Application of 27-hydroxycholesterol to preparation of product for diagnosing schizophrenia

[0058] Embodiment 1: the collection of peripheral blood and the method for measuring the concentration of peripheral blood 27OHC

[0059] 1. According to the experimental protocol and diagnostic criteria that have passed the ethical review, the healthy control population (HC), the genetically high risk of schizophrenia (first-degree relatives of the patient, FDR), the clinical ultra-high risk (UHR) and the patients with schizophrenia ( SCZ) 4 groups with a total of 312 people. The entry criteria for each group are as follows:

[0060] Healthy controls: using the Structured Clinical Interview for DSM Disorders-Fourth Edition (DSM-IV) clinical interview (Structured Clinical Interview for DSM-IV Axis I disorders-Patient Edition, SCID-I/P ) and SIPS screening showed no mental illness and no family history of mental illness.

[0061] Inclusion criteria for patients with schizophrenia: in line with the diagnostic criteria of the fourth edition of the American Diagnostic and Statistical Manual of Mental Disorders (DSM-IV), without a history of clearly diagnosed mental illness, nervous system and other serious physical diseases except schizophrenia; Family history of neurological disorders. Exclude those who are extremely excited, impulsive, and uncooperative; patients who have participated in other research treatments or received MECT treatment within three months. First-degree relatives of patients who exclude neurological, psychiatric and other serious physical diseases can be included in the genetic high-risk group.

[0062] One or more of the three conditions that meet the criteria of psychotic risk syndrome after screening by the psychiatric risk syndrome stereotyped interview (SIPS).

[0063] All subjects were 14-55 years old, and pregnant or breastfeeding women, those with a history of food and drug allergy, those who could not discontinue drug use due to other physical diseases, those with a history of alcohol and drug abuse, and those with a history of head trauma were excluded.

[0064] The patients and normal subjects were screened using the DSM-IV Clinical Interview (Structured Clinical Interview for DSM Disorders, SCID), and the clinical psychopathological symptoms of the patients were assessed using the Positive and Negative Scale (PANSS). At the same time, 5 ml of peripheral blood was drawn into an EDTA anticoagulant tube, centrifuged at 3000 rpm for 10 minutes, and the upper layer of plasma was separated, and immediately aliquoted, and stored in a -80°C ultra-low temperature refrigerator for future use.

[0065] 2. After the plasma is extracted, the 27OHC content is detected by high performance liquid chromatography-mass spectrometry. The specific method is as follows:

[0066] (1) 5% bovine serum albumin solution is used to prepare a standard solution with a concentration of 27OHC of 1-2000 ng/ml, and 5% bovine serum albumin solution is used as a blank sample.

[0067] (2) Add 50μl standard/blank sample/plasma to be tested in 1.5ml EP tube, then add 50μl internal standard (D5 deuterium cholesterol + D7 deuterium cholesterol 50ng/ml), then add 200μl acidic buffer (50mM ammonium acetate, 1% formic acid) and 1 ml methyl tert-butyl ether.

[0068] (3) After fully mixing, put the sample in a -80°C refrigerator, and after the water phase in it cools and crystallizes, transfer 0.5ml of methyl tert-butyl ether phase (supernatant) to a 1.5ml centrifuge tube .

[0069] (4) Dry the methyl tert-butyl ether at 35°C with nitrogen.

[0070] (5) Add 50 μl of derivatization reagent (63mg N,N'-diisopropylcarbimide, 62mg nicotinamide and 61mg 4-dimethylaminopyridine dissolved in 5ml chloroform) to each EP tube, Heat mixing at 35°C for 2 hours.

[0071] (6) Dry dichloromethane with 35°C nitrogen gas.

[0072] (7) After reconstitution in 100 μl of methanol, inject the sample for detection.

[0073] In the following examples, the method described in this example was used to collect peripheral blood and measure the concentration of 27OHC in peripheral blood.

[0074] Example 2: Peripheral blood 24OHC or 27OHC is used as a biomarker for the diagnosis of schizophrenia

[0075] 1. A total of 312 gender-matched subjects were included in this study. The basic demographic information and patient symptom assessment of each group are shown in Table 2.

[0076] Table 2: Basic demographics of subjects and evaluation of clinical symptoms of patients

[0077]

[0078] Note: HC: healthy controls; FDR: first-degree relatives of schizophrenia; UHR: clinical ultra-high risk of schizophrenia; SCZ: patients with schizophrenia; SIPs: schizophrenia risk syndrome structured interviews; PANSS: positive and negative symptoms scale; P: positive symptoms; N: negative symptoms; G: general psychotic symptoms: T: total score; NA: missing value.

[0079] After the detection of plasma 27OHC, it was found that compared with the healthy control group, the plasma 27OHC level of patients with schizophrenia was significantly lower ( figure 1 , p<0.0001), which suggested that the cholesterol metabolism was significantly reduced in patients, which may become a potential biomarker for the diagnosis of schizophrenia. However, compared with the healthy control group, first-degree relatives of schizophrenia and clinical ultra-high-risk groups did not change significantly ( figure 1 ), indicating that the reduction of 27OHC in schizophrenia does not appear before the onset. The results of correlation analysis showed that the clinical symptoms of schizophrenic patients were significantly correlated with the level of 27OHC ( Figure 2-1 to Figure 2-4 ), where 27OHC is negatively correlated with positive symptoms ( Figure 2-1 ), which further proved the relationship between the change of 27OHC level and the occurrence and development of the disease. The receiver operating characteristic curve (Receiver Operating Characteristic, ROC) curve ( image 3 ), the area under the curve was 0.741, and the diagnostic sensitivity and specificity were 48.0% and 88.9%, respectively.

[0080] After the detection of plasma 24OHC, it was found that compared with the healthy control group, the plasma 24OHC of patients with schizophrenia increased significantly ( Figure 4, p<0.05), which suggested that the cholesterol metabolism in the brain of patients was significantly increased, which may become a potential biomarker for the diagnosis of schizophrenia. The results of correlation analysis showed that the clinical symptoms of schizophrenia patients were significantly correlated with the level of hydroxycholesterol ( Figure 5-1 to Figure 5-4 ), among them, 24OHC was positively correlated with negative symptoms and PANSS total score, which further proved the relationship between the change of hydroxycholesterol level and the occurrence and development of the disease. Combined with the subject's family history indicators, the concentration of 24OHC was used as a diagnostic indicator to make a receiver operating characteristic curve (Receiver Operating Characteristic, ROC) curve ( Image 6 ), the area under the curve was 0.703, and the diagnostic sensitivity and specificity were 71.6% and 61.1%, respectively.

[0081] As can be seen from the above examples, the present invention diagnoses the condition of patients with schizophrenia by selecting peripheral blood 27OHC or 24OHC as a biomarker, and realizes an objective diagnosis method for schizophrenia, which has a higher impact on the diagnosis of schizophrenia. sensitivity and specificity.