Application of ethanol and pyrrole in exhaled gas as markers in preparation of products for diagnosis or auxiliary diagnosis of Alzheimer's disease

Abstract

The invention relates to the technical field of biological medicine. The invention provides application of ethanol and pyrrole in exhaled gas as markers in preparation of products for diagnosis or auxiliary diagnosis of Alzheimer's disease. The markers are ethanol and pyrrole. According to the method, a brand new AD diagnosis marker is mainly found from expired gas of a human body, an expired gas sample is easier to obtain compared with a body fluid sample, the patient and family member matching degree is higher, due to the fact that expired gas collection is completely noninvasive, the corresponding detection means is high in detection speed and easy to operate, and the method has a wider application prospect in AD patient screening.

Description

[0001] The present application is a divisional application of the original application with the application number 202411064129.9 and the filing date of August 5, 2024, and the invention name of Alzheimer's disease diagnostic marker based on volatile organic compounds in exhaled breath and its application. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to the application of ethanol and pyrrole in exhaled breath as markers in the preparation of products for diagnosing or assisting in the diagnosis of Alzheimer's disease. BACKGROUND

[0003] Early diagnosis of Alzheimer's disease (AD) provides an important opportunity for early intervention of the disease. One of the currently widely recognized AD diagnostic criteria is the 2011 NIA-AA diagnostic criteria released by the National Institute of Aging (NIA) and the Alzheimer's Association (AA) in 2011. This standard regards AD as a continuous disease process including mild cognitive impairment (MCI), and includes biomarkers in the diagnostic criteria for MCI and dementia caused by AD, which mainly includes amyloid deposition (Aβ, cerebrospinal fluid or PET detection), neuronal damage (tau, PET brain metabolism detection, structural magnetic resonance imaging, etc.).

[0004] In 2018, NIA-AA further proposed to define AD by detecting Aβ deposition and tau abnormalities biologically, to study dementia and changes before dementia under a unified biological framework, and proposed a characteristic biomarker AT(N) diagnostic framework, A for Aβ deposition, T for pathological tau protein, and (N) for neurodegeneration. The representative marker of A is the positive marker of brain Aβ-PET imaging and / or the decrease of cerebrospinal fluid Aβ42 level and the Aβ42 / Aβ40 ratio below the threshold value; the representative marker of T is the positive marker of brain tau-PET and / or the increase of cerebrospinal fluid phosphorylated tau level; the representative marker of (N) is brain atrophy shown by head MRI and / or the increase of cerebrospinal fluid total tau level and / or the decrease of brain fluorodeoxyglucose (FDG) PET metabolism. Under this framework, the diagnostic value of biomarkers in AD has been improved to a new level, and among them, PET and cerebrospinal fluid detection occupy an important position.

[0005] PET-CT can provide the function and metabolism of lesions and accurate positioning through imaging agents, and has the characteristics of sensitivity, accuracy, specificity and precision, and is relatively non-invasive and convenient. The Aβ-PET which can specifically recognize Aβ protein can effectively show the distribution of Aβ protein in the brain. However, the development of PET-CT examination and the preparation of specific imaging agents have technical threshold, and the examination cost is high, which is often difficult for patients and their families to accept. Although the detection of Aβ and tau in cerebrospinal fluid is relatively low in price and easy to operate, it is relatively invasive, and some patients have headache and other reactions after the operation, which makes patients and their families have concerns.

[0006] Therefore, the defects existing in the prior art can be summarized as follows: 1. The PET-CT detection cost is high; 2. The cerebrospinal fluid examination is an invasive examination, and the patient's cooperation degree is low; 3. The current technology pays more attention to new markers related to body fluids, and the cooperation degree and detection means are low in the process of obtaining and detecting the body fluid sample; 4. The previous related technology is not suitable for large-scale population screening.

[0007] In summary, developing a simple and non-invasive new marker has become a problem that needs to be solved in the current AD research field. SUMMARY

[0008] The purpose of the present application is to provide an Alzheimer's disease diagnostic marker based on volatile organic compounds VOCs in exhaled breath and its application.

[0009] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0010] The present application provides an Alzheimer's disease diagnostic marker based on volatile organic compounds in exhaled breath.

[0011] As a preferred, the marker is one or more of ethanol, isopropanol, chloroethane, pyrrole, n-butanol and benzene.

[0012] The present application also provides an application of the marker in a product for diagnosing or assisting in diagnosing Alzheimer's disease.

[0013] The present application also provides a product for diagnosing or assisting in diagnosing Alzheimer's disease, which contains the marker.

[0014] The present application has the following advantages:

[0015] The application provides an Alzheimer's disease diagnostic marker based on volatile organic compounds in exhaled breath, the marker being one or more of ethanol, isopropanol, chloroethane, pyrrole, n-butanol and benzene. Prior research in the field has focused on the development of markers in body fluids, such as detection of Aβ and p-tau protein in peripheral blood, detection of metabolites, detection of nerve-derived exosomes, detection of flora and metabolites in saliva, detection of metabolites in urine, etc. The present application, however, seeks new AD diagnostic markers in human exhaled breath. Exhaled breath samples are easier to obtain than body fluid samples, and patients and their families have a higher degree of cooperation. Since exhaled breath collection is completely non-invasive, the corresponding detection method is fast, easy to operate, and has a wider application prospect in AD patient screening. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a flowchart of the screening process for Alzheimer's disease diagnostic marker VOC molecules.

[0017] Figure 2 Figure 2 shows the distribution and diagnostic performance of the six different VOC molecules in dataset 1 and 2, wherein Figure 2 Figure 2A shows the distribution of VOC molecules in dataset 1 and 2; Figure 2 Figure 2B shows the diagnostic performance of the six VOC molecules in dataset 1 and 2, dataset 3 (Note: the peak area in the figure reflects the VOC content; AUC, area under curve, is the area under the ROC curve).

[0018] Figure 3 Figure 3 shows the relationship between the six VOC molecules and the cognitive function level and the plasma marker p-tau181, wherein Figure 3 Figure 3A shows that the six VOC molecules are all correlated with the Mini-mental State Examination (MMSE) score after correction for education level; Figure 3 Figure 3B shows that two VOC molecules (n-butanol m / z = 74, benzene m / z = 78) are correlated with the Clinical Dementia Rating (CDR) score; Figure 3 Figure 3C shows the correlation of five VOC molecules (ethanol m / z = 46, chloroethane m / z = 64, pyrrole m / z = 67, n-butanol m / z = 74, benzene m / z = 78) with the plasma marker p-tau181. DETAILED DESCRIPTION

[0019] The application provides an Alzheimer's disease diagnostic marker based on volatile organic compounds in exhaled breath, the marker being one or more of ethanol, isopropanol, chloroethane, pyrrole, n-butanol and benzene.

[0020] In the present application, the marker is further preferably one or both of ethanol and pyrrole.

[0021] The present application also provides a product for diagnosing or assisting in the diagnosis of Alzheimer's disease, the product comprising the marker, which is preferably one or more of ethanol, isopropanol, chloroethane, pyrrole, n-butanol and benzene.

[0022] In the present application, the marker is further preferably one or both of ethanol and pyrrole.

[0023] The present application also provides a product for diagnosing or assisting in the diagnosis of Alzheimer's disease, the product comprising the marker, which is preferably one or more of ethanol, isopropanol, chloroethane, pyrrole, n-butanol and benzene.

[0024] In the present application, the marker is further preferably one or both of ethanol and pyrrole.

[0025] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0026] Example 1 Identification of 6 different VOC molecules in the exhaled breath of Alzheimer's disease patients

[0027] After excluding subjects with a history of respiratory diseases and other neurodegenerative diseases, a total of 861 subjects from Xiangya Hospital of Central South University and Jili community cohort in Liuyang City, Changsha City, Hunan Province were recruited, including AD patients and controls. Their demographic characteristics (including gender, age, education level, smoking, drinking, MMSE score, etc.) were recorded, as shown in the following table. The exhaled breath samples were collected according to the aforementioned standard, and the blood samples of the subjects were collected to obtain the plasma component (all operations and processes were approved by the Ethics Committee of Xiangya Hospital of Central South University).

[0028] Table 1 Demographic characteristics statistical table

[0029]

[0030]

[0031] Note: (IQR: interquartile range, interquartile range)

[0032] To strictly demonstrate the reproducibility of VOC for diagnosing AD, the 861 subjects were divided into three data sets (training set, validation set and test set) according to the ratio of 6:2:2. Figure 1), wherein dataset 1 contains 30 AD patients and 30 normal controls confirmed by Aβ-PET or cerebrospinal fluid (Aβ42, Aβ40, t-tau, p-tau); dataset 2 contains 82 AD patients and 82 normal controls diagnosed as probable AD by 2011 NIA-AA diagnostic criteria; dataset 3 contains 31 AD patients and 606 normal controls diagnosed as probable AD by 2011 NIA-AA diagnostic criteria from the Chuxiong community cohort. Dataset 1 and 2 are used to identify VOC molecules in exhaled breath of AD patients that can be used as diagnostic markers, and dataset 3 is used for community cohort validation to test the effectiveness of exhaled breath detection in screening AD in community cohorts.

[0033] After collection of exhaled breath, the VOC components were detected within 4 hours using a mass spectrometer HPPI-TOFMS developed by Shenzhen Buxile Biological Technology Co., Ltd. Through the above detection and analysis, it was found that the 6 VOC molecules (including ethanol m / z = 46, isopropanol m / z = 60, chloroethane m / z = 64, pyrrole m / z = 67, n-butanol m / z = 74, benzene m / z = 78) found in dataset 1 were all verified in dataset 2 Figure 2 A). The diagnostic performance of these 6 VOC molecules in datasets 1 and 2 was high, with an AUC of more than 0.7; ethanol (m / z = 46) and pyrrole (m / z = 67) had the highest diagnostic performance when used alone Figure 2 B). This suggests that the 6 VOC components in exhaled breath can be used as markers for AD diagnosis, and ethanol (m / z = 46) and pyrrole (m / z = 67) have the best diagnostic performance.

[0034] Community validation: The 6 VOC molecules found in datasets 1 and 2 were used in dataset 3 from the community cohort, and ethanol (m / z = 46) and pyrrole (m / z = 67) still had excellent diagnostic performance Figure 2 B), further indicating that the 6 VOC molecules can be used as markers for diagnosing AD and may be used as new markers for community AD screening.

[0035] Example 26 Correlation between 6 VOC molecules and cognitive function of subjects

[0036] The subjects in the present case were subjected to cognitive function evaluation by a neuropsychological evaluation expert, including MMSE and CDR. Linear correlation analysis found that the above-mentioned 6 VOC molecules were highly correlated with the MMSE score after correcting the education level (P<0.05), which suggests that the decrease in the abundance of the 6 VOC molecules in the exhaled breath is related to the decrease in cognitive function. In addition, the CDR scale for rating the severity of dementia also shows a certain correlation. Two VOC molecules (n-butanol m / z=74, benzene m / z=78) are highly correlated with the CDR score (P<0.05), indicating that VOC detection can be used to assess the severity of dementia.

[0037] Correlation between Example 3 and plasma marker p-tau181

[0038] In order to compare the VOC markers found in the present application with the traditional and recognized AD diagnostic markers, plasma p-tau181 detection was performed on some subjects in Example 1 of the present application.

[0039] The p-tau181 detection was performed using the internationally recognized single molecular array technology (Single Molecular Array, Simoa), using the simoa SR-X instrument, to detect p-tau181 in the plasma of the subjects. The obtained p-tau181 was subjected to correlation analysis with the VOC molecules in the exhaled breath of the corresponding subjects, and 5 VOC molecules (ethanol m / z=46, chloroethane m / z=64, pyrrole m / z=67, n-butanol m / z=74, benzene m / z=78) were highly correlated with the plasma marker p-tau181 (p<0.05), suggesting that the VOC molecules have high consistency with the classic marker p-tau181.

[0040] From the above examples, the present application provides an Alzheimer's disease diagnostic marker based on volatile organic compounds VOCs in exhaled breath and its application, the marker being one or more of ethanol, isopropanol, chloroethane, pyrrole, n-butanol and benzene. The present application mainly seeks new AD diagnostic markers from human exhaled breath, which is easier to obtain than body fluid samples, and has higher patient and family compliance. Since the exhaled breath collection is completely non-invasive, the corresponding detection method is fast, easy to operate, and has a broader application prospect in AD patient screening.

[0041] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A diagnostic biomarker for Alzheimer's disease based on volatile organic compounds in exhaled breath, characterized in that, The markers are ethanol and pyrrole.

2. The use of the biomarker of claim 1 in the preparation of products for the diagnosis or auxiliary diagnosis of Alzheimer's disease.

3. A product for diagnosing or assisting in the diagnosis of Alzheimer's disease, characterized in that, The product includes the marker as described in claim 1.

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