally, these marker sequences were quantified in metagenomic samples, and the quantitative data were standardized using RPKM (Reads Per Kilobase per Million mapped reads). Simultaneously, MetaPhlAn2 (version 2.7.7) was used to quantify species abundance.

[0048] Further, targeted metabolomics analysis was performed on serum samples, targeting phenylethylamine, a metabolite of aromatic amino acid decarboxylase. Sample processing and detection methods were as follows: Serum samples were diluted in methanol (1:4) and vortexed for 10 minutes. The samples were then centrifuged to collect the supernatant (14,000 rpm, 10 minutes, 4°C) and filtered through a 0.22 μm filter. The flow-through was incubated overnight at -80°C before HPLC-MS / MS measurement of the monoamine level. These steps were used to purify the target substance. Next, the target substance was detected using a mass spectrometry-chromatography system. Specifically, 100 μL of sample was separated at 40°C using a Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) on a Prelude SPLC system. The following mobile phases were used: (A) 1 mM ammonium formate and 0.1% aqueous formic acid, and (B) 0.1% formic acid acetonitrile solution. The gradients were 0 min (5% B), 1 min (85% B), 5 min (95% B), 5.5 min (95% B), and 6 min (5% B), with a flow rate of 0.3 mL / min. MS analysis was performed using multiple reaction monitoring (MRM) on a TSQ Quantiva triple quadrupole mass spectrometer. A positive-mode H-ESI source was used. The parameters are as follows: ion pair (122.152 m / z, 77.04 m / z) 28.55 V, ion pair (138.152 m / z, 77.04 m / z) 27.494 V, ion pair 10.253 V (161.152 m / z, 144.11 m / z), 10 V for ion pair (177.152 m / z, 160.054 m / z). Peak detection was visualized using Thermo Xcalibur software. The peak and corresponding concentration of phenethylamine in each serum sample were confirmed.

[0049] Figure 1 shows that the abundance of the AADC gene and AADC-expressing bacteria was increased in patients with hepatic encephalopathy and correlated with serum phenylethylamine levels. (A) Alpha diversity of fecal microbiota in the control group (n=10), patients with cirrhosis (n=26), and patients with hepatic encephalopathy (n=21). Alpha diversity was measured by the Shannon index. (B) β-diversity (Bray-Curtis distance, measured by pairwise PERMANOVA) among the control group (n=10), patients with cirrhosis (n=26), and patients with hepatic encephalopathy (n=21). (C) LEfSe linear discriminant analysis plot of gut microbiota in the control group (n=10), patients with compensated cirrhosis (n=26), and patients with hepatic encephalopathy (n=21). (DE) Gene abundance of AADC (C) or total AADC (D) derived from Ruminococcus gnavus in the healthy control group (n=10), patients with compensated cirrhosis (n=26), or patients with hepatic encephalopathy (n=21). (F) Clinical and microbiome factors associated with serum phenylethylamine levels (only the top 5 species are shown). This association was analyzed using S-phenylethylamine RMAN correlation analysis. *p<0.05; **p<0.01.

[0050] Metagenomic analysis revealed significant differences in α- and β-diversity in the gut microbiota of healthy subjects, non-hepatic encephalopathy cirrhosis patients, and hepatic encephalopathy patients (Figure 1A-B). The species *Ruminococcus gnavus*, which primarily encodes the AADC gene, was significantly enriched in hepatic encephalopathy patients (Figure 1C). The abundance of AADC genes derived from gut microbiota and from *Ruminococcus gnavus* gradually increased among healthy subjects, non-hepatic encephalopathy cirrhosis patients, and hepatic encephalopathy patients (Figure 1D-E). Importantly, the abundance of *Ruminococcus gnavus* species and AADC genes was significantly positively correlated with serum phenethylamine levels (Figure 1F), suggesting that serum phenethylamine concentration can reflect the progression of hepatic encephalopathy.

[0051] Figures 2 and 3 show that phenylethylamine levels were increased in the serum of patients with hepatic encephalopathy. Specifically, (AF) quantifications of phenylethylamine (A), tryptamine (B), tyramine (C), serotonin (D), octopamine (E), and ammonia (F) in the serum of healthy controls (n=44), patients with cirrhosis (n=37), or patients with hepatic encephalopathy (n=40). (Figure 3, G) Accuracy of ROC curves for serum monoamines and ammonia in distinguishing between hepatic encephalopathy and cirrhosis. **p<0.01, ***p<0.001.

[0052] Table 1. Demographic and clinical parameters of patients with liver disease

[0053] (Demographic and clinical parameters of patients with liver disease)

[0054] Metabolomics analysis revealed that serum phenylethylamine levels in HE patients were significantly higher than in healthy controls and cirrhotic patients without hepatic encephalopathy compared to other monoamines (Figures 2A-F, Table 1). Although serum ammonia levels were also elevated in patients with hepatic encephalopathy, receiver operating characteristic (ROC) curve analysis showed that phenylethylamine was superior to serum ammonia in distinguishing between hepatic encephalopathy and cirrhotic patients without hepatic encephalopathy (AUC: 0.681 vs. 0.743) (Figure 3, Table 1).

[0055] To further verify the predictive ability of serum phenylethylamine for the risk of hepatic encephalopathy (HEEE), this application also conducted a longitudinal comparison, including 60 patients with cirrhosis who underwent transjugular intrahepatic portosystemic shunt (TIPS) surgery (HEEE is the most common complication after TIPS). Detailed clinical information and peripheral blood samples were collected before surgery. Patients were followed up for 3 months post-surgery, and the incidence of HEEE was observed and recorded. Serum phenylethylamine concentration was measured using the aforementioned metabolomics methods. Results showed that 18 patients developed HEEE within 3 months post-surgery. Patients with HEEE had significantly higher baseline serum phenylethylamine concentrations than those without HEEE (Figure 4); and the area under the ROC curve for diagnosing HEEE using baseline serum phenylethylamine levels reached 0.70 (Figure 5), significantly superior to other clinical indicators.

[0056] In summary, this application of aromatic amino acid decarboxylases as biomarkers in the detection product for hepatic encephalopathy (HEEE) confirms the increased abundance of the AADC gene encoded by intestinal flora and the increased presence of the AADC product phenylethylamine in HEEE patients. Furthermore, phenylethylamine, compared to serum ammonia, provides a more accurate diagnosis of HEEE, suggesting that the presence and progression of HEEE can be rapidly confirmed by testing patients' stool and serum, thus increasing the demand for clinical detection of HEEE. This application also confirms that baseline serum phenylethylamine levels can effectively predict the incidence of HEEE 3 months after Tips surgery, providing a detection method for early warning of HEEE.

[0057] The above embodiments are preferred embodiments of this application, but are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and shall be included within the protection scope of this application.

Claims

1. Use of aromatic amino acid decarboxylase as a marker in a product for detecting hepatic encephalopathy.

2. The use of claim 1, wherein the aromatic amino acid decarboxylase as a marker, the detection item comprises at least one of the following: detecting the abundance of the gene encoding aromatic amino acid decarboxylase in the sample; detecting the species of microorganisms producing aromatic amino acid decarboxylase and the corresponding abundance in the sample; detecting the concentration of the metabolic product of aromatic amino acid decarboxylase in the sample.

3. The use of claim 2, wherein the sample is derived from feces and / or serum.

4. The use of claim 3, wherein the sample derived from feces is processed as follows: extracting genomic DNA in the sample and detecting the concentration and purity of the genomic DNA; preparing a DNA library from the extracted genomic DNA; quantitatively analyzing the abundance of the gene encoding aromatic amino acid decarboxylase using the DNA library; quantitatively analyzing the abundance of the microorganism producing aromatic amino acid decarboxylase using the DNA library; confirming the species of the microorganism producing aromatic amino acid decarboxylase using the DNA library.

5. The use of claim 4, wherein the abundance of the gene encoding aromatic amino acid decarboxylase in the test sample is increased compared to the control sample.

6. The use of claim 4, wherein the microorganism with the highest abundance producing aromatic amino acid decarboxylase in the test sample is Ruminococcus gnavus compared to the control sample.

7. The use of claim 3, wherein the sample derived from serum is processed as follows: purifying the target substance in the serum sample; confirming the concentration of the target substance using a mass spectrometry-chromatography system; the target substance is the metabolic product of aromatic amino acid decarboxylase.

8. The use of claim 7, wherein the target substance is phenethylamine.

9. The use of claim 7, wherein the concentration of the target substance in the test sample is increased compared to the control sample.

10. The use of claim 2, wherein the abundance of the gene encoding aromatic amino acid decarboxylase, the species of microorganism producing aromatic amino acid decarboxylase and the corresponding abundance, and the concentration of the metabolic product of aromatic amino acid decarboxylase in the sample are significantly positively correlated.

11. The use of claim 2, wherein the concentration difference of the metabolic product of aromatic amino acid decarboxylase is used to distinguish cirrhosis with hepatic encephalopathy from cirrhosis without hepatic encephalopathy.

Images