CN110702929B

CN110702929B - Use of 27-hydroxycholesterols for the preparation of products for diagnosing schizophrenia

Info

Publication number: CN110702929B
Application number: CN201910837972.9A
Authority: CN
Inventors: 李人; 孙作厘; 王传跃
Original assignee: Beijing Anding Hospital
Current assignee: Beijing Anding Hospital
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2023-08-15
Anticipated expiration: 2039-09-05
Also published as: CN110702929A

Abstract

The invention provides the use of 27-hydroxycholesterol or a reagent for detecting 27-hydroxycholesterol in the preparation of a reagent or kit for diagnosing schizophrenia. The 27-hydroxy cholesterol can be used as a biomarker for diagnosing the schizophrenia, has higher sensitivity and specificity for diagnosing the schizophrenia, can diagnose and predict the level of the 27-hydroxy cholesterol in the peripheral blood only by measuring one index, and is convenient to popularize and use.

Description

Use of 27-hydroxycholesterols for the preparation of products for diagnosing schizophrenia

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of brain cholesterol metabolite 27-hydroxycholesterol (27 OHC) in diagnosis of schizophrenia and application of preparation of corresponding reagents.

Background

Schizophrenia is a common mental disorder and is a major public health problem affecting the development of economy and society. The cause of the schizophrenia is unknown, the prognosis is poor, the disability rate is high, the schizophrenia is one of the most serious and complex neuropsychiatric diseases, and the clinical symptoms of the schizophrenia mainly comprise hallucinations, delusions, neurophysiologic dysfunction, neurocognitive dysfunction and the like. In recent years, development of discipline technologies such as neuroimaging, genomics, proteomics, and immunohistochemistry has provided an important tool for understanding the etiology of schizophrenia at the gene and cell level, but the pathophysiological mechanism of schizophrenia has not yet been clarified.

At present, most mental diseases, especially schizophrenia, lack specific pathological diagnosis indexes, clinical diagnosis can be judged by clinical experience only by means of symptom description and duration and after other diseases are eliminated, and diagnosis delay or misdiagnosis is very unfavorable for prognosis of the diseases. Therefore, the search for a biomarker with high stability, specificity and sensitivity has become an important direction for basic and clinical studies of schizophrenia.

Epidemiological results show that 60% of schizophrenic patients die from somatic diseases, while the increase in the incidence of cardiovascular disease is particularly pronounced, accounting for more than one third of the causes of mortality in schizophrenic patients. It is reported that the risk of coronary artery disease increases 1.2 to 3.6 times and the risk of sudden cardiac death increases 6 to 8 times, 3 times that of the general population in schizophrenic patients. The cardiovascular metabolism risk spectrum of patients with schizophrenia is complex, and particularly, with the wide application of atypical antipsychotics, the risk of obesity, hyperglycemia, type II diabetes, lipid metabolism disorder and the like of patients with schizophrenia is obviously increased. This suggests that schizophrenia itself may be associated with lipid metabolism disorders, and that antipsychotic therapy may also cause lipid metabolism disorders, suggesting an important role for lipid metabolism disorders in schizophrenia.

Disclosure of Invention

The technical problem to be solved by the invention is that no blood biomarker is uniformly approved in the aspect of diagnosing the schizophrenia at present, that is, a diagnosis method for objectively judging the schizophrenia is lacked in the prior art.

In order to solve the above technical problems, it is necessary to screen biomarkers related to schizophrenia, thereby helping to reveal the underlying pathophysiological mechanisms and develop objective diagnostic methods. Researchers believe that peripheral blood total cholesterol changes abnormally in schizophrenic patients and after treatment, and can be used as a biomarker, but the conclusions of different researchers are inconsistent, and cholesterol cannot pass through the blood brain barrier, so that the level change and central damage of the cholesterol cannot be directly related. It is therefore also difficult to use as an objective biomarker for diagnosis of schizophrenia.

The present inventors have found that peripheral cholesterol cannot freely pass through the blood brain barrier into the central nervous system, but that the cholesterol metabolite 27-hydroxycholesterol (27 OHC) can freely pass through the blood brain barrier, and that changes in its concentration can also cause central cholesterol metabolic disorders. Thus, cholesterol metabolite 27OHC in peripheral blood has potential as a biomarker associated with depression.

Further studies by the present inventors have found that 27OHC concentrations in peripheral blood plasma are significantly reduced in schizophrenic patients relative to healthy persons, and that clinical symptoms in schizophrenic patients are significantly correlated with hydroxycholesterols levels. Plasma 27OHC levels can therefore be used as biomarkers for diagnosis of schizophrenic patients.

Based on the research findings, the invention provides the following technical scheme:

in one aspect, the invention provides the use of 27-hydroxycholesterol or a reagent for detecting 27-hydroxycholesterol in the manufacture of a reagent or kit for diagnosing schizophrenia.

Preferably, the use as described above, wherein the kit is selected from a biochemical diagnostic kit, an immunodiagnostic kit or a molecular diagnostic kit.

Preferably, the use as described above, wherein the kit is selected from one or more of Western blot kit, enzyme-linked immunosorbent assay (ELISA) kit, radioimmunoassay (RIA) kit, radioimmunodiffusion kit, two-dimensional biphasic immunodiffusion kit, rocket immunoelectrophoresis kit, immunohistochemical staining kit, immunoprecipitation assay kit, complement fixation assay kit, fluorescence Activated Cell Sorting (FACS) kit, aptamer chip kit, microarray kit, and protein chip kit.

Preferably, the use as described above, wherein the kit comprises a schizophrenia rating scale; preferably, the schizophrenia evaluation scale is selected from one or more than two of a karcaliy schizophrenia depression scale (CDSS), a positive symptom scale, a negative symptom scale (SANS), a concise negative symptom scale (BNSS), a general psychosis shape scale and a positive and negative symptom scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptom scale (PANSS).

Preferably, the above use, wherein the diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 27-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of 27-hydroxycholesterol measured in step 1 with a standard concentration; and

step 3: determining the risk of the subject for developing schizophrenia, e.g., if the plasma 27-hydroxycholesterol concentration is less than 41ng/ml, the probability of developing schizophrenia is about 48%;

preferably, in step 2, the standard concentration is the concentration of 27-hydroxycholesterol in peripheral blood of a normal population.

In another aspect, the invention provides a kit for diagnosing schizophrenia, the kit comprising 27-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia evaluation scale is selected from one or more than two of a karcaliy schizophrenia depression scale (CDSS), a positive symptom scale, a negative symptom scale (SANS), a concise negative symptom scale (BNSS), a general psychosis shape scale and a positive and negative symptom scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptom scale (PANSS).

In another aspect, the invention provides a kit for diagnosing schizophrenia, the kit comprising reagents for detecting 27-hydroxycholesterol and a schizophrenia assessment scale; preferably, the schizophrenia evaluation scale is selected from one or more than two of a karcaliy schizophrenia depression scale (CDSS), a positive symptom scale, a negative symptom scale (SANS), a concise negative symptom scale (BNSS), a general psychosis shape scale and a positive and negative symptom scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptom scale (PANSS).

Preferably, the above use or the above kit, wherein the reagent for detecting 27-hydroxycholesterol is selected from one or more of 27-hydroxycholesterol ligand or an antibody to 27-hydroxycholesterol ligand, an antibody to 27-hydroxycholesterol, a chromogenic reagent, a luminescent marker, a dye and a fluorescent marker.

Preferably, the above-mentioned use or the above-mentioned kit, wherein the reagent for detecting 27-hydroxycholesterol is selected from one or more of a standard, an alkaline hydrolysis reagent, a derivatization reagent, an internal standard and an external standard; preferably, the standard is selected from serum albumin; further preferably, the alkaline hydrolysis reagent is selected from one or more of butylhydroxytoluene, potassium hydroxide, ethylenediamine tetraacetic acid and ethanol; further preferably, the derivatizing agent is selected from one or more of N' -diisopropylcarbodiimide, nicotinamide and 4-dimethylaminopyridine; further preferably, the internal standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol; further preferably, the external standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol.

Preferably, the use as described above or the kit as described above, wherein the kit further comprises a reagent for detecting 24-hydroxycholesterol.

The beneficial effects of the invention include:

the invention uses the level of 27-hydroxy cholesterol as the basis of diagnosing the schizophrenia, and provides a corresponding diagnosis reagent, which has higher sensitivity and specificity for diagnosing the schizophrenia compared with the traditional diagnosis method which can only be judged by clinical experience after relying on symptom description and duration and excluding other diseases.

On the other hand, the 27-hydroxycholesterol biomarker belongs to the peripheral blood index, can be conveniently obtained, and can be diagnosed and predicted by only measuring one index of the peripheral blood 27-hydroxycholesterol level, thereby being convenient to popularize and use. Meanwhile, the 27-hydroxycholesterol biomarker is used as a diagnosis method, and more reliable results can be obtained in combination with other detection modes.

Drawings

In order to more clearly illustrate the technical solutions of the present invention, the drawings that are needed to be used are briefly described below, it being obvious that the drawings in the following description are only some embodiments described in the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1: plasma 27OHC concentrations in healthy controls, primary relatives, clinically ultra-high risk populations, and schizophrenic patients.

Fig. 2-1: correlation of positive symptom score of schizophrenic patients with plasma 27OHC concentration, correlation coefficient r= -0.301, concomitance probability p=0.011.

Fig. 2-2: correlation of negative symptoms score with plasma 27OHC concentration in schizophrenic patients, r=0.064, p=0.597.

Fig. 2-3: correlation of general psychotic disorder scores with plasma 27OHC concentration in schizophrenic patients, r=0.062, p=0.609.

Fig. 2-4: correlation of positive and negative symptom scale scores (PANSS scores) with plasma 27OHC concentration for schizophrenic patients, r= -0.051, p=0.675.

Fig. 3: ROC curve of 27OHC as diagnostic index for schizophrenia.

Fig. 4: healthy controls and schizophrenic patients plasma 24OHC concentrations.

Fig. 5-1: correlation of positive symptoms score in schizophrenic patients with plasma 24OHC concentration.

Fig. 5-2: correlation of negative symptoms score in schizophrenic patients with plasma 24OHC concentration.

Fig. 5-3: the correlation of a general psychotic disorder-like score with plasma 24OHC concentration in schizophrenic patients.

Fig. 5-4: correlation of positive and negative symptom scale scores (PANSS scores) with plasma 24OHC concentrations in schizophrenic patients.

Fig. 6: ROC curve of 24OHC as diagnostic index for schizophrenia.

Detailed Description

In order that those skilled in the art will better understand the present invention, a detailed description of embodiments of the present invention will be provided below, together with the accompanying drawings, wherein it is evident that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In one embodiment of the invention 27-hydroxycholesterol is used as biomarker in a reagent or kit for diagnosing schizophrenia. The concentration of 27OHC in the peripheral blood of the subject is detected by using the above-described reagent or kit, thereby diagnosing schizophrenia. Specifically, peripheral blood of the subject to be tested can be extracted, plasma is separated and the 27OHC concentration is measured, the disease state of the subject can be diagnosed before receiving treatment, and meanwhile, the blood extraction time can be carried out along with the treatment process or after the treatment is finished so as to monitor the treatment effect.

Herein, the term "schizophrenia" is a mental disorder characterized by hallucinations, delusions, neurophysiologic dysfunction and neurocognitive dysfunction as the main clinical features.

Herein, the term "27-hydroxycholesterol" (27 OHC) is a metabolite of cholesterol via mitochondrial cholesterol 27-hydroxylase (CYP 27 A1), which has the structural formula:

27OHC is the cholesterol metabolite with the highest content in the peripheral circulatory system, and finally is converted into bile acid to be discharged out of the body, thereby playing an important role in maintaining the cholesterol balance in the body.

The term "diagnosis" refers to a method of detecting and/or identifying a pathological state in a subject, identifying and/or determining whether the subject has a given disease or disorder, assessing or determining future clinical progression in the subject, either before or after symptoms appear.

In a specific embodiment of the present invention, the reagent for diagnosing schizophrenia is prepared as a kit, which may be a biochemical diagnosis kit, an immunodiagnosis kit, a molecular diagnosis kit, or the like, preferably as the immunodiagnosis kit.

The diagnosis kit is prepared by adopting principles or methods of immunology, microbiology, molecular biology and the like, and is used for diagnosing, detecting, epidemiologically investigating and the like of human diseases in vitro.

The diagnostic kit may be of a type well known in the art including, but not limited to, western blot kits, enzyme-linked immunosorbent assay (ELISA) kits, radioimmunoassay (RIA) kits, radioimmunoassay, ouchterlony immunodiffusion kits, rocket immunoelectrophoresis kits, immunohistochemical staining kits, immunoprecipitation assay kits, complement fixation assay kits, fluorescence activated cell sorting (FACS kits, aptamer chip kits, microarray kits, protein chips, and the like.

The kit comprises the reagent for detecting 27OHC, wherein the reagent can specifically detect the concentration of 27OHC, and comprises but is not limited to nucleic acid molecules, proteins, compounds and the like which can specifically bind to 27 OHC.

In one embodiment of the present invention, a protein capable of specifically binding to 27OHC may be selected as the reagent for detecting 27OHC, and in another embodiment of the present invention, an antibody, which may be an antibody capable of specifically binding to a marker protein, including but not limited to polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and antigen binding fragments thereof, is preferred as the reagent for detecting 27OHC, as long as it retains antigen binding function.

In the present invention, a labeling molecule capable of being detected may be attached to the reagent for detecting 27 OHC.

The invention will now be described in more detail with reference to specific examples, which are, however, given for illustrative purposes only and are not limiting of the invention.

The reagents and instrument sources used in the examples below are shown in Table 1 below, and the reagents or instruments or procedures not described herein are those routinely determinable by one of ordinary skill in the art:

table 1 reagents and apparatus used in the examples

Reagents/apparatus	Model/specification	Manufacturer' s
			Ammonium acetate	500g content is more than or equal to 98%	National medicine group chemical reagent
Formic acid	50ml	Fluka
			Methyl tert-butyl ether	The 1L content is more than or equal to 99.9 percent	Merck
N' -diisopropylcarbodiimide	D125407 25g content is more than or equal to 99 percent	Merck
			Nicotinamide	72340 100g content is more than or equal to 99 percent	Merck
4-dimethylaminopyridine	107700 25g content is more than or equal to 99 percent	Merck
			Methanol	34860 The content of 4L is more than or equal to 99.9 percent	Merck
High performance liquid chromatograph	Agilent G1312B	Agilent
			Mass spectrometer	AB Sciex Qtrap 6500	ABI Co., ltd

Examples

Example 1: peripheral blood collection and method for measuring concentration of peripheral blood 27OHC

1. According to the passed ethical examination experimental scheme and diagnosis standard, 312 people in the healthy control group (HC), the high risk of hereditary schizophrenia (first-class relatives of patients, FDR), the ultra-high risk of clinical (UHR) and the 4 groups of schizophrenic patients (SCZ) are screened out. The row entry criteria for each group are as follows:

healthy controls: screening with the clinical diagnostic and statistical Manual, fourth Edition (Structured Clinical Interview for DSM Disorders-Fourier Edition, DSM-IV) of American psychiatric disorders (Structured Clinical Interview for DSM-IV Axis I disorders-Patent Edition, SCID-I/P) and SIPS showed no mental disorders, no mental illness family history.

Schizophrenic patients entered group criteria: meets the diagnostic criteria of the four-edition of the manual for diagnosis and statistics of mental diseases in the united states (DSM-IV), without a clear history of mental diseases, nervous system and other serious somatic diseases other than schizophrenia; there is no family history of neurological disease. Eliminating extreme excitement, impulse and non-collaborators; patients who participated in other scientific research treatments or received MECT treatment within three months. The first relatives of the patients exclude the genetic high-risk group of the possible group of the nerve, mental diseases and other serious somatic diseases.

Clinical high risk menstrual risk Syndrome Interview (SIPS) screening meets one or more of three conditions of psychotic risk syndrome criteria.

All subjects were 14-55 years old, excluding pregnant or lactating women, with food-drug allergy history, who could not discontinue use of the drug due to other somatic diseases, alcohol and drug abuse history, and head trauma history population.

Patients were screened and normal tested using a DSM-IV clinical interview (Structured Clinical Interview for DSM Disorders, SCID) and patients were assessed for clinical psychopathological symptoms using the positive and negative scales (PANSS). And simultaneously, 5 milliliters of peripheral blood is extracted in an EDTA anticoagulation tube, the upper plasma is separated after 3000 revolutions per minute for 10 minutes, and the upper plasma is immediately packaged, stored and kept in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for later use.

2. The 27OHC content of the plasma is detected by a high performance liquid chromatography-mass spectrometry method after the plasma is extracted, and the specific method is as follows:

(1) A standard solution of 27OHC at a concentration of 1-2000ng/ml was prepared with a 5% bovine serum albumin solution, and a 5% bovine serum albumin solution was used as a blank sample.

(2) 50 μl of standard/blank/test plasma was added to a 1.5ml EP tube, followed by 50 μl of internal standard (D5 deuterium cholesterol+D7 deuterium cholesterol 50 ng/ml) followed by 200 μl of acidic buffer (50 mM ammonium acetate, 1% formic acid) and 1ml of methyl tert-butyl ether.

(3) After thoroughly mixing, the sample was placed in a-80 ℃ refrigerator, and after cooling the aqueous phase therein to crystallize, 0.5ml of methyl tert-butyl ether phase (supernatant) was transferred to a 1.5ml centrifuge tube.

(4) The methyl tert-butyl ether was blow-dried with nitrogen at 35 ℃.

(5) Mu.l of derivatizing reagent (63 mg of N, N' -diisopropylcarbodiimide, 62mg of nicotinamide and 61mg of 4-dimethylaminopyridine in 5ml of chloroform) was added to each EP tube and mixed with heating at 35℃for 2 hours.

(6) The methylene chloride was dried with nitrogen at 35 ℃.

(7) 100 μl methanol was reconstituted and then assayed by sample injection.

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

Example 2: peripheral blood 24OHC or 27OHC as biomarker for diagnosis of schizophrenia

1. The study included a total of 312 gender-matched subjects, with the basic demographics and patient symptom assessments for each group shown in table 2.

Table 2: subject basic population and patient clinical symptom assessment

Note that: HC: healthy controls; FDR: first-degree relatives of schizophrenia; UHR: clinical ultra-high risk of schizophrenia; SCZ: patients with schizophrenia; SIPs: psychosis risk syndrome interviews; PANSS: positive and negative symptom scales; p: positive symptoms; n: negative symptoms; g: general psychotic symptoms: t: a total score; NA: missing values.

Through plasma 27OHC detection, plasma 27OHC levels were found to be significantly reduced in schizophrenic patients compared to healthy controls (fig. 1, p < 0.0001), suggesting that cholesterol metabolism was significantly reduced in patients, potentially becoming a potential biomarker for schizophrenic diagnosis. However, there was no significant change in the first-order relatives of schizophrenia and clinically ultra-high risk groups compared to healthy controls (fig. 1), indicating that a decrease in 27OHC in schizophrenia did not occur prior to onset. The correlation analysis results show that the clinical symptoms of the patients with schizophrenia are obviously correlated with the 27OHC level (figures 2-1 to 2-4), wherein the 27OHC is inversely correlated with the positive symptoms (figure 2-1), and the relationship between the change of the 27OHC level and the occurrence and the development of diseases is further demonstrated. The receiver operating characteristic curve (Receiver Operating Characteristic, ROC) curve (fig. 3) was taken as a combination of subject family history index and 27OHC as diagnostic index, with an area under the curve of 0.741, and diagnostic sensitivity and specificity of 48.0% and 88.9%, respectively.

Through plasma 24OHC detection, it was found that plasma 24OHC was significantly increased in schizophrenic patients compared to healthy controls (fig. 4, p < 0.05), suggesting that cholesterol metabolism in the brain of patients was significantly increased, potentially becoming a potential biomarker for schizophrenic diagnosis. The correlation analysis results show that the clinical symptoms of the patients with schizophrenia are obviously correlated with the level of the hydroxycholesterol (figures 5-1 to 5-4), wherein 24OHC is positively correlated with the negative symptoms and PANSS total division, and further the relation between the change of the level of the hydroxycholesterol and the occurrence and the development of diseases is proved. In combination with the subject family history index, the concentration of 24OHC was used as a diagnostic index to make a receiver operating characteristic curve (Receiver Operating Characteristic, ROC) curve (fig. 6) with an area under the curve of 0.703 and diagnostic sensitivity and specificity of 71.6% and 61.1%, respectively.

According to the embodiment, the invention can diagnose the condition of the patient suffering from the schizophrenia by selecting the peripheral blood 27OHC or 24OHC as a biomarker, thereby realizing an objective diagnosis method for the schizophrenia and having higher sensitivity and specificity for diagnosing the schizophrenia.

Claims

Use of 27-hydroxycholesterol or a reagent for detecting 27-hydroxycholesterol in the preparation of a reagent or kit for diagnosing schizophrenia using the schizophreniform scale.
2. Use according to claim 1, wherein the kit is selected from a biochemical diagnostic kit or an immunodiagnostic kit.
3. The use of claim 1, wherein the kit is selected from one or more of a Western blot kit, an enzyme-linked immunosorbent assay (ELISA) kit, a Radioimmunoassay (RIA) kit, a radioimmunodiffusion kit, a two-dimensional biphasic immunodiffusion kit, a rocket immunoelectrophoresis kit, and an immunoprecipitation assay kit.
4. The use according to claim 1, wherein the schizophrenia evaluation scale is selected from one or more of the karcaliy schizophrenia depression scale (CDSS), positive symptom scale, negative symptom scale (SANS), brief Negative Symptom Scale (BNSS), general psychotic shape scale and positive and negative symptom scale (PANSS).
5. The use of claim 4, wherein the schizophrenia rating scale is the positive and negative symptom scale (PANSS).
6. The use according to claim 1, wherein the diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 27-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of 27-hydroxycholesterol measured in step 1 with a standard concentration; and step 3: the subject is judged to be at risk for developing schizophrenia, and if the plasma 27-hydroxycholesterol concentration is below 41ng/ml, the probability of developing schizophrenia is about 48%.
7. The use according to claim 6, wherein in step 2, the standard concentration is the concentration of 27-hydroxycholesterol in peripheral blood of a normal population.
8. A kit for diagnosing schizophrenia, said kit comprising 27-hydroxycholesterol and an evaluation scale for schizophrenia.
9. A kit for diagnosing schizophrenia, the kit comprising reagents for detecting 27-hydroxycholesterol and a schizophrenia assessment scale.
10. The kit according to claim 8 or 9, wherein the schizophrenia evaluation scale is selected from one or more of the kargari schizophrenia depression scale (CDSS), positive symptom scale, negative symptom scale (SANS), brief Negative Symptom Scale (BNSS), general psychosis shape scale and positive and negative symptom scale (PANSS).
11. The kit of claim 8 or 9, wherein the schizophrenia rating scale is positive and negative symptoms scale (PANSS).
12. The use according to any one of claims 1 to 7 or the kit according to claim 9, wherein the reagent for detecting 27-hydroxycholesterol is selected from 27-hydroxycholesterol ligand or one or more of an antibody, a chromogenic reagent, a luminescent marker and a dye of 27-hydroxycholesterol ligand.
13. The kit of claim 12, wherein the luminescent marker is a fluorescent marker.
14. The use according to any one of claims 1 to 7 or the kit according to claim 9, wherein the reagent for detecting 27-hydroxycholesterol is selected from one or more of a standard, a derivatizing reagent and an internal standard.
15. The kit of claim 14, wherein the standard is selected from serum albumin.
16. The kit of claim 14, wherein the derivatizing reagent is selected from one or more of N' -diisopropylcarbodiimide, nicotinamide, and 4-dimethylaminopyridine.
17. Kit according to claim 14, wherein the internal standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol.
18. The use according to any one of claims 1-7 or the kit according to claim 8 or 9, wherein the kit further comprises a reagent for detecting 24-hydroxycholesterol.