Vitamin D metabolic deficiency syndrome early typing system and early screening kit thereof

Abstract

The invention discloses a vitamin D metabolic deficiency syndrome early typing system and an early screening kit thereof. The typing system comprises a data acquisition module and a vitamin D metabolic deficiency syndrome typing module. The data acquisition module is used for acquiring the concentration of vitamin D metabolites in a blood sample of a subject according to biomass energy product detection and submitting the concentration to the typing module, and the vitamin D metabolites comprise 1, 25 (OH) 2D and 24, 25 (OH) 2D; the typing module can distinguish type I vitamin D dependent rickets, type II vitamin D dependent rickets and idiopathic hypercalcemia according to the comparison of the concentration of 1, 25 (OH) 2D and 24, 25 (OH) 2D of a subject and the concentration of metabolite corresponding to a built-in healthy person; the screening kit is used for biomass energy product detection service, and can be used for early screening of vitamin D metabolic deficiency syndrome according to biomass energy product detection such as vitamin D metabolite detection.

Description

Technical Field

[0001] This invention belongs to the field of biomass energy product testing services and application technology, and more specifically, relates to an early typing system for vitamin D metabolism deficiency syndrome and its early screening kit. Background Technology

[0002] Vitamin D is a fat-soluble, open-ring sterol that is metabolized in the human body into the active form 1,25-hydroxyvitamin D (1,25(OH)₂D). This active form binds to vitamin D receptors (VDRs) and plays a vital role in promoting calcium and phosphorus absorption, inhibiting cell growth, and stimulating cell differentiation. Therefore, it is closely related to bone development, immunity, and cardiovascular health. Vitamin D and its metabolites are essential biomass energy products for the human body. Vitamin D deficiency, metabolic abnormalities, or excess can all affect human health, primarily impacting bones, but also broadly affecting other organ systems such as the immune, cardiovascular, metabolic, cell growth and differentiation, and nervous systems. It may also be associated with the risk of various chronic diseases.

[0003] Vitamin D metabolism deficiency syndromes refer to a collective term for diseases caused by abnormal function of macromolecules in the body's vitamin D metabolism process. These macromolecules include not only key enzymes responsible for catalytic reactions, but also transporters and nuclear receptors. Currently identified vitamin D metabolism deficiency syndromes include type I vitamin D-dependent rickets (VDDR I), type II vitamin D-dependent rickets (VDDR II), and idiopathic infantile hypercalcemia (IIH). VDDR I is further subdivided into type IA vitamin D-dependent rickets (VDDR IA) and type IB vitamin D-dependent rickets (VDDR IB) based on the different deficient enzymes. However, because these types of vitamin D metabolism deficiency syndromes have different causes, their treatment plans also differ. However, there is no research on accurately classifying vitamin D metabolic deficiency syndrome by testing biomass energy products, such as vitamin D and its metabolites. How to accurately classify vitamin D metabolic deficiency syndrome based on biomass energy product testing services, especially for early-stage vitamin D metabolic deficiency syndrome, is an urgent problem to be solved.

[0004] Currently, the diagnosis of vitamin D metabolism deficiency syndrome in clinical practice mainly relies on clinical symptoms combined with 25(OH)D. However, the assessment of clinical symptoms depends on the individual diagnostic experience of doctors, making it difficult to establish a unified diagnostic standard. Moreover, the clinical symptoms of different subtypes of vitamin D metabolism deficiency syndrome are very similar; for example, the clinical symptoms of VDDR II are similar to those of VDDR I. Relying solely on the level of 25(OH)D, a single vitamin D metabolite, in the blood is insufficient to accurately diagnose different subtypes. Furthermore, vitamin D metabolites tend to undergo C3 isomerization. 3-epi-25-hydroxyvitamin D (3-epi-25(OH)D) is an epimer of 25(OH)D. Without effective separation, false positives in 25(OH)D testing can easily occur, leading to misdiagnosis. Although patent CN 117147712 A uses optimized chromatographic conditions to ensure the absolute separation of the epimer (3-epi-25(OH)D) and the isomer (25(OH)D), it only detects the content of various vitamin D metabolites in the blood. There is no research on whether there is a correlation between changes in the content of these biomass energy products, such as changes in the content of vitamin D metabolites, and the classification of vitamin D metabolism deficiency syndrome.

[0005] To accurately differentiate between different subtypes of vitamin D metabolic deficiency syndrome (VDS), genetic testing is necessary in clinical practice, but this requires the presence of obvious rickets symptoms. However, early-stage VDS often presents with subtle clinical symptoms, or even no rickets-related symptoms at all. Although VDS is caused by pathogenic gene mutations, it is difficult to classify early-stage VDS using gene, protein, or enzyme activity testing. For example, in VDDRIA (a pathogenic mutation in CYP27B1), testing for CYP27B1 gene mutations is insufficient due to complexities such as gene polymorphism and nonsense mutations; detecting a mutation does not necessarily indicate a pathogenic mutation (causing changes in protein structure and function). Therefore, testing CYP27B1 expression or enzyme activity cannot definitively diagnose the disease. Similar situations exist for other subtypes of VDS. It is evident that existing typing technologies cannot be used to categorize early vitamin D metabolism deficiency syndrome based on biomass energy product testing services, and there are currently no research reports on the categorization of early vitamin D metabolism deficiency syndrome. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides an early classification system and early screening kit for Vitamin D Metabolic Deficiency Syndrome (VDDR). The aim is to discover, based on biomass energy product testing services and the concentrations of 1,25(OH)₂D and 24,25(OH)₂D in the blood samples of subjects, that patients with early VDDR I, II, and IIH vitamin D metabolic deficiency syndrome can be distinguished. In particular, by combining 25(OH)D, 1,25(OH)₂D, and 24,25(OH)₂D, early VDDR IA and IB vitamin D metabolic deficiency syndrome can be distinguished. This solves the problem that existing technologies based on clinical symptoms and 25(OH)D cannot classify early VDDR I metabolic deficiency syndrome.

[0007] To achieve the above objectives, according to one aspect of the present invention, an early typing system for vitamin D metabolism deficiency syndrome is provided, comprising a data acquisition module and a vitamin D metabolism deficiency syndrome typing module;

[0008] The data acquisition module, based on biomass energy product testing, is used to obtain the concentration of vitamin D metabolites in the subject's blood sample and submit it to the vitamin D metabolism deficiency syndrome typing module; the vitamin D metabolites include 1,25(OH)2D and 24,25(OH)2D;

[0009] The vitamin D metabolism deficiency syndrome typing module compares the concentrations of 1,25(OH)₂D and 24,25(OH)₂D in the subject with the corresponding metabolite concentrations in healthy individuals. The concentrations of 1,25(OH)₂D and 24,25(OH)₂D in healthy individuals are selected from the average concentrations of 1,25(OH)₂D and 24,25(OH)₂D in healthy individuals. Typing is performed according to the following method:

[0010] Compared to the 1,25(OH)2D and 24,25(OH)2D in the blood samples of healthy individuals, if the concentrations of both 1,25(OH)2D and 24,25(OH)2D in the subject are decreased, then the subject is classified as having type I vitamin D-dependent rickets.

[0011] Compared to the 1,25(OH)2D and 24,25(OH)2D in the blood samples of healthy individuals, if the concentrations of both 1,25(OH)2D and 24,25(OH)2D in the subject are elevated, then the subject is classified as having type II vitamin D-dependent rickets.

[0012] Compared to the 1,25(OH)2D and 24,25(OH)2D in the blood samples of healthy individuals, if the concentration of 1,25(OH)2D in a subject increases while the concentration of 24,25(OH)2D decreases, the subject is classified as having idiopathic hypercalcemia.

[0013] Preferably, in the early typing system, the data acquisition module is further used to acquire the concentration of 25(OH)D in the subject's blood sample;

[0014] The vitamin D metabolism deficiency syndrome typing module further types the syndrome based on a comparison of the subject's concentrations of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D with the concentrations of corresponding metabolites (25(OH)D, 1,25(OH)2D, and 24,25(OH)2D) in healthy individuals. The concentration of the corresponding metabolite 25(OH)D in healthy individuals is selected from the normal concentration or the average concentration of 25(OH)D in healthy individuals. The typing is performed as follows:

[0015] Compared to the corresponding metabolite concentrations in blood samples from healthy individuals, if the concentrations of 1,25(OH)2D and 24,25(OH)2D in the subject are both decreased, while the concentration of 25(OH)D is not statistically different from that of healthy individuals or falls within the normal concentration range, then the subject is classified as having type IA vitamin D-dependent rickets.

[0016] Compared to the corresponding metabolite concentrations in the blood samples of healthy individuals, if the concentrations of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D in a subject are all decreased, then the subject is classified as having type IB vitamin D-dependent rickets.

[0017] Preferably, in the early typing system, the data acquisition module is used to acquire the D3 metabolites 25(OH)D3, 1,25(OH)2D3, 24,25(OH)2D3 and the D2 metabolites 25(OH)D2, 1,25(OH)2D2, 24,25(OH)2D2 in the subject's blood sample, calculate the concentrations of 25(OH)D, 1,25(OH)2D and 24,25(OH)2D by summing the concentrations of the D3 metabolites and the D2 metabolites, and submit the results to the vitamin D metabolism deficiency syndrome typing module.

[0018] Preferably, in the early typing system, the concentration of 25(OH)D acquired by the data acquisition module is the concentration after elution separation to exclude interference from the diastereomer 3-epi-25(OH)D.

[0019] Preferably, the data acquisition module of the early typing system is further used to acquire the concentrations of D3 metabolite 3-epi-25(OH)D3 and D2 metabolite 3-epi-25(OH)D2 in the subject's blood sample, in order to exclude false positives of 25(OH)D3 and 25(OH)D2.

[0020] Preferably, in the early typing system, the blood sample is whole blood, plasma, or serum, and the D3 and D2 metabolites are detected using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). The sample processing is as follows:

[0021] Mix the blood sample to be tested, internal standard solution and protein precipitant, precipitate the protein, take the supernatant, blow dry with nitrogen, add derivatization solution to reconstitute, and perform derivatization treatment; after the reaction is completed, blow dry with nitrogen, add reconstitution solution containing formic acid, centrifuge and take the supernatant as the injection sample.

[0022] The derivatization solution uses DAPTAD or PTAD as the derivatizing agent, and the solvent is selected from ethyl acetate or acetonitrile; the derivatization reaction takes 1.5 to 2 hours;

[0023] The injected samples were separated by chromatographic elution of derivatized 1,25(OH)2D3, 24,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D2, 25(OH)D3, 25(OH)D2, and epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2. The D3 and D2 metabolites were quantitatively detected by mass spectrometry.

[0024] According to another aspect of the present invention, an early screening kit for vitamin D metabolism deficiency syndrome is also provided. This early screening kit is used for biomass energy product testing services. It includes internal standards for the quantitative detection of isotopes 1,25(OH)₂D₃, 1,25(OH)₂D₂, 24,25(OH)₂D₃, and 24,25(OH)₂D₂ in blood samples, and a derivatization reagent used to derivatize vitamin D metabolites and their isotope internal standards extracted from blood samples. The kit is used to screen for vitamin D metabolism deficiency syndrome patients, especially those with early-stage vitamin D metabolism deficiency syndrome, based on the detection of biomass energy products in blood samples.

[0025] Preferably, the early screening kit further includes isotopic internal standards for the quantitative detection of 25(OH)D3 and 25(OH)D2.

[0026] Preferably, in the early screening kit, the derivatization reagent is selected from DAPTAD.

[0027] Preferably, the early screening kit further includes mobile phase A and mobile phase B for chromatographic elution separation of 25(OH)D3, 25(OH)D2 and epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2.

[0028] Preferably, in the early screening kit, the mobile phase A is selected from an aqueous solution containing 5 mM ammonium bicarbonate and 0.004% formic acid, and the mobile phase B is selected from a methanol solution containing 2 mM ammonium formate and 0.16% formic acid.

[0029] Preferably, the early screening kit may further include a protein precipitant for extracting vitamin D metabolites from blood samples, the protein precipitant being selected from acetonitrile, methanol, or anhydrous ethanol; the vitamin D metabolites include 1,25(OH)₂D₃, 1,25(OH)₂D₂, 24,25(OH)₂D₃, and 24,25(OH)₂D₂. In some embodiments, the early screening kit may further include a reconstituted solution for rehydrating the extracted vitamin D metabolites, for example, an aqueous methanol solution containing 0.1% formic acid (volume ratio 4:1).

[0030] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects:

[0031] The vitamin D metabolism deficiency syndrome early typing system provided by this invention, based on biomass energy product testing services, is used to obtain the concentration of vitamin D metabolites (including 1,25(OH)₂D and 24,25(OH)₂D) in the blood samples of subjects. Based on a comparison of the vitamin D metabolite concentrations in the subject's blood sample with the corresponding metabolite concentrations in healthy individuals, and according to changes in the concentrations of 1,25(OH)₂D and 24,25(OH)₂D, it can distinguish between type I vitamin D-dependent rickets, type II vitamin D-dependent rickets, and idiopathic hypercalcemia. In particular, by combining 25(OH)D, it can further distinguish between type IA vitamin D-dependent rickets and type IB vitamin D-dependent rickets, and can be used for early screening of vitamin D metabolism deficiency syndrome based on biomass energy product testing. Attached Figure Description

[0032] Figure 1 It refers to the metabolic process and related metabolites of vitamin D in the human body;

[0033] Figure 2 This is a representative chromatogram of vitamin D metabolites in the plasma of healthy individuals;

[0034] Figure 3 This is an analysis of the concentration differences of 25(OH)D in the plasma of healthy individuals and patients with vitamin D metabolism deficiency syndrome;

[0035] Figure 4 This study analyzed the difference in plasma concentrations of 1,25(OH)2D between healthy individuals and patients with vitamin D metabolism deficiency syndrome.

[0036] Figure 5This study analyzed the difference in plasma concentrations of 24,25(OH)2D between healthy individuals and patients with vitamin D metabolism deficiency syndrome. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0038] Vitamin D in the human body needs to be metabolized into active 1,25-hydroxyvitamin D (1,25(OH)2D), which then binds to vitamin D receptors (VDRs) to exert its important physiological functions. The metabolism of vitamin D in the body involves both classical and non-classical (local tissue) metabolic pathways. The classical metabolic pathway of vitamin D in the human body is as follows:

[0039] Hepatic hydroxylation (first step of activation): Vitamin D is transported to the liver via the bloodstream through vitamin D binding protein (DBP). Within hepatocytes, it is converted to 25-hydroxyvitamin D (25(OH)D) by vitamin D-25 hydroxylase (encoded by CYP2R1 and CYP27A1). This is the primary circulating storage form and the gold standard for clinically assessing vitamin D status; a range of 25-100 ng / mL (or 50-75 nmol / L) is generally considered normal or adequate.

[0040] Renal hydroxylation (second step activation - key regulatory point): 25(OH)D is transported to the kidney, enters the renal tubular cells with the assistance of DBP, and is converted into active 1,25-hydroxyvitamin D (1,25(OH)2D) under the catalysis of 25(OH)D-1-α-hydroxylase (1-α-hydroxylase, encoded by CYP27B1).

[0041] Vitamin D metabolites produced during the vitamin D metabolism process include 25-hydroxyvitamin D (25(OH)D), 1,25-hydroxyvitamin D (1,25(OH)2D), 24,25-hydroxyvitamin D (24,25(OH)2D), 3-epi-25-hydroxyvitamin D (3-epi-25(OH)D), and 1,24,25-trihydroxyvitamin D (1,24,25(OH)3D). Each vitamin D metabolite exists in two subtypes, D3 and D2. For example, 25-hydroxyvitamin D includes 25-hydroxyvitamin D3 (25(OH)D3) and 25-hydroxyvitamin D2 (25(OH)D2).

[0042] 1,25(OH)2D is transported by DBP to target organs and tissues, such as the intestines, kidneys, and bones, where it binds to vitamin D receptors in the cells of these tissues and plays an important physiological role in promoting calcium and phosphorus absorption, inhibiting cell growth, and stimulating cell differentiation. Thus, it is closely related to human bone development, autoimmunity, and cardiovascular health.

[0043] However, vitamin D deficiency, metabolic disorders, or excess can all affect human health, primarily impacting bones, but also broadly affecting other organ systems such as the immune, cardiovascular, metabolic, cell growth and differentiation, and nervous systems. It may also be associated with the risk of various chronic diseases. For example, excessive vitamin D leads to excessive intestinal calcium absorption and increased bone resorption, ultimately resulting in hypercalcemia; vitamin D deficiency leads to deficiency rickets, osteomalacia, and osteoporosis. Although vitamin D deficiency can cause deficiency rickets, some rickets patients have been found clinically to exhibit symptoms of vitamin D deficiency without actually lacking 25(OH)D. Current technology mainly focuses on the quantitative detection of vitamin D metabolites, with limited research on the classification of early vitamin D metabolic deficiency syndromes.

[0044] To investigate the classification techniques for early vitamin D metabolism deficiency syndrome, this invention, based on biomass energy product testing, examines whether changes in vitamin D metabolites are related to the classification of vitamin D metabolism deficiency syndrome. We detected multiple vitamin D metabolites in blood samples from healthy individuals and patients with different subtypes of vitamin D metabolism deficiency syndrome, analyzing the changes in these metabolites among these subtypes. The results showed that 1,25(OH)₂D and 24,25(OH)₂D can be used to distinguish different subtypes of early vitamin D metabolism deficiency syndrome. Compared to healthy individuals, subjects with type I vitamin D-dependent rickets (VDDR I) showed decreased concentrations of both 1,25(OH)2D and 24,25(OH)2D; subjects with type II vitamin D-dependent rickets (VDDR II) showed increased concentrations of both 1,25(OH)2D and 24,25(OH)2D; and subjects with idiopathic hypercalcemia (IIH) showed increased concentrations of 1,25(OH)2D and decreased concentrations of 24,25(OH)2D.

[0045] Furthermore, it was found that the combination of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D could be used to differentiate between type IA and type I vitamin D-dependent rickets. Compared to healthy individuals, subjects with type IA vitamin D-dependent rickets (VDDR IA) showed decreased concentrations of both 1,25(OH)2D and 24,25(OH)2D, while the concentration of 25(OH)D remained within the normal range. In subjects with type IB vitamin D-dependent rickets (VDDR IB), the concentrations of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D were all decreased.

[0046] Two batch cohorts were used for validation. Predictive models were constructed using 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D as classification indicators, and the diagnostic efficacy of the predictive models for these four subtypes was evaluated. The validation results showed that the models had good differentiation ability for VDDR IA and VDDR IB (AUC 0.85-0.88) and good differentiation ability for VDDR II and IIH (AUC 0.79-0.82). As the patient sample size of these four subtypes increased, the model's differentiation ability for the four subtypes of vitamin D metabolism deficiency syndrome improved (AUC 0.85-0.93).

[0047] In this invention, 25(OH)D is the sum of 25(OH)D3 and 25(OH)D2; 1,25(OH)2D is the sum of 1,25(OH)2D3 and 1,25(OH)2D2; and 24,25(OH)2D is the sum of 24,25(OH)2D3 and 24,25(OH)2D2.

[0048] Based on this discovery, the present invention provides an early classification system for vitamin D metabolism deficiency syndrome, which includes a data acquisition module and a vitamin D metabolism deficiency syndrome classification module.

[0049] The data acquisition module, based on biomass energy product testing, is used to obtain the concentration of vitamin D metabolites in the subject's blood sample and submit it to the vitamin D metabolism deficiency syndrome typing module; the vitamin D metabolites include 1,25(OH)2D and 24,25(OH)2D;

[0050] The vitamin D metabolism deficiency syndrome typing module compares the concentrations of 1,25(OH)₂D and 24,25(OH)₂D in the subject with the corresponding metabolite concentrations in healthy individuals. The concentrations of 1,25(OH)₂D and 24,25(OH)₂D in healthy individuals are selected from the average concentrations of 1,25(OH)₂D and 24,25(OH)₂D in healthy individuals. Typing is performed according to the following method:

[0051] Compared to the concentrations of the metabolites 1,25(OH)2D and 24,25(OH)2D in the blood samples of healthy individuals, if the concentrations of both 1,25(OH)2D and 24,25(OH)2D in a subject decrease, the subject is classified as having type I vitamin D-dependent rickets.

[0052] Compared to the concentrations of the metabolites 1,25(OH)2D and 24,25(OH)2D in the blood samples of healthy individuals, if the concentrations of both 1,25(OH)2D and 24,25(OH)2D in a subject are elevated, the subject is classified as having type II vitamin D-dependent rickets.

[0053] Compared to the concentrations of the metabolites 1,25(OH)2D and 24,25(OH)2D in the blood samples of healthy individuals, if the concentration of 1,25(OH)2D in a subject increases while the concentration of 24,25(OH)2D decreases, the subject is classified as having idiopathic hypercalcemia.

[0054] If the concentrations of the metabolites 1,25(OH)2D and 24,25(OH)2D in a subject's blood sample are not statistically different from those in a healthy individual or fall within the normal range, then the subject is classified as not having vitamin D metabolism deficiency syndrome.

[0055] Furthermore, the data acquisition module is also used to acquire the concentration of 25(OH)D in the subject's blood sample; the vitamin D metabolism deficiency syndrome typing module, based on the comparison of the subject's concentrations of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D with the built-in concentrations of corresponding metabolites in healthy individuals, wherein the concentration of the corresponding metabolite 25(OH)D in healthy individuals is selected from the normal concentration of 25(OH)D in healthy individuals or the average concentration of 25(OH)D in healthy individuals, is further typed according to the following method:

[0056] Compared to the concentrations of the metabolites 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D in the blood samples of healthy individuals, if the concentrations of 1,25(OH)2D and 24,25(OH)2D in the subject are both decreased, while the concentration of 25(OH)D is not statistically different from that of healthy individuals or is within the normal concentration range, then the subject is classified as having type IA vitamin D-dependent rickets.

[0057] Compared to the concentrations of the metabolites 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D in the blood samples of healthy individuals, if the concentrations of all three metabolites in a subject are decreased, the subject is classified as having type IB vitamin D-dependent rickets.

[0058] In some embodiments, the average concentration of 25(OH)D in the plasma of healthy individuals is 51–52 nmol / L, the average concentration of 1,25(OH)2D in the plasma of healthy individuals is 0.8–0.9 nmol / L, and the average concentration of 24,25(OH)2D in the plasma of healthy individuals is 3.5–3.6 nmol / L.

[0059] Preferably, the obtained 25(OH)D concentration is the concentration after separating and excluding the interference of the diastereomer 3-epi-25(OH)D. This can eliminate the false positive effect of 3-epi-25(OH)D on 25(OH)D, making the detection result of 25(OH)D more accurate and more conducive to distinguishing between patients with early type IA vitamin D-dependent rickets and early type IB vitamin D-dependent rickets. In this invention, vitamin D metabolites refer to the sum of D3-type metabolites and D2-type metabolites. For example, 25(OH)D is the sum of 25(OH)D3 and 25(OH)D2; 1,25(OH)2D is the sum of 1,25(OH)2D3 and 1,25(OH)2D2; 24,25(OH)2D is the sum of 24,25(OH)2D3 and 24,25(OH)2D2; and 3-epi-25(OH)D is the sum of 3-epi-25(OH)D3 and 3-epi-25(OH)D2.

[0060] In some embodiments, the data acquisition module is used to acquire the concentrations of at least four vitamin D metabolites in the blood sample of the subject. The vitamin D metabolites include the D3 type metabolites 1,25-hydroxyvitamin D3 (1,25(OH)2D3) and 24,25-hydroxyvitamin D3 (24,25(OH)2D3) and the D2 type metabolites 1,25-hydroxyvitamin D2 (1,25(OH)2D2) and 24,25-hydroxyvitamin D2 (24,25(OH)2D2). The total concentration of the corresponding vitamin D metabolites is calculated by summing the concentrations of the D3 type metabolites and the D2 type metabolites, and two judgment indicators, 1,25(OH)2D and 24,25(OH)2D, are obtained and submitted to the vitamin D metabolism deficiency syndrome classification module.

[0061] Preferably, the data acquisition module is used to acquire the concentrations of six vitamin D metabolites, which include D3 type metabolites 25-hydroxyvitamin D3 (25(OH)D3), 1,25-hydroxyvitamin D3 (1,25(OH)2D3), 24,25-hydroxyvitamin D3 (24,25(OH)2D3) and D2 type metabolites 25-hydroxyvitamin D2 (25(OH)D2), 1,25-hydroxyvitamin D2 (1,25(OH)2D2), 24,25-hydroxyvitamin D2 (24,25(OH)2D2).

[0062] We considered that the results of 3-epig-25-hydroxyvitamin D (3-epi-25(OH)D) on 25(OH)D are prone to false positives, and should also be excluded after testing as an interfering item. More preferably, the data acquisition module is used to acquire the concentrations of eight vitamin D metabolites in the subject, including the D3 type metabolites 25-hydroxyvitamin D3 (25(OH)D3), 1,25-hydroxyvitamin D3 (1,25(OH)2D3), 24,25-hydroxyvitamin D3 (24,25(OH)2D3), 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3) and the D2 type metabolites 25-hydroxyvitamin D2 (25(OH)D2), 1,25-hydroxyvitamin D2 (1,25(OH)2D2), 24,25-hydroxyvitamin D2 (24,25(OH)2D2), 3-epi-25-hydroxyvitamin D2 (3-epi-25(OH)D2). The detection of 3-epi-25(OH)D was used to verify that 3-epi-25(OH)D3 and 3-epi-25(OH)D2 were successfully separated, so as to eliminate their interference with the detection of 25(OH)D3 and 25(OH)D2 and avoid false positives of 25(OH)D3 and 25(OH)D2.

[0063] In some embodiments, the blood sample is whole blood, serum, or plasma. The concentration of the vitamin D metabolites can be detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The liquid chromatography can be selected from high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UHPLC), and nano-liquid chromatography (NLC), and the tandem mass spectrometry can be selected from quadrupole mass spectrometry (QLS), time-of-flight mass spectrometry (TOF-MS), ion hydrazine mass spectrometry (IHS-MS), and high-resolution orbital hydrazine mass spectrometry (HROSM). For example, if the blood sample is plasma, the concentrations of eight vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, 24,25(OH)2D2, 3-epi-25(OH)D3, and 3-epi-25(OH)D2) in the plasma can be detected using UHPLC-MS / MS. In some embodiments, ultra-high performance liquid chromatography-mass spectrometry (Agilent 1290-6470UPLC-MS / MS) was used for detection. The chromatographic column was an Agilent ZORBAX Eclipse Plus C18 (2.1×100mm, 1.8μm). The mobile phase A for elution was an aqueous solution containing 5mM ammonium bicarbonate and 0.004% formic acid; the mobile phase B was a methanol solution containing 2mM ammonium formate and 0.16% formic acid. The column temperature was 45℃, and the injection volume was 5μL. The preferred elution conditions are as follows:

[0064] Time (min) Phase A Phase B Flow rate 0.0 30％ 70％ 0.50 2.0 25％ 75％ 0.50 3 20％ 80％ 0.50 3.2 15％ 85％ 0.50 5.0 15％ 85％ 0.50 6.0 10.0％ 90.0％ 0.50 6.1 0.0％ 100.0％ 0.6 9.0 0.0％ 100.0％ 0.6 9.1 30.0％ 70.0％ 0.5

[0065] An electrospray ionization source in positive ion mode with multiple reaction monitoring (MRM) was used. The parameters were: dry gas flow rate 10 L / min, dry gas temperature 315 °C, nebulizer pressure 50 psi, sheath gas temperature 350 °C, sheath gas flow rate 10 L / min, nozzle voltage 500 V, and capillary voltage 4000 V. The optimal mass spectrometry ion pairing parameters are as follows:

[0066]

[0067] In some embodiments, a Shimadzu LCMS 8060RX triple quadrupole LC-MS system was used for detection. The chromatographic column was an Agilent ZORBAX Eclipse Plus C18 (2.1 × 100 mm, 1.8 μm). The mobile phase A for elution was an aqueous solution containing 5 mM ammonium bicarbonate and 0.004% formic acid; the mobile phase B was a methanol solution containing 2 mM ammonium formate and 0.16% formic acid. The column temperature was 45 °C, and the injection volume was 3 μL. The specific elution conditions were as follows:

[0068] Time (min) Phase A Phase B Flow rate 0.0 30％ 70％ 0.50 2.0 25％ 75％ 0.50 2.5 20％ 80％ 0.50 3.0 20％ 80％ 0.50 3.2 18％ 82％ 0.50 4.0 16.3％ 83.7％ 0.50 6.0 13％ 87％ 0.55 7.0 11％ 89％ 0.55 9.0 8％ 92％ 0.60 9.1 30％ 70％ 0.50

[0069] An electrospray ionization source was used in positive ion mode with multiple reaction monitoring. The nebulizer gas flow rate was 3 L / min, the heating gas flow rate was 10 L / min, the drying gas flow rate was 10 L / min, the interface temperature was 300℃, the desolventizing temperature was 526℃, the DL temperature was 250℃, and the heating block temperature was 400℃. The mass spectrometry ion pair parameters are as follows:

[0070]

[0071] Alternatively, the concentration of vitamin D metabolites can be detected using the liquid chromatography-mass spectrometry analysis method for vitamin D disclosed in patent CN 117147712 A.

[0072] In addition, the present invention also provides an early screening kit for vitamin D metabolism deficiency syndrome. The early screening kit is used for the detection of biomass energy products. It includes an internal standard for the quantitative detection of isotopes of 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2 in a blood sample, and a derivatization reagent for derivatizing vitamin D metabolites and their isotope internal standards extracted from the blood sample.

[0073] Preferably, the early screening kit also includes isotopic internal standards for quantitative detection of 25(OH)D3 and 25(OH)D2, used in conjunction with 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D to further classify type I vitamin D-dependent rickets into type IA or type IB vitamin D-dependent rickets. Specifically, compared to the corresponding metabolite concentrations in healthy individuals, if the concentrations of both 1,25(OH)2D and 24,25(OH)2D in the subject are decreased, while the 25(OH)D concentration remains within the normal range for healthy individuals, the subject is classified as having type IA vitamin D-dependent rickets; if the concentrations of all three metabolites in the subject are decreased, the subject is classified as having type IB vitamin D-dependent rickets.

[0074] In some embodiments, the derivatizing agent is selected from PTAD or DAPTAD. PTAD or DAPTAD can significantly enhance the ionic strength by derivatizing the conjugated dienes of vitamin D metabolites, with DAPTAD being preferred. Compared to the derivatizing agent PTAD, DAPTAD is more effective in improving sensitivity and can derivatize a variety of vitamin D metabolites (including 24,25(OH)2D and 1,25(OH)2D).

[0075] The early screening kit also includes mobile phases A and B for chromatographic elution separation of 25(OH)D3, 25(OH)D2 and their epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2. Pre-separating 25(OH)D3, 25(OH)D2 with their epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2 via chromatographic separation (baseline separation) followed by mass spectrometry quantitative detection avoids interference from 3-epi-25(OH)D3 and 3-epi-25(OH)D2 in the detection of 25(OH)D3 and 25(OH)D2, preventing false positives for 25(OH)D and thus improving the accuracy of 25(OH)D detection results. This is more conducive to early screening for vitamin D metabolism deficiency syndrome based on biomass energy product testing services. In some embodiments, mobile phase A is selected from an aqueous solution containing 5 mM ammonium bicarbonate and 0.004% formic acid, and mobile phase B is selected from a methanol solution containing 2 mM ammonium formate and 0.16% formic acid. Preferably, the early screening kit also includes isotopic internal standards for the quantitative detection of 3-epi-25(OH)D3 and 3-epi-25(OH)D2, which can be used to verify the effectiveness of chromatographic elution in separating 25(OH)D3 and 25(OH)D2 from the epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2.

[0076] Preferably, the early screening kit may further include a protein precipitant for extracting vitamin D metabolites from blood samples, the protein precipitant being selected from acetonitrile, methanol, or anhydrous ethanol; the vitamin D metabolites include 1,25(OH)₂D₃, 1,25(OH)₂D₂, 24,25(OH)₂D₃, and 24,25(OH)₂D₂. In some embodiments, the early screening kit may further include a reconstituted solution for rehydrating the extracted vitamin D metabolites, for example, an aqueous methanol solution containing 0.1% formic acid (volume ratio 4:1).

[0077] The following are examples.

[0078] All recruiters listed below have provided informed consent forms and were recruited according to the following inclusion and exclusion criteria:

[0079] ①Inclusion and exclusion criteria for healthy individuals:

[0080] Healthy individuals were included based on the criteria of normal levels and good health in plasma samples for testing eight vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, 24,25(OH)2D2, 3-epi-25(OH)D3, and 3-epi-25(OH)D2). Individuals who had received any form of vitamin D supplementation or used known vitamin D metabolism-altering drugs (such as rifampin, corticosteroids, antiepileptic drugs, phosphate binders, and calcium-sensitive receptor agonists) within the past three months were excluded.

[0081] ②Inclusion and exclusion criteria for patients with vitamin D metabolism deficiency syndrome:

[0082] Patients diagnosed with vitamin D metabolism deficiency syndrome (including early-stage patients) were included according to clinical diagnostic criteria, specifically VDDR IA, VDDR IB, VDDR II, and IIH patients; patients using known vitamin D metabolism-altering drugs (such as rifampin, corticosteroids, antiepileptic drugs, phosphate binders, and calcium-sensitive receptor agonists) or chronic kidney disease (CKD) were excluded.

[0083] Example 1

[0084] The metabolic process of vitamin D in the human body and related metabolites such as Figure 1 As shown. To evaluate the clinical efficacy of the early subtyping system for vitamin D metabolism deficiency syndrome described in this invention, a discovery cohort comprising 1142 clinical plasma samples was constructed. The discovery cohort was randomly split into a training set (n = 800 cases) and a validation set (n = 342 cases) at a 7:3 ratio. The training set included 30 patients with VDDR IA, 42 patients with VDDR IB, 20 patients with VDDR II, 13 patients with IIH, and the remainder being healthy controls. The validation set included 12 patients with VDDR IA, 19 patients with VDDR IB, 6 patients with VDDR II, 5 patients with IIH, and the remainder being healthy controls.

[0085] The concentrations of eight vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, 24,25(OH)2D2, 3-epi-25(OH)D3, and 3-epi-25(OH)D2) in the plasma of 1142 clinical cases were measured, and four evaluation indicators (25(OH)D, 1,25(OH)2D, 24,25(OH)2D, and 3-epi-25(OH)D) were obtained. D is the sum of D3 type metabolite 25(OH)D3 and D2 type metabolite 25(OH)D2; 1,25(OH)2D is the sum of D3 type metabolite 1,25(OH)2D3 and D2 type metabolite 1,25(OH)2D2; 24,25(OH)2D is the sum of D3 type metabolite 24,25(OH)2D3 and D2 type metabolite 24,25(OH)2D2; 3-epi-25(OH)D is the sum of D3 type metabolite 3-epi-25(OH)D3 and D2 type metabolite 3-epi-25(OH)D2.

[0086] The detection of vitamin D metabolites is referenced in "LC-MS / MS analysis of vitamin D3 metabolites in human serum using a salting-out based liquid-liquid extraction and DAPTAD derivatization" (Journal of Chromatography B1173(2021)122654). In this example, the internal standards used were isotopically labeled deuterated internal standards (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, 24,25(OH)2D2, 3-epi-25(OH)D3, and 3-epi-25(OH)D2). The solvent for dissolving the derivatization reagent DAPTAD was replaced with acetonitrile instead of ethyl acetate. The specific detection method is as follows:

[0087] Transfer 50 μL of plasma sample, add 5 μL of internal standard solution and 245 μL of cold acetonitrile to precipitate proteins, vortex to mix, centrifuge (4℃, 12000 rpm, 10 min), collect the supernatant and dry it under nitrogen, redissolve it with 50 μL of 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) solution, react at room temperature for 1.5 h, and then add 20 μL of anhydrous ethanol to terminate the reaction. Dry the sample again under nitrogen, redissolve it with 50 μL of methanol-water solution containing 0.1% formic acid (4:1, v / v), centrifuge (4℃, 12000 rpm, 10 min), collect the supernatant, filter it through a 0.22 μM filter membrane, and analyze it using an ultra-high performance liquid chromatography-mass spectrometry (Agilent 1290-6470UPLC-MS / MS) system as follows:

[0088] The optimal chromatographic conditions are as follows: the column is an Agilent ZORBAX Eclipse Plus C18 (2.1 × 100 mm, 1.8 μm); mobile phase A is ultrapure water (containing 5 mM ammonium bicarbonate and 0.004% formic acid); mobile phase B is chromatographically pure methanol (containing 2 mM ammonium formate and 0.16% formic acid); the column temperature is 45 °C; the injection volume is 5 μL; and the chromatographic gradient elution conditions are shown in Table 1.

[0089] Table 1 Chromatographic gradient elution conditions

[0090]

[0091]

[0092] The optimal mass spectrometry conditions are as follows: electrospray ionization source, positive ion mode, multiple reaction monitoring, drying gas flow rate 10 L / min, drying gas temperature 315 °C, nebulizer pressure 50 psi, sheath gas temperature 350 °C, sheath gas flow rate 10 L / min, nozzle voltage 500 V, and capillary voltage 4000 V. The mass spectrometry ion pair parameters are shown in Table 2.

[0093] Table 2 Mass spectrometry ion pair parameters

[0094]

[0095] In this embodiment, the liquid chromatography can also be selected from high performance liquid chromatography, ultra-high performance liquid chromatography, and nanoliter liquid chromatography, and the tandem mass spectrometry can be selected from quadrupole mass spectrometry, time-of-flight mass spectrometry, ion hydrazine mass spectrometry, and high-resolution orbital hydrazine mass spectrometry. The detection method for vitamin D metabolites can also employ other existing detection methods capable of detecting these vitamin D metabolites, such as the vitamin D analysis method disclosed in patent CN 117147712 A.

[0096] In this embodiment, a representative chromatogram of vitamin D metabolites in the plasma of healthy individuals is shown below. Figure 2 As shown; and the differences in (25(OH)D, 1,25(OH)2D, and 24,25(OH)2D) between VDDR IA patients, VDDR IB patients, VDDR II patients, and IIH patients and healthy controls were compared and analyzed. 25(OH)D was the concentration after chromatographic elution to exclude interference from the diastereomer 3-epi-25(OH)D. The analytical results are as follows. Figures 3 to 5 As shown.

[0097] Depend on Figures 3 to 5 The results showed that, compared with healthy individuals, the concentrations of 1,25(OH)₂D and 24,25(OH)₂D were significantly decreased in VDDR IA patients, while the concentrations of 25(OH)D and 3-epi-25(OH)D were not statistically different from those in healthy individuals. Conversely, compared with healthy individuals, the concentrations of 1,25(OH)₂D and 24,25(OH)₂D were significantly increased in VDDR II patients, while the concentrations of 25(OH)D and 3-epi-25(OH)D were not statistically different from those in healthy individuals. Compared with healthy individuals, the concentrations of 25(OH)D, 1,25(OH)₂D, 24,25(OH)₂D, and 3-epi-25(OH)D were all significantly decreased in VDDR IB patients. It is evident that, compared to the healthy control group, patients with VDDR IA, VDDR IB, VDDR II, and IIH exhibited significantly different changes in 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D. It is speculated that these three indicators can be used to differentiate between patients with type IA vitamin D-dependent rickets (VDDR IA), type IB vitamin D-dependent rickets (VDDR IB), type II vitamin D-dependent rickets (VDDR II), and idiopathic hypercalcemia (IIH). However, the results of 3-epi-25(OH)D on 25(OH)D are prone to false positives and, as a confounding factor, need to be tested and excluded.

[0098] To evaluate the use of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D to distinguish different subtypes of vitamin D metabolic deficiency syndrome, we constructed a predictive model using XGBoost ensemble learning on the training set and performed blinded testing on the validation set. In machine learning and statistics, the confusion matrix is ​​used to evaluate the performance of a classification model. Its core parameters include True Positive (TP), False Positive (FP), False Negative (FN), and True Negative (TN). The parameters of these four subtype confusion matrices are shown in Table 3 below.

[0099] Table 3. Parameters of the Confusion Matrix for Four Subtypes

[0100]

[0101]

[0102] In disease diagnosis, sensitivity and specificity are core indicators for evaluating the accuracy of diagnostic tests. Sensitivity measures the ability of a diagnostic test to correctly identify individuals with the disease, i.e., the proportion of individuals correctly diagnosed as positive in the actual disease population, and is calculated using the following formula:

[0103]

[0104] In the formula, true positive (TP) is the number of individuals who actually have the disease and are diagnosed as positive; false negative (FN) is the number of individuals who actually have the disease but are diagnosed as negative.

[0105] Specificity measures the ability of a diagnostic test to correctly identify individuals without the disease, that is, the proportion of people in the actual population without the disease who are correctly diagnosed as negative. It is calculated using the following formula:

[0106]

[0107] In the formula, true negative (TN) is the number of individuals who are actually not infected but are diagnosed as negative; false positive (FP) is the number of individuals who are actually not infected but are diagnosed as positive.

[0108] The AUC was calculated using the DeLong method, and the confidence interval was obtained through 2000 Bootstrap resampling. The sensitivity / specificity confidence interval was calculated using the Clopper-Pearson exact method. The diagnostic efficacy evaluation results for the four subtypes are shown in Table 4 below.

[0109] Table 4 Diagnostic efficacy of the four subtypes

[0110] Subtype AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) VDDR IA 0.85(0.76-0.91) 83.3％(62.2-94.5％) 92.4(88.1-95.4％) VDDR IB 0.88(0.80-0.93) 78.9％(61.1-90.0％) 93.8(89.7-96.5％) VDDR II 0.82(0.68-0.92) 66.7％(35.4-88.7％) 95.2(91.8-97.4％) IIH 0.79(0.63-0.90) 60.0％(23.1-88.2％) 96.7(93.7-98.5％)

[0111] As shown in Table 4, in the validation set of 342 samples, the model is effective for the dominant subtype.

[0112] VDDR IA and VDDR IB exhibited stable discriminative power (AUC 0.85-0.88), and their predictive power for small sample subtypes VDDR II and IIH met expectations (AUC > 0.75). Validation using patients diagnosed with subtype IIH in conjunction with clinical symptoms further improved the AUC of this classification system to 0.86 for subtype IIH. This embodiment demonstrates that using the concentrations of six vitamin D metabolites as discriminative criteria, this invention maintains clinical usability even in the early stages of these extremely rare subtypes.

[0113] Example 2

[0114] To further evaluate the clinical efficacy of the early subtyping system for vitamin D metabolism deficiency syndrome described in this invention, an independent validation cohort (different from the samples in Example 1) containing 3025 clinical plasma samples was constructed. This independent validation cohort was randomly split into a training set (discovery cohort, n = 2118 cases) and a validation set (validation cohort, n = 907 cases) at a 7:3 ratio. The training set included 63 patients with early VDDR IA, 85 patients with early VDDR IB, 51 patients with early VDDR II, 29 patients with early IIH, and the remainder were healthy controls. The validation set included 32 patients with early VDDR IA, 51 patients with early VDDR IB, 34 patients with early VDDR II, 17 patients with early IIH, and the remainder were healthy controls.

[0115] The concentrations of eight vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, 24,25(OH)2D2, 3-epi-25(OH)D3, and 3-epi-25(OH)D2) in the plasma of 3025 clinical cases were measured, and four evaluation indicators (25(OH)D, 1,25(OH)2D, 24,25(OH)2D, and...) were obtained.

[0116] 3-epi-25(OH)D). The specific detection method is as follows:

[0117] Transfer 50 μL of plasma sample, add 5 μL of internal standard solution and 245 μL of cold acetonitrile to precipitate proteins, vortex to mix, centrifuge (4℃, 12000 rpm, 10 min), collect the supernatant and dry it under nitrogen, redissolve it with 50 μL of 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) solution, react at room temperature for 1.5 h, and then add 20 μL of anhydrous ethanol to terminate the reaction. Dry the sample again under nitrogen, redissolve it with 50 μL of methanol-water solution containing 0.1% formic acid (4:1, v / v), centrifuge (4℃, 12000 rpm, 10 min), collect the supernatant, filter it through a 0.22 μM filter membrane, and analyze it using a triple quadrupole LC-MS / MS (Shimadzu LCMS 8060RX). Details are as follows:

[0118] The optimal chromatographic conditions are as follows: the column is an Agilent ZORBAX Eclipse Plus C18 (2.1 × 100 mm, 1.8 μm); mobile phase A is ultrapure water (containing 5 mM ammonium bicarbonate and 0.004% formic acid); mobile phase B is chromatographically pure methanol (containing 2 mM ammonium formate and 0.16% formic acid). The column temperature is 45 °C, and the injection volume is 3 μL. The gradient elution conditions are shown in Table 5.

[0119] Table 5 Chromatographic gradient elution conditions

[0120] Time (min) Phase A Phase B Flow rate 0.0 30％ 70％ 0.50 2.0 25％ 75％ 0.50 2.5 20％ 80％ 0.50 3.0 20％ 80％ 0.50 3.2 18％ 82％ 0.50 4.0 16.3％ 83.7％ 0.50 6.0 13％ 87％ 0.55 7.0 11％ 89％ 0.55 9.0 8％ 92％ 0.60 9.1 30％ 70％ 0.50

[0121] The optimal mass spectrometry conditions are as follows: electrospray ionization source, positive ion mode, multiple reaction monitoring, nebulizer gas flow rate 3 L / min, heating gas flow rate 10 L / min, drying gas flow rate 10 L / min, interface temperature 300 °C, desolventizing temperature 526 °C, DL temperature 250 °C, and heating block temperature 400 °C. The mass spectrometry ion pair parameters are shown in Table 6.

[0122] Table 6 Mass spectrometry ion pair parameters

[0123]

[0124] The discovery cohort included 63 patients with VDDR IA, 85 patients with VDDR IB, 51 patients with VDDR II, 29 patients with IIH, and the remainder were healthy controls. The validation cohort included 32 patients with VDDR IA, 51 patients with VDDR IB, 34 patients with VDDR II, 17 patients with IIH, and the remainder were healthy controls. XGBoost ensemble learning was used to build the prediction model on the training set, and blind testing was performed on the validation set. The confusion matrix parameters for these four subtypes are shown in Table 7 below. AUC was calculated using the DeLong method, and confidence intervals were obtained through 2000 Bootstrap resampling iterations. Sensitivity / specificity confidence intervals were...

[0125] The Clopper-Pearson exact method was used to calculate the results, which are shown in Table 8.

[0126] Table 7. Parameters of the Confusion Matrix for Four Subtypes

[0127] Subtype Validation set (907 samples) TP FP FN TN VDDR IA There were 32 patients 28 54 4 821 VDDR IB There were 51 patients 44 42 7 814 VDDR II There were 34 patients 28 52 6 821 IIH There were 17 patients 13 33 4 857

[0128] Table 8 validates the diagnostic efficacy of the four subtypes.

[0129] Subtype AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) VDDR IA 0.91(0.86-0.95) 87.5％(73.2-95.0％) 93.8％(91.6-95.6％) VDDR IB 0.93(0.89-0.96) 86.3％(75.3-93.1％) 95.1％(93.2-96.6％) VDDR II 0.89(0.83-0.94) 82.4％(68.2-91.3％) 94.0％(91.8-95.8％) IIH 0.85(0.77-0.91) 76.5％(56.3-89.7％) 96.3(94.7-97.5％)

[0130] As shown in Table 8, when the sample size of patients with subtype IIH was expanded, the AUC of all subtypes exceeded 0.80, which met the requirements for clinical auxiliary diagnosis.

[0131] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An early classification system for vitamin D metabolism deficiency syndrome, characterized in that, Includes a data acquisition module and a vitamin D metabolism deficiency syndrome subtyping module; The data acquisition module, based on biomass energy product testing, is used to obtain the concentration of vitamin D metabolites in the subject's blood sample and submit it to the vitamin D metabolism deficiency syndrome typing module; the vitamin D metabolites include 1,25(OH)2D and 24,25(OH)2D; The vitamin D metabolism deficiency syndrome typing module classifies subjects based on a comparison of their 1,25(OH)2D and 24,25(OH)2D concentrations with the corresponding metabolite concentrations in healthy individuals, according to the following method: Compared to the corresponding metabolite concentrations in the blood samples of healthy individuals, if the concentrations of both 1,25(OH)2D and 24,25(OH)2D in a subject decrease, the subject is classified as having type I vitamin D-dependent rickets. Compared to the corresponding metabolite concentrations in blood samples from healthy individuals, if the concentrations of both 1,25(OH)2D and 24,25(OH)2D in a subject are elevated, the subject is classified as having type II vitamin D-dependent rickets. Compared to the corresponding metabolite concentrations in blood samples from healthy individuals, if a subject's 1,25(OH)2D concentration increases while their 24,25(OH)2D concentration decreases, the subject is classified as having idiopathic hypercalcemia.

2. The early classification system as described in claim 1, characterized in that, The data acquisition module is also used to acquire the concentration of 25(OH)D in the subject's blood sample; The vitamin D metabolism deficiency syndrome typing module further types the syndrome based on a comparison of the subject's concentrations of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D with the corresponding metabolite concentrations of healthy individuals, as follows: Compared to the corresponding metabolite concentrations in blood samples from healthy individuals, if the concentrations of 1,25(OH)2D and 24,25(OH)2D in the subject are both decreased, while the concentration of 25(OH)D is not statistically different from that of healthy individuals or falls within the normal concentration range, then the subject is classified as having type IA vitamin D-dependent rickets. Compared to the corresponding metabolite concentrations in the blood samples of healthy individuals, if the concentrations of 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D in a subject are all decreased, then the subject is classified as having type IB vitamin D-dependent rickets.

3. The early classification system as described in claim 2, characterized in that, The data acquisition module is used to acquire the concentrations of D3 metabolites 25(OH)D3, 1,25(OH)2D3, 24,25(OH)2D3 and D2 metabolites 25(OH)D2, 1,25(OH)2D2, 24,25(OH)2D2 in the subject's blood sample, and to calculate the concentrations of 25(OH)D, 1,25(OH)2D and 24,25(OH)2D by summing the concentrations of D3 metabolites and D2 metabolites.

4. The early classification system as described in claim 3, characterized in that, The data acquisition module also acquires the concentrations of D3 metabolite 3-epi-25(OH)D3 and D2 metabolite 3-epi-25(OH)D2 in the subject's blood sample to exclude false positives for 25(OH)D3 and 25(OH)D2.

5. The early classification system as described in claim 4, characterized in that, The blood sample was whole blood, plasma, or serum. The D3 and D2 metabolites were detected using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) as follows: The blood sample to be tested, internal standard solution, and protein precipitant were mixed thoroughly. After protein precipitation, the supernatant was collected, dried under nitrogen, and then reconstituted with a derivatization solution for derivatization. After the reaction was completed, the sample was dried under nitrogen, and a reconstituted solution containing formic acid was added. The supernatant was collected by centrifugation and used as the injection sample. The derivatization solution used DAPTAD or PTAD as the derivatizing agent, and the solvent was selected from ethyl acetate or acetonitrile. The derivatization reaction lasted 1.5 to 2 hours. The injected samples were separated by chromatographic elution of derivatized 1,25(OH)2D3, 24,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D2, 25(OH)D3, 25(OH)D2, and epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2. The D3 and D2 metabolites were quantitatively detected by mass spectrometry.

6. A screening kit for early vitamin D metabolism deficiency syndrome, characterized in that, The early screening kit includes an internal standard for the quantitative detection of isotopic 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2 in blood samples, as well as a derivatization reagent.

7. The early screening kit as described in claim 6, characterized in that, The early screening kit also includes isotopic internal standards for the quantitative detection of 25(OH)D3 and 25(OH)D2.

8. The early screening kit as described in claim 7, characterized in that, The derivatizing reagent is DAPTAD.

9. The early screening kit as described in claim 8, characterized in that, The early screening kit also includes mobile phase A and mobile phase B for chromatographic elution separation of 25(OH)D3, 25(OH)D2 and epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2.

10. The early screening kit as described in claim 9, characterized in that, The mobile phase A is an aqueous solution containing 5 mM ammonium bicarbonate and 0.004% formic acid, and the mobile phase B is a methanol solution containing 2 mM ammonium formate and 0.16% formic acid.

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