Serum metabolite composition, kit and application

Abstract

The invention discloses a novel serum metabolite composition based on dried blood spot detection and elaborates application of the serum metabolite composition as a marker in preparation of a congenital hypothyroidism diagnostic reagent or kit. The serum metabolite composition comprises thyroxine, 2-piperidone and phosphorylcholine PC (14: 0 / 20: 4). The serum metabolite composition can be used for screening and diagnosing patients with the congenital hypothyroidism, and has the characteristics of low detection cost, good repeatability and relatively high sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the application of a novel serum combined biomarker in congenital hypothyroidism. It belongs to the fields of analytical chemistry, clinical medicine, and medicine. Background Technology

[0002] Congenital hypothyroidism (CH) is the most common congenital endocrine disorder affecting newborns, with an incidence of approximately 1 in 2,000 to 3,000 live births (depending on race and region) (Reference 1: Rose SR, Wassner AJ, Wintergerst KA, et al. Congenital Hypothyroidism: Screening and Management[J]. PEDIATRICS, 2023, 151(1)). CH results in decreased levels of thyroid hormones in the blood of infants. Given the crucial role of thyroid hormones in neonatal growth and development, particularly intellectual development, decreased thyroid hormone levels due to CH have been identified as a leading cause of preventable intellectual disability worldwide (Reference 2: Hypothyroidism[J]. Nature Reviews Disease Primers, 2022, 8(1)). To mitigate the harmful effects of neonatal hypothyroidism (CH), newborn screening programs began incorporating CH testing in 1973, a practice that continues to this day (Reference 3: Dussault JH, Laberge C. Thyroxine (T4) determination in dried blood by radioimmunoassay: A screening method for neonatal hypothyroidism[J]. L'uniónmédicaledu Canada, 1973, 102(10):2062-2064.). Currently, the main methods for diagnosing neonatal CH include initial screening for thyroid-stimulating hormone (TSH), followed by secondary screening to measure TSH and thyroid hormone levels. This two-tiered approach allows for early detection and subsequent treatment of CH (Reference 4: Van Trotsenburg P, Stoupa A, Leger J, et al. Congenital Hypothyroidism: A 2020-2021 Consensus Guidelines Update - An ENDO - European Reference Network Initiative Endorsed by the European Society for Pediatric Endocrinology and the European Society for Endocrinology[J]. Thyroid: official journal of the American Thyroid Association, 2021(3):31).

[0003] The introduction of newborn screening can diagnose congenital hypothyroidism (CH) in a timely manner, thereby reducing its impact on newborn development. However, the TSH cutoff level varies in different regions. Too high a cutoff level may cause newborns with CH to miss the optimal treatment time, while too low a cutoff level may lead to more follow-up cases, increasing the burden on patients' families (Reference 5: Levaillant L, Huet F, Bretones P, et al. Neonatal screening for congenital hypothyroidism: Time to lower the TSH threshold in France[J]. Archives de pediatrie:organe officiel de la Societe francaise depediatrie,2022,29(4):253-257). Therefore, it is necessary to use metabolomics to study the metabolic pathways affected by CH and to find new biomarkers for CH screening.

[0004] This invention utilizes ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QFS-MS) to perform non-targeted metabolomics analysis on dried blood smears from newborns with congenital hypothyroidism. Using binary logistic regression analysis, potential combined biomarkers distinguishing congenital hypothyroidism from hyperthyroidism and healthy newborns were screened. These biomarkers include thyroxine, 2-piperidone, and phosphocholine PC (14:0 / 20:4), and this combined biomarker can effectively diagnose congenital hypothyroidism in newborns. Summary of the Invention

[0005] The purpose of this invention is to address the screening problem of congenital hypothyroidism in newborns by providing a novel serum metabolite combined biomarker based on dried blood smear detection for the diagnosis of congenital hypothyroidism, and to provide a kit and analytical detection method for the serum metabolites used in the above combination.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] (1) Metabolomics analysis was performed on dried blood smears of patients with congenital hypothyroidism, including healthy neonatal controls, hyperthyroidism, and patients with congenital hypothyroidism, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0008] The metabolomics analysis method based on ultra-high performance liquid chromatography-tandem mass spectrometry is as follows:

[0009] Liquid chromatography conditions: The metabolite separation system was an ultra-high performance liquid chromatography (UPLC) system (Shimadzu, Kyoto City, Kyoto Prefecture, Japan), using a Waters BEH C8 column (2.1 × 50 mm, 1.7 μm, Waters, Milford, MA, USA). The flow rate was set to 0.40 mL / min. The column temperature and sample chamber temperature were set to 60 °C and 4 °C, respectively. The eluent solutions were: mobile phase A, 0.1%–0.5% formic acid / water solution (v / v); mobile phase B, 0.1%–0.5% formic acid / acetonitrile solution (v / v).

[0010] Mass spectrometry conditions: The mass spectrometer was an AB SCIEX Triple TOF 7600 system (AB SCIEX, Framingham, MA). Electrospray ionization in positive ion mode was used for detection, with a scan range of m / z 60-1000. The operating parameters of the mass spectrometer were as follows: ion ejection voltage 5500 V; declustering voltage 30 V; collision energy 10 V; interface heater temperature 550 °C. Quantification of metabolites: The relative concentration of each metabolite was obtained by dividing the peak area of ​​each metabolite in the chromatogram by the peak area of ​​the corresponding internal standard. The internal standard for thyroxine was tryptophan-d5, the internal standard for 2-piperidinone was tryptophan-d5, and the internal standard for phosphocholine PC (14:0 / 20:4) was hexadecylcarnitine-d3.

[0011] (2) Data statistics were performed using Python. Binary logistic regression (R language, glm function) was employed to randomly combine candidate differentially expressed metabolites to determine the combination of joint biomarkers. ROC (Receiver Operating Characteristic) curves were used to evaluate the sensitivity and specificity of the joint biomarkers. Considering both high sensitivity and high specificity, the joint biomarkers were determined to be thyroxine, 2-piperidone, and phosphoric acid choline PC (14:0 / 20:4).

[0012] (3) Use of combined biomarkers in the diagnosis of congenital hypothyroidism: Compared with newborns without congenital hypothyroidism (healthy + hyperthyroidism), patients with congenital hypothyroidism have decreased thyroxine and phosphocholine PC (14:0 / 20:4), while 2-piperidone concentration is increased. Using statistical software, these three metabolites were regressed to the combined biomarker variable P through binary logistic regression. Preferably, the binary logistic regression equation is as follows: P = 1 / (1 + e- (2.646-354.995*a+18.494*b-7.203*c) ),

[0013] Where a is the relative concentration of thyroxine in the dried blood sample, b is the relative concentration of 2-piperidone in the dried blood sample, and c is the relative concentration of phosphocholine PC (14:0 / 20:4) in the dried blood sample; where * represents a multiplicative relationship.

[0014] The obtained variable P was elevated in patients with congenital hypothyroidism, and its value can be used to assist in the diagnosis of congenital hypothyroidism. Based on the sample involved in the experiment, and according to the principle that the diagnostic sensitivity should not be less than 0.9, the cutoff value of this combined biomarker variable was set at 0.296. Newborns with a value greater than the cutoff value are likely to have congenital hypothyroidism. ROC analysis showed that this biomarker has good discriminative power.

[0015] (4) The advantages of this invention are: the small molecule metabolites thyroxine, 2-piperidone, and phosphocholine PC (14:0 / 20:4) in dried blood smear samples can be used in combination to differentiate between congenital hypothyroidism and non-congenital hypothyroidism (healthy individuals with hyperthyroidism), exhibiting high sensitivity and specificity. This has significant practical implications for guiding clinical screening of congenital hypothyroidism.

[0016] The kit described herein enables highly sensitive and efficient detection. The three small molecule metabolites involved in this invention are characterized by low detection cost, good reproducibility, and high sensitivity and specificity. This invention can be applied to assist in the clinical diagnosis of congenital hypothyroidism and has promising application prospects. Attached Figure Description

[0017] The above-described features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein: Figure 1 List of 27 candidate metabolites in Example 1 and their correlations.

[0018] Figure 2 The changes in the levels of metabolites thyroxine, 2-piperidone, and phosphocholine PC (14:0 / 20:4) in non-congenital hypothyroid newborns (healthy + hyperthyroidism) and patients with congenital hypothyroidism in Example 1 (mean ± standard error). In the comparison between the test groups, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0019] Figure 3 Example 1: ROC curves for distinguishing between patients with congenital hypothyroidism and those without congenital hypothyroidism (healthy individuals with hyperthyroidism).

[0020] Figure 4In Case 1, based on the principle that the sensitivity should not be less than 0.9, the sensitivity of the new serum biomarker combination was determined to be 0.907, the specificity to be 0.658, and the cutoff value to be 0.296.

[0021] Figure 5 The changes in the levels of metabolites thyroxine, 2-piperidone, and phosphocholine PC (14:0 / 20:4) in non-congenital hypothyroidism newborns (healthy + hyperthyroidism) and patients with congenital hypothyroidism in Case 2 (mean ± standard error). In the comparison between the test groups, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. Detailed Implementation

[0022] Example 1

[0023] 1. Collection of dried blood smear samples

[0024] All volunteers included in the study signed informed consent forms before sampling. Dried blood samples were collected from 45 healthy newborns, 34 patients with hyperthyroidism, and 75 patients with congenital hypothyroidism. Heel prick blood was collected from newborns within 72 hours of birth to prepare dried blood samples, which were stored at 4°C and then stored at -80°C after laboratory acceptance.

[0025] 2. Analytical Methods

[0026] 2.1 Pretreatment of dried blood smear samples

[0027] Dried blood smears were thawed at room temperature for 15 min. Three 3.175 mm diameter discs were punched into each sample, and the samples were transferred to 2 mL test tubes. The puncher was cleaned three times with blank filter paper. 150 μL of extraction buffer (methanol / acetonitrile / water, 4:4:2, v / v / v) containing 1.558 μg / mL tryptophan-d5 and 0.0825 μg / mL hexadecylcarnitine-d3 (Carnitine C16:0-d3) was added to each tube, and the mixture was extracted with shaking at room temperature for 30 min. The samples were then centrifuged (15076 g, 10 min, 4 °C), and 120 μL of the supernatant was transferred to a new test tube for lyophilization (overnight drying for 12 h). Additionally, 20 μL of the supernatant was used to prepare quality control (QC) samples. For instrumental analysis, the lyophilized samples were reconstituted with 50 μL of acetonitrile / water (1:3, v / v) solution. Finally, the sample was analyzed in positive ion mode.

[0028] 2.2 Ultra-high performance liquid chromatography-mass spectrometry analysis

[0029] (1) Liquid Chromatography Conditions: Data were analyzed using an ultra-high performance liquid chromatograph (UPLC) (Waters, Milford, MA) coupled with a Triple TOF 7600 mass spectrometer (AB SCIEX, Framingham, MA). For metabolomics analysis in positive ion mode, a Waters BEH C8 column (2.1 × 50 mm, 1.7 μm) was used for metabolite separation at a flow rate of 400 μL / min. The mobile phase consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). Gradient elution was performed for 12 minutes. The gradient started at 5% B and was maintained for 0.5 minutes; then it was linearly increased to 40% B within 1.5 minutes; and increased to 100% B within 6 minutes; it was maintained at 100% B for 2 minutes; finally, it was restored to 5% B at 10.1 minutes and reequilibrated for 2 minutes.

[0030] (2) Mass spectrometry conditions: Triple TOF 7600 mass spectrometer (AB SCIEX, Framingham, MA). Electrospray ionization positive ion mode was used for detection, with a scan range of m / z 60-1000. The operating parameters of the mass spectrometer were as follows: ion ejection voltage 5500 V; declustering voltage 30 V; collision energy 10 V; interface heater temperature 550 °C.

[0031] 2.3 Dried blood smear test results and auxiliary diagnostic methods

[0032] Metabolomics analysis was performed on dried blood smears from patients with congenital hypothyroidism, healthy individuals, and patients with hyperthyroidism using the analytical conditions described above for high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Based on retention time and primary and secondary mass spectrometry information, 239 metabolites were qualitatively identified in positive ion mode and 103 metabolites in negative ion mode. Following the 80% rule, 238 and 102 metabolites were ultimately retained in positive and negative ion modes, respectively. Seventeen metabolites were detected in both modes. After removing duplicates, the final 323 metabolites were used for subsequent metabolomics analysis. In the variable analysis between patients with congenital hypothyroidism and the control group (healthy individuals with hyperthyroidism), multivariate analysis screened 80 metabolites meeting the criteria (VIP > 1, 95% CI > 0, |P(corr)| > 0.3). In univariate analysis, 27 candidate biomarkers were finally identified from the 80 metabolites (non-parametric test, p < 0.05, FDR < 0.05). Figure 1 As shown. Using a binary logistic regression analysis method, the final combination of serum biomarkers was determined by randomly combining 27 differentially expressed metabolites, namely thyroxine, 2-piperidone, and phosphocholine PC (14:0 / 20:4).

[0033] The relative concentration of each metabolite is obtained by dividing the peak area of ​​each metabolite in the chromatogram by the peak area of ​​the corresponding internal standard.

[0034] The internal standard for thyroxine is tryptophan-d5. After dividing by the internal standard, the relative concentration range in the healthy group is greater than 0.0065 and less than 0.0207, the relative concentration range in the hyperthyroidism group is greater than 0.0019 and less than 0.0228, and the relative concentration range in the congenital hypothyroidism group is greater than 0.000083 and less than 0.0192.

[0035] The internal standard for 2-piperidinone is tryptophan-d5. After dividing by the internal standard, the relative concentration range in the healthy group is greater than 0.0045 and less than 0.205, the relative concentration range in the hyperthyroidism group is greater than 0.0051 and less than 0.134, and the relative concentration range in the congenital hypothyroidism group is greater than 0.0080 and less than 0.143.

[0036] The internal standard corresponding to phosphocholine PC (14:0 / 20:4) is hexadecylcarnitine-d3. After dividing by the internal standard, the relative concentration range in the healthy group is greater than 0.0264 and less than 0.318, the relative concentration range in the hyperthyroidism group is greater than 0.0260 and less than 0.440, and the relative concentration range in the congenital hypothyroidism group is greater than 0.0380 and less than 0.322.

[0037] Quantitative analysis of the relative concentrations of thyroxine, 2-piperidinone, and phosphoric acid choline (PC) (14:0 / 20:4) in individuals with non-congenital hypothyroidism (healthy individuals with hyperthyroidism) and those with congenital hypothyroidism is shown in the table below. Figure 1 CH stands for congenital hypothyroidism; High TSH stands for hyperthyroidism; and Health stands for healthy individuals. Compared to patients without congenital hypothyroidism (healthy individuals with hyperthyroidism), patients with congenital hypothyroidism had decreased levels of thyroxine and phosphocholine (PC) (14:0 / 20:4), while their 2-piperidone concentration was increased.

[0038] Data statistics were performed using Python. The three metabolites were regressed into a joint biomarker variable P using binary logistic regression (R language, glm function). Preferably, the binary logistic regression equation is as follows:

[0039] P = 1 / (1 + e-) (2.646-354.995*a+18.494*b-7.203*c) ),

[0040] Where a is the relative concentration of thyroxine in the dried blood sample, b is the relative concentration of 2-piperidone in the dried blood sample, and c is the relative concentration of phosphocholine PC (14:0 / 20:4) in the dried blood sample; where * represents a multiplicative relationship.

[0041] Figure 2 In the figure, the area under the ROC curve is 0.907. Figure 3 In this study, the variable P obtained from discriminant analysis of the combined biomarker was elevated in patients with congenital hypothyroidism. When the P variable was used to distinguish between patients with congenital hypothyroidism and controls, the cutoff value for the combined biomarker variable was set at 0.296. The sensitivity was 0.907 and the specificity was 0.658. Figure 3 This demonstrates that the combined biomarker has a good screening effect on congenital hypothyroidism.

[0042] Example 2

[0043] To verify the diagnostic efficacy of the combined biomarker for patients with congenital hypothyroidism, 53 healthy newborn samples, 38 patients with hyperthyroidism, and 84 newborn samples with congenital hypothyroidism were added for validation. The analytical methods, instruments, and reagents used in this experiment were completely consistent with those in Example 1.

[0044] 1. Before collecting dried blood smear samples, volunteers must sign an informed consent form.

[0045] Under the same conditions, samples from 53 healthy newborns, 38 patients with hyperthyroidism, and 84 newborns with congenital hypothyroidism were collected, preserved, and prepared for testing according to the method described in Example 1.

[0046] 2. Verification of diagnostic results from dried blood smear samples

[0047] Quantitative analysis of the relative concentrations of thyroxine, 2-piperidinone, and phosphoric acid choline (PC) (14:0 / 20:4) in individuals with non-congenital hypothyroidism (healthy individuals with hyperthyroidism) and those with congenital hypothyroidism is shown in the table below. Figure 4 CH stands for congenital hypothyroidism; High TSH stands for hyperthyroidism; and Health stands for healthy individuals. Compared to individuals without congenital hypothyroidism (healthy individuals with hyperthyroidism), patients with congenital hypothyroidism had decreased levels of thyroxine and phosphocholine (PC) (14:0 / 20:4), while their 2-piperidone levels were increased.

[0048] The levels of each biomarker were substituted into the regression equation described in Example 1 to calculate the probability P. When this combined biomarker was used for the diagnosis of early-stage lung cancer patients through the discriminant variable P, the optimal cutoff value of 0.296 described in Example 1 was still used. That is, when the probability P value of the combined biomarker was greater than 0.296, it was considered to be congenital hypothyroidism. The diagnostic results are as follows: the area under the ROC curve was 0.814, the sensitivity was 0.821, and the specificity was 0.648; it can be seen that this group of biomarkers has a good diagnostic effect on congenital hypothyroidism.

[0049] The reagent kit described in this invention features low detection cost and good stability in the diagnosis of congenital hypothyroidism. This invention can also be applied to the clinical diagnosis of congenital hypothyroidism with good sensitivity and specificity. In summary, this invention has high development value.

[0050] It should be understood that although the invention has been specifically shown and described with reference to its exemplary embodiments, those skilled in the art will understand that various changes in form and detail may be made therein, and various combinations of embodiments may be made, without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A serum metabolite composition, said serum metabolite composition comprising: Thyroxine (also known as tetraiodothyronine), 2-piperidinone, and phosphocholine PC (14:0 / 20:4).

2. The serum metabolite composition according to claim 1, characterized in that, The mass ratio of each component in the serum metabolite composition is: thyroxine, 2-piperidone and phosphocholine PC (14:0 / 20:4) = 3.5:3.5:1-2.2:2.2:

1.

3. The use of the serum metabolite composition of claim 1 or 2 in the preparation of a diagnostic reagent or kit for diagnosing congenital hypothyroidism.

4. The application according to claim 3, characterized in that, The diagnostic reagent or kit includes: standards for each component, dried blood smear sample extraction solution, internal standard required for detecting the concentration of the serum metabolite, and extraction solution containing the internal standard; (1) The standard products include: thyroxine; 2-piperidone; phosphoric acid choline PC (14:0 / 20:4); (2) Extraction solution containing internal standard: The extraction solution is a methanol / acetonitrile / ultrapure aqueous solution containing 1.8-1.4 μg / mL tryptophan-d5 and 0.12-0.07 μg / mL hexadecylcarnitine-d3 in a volume ratio of 4.5:4.5:1-3.5:3.5:

3.

5. The application according to claim 4, characterized in that, The diagnostic reagent or kit also includes a chromatographic column and eluent. The chromatographic column is a Waters BEH C8 column, 2.1 mm × 50 mm, 1.7 μm. The eluent composition is: mobile A, 0.1%-0.5% formic acid / water solution (v / v); mobile B, 0.1%-0.5% formic acid / acetonitrile solution (v / v).

6. The application according to claim 4 or 5, characterized in that, First, dried blood samples from subjects were processed with a dried blood sample extraction solution. After shaking extraction, the supernatant containing metabolites was collected by centrifugation, then lyophilized, reconstituted, and subjected to metabolomics analysis based on liquid chromatography-tandem mass spectrometry. Chromatographic elution was performed using eluents. The chromatographic column was a Waters BEH C8 column, 2.1 mm × 50 mm, 1.7 μm. The eluent composition was: mobile A, 0.5%–0.1% formic acid / water solution (v / v); and mobile B, 0.5%–0.1% formic acid / acetonitrile solution (v / v).

7. The application according to any one of claims 3-6, using the diagnostic reagent or kit to detect various serum metabolites according to claim 1, comprising the following steps: (1) By detecting the relative concentrations of thyroxine, 2-piperidone, and phosphocholine PC (14:0 / 20:4) in dried blood smears of newborns, the probability P-value of the joint biomarker variable was calculated based on the relative concentrations of the three metabolites using a binary logistic regression equation. Then, based on the determined cutoff values, patients with congenital hypothyroidism and those without congenital hypothyroidism (newborn healthy controls + hyperthyroidism) were distinguished; and (2) By detecting the relative concentrations of thyroxine, 2-piperidone and phosphocholine PC (14:0 / 20:4) in dried blood samples from patients with congenital hypothyroidism, the P value is calculated by substituting the relative concentration values ​​of the three metabolites into the above equation, and then the patients with congenital hypothyroidism are diagnosed based on the cutoff value determined above.

8. The application according to claim 7, characterized in that, The logistic regression discriminant formula for distinguishing between patients with congenital hypothyroidism and those without (healthy newborn controls + hyperthyroidism) is as follows: P=1 / (1+e -(2.646-354.995*a+18.494*b-7.203*c) ), Where a is the relative concentration of thyroxine in the dried blood sample, b is the relative concentration of 2-piperidinone in the dried blood sample, and c is the relative concentration of phosphocholine PC (14:0 / 20:4) in the dried blood sample; where * represents a multiplicative relationship. The cutoff value is 0.

296. A P-value greater than this cutoff value indicates that the subject has congenital hypothyroidism, while a P-value less than or equal to this cutoff value indicates that the subject does not have congenital hypothyroidism and is a non-congenital hypothyroidism subject.

9. A reagent kit for diagnosing congenital hypothyroidism in newborns, characterized in that, The kit contains standards: thyroxine, 2-piperidinone, and phosphocholine PC (14:0 / 20:4), with a mass ratio of thyroxine, 2-piperidinone, and phosphocholine PC (14:0 / 20:4) of 3.5:3.5:1 to 2.2:2.2:

1.

10. The reagent kit according to claim 9, characterized in that, It also includes an extraction solution containing an internal standard: the extraction solution is a methanol / acetonitrile / ultrapure aqueous solution containing 1.8-1.4 μg / mL tryptophan-d5 and 0.12-0.07 μg / mL hexadecylcarnitine-d3 in a volume ratio of 4.5:4.5:1 to 3.5:3.5:3.

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